U.S. patent application number 10/023523 was filed with the patent office on 2002-10-17 for novel low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis.
This patent application is currently assigned to Boston Heart Foundation, Inc., a Massachusetts corporation. Invention is credited to Arjona, Anibal A., Law, Simon W., Lees, Ann M., Lees, Robert S..
Application Number | 20020152485 10/023523 |
Document ID | / |
Family ID | 27059254 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020152485 |
Kind Code |
A1 |
Lees, Ann M. ; et
al. |
October 17, 2002 |
Novel low density lipoprotein binding proteins and their use in
diagnosing and treating atherosclerosis
Abstract
Isolated polynucleotides encoding novel polypeptides which are
capable of binding to native and methylated LDL (low density
lipoprotein), the isolated polypeptides, called LBPs (LDL binding
proteins), and biologically active fragments and analogs thereof,
are described. Also described are methods for determining if an
animal is at risk for atherosclerosis, methods for evaluating an
agent for use in treating atherosclerosis, methods for treating
atherosclerosis, and methods for treating a cell having an
abnormality in structure or metabolism of LBP. Pharmaceutical
compositions and vaccine compositions are also provided.
Inventors: |
Lees, Ann M.; (Brookline,
MA) ; Lees, Robert S.; (Brookline, MA) ; Law,
Simon W.; (Lexington, MA) ; Arjona, Anibal A.;
(Boston, MA) |
Correspondence
Address: |
LOUIS MYERS
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Assignee: |
Boston Heart Foundation, Inc., a
Massachusetts corporation
|
Family ID: |
27059254 |
Appl. No.: |
10/023523 |
Filed: |
December 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10023523 |
Dec 17, 2001 |
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09616289 |
Jul 14, 2000 |
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09616289 |
Jul 14, 2000 |
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09517849 |
Mar 2, 2000 |
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09517849 |
Mar 2, 2000 |
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08979608 |
Nov 26, 1997 |
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60031930 |
Nov 27, 1996 |
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60048547 |
Jun 3, 1997 |
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Current U.S.
Class: |
800/13 ; 435/7.1;
514/1; 530/359; 530/388.23; 536/23.5 |
Current CPC
Class: |
G01N 2800/323 20130101;
A61K 49/0004 20130101; C07K 14/705 20130101; G01N 33/92 20130101;
G01N 2800/044 20130101; A61P 9/10 20180101; A61K 38/00 20130101;
A61K 39/00 20130101; G01N 33/6893 20130101; C07K 14/47 20130101;
A61K 48/00 20130101 |
Class at
Publication: |
800/13 ;
536/23.5; 530/359; 530/388.23; 435/7.1; 514/1 |
International
Class: |
A01K 067/00; G01N
033/53; A61K 031/00 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a member selected from the
group consisting of: (a) a polynucleotide encoding the polypeptide
comprising the amino acid sequence as set forth in SEQ ID NO:1; (b)
a polynucleotide encoding the polypeptide comprising the amino acid
sequence as set forth in SEQ ID NO:2; (c) a polynucleotide encoding
the polypeptide comprising the amino acid sequence as set forth in
SEQ ID NO:3; (d) a polynucleotide encoding the polypeptide
comprising the amino acid sequence as set forth in SEQ ID NO:4; (e)
a polynucleotide encoding the polypeptide comprising the amino acid
sequence as set forth in SEQ ID NO:5; (f) a polynucleotide encoding
the polypeptide comprising the amino acid sequence as set forth in
SEQ ID NO:6; (g) a polynucleotide encoding the polypeptide
comprising the amino acid sequence as set forth in SEQ ID NO:7; (h)
a polynucleotide encoding the polypeptide comprising the amino acid
sequence as set forth in SEQ ID NO:8; (i) a polynucleotide encoding
the polypeptide comprising the amino acid sequence as set forth in
SEQ ID NO:9; (j) a polynucleotide encoding the polypeptide
comprising the amino acid sequence as set forth in SEQ ID NO:43;
(k) a polynucleotide encoding the polypeptide comprising the amino
acid sequence as set forth in SEQ ID NO:44; (l) a polynucleotide
encoding the polypeptide comprising the amino acid sequence as set
forth in SEQ ID NO:47; (m) a polynucleotide capable of hybridizing
to and which is at least about 95% identical to the polynucleotide
of (a)-(k) or (l) wherein the encoded polypeptide is capable of
binding to LDL; and (n) a biologically active fragment of
polynucleotide (a)-(l) or (m) wherein the encoded polypeptide is
capable of binding to LDL.
2. The isolated polynucleotide of claim 1 wherein said member is
selected from the group consisting of: (a) a polynucleotide
encoding the polypeptide comprising the amino acid residues 329-343
(SEQ ID NO:19), 329-354 (SEQ ID NO:20), 344-354 (SEQ ID NO:21) or
529-538 (SEQ ID NO:22) of the amino acid sequence as set forth in
SEQ ID NO:43; (b) a polynucleotide encoding the polypeptide
comprising the amino acid residues 14-43 (SEQ ID NO:23) or 38-43
(SEQ ID NO:24) of the amino acid sequence as set forth in SEQ ID
NO: 1 and SEQ ID NO:6; (c) a polynucleotide encoding the
polypeptide comprising the amino acid residues 338-353 (SEQ ID
NO:25), 338-365 (SEQ ID NO:26), 354-365 (SEQ ID NO:27) or 444-453
(SEQ ID NO:28) of the amino acid sequence as set forth in SEQ ID
NO:47; (d) a polynucleotide encoding the polypeptide comprising the
amino acid residues 96-110 (SEQ ID NO:29) of the amino acid
sequence as set forth in SEQ ID NO:5; (e) a polynucleotide encoding
the polypeptide comprising the amino acid residues 69-75 (SEQ ID
NO:41) of the amino acid sequence as set forth in SEQ ID NO: 44;
(f) a polynucleotide capable of hybridizing to and which is at
least about 95% identical to the polynucleotide of (a)-(d) or (e)
wherein the encoded polypeptide is capable of binding to LDL; and
(g) a biologically active fragment of polynucleotide (a)-(e) or (f)
wherein the encoded polypeptide is capable of binding to LDL.
3. The polynucleotide of claim 1 wherein said polynucleotide
comprises the nucleic acid as set forth in SEQ ID NO:10.
4. The polynucleotide of claim 1 wherein said polynucleotide
comprises the nucleic acid as set forth in SEQ ID NO:48.
5. The polynucleotide of claim 1 wherein said polynucleotide
comprises the nucleic acid as set forth in SEQ ID NO:14.
6. The polynucleotide of claim 1 wherein said polynucleotide
comprises the nucleic acid as set forth in SEQ ID NO:15.
7. The polynucleotide of claim 1 wherein said polynucleotide
comprises the nucleic acid as set forth in SEQ ID NO:45.
8. The polynucleotide of claim 1 wherein said polynucleotide
comprises the nucleic acid as set forth in SEQ ID NO:46.
9. The polynucleotide of claim 1 wherein said polynucleotide is
genomic DNA.
10. A recombinant vector comprising said polynucleotide of claim
1.
11. A cell comprising said recombinant vector of claim 10.
12. A method for producing an LDL binding protein comprising
culturing a cell of claim 11 under conditions that permit
expression of said LDL binding protein.
13. An isolated polypeptide comprising a member selected from the
group consisting of: (a) a polypeptide having the amino acid
sequence as set forth in SEQ ID NO:1; (b) a polypeptide having the
amino acid sequence as set forth in SEQ ID NO:2; (c) a polypeptide
having the amino acid sequence as set forth in SEQ ID NO:3; (d) a
polypeptide having the amino acid sequence as set forth in SEQ ID
NO:4; (e) a polypeptide having the amino acid sequence as set forth
in SEQ ID NO:5; (f) a polypeptide having the amino acid sequence as
set forth in SEQ ID NO:6; (g) a polypeptide having the amino acid
sequence as set forth in SEQ ID NO:7; (h) a polypeptide having the
amino acid sequence as set forth in SEQ ID NO:8; (i) a polypeptide
having the amino acid sequence as set forth in SEQ ID NO:9; (j) a
polypeptide having the amino acid sequence as set forth in SEQ ID
NO:43; (k) a polypeptide having the amino acid sequence as set
forth in SEQ ID NO:44; (l) a polypeptide having the amino acid
sequence as set forth in SEQ ID NO:47; (m) a polypeptide which is
at least about 95% identical to the polypeptide of (a)-(k) or (l)
wherein said polypeptide is capable of binding to LDL; and (n) a
biologically active fragment of polypeptide (a)-(l) or (m) wherein
said fragment is capable of binding to LDL.
14. The isolated polypeptide of claim 13 wherein said member is
selected from the group consisting of: (a) a polypeptide having the
amino acid residues 329-343 (SEQ ID NO:19), 329-354 (SEQ ID NO:20),
344-354 (SEQ ID NO:21) or 529-538 (SEQ ID NO:22) of the amino acid
sequence as set forth in SEQ ID NO: 43; (b) a polypeptide having
the amino acid residues 14-43 (SEQ ID NO:23) or 38-43 (SEQ ID
NO:24) of the amino acid sequence as set forth in SEQ ID NO: 1 and
SEQ ID NO:6; (c) a polypeptide having the amino acid residues
338-353 (SEQ ID NO:25), 338-365 (SEQ ID NO:26), 354-365 (SEQ ID
NO:27) or 444-453 (SEQ ID NO:28) of the amino acid sequence as set
forth in SEQ ID NO:47; (d) a polypeptide having the amino acid
residues 96-110 (SEQ ID NO:29) of the amino acid sequence as set
forth in SEQ ID NO:5; (e) a polypeptide having the amino acid
residues 69-75 (SEQ ID NO:41) of the amino acid sequence as set
forth in SEQ ID NO:44; (f) a polypeptide which is at least about
95% identical to the polypeptide of (a)-(d) or (e) wherein said
polypeptide is capable of binding to LDL; and (g) a biologically
active fragment of polypeptide (a)-(e) or (f) wherein said fragment
is capable of binding to LDL.
15. A method for determining if an animal is at risk for
atherosclerosis, comprising: providing an animal; and evaluating an
aspect of LBP metabolism or structure in said animal, an
abnormality in said aspect of LBP metabolism or structure being
diagnostic of being at risk for atherosclerosis.
16. The method of claim 15 wherein said LBP is selected from the
group consisting of LBP-1, LBP-2 and LBP-3.
17. A method for evaluating an agent for use in treating
atherosclerosis, comprising: providing a test cell, cell-free
system or animal; providing an agent; administering said agent to
said test cell, cell-free system or animal in a therapeutically
effective amount; and evaluating the effect of said agent on an
aspect of LBP metabolism or structure, a change in said aspect of
LBP metabolism or structure being indicative of the usefulness of
said agent in treating atherosclerosis.
18. The method of claim 17 wherein said LBP is selected from the
group consisting of LBP-1, LBP-2 and LBP-3.
19. The agent identified in claim 17.
20. A method for evaluating an agent for the ability to alter the
binding of LBP polypeptide to a binding molecule, comprising:
providing an agent; providing LBP polypeptide; providing a binding
molecule; combining said agent, said LBP polypeptide and said
binding molecule; and detecting the formation of a complex
comprising said LBP polypeptide and said binding molecule, an
alteration in the formation of said complex in the presence of said
agent as compared to in the absence of said agent being indicative
of said agent altering the binding of said LBP binding
molecule.
21. The method of claim 20 wherein said LBP polypeptide is selected
from the group consisting of LBP-1, LBP-2 and LBP-3
polypeptide.
22. A method for evaluating an agent for the ability to bind to an
LBP polypeptide, comprising: providing an agent; providing an LBP
polypeptide; contacting said agent with said LBP polypeptide; and
evaluating the ability of said agent to bind to said LBP
polypeptide.
23. The agent identified in claim 22.
24. A method for evaluating an agent for the ability to bind to a
nucleic acid encoding an LBP regulatory sequence, comprising:
providing an agent; providing a nucleic acid encoding an LBP
regulatory sequence; contacting said agent with said nucleic acid;
and evaluating the ability of said agent to bind to said nucleic
acid.
25. The agent identified in claim 24.
26. A method for treating atherosclerosis in an animal, comprising:
providing an animal in need of treatment for atherosclerosis;
providing an agent capable of altering an aspect of LBP structure
or metabolism; administering said agent to said animal in a
therapeutically effective amount such that treatment of said
atherosclerosis occurs.
27. The method of claim 26 wherein said LBP is LBP-1, LBP-2 or
LBP-3 polypeptide or a biologically active fragment or analog
thereof.
28. A method for treating an animal at risk for atherosclerosis,
comprising: providing an animal at risk for atherosclerosis;
providing an agent capable of altering an aspect of LBP structure
or metabolism; and administering said agent to said animal in a
therapeutically effective amount such that treatment of said animal
occurs.
29. A method for treating a cell having an abnormality in structure
or metabolism of LBP, comprising: providing a cell having an
abnormality in structure or metabolism of LBP; providing an agent
capable of altering an aspect of LBP structure or metabolism; and
administering said agent to said cell in a therapeutically
effective amount such that treatment of said cell occurs.
30. A pharmaceutical composition for treating atherosclerosis in an
animal, comprising: a therapeutically effective amount of an agent,
said agent being capable of altering an aspect of LBP metabolism or
structure in said animal so as to result in treatment of said
atherosclerosis; and a pharmaceutically acceptable carrier.
31. The pharmaceutical composition of claim 30 wherein said agent
is an LBP polypeptide or nucleic acid, or active fragment or analog
thereof.
32. A vaccine composition for treating atherosclerosis in an
animal, comprising: a therapeutically effective amount of an agent,
said agent being capable of altering an aspect of LBP metabolism or
structure in said animal so as to result in treatment of said
atherosclerosis; and a pharmaceutically acceptable carrier.
33. A method for diagnosing atherosclerotic lesions in an animal,
comprising: providing an animal; providing a labeled agent capable
of binding to LBP present in atherosclerotic lesions; administering
said labeled agent to said animal under conditions which allow said
labeled agent to interact with said LBP so as to result in labeled
LBP; and determining the localization or quantification of said
labeled LBP by imaging so as to diagnose the presence of
atherosclerotic lesions in said animal.
34. A method for immunizing an animal against an LBP or fragment or
analog thereof, comprising: providing an animal having LDL;
providing an LBP or fragment or analog thereof; administering said
LBP or fragment or analog thereof to said animal so as to stimulate
antibody production by said animal to said LBP or fragment or
analog thereof such that binding of said LBP to said LDL is
altered.
35. A method of making a fragment or analog of LBP polypeptide,
said fragment or analog having the ability to bind to modified LDL
and native LDL, comprising: providing an LBP polypeptide; altering
the sequence of said LBP polypeptide; and testing said altered LBP
polypeptide for the ability to bind to modified LDL and native
LDL.
36. A method of treating a subject at risk for atherosclerosis,
comprising providing a subject at risk for atherosclerosis and
administering to the subject one or more of the following: an LBP
protein or fragment or analog thereof and an adjuvant; a nucleic
acid encoding an LBP protein; a virus or bacteria comprising a
nucleic acid encoding an LBP protein; and an edible plant
comprising a nucleic acid encoding an LBP protein.
37. A method of treating a subject at risk for atherosclerosis,
comprising: providing a subject at risk for atherosclerosis;
identifying one or more autologous LBP proteins produced by the
subject; administering to the subject an non-autologous LBP
protein, wherein the non-autologous LBP protein induces an immune
response against one or more autologous LBP proteins when
administered to the subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/517,849, filed Mar. 2, 2000, which was a continuation-in part of
U.S. Ser. No. 08/979,608, filed Nov. 26, 1997, which claimed
priority from U.S. Ser. No. 60/031,930, filed Nov. 27, 1996, and
U.S. Ser. No. 60/048,547, filed Jun. 3, 1997.
FIELD OF THE INVENTION
[0002] This invention relates to novel polypeptides (LBPs) which
bind to low density lipoprotein (LDL), polynucleotides which encode
these polypeptides, and treatments, diagnoses and therapeutic
agents for atherosclerosis.
BACKGROUND OF THE INVENTION
[0003] Atherosclerosis is the principal cause of heart attacks and
strokes. It has been reported that about 50% of all deaths in the
United States, Europe and Japan are due to atherosclerosis.
Atherosclerotic lesions in the arterial wall characterize
atherosclerosis. Cholesteryl esters (CE) are present in these
atherosclerotic lesions. Low density lipoprotein (LDL) has been
shown to be the major carrier of plasma CE, and has been implicated
as the agent by which CE enter the atherosclerotic lesions.
[0004] Scattered groups of lipid-filled macrophages, called foam
cells, are the first visible signs of atherosclerosis and are
described as type I lesions. These macrophages are reported to
contain CE derived from LDL. The macrophages recognize oxidized
LDL, but not native LDL, and become foam cells by phagocytosing
oxidized LDL. Larger, more organized collections of foam cells,
fatty streaks, represent type II lesions. These lesions further
develop into complex lesions called plaques, which can result in
impeding the flow of blood in the artery.
[0005] It is widely believed that accumulation of LDL in the artery
depends on the presence of functionally modified endothelial cells
in the arterial wall. It has been reported in animal models of
atherosclerosis that LDL, both native LDL and methylated LDL,
accumulates focally and irreversibly only at the edges of
regenerating endothelial islands in aortic lesions, where
functionally modified endothelial cells are present, but not in the
centers of these islands where endothelial regeneration is
completed. Similarly, LDL accumulates in human atherosclerotic
lesions. The mechanism by which the LDL accumulates focally and
irreversibly in arterial lesions has not heretofore been
understood.
SUMMARY OF THE INVENTION
[0006] It is an object of the invention to provide polypeptides
which bind to LDL.
[0007] It is yet another object of the invention to provide a
method for determining if an animal is at risk for
atherosclerosis.
[0008] It is yet another object of the invention to provide a
method for evaluating an agent for use in treating atherosclerosis.
It is yet another object of the invention to provide a method for
treating atherosclerosis.
[0009] Still another object of the invention is to utilize an LBP
(low density lipoprotein binding protein) gene and/or polypeptide,
or fragments, analogs and variants thereof, to aid in the
treatment, diagnosis and/or identification of therapeutic agents
for atherosclerosis.
[0010] In one aspect, the invention features an isolated
polynucleotide comprising a polynucleotide encoding the polypeptide
comprising the amino acid sequence as set forth in SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO:43; SEQ ID
NO:44; SEQ ID NO:47 or a polynucleotide capable of hybridizing to
and which is at least about 95% identical to any of the above
polynucleotides and wherein the encoded polypeptide is capable of
binding to LDL; or a biologically active fragment of any of the
above polynucleotides wherein the encoded polypeptide is capable of
binding to LDL.
[0011] In certain embodiments, the polynucleotide comprises the
nucleic acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO:45; SEQ ID NO:46;
SEQ ID NO:48.
[0012] Another aspect of the invention is an isolated polypeptide
comprising a polypeptide having the amino acid sequence as set
forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NO:
9; SEQ ID NO: 43; SEQ ID NO:44; SEQ ID NO:47; or a polypeptide
which is at least about 95% identical to any of the above
polypeptides and wherein the polypeptide is capable of binding to
LDL; or a biologically active fragment of any of the above
polypeptides wherein the fragment is capable of binding to LDL.
[0013] Another aspect of the invention is a method for determining
if an animal is at risk for atherosclerosis. An animal is provided.
An aspect of LBP metabolism or structure is evaluated in the
animal. An abnormality in the aspect of LBP metabolism or structure
is diagnostic of being at risk for atherosclerosis. Another aspect
of the invention is a method for evaluating an agent for use in
treating atherosclerosis. A test cell, cell-free system or animal
is provided. An agent is provided. The agent is administered to the
test cell, cell-free system or animal in a therapeutically
effective amount. The effect of the agent on an aspect of LBP
metabolism or structure is evaluated. A change in the aspect of LBP
metabolism or structure is indicative of the usefulness of the
agent in treating atherosclerosis.
[0014] Another aspect of the invention is a method for evaluating
an agent for the ability to alter the binding of LBP polypeptide to
a binding molecule, e.g., native LDL, modified LDL, e.g.,
methylated LDL or oxidized LDL, or an arterial extracellular matrix
structural component. An agent is provided. An LBP polypeptide is
provided. A binding molecule is provided. The agent, LBP
polypeptide and binding molecule are combined. The formation of a
complex comprising the LBP polypeptide and binding molecule is
detected. An alteration in the formation of the complex in the
presence of the agent as compared to in the absence of the agent is
indicative of the agent altering the binding of the LBP polypeptide
to the binding molecule.
[0015] Another aspect of the invention is a method for evaluating
an agent for the ability to bind to an LBP polypeptide. An agent is
provided. An LBP polypeptide is provided. The agent is contacted
with the LBP polypeptide. The ability of the agent to bind to the
LBP polypeptide is evaluated.
[0016] Another aspect of the invention is a method for evaluating
an agent for the ability to bind to a nucleic acid encoding an LBP
regulatory sequence. An agent is provided. A nucleic acid encoding
an LBP regulatory sequence is provided. The agent is contacted with
the nucleic acid. The ability of the agent to bind to the nucleic
acid is evaluated.
[0017] Another aspect of the invention is a method for treating
atherosclerosis in an animal. An animal in need of treatment for
atherosclerosis is provided. An agent capable of altering an aspect
of LBP structure or metabolism is provided. The agent is
administered to the animal in a therapeutically effective amount
such that treatment of the atherosclerosis occurs. In certain
embodiments, the agent is an LBP polypeptide, e.g., LBP-1, LBP-2 or
LBP-3, or a biologically active fragment or analog thereof. In
certain embodiments, the agent is a polypeptide of no more than
about 100, 50, 30, 20, 10, 5, 4, 3 or 2 amino acid residues in
length. In certain embodiments, the agent is a polypeptide having
an amino acid sequence that includes at least about 20%, 40%, 60%,
80%, 90%, 95% or 98% acidic amino acid residues.
[0018] Another aspect of the invention is a method for treating an
animal at risk for atherosclerosis. An animal at risk for
atherosclerosis is provided. An agent capable of altering an aspect
of LBP structure or metabolism is provided. The agent is
administered to the animal in a therapeutically effective amount
such that treatment of the animal occurs.
[0019] Another aspect of the invention is a method for treating a
cell having an abnormality in structure or metabolism of LBP. A
cell having an abnormality in structure or metabolism of LBP is
provided. An agent capable of altering an aspect of LBP structure
or metabolism is provided. The agent is administered to the cell in
a therapeutically effective amount such that treatment of the cell
occurs.
[0020] Another aspect of the invention is a pharmaceutical
composition for treating atherosclerosis in an animal comprising a
therapeutically effective amount of an agent, the agent being
capable of altering an aspect of LBP metabolism or structure in the
animal so as to result in treatment of the atherosclerosis, and a
pharmaceutically acceptable carrier.
[0021] Another aspect of the invention is a vaccine composition for
treating atherosclerosis in an animal comprising a therapeutically
effective amount of an agent, the agent being capable of altering
an aspect of LBP metabolism or structure in the animal so as to
result in treatment of the atherosclerosis, and a pharmaceutically
acceptable carrier.
[0022] Another aspect of the invention is a method for diagnosing
atherosclerotic lesions in an animal. An animal is provided. A
labeled agent capable of binding to LBP, e.g., LBP-1, LBP-2 or
LBP-3, present in atherosclerotic lesions is provided. The labeled
agent is administered to the animal under conditions which allow
the labeled agent to interact with the LBP so as to result in
labeled LBP. The localization or quantification of the labeled LBP
is determined by imaging so as to diagnose the presence of
atherosclerotic lesions in the animal.
[0023] Another aspect of the invention is a method for immunizing
an animal against an LBP, e.g., LBP-1, LBP-2 or LBP-3, or fragment
or analog thereof. An animal having LDL is provided. The LBP or
fragment or analog thereof is administered to the animal so as to
stimulate antibody production by the animal to the LBP or fragment
or analog thereof such that binding of the LBP to the LDL is
altered, e.g., decreased or increased.
[0024] Another aspect of the invention is a method of making a
fragment or analog of LBP polypeptide, the fragment or analog
having the ability to bind to native LDL and to modified LDL, e.g.,
methylated LDL, oxidized LDL, acetylated LDL, or
cyclohexanedione-treated LDL. An LBP polypeptide is provided. The
sequence of the LBP polypeptide is altered. The altered LBP
polypeptide is tested for the ability to bind to modified LDL and
native LDL.
[0025] Yet another aspect of the invention is a method for
isolating a cDNA encoding an LBP. A cDNA library is provided. The
cDNA library is screened for a cDNA encoding a polypeptide which
binds to native LDL and modified LDL, e.g., methylated LDL or
oxidized LDL. The cDNA which encodes the polypeptide is isolated,
the cDNA encoding an LBP.
[0026] The above and other features, objects and advantages of the
present invention will be better understood by a reading of the
following specification in conjunction with the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 depicts the amino acid sequence of rabbit LBP-1 (SEQ
ID NO: 1). Differences in amino acids between rabbit and human
LBP-1 are depicted in bold type.
[0028] FIG. 2A depicts the nucleotide sequence (SEQ ID NO: 48) and
amino acid sequence (SEQ ID NO: 47) of rabbit LBP-2.
[0029] FIG. 2B depicts a portion of the amino acid sequence of
rabbit LBP-2 (SEQ ID NO: 2). Differences in amino acids between
rabbit and human LBP-2 are depicted in bold type. Where the
sequences depicted in FIG. 2A and FIG. 2B differ, FIG. 2A
represents the rabbit LBP-2 sequence.
[0030] FIG. 3 depicts the amino acid sequence of amino acids 319 to
350 of rabbit LBP-2 (SEQ ID NO: 3).
[0031] FIG. 4 depicts the amino acid sequence of amino acids 299 to
350 of rabbit LBP-2 (SEQ ID No: 4).
[0032] FIG. 5 depicts the amino acid sequence of rabbit LBP-3 (SEQ
ID NO: 5). Differences in amino acids between rabbit and human
LBP-3 are depicted in bold type.
[0033] FIG. 6 depicts the amino acid sequence of human LBP-1 (SEQ
ID NO: 6). Differences in amino acids between rabbit and human
LBP-1 are depicted in bold type.
[0034] FIG. 7A depicts the nucleotide sequence (SEQ ID NO: 45) and
amino acid sequence (SEQ ID NO: 43) of human LBP-2.
[0035] FIG. 7B depicts the amino acid sequence of amino acids 322
to 538 of human LBP-2 (SEQ ID NO: 7). Differences in amino acids
between rabbit and human LBP-2 are depicted in bold type.
[0036] FIG. 8A depicts the nucleotide sequence (SEQ ID NO: 46) and
amino acid sequence (SEQ ID NO: 44) of human LBP-3.
[0037] FIG. 8B depicts the amino acid sequence of amino acids 17 to
546 of human LBP-3 (SEQ ID NO: 8). Differences in amino acids
between rabbit and human LBP-3 are depicted in bold type. Where the
sequences depicted in FIG. 8A and FIG. 8B differ, FIG. 8A
represents the human LBP-3 sequence.
[0038] FIG. 9 depicts the amino acid sequence of amino acids 14 to
33 of human or rabbit LBP-1, called BHF-1 (SEQ ID NO: 9).
[0039] FIG. 10 depicts the cDNA sequence encoding rabbit LBP-1 (SEQ
ID NO: 10) and the corresponding amino acid sequence. Differences
in amino acids between rabbit and human LBP-1 are depicted in bold
type.
[0040] FIG. 11 depicts a cDNA sequence encoding a portion of rabbit
LBP-2 (SEQ ID NO: 11) and the corresponding amino acid sequence.
Differences in amino acids between rabbit and human LBP-2 are
depicted in bold type. Where the sequences depicted in FIG. 2A and
FIG. 11 differ, FIG. 2A represents the rabbit LBP-2 sequence.
[0041] FIG. 12 depicts a cDNA sequence of nucleotides 256 to 1617
(SEQ ID NO: 12) of SEQ ID NO: 11 of rabbit LBP-2 and the
corresponding amino acid sequence.
[0042] FIG. 13 depicts a cDNA sequence of nucleotides 196 to 1617
(SEQ ID NO: 13) of SEQ ID NO: 11 of rabbit LBP-2 and the
corresponding amino acid sequence.
[0043] FIG. 14 depicts the cDNA sequence encoding rabbit LBP-3 (SEQ
ID NO: 14) and the corresponding amino acid sequence. Differences
in amino acids between rabbit and human LBP-3 are depicted in bold
type.
[0044] FIG. 15 depicts the cDNA sequence encoding human LBP-1 (SEQ
ID NO: 15) and the corresponding amino acid sequence. Differences
in amino acids between rabbit and human LBP-1 are depicted in bold
type.
[0045] FIG. 16 depicts a cDNA sequence encoding a portion of human
LBP-2 (SEQ ID NO: 16) and the corresponding amino acid sequence.
Differences in amino acids between rabbit and human LBP-2 are
depicted in bold type.
[0046] FIG. 17 depicts a cDNA sequence encoding a portion of human
LBP-3 (SEQ ID NO: 17) and the corresponding amino acid sequence.
Differences in amino acids between rabbit and human LBP-3 are
depicted in bold type. Where the sequences depicted in FIG. 8A and
FIG. 17 differ, FIG. 8A represents the human LBP-3 sequence.
[0047] FIG. 18 depicts the cDNA sequence encoding BHF-1 (SEQ ID NO:
18).
[0048] FIG. 19 corresponds to the amino acid sequence of rabbit
LBP-1 (top sequence) in alignment with the amino acid sequence of
human LBP-1 (bottom sequence).
[0049] FIG. 20 corresponds to the amino acid sequence of a portion
of the amino acid sequence of rabbit LBP-2 (top sequence) in
alignment with a portion of the amino acid sequence of human LBP-2
(bottom sequence).
[0050] FIG. 21 corresponds to the amino acid sequence of rabbit
LBP-3 (top sequence) in alignment with the amino acid sequence of a
portion of human LBP-3 (bottom sequence).
[0051] FIG. 22 depicts the genomic sequence of human LBP-1.
[0052] FIG. 23 depicts the genomic sequence of human LBP-2.
[0053] FIG. 24 depicts the genomic sequence of human LBP-3.
DETAILED DESCRIPTION
[0054] In accordance with aspects of the present invention, there
are provided novel mature human and rabbit polypeptides, LBP-1,
LBP-2 and LBP-3, and biologically active analogs and fragments
thereof, and there are provided isolated polynucleotides which
encode such polypeptides. LBP is an abbreviation for low density
lipoprotein (LDL) binding protein. The terms polynucleotide,
nucleotide and oligonucleotide are used interchangeably herein, and
the terms polypeptides, proteins and peptides are used
interchangeably herein.
[0055] This invention provides for an isolated polynucleotide
comprising a polynucleotide encoding the polypeptide having the
amino acid sequence of rabbit LBP-1 as set forth in FIG. 1 (SEQ ID
NO: 1); rabbit LBP-2 as set forth in FIG. 2A (SEQ ID NO: 47); a
portion of rabbit LBP-2 as set forth in FIG. 2B (SEQ ID NO: 2); 319
to 350 of rabbit LBP-2 as set forth in FIG. 3 (SEQ ID NO: 3); 299
to 350 of rabbit LBP-2 as set forth in FIG. 4 (SEQ ID NO: 4);
rabbit LBP-3 as set forth in FIG. 5 (SEQ ID NO: 5); human LBP-1 as
set forth in FIG. 6 (SEQ ID NO: 6); human LBP-2 as set forth in
FIG. 7A (SEQ ID NO: 43); 322 to 538 of human LBP-2 as set forth in
FIG. 7B (SEQ ID NO: 7); human LBP-3 as set forth in FIG. 8A (SEQ ID
NO: 44); 17-546 of human LBP-3 as set forth in FIG. 8B (SEQ ID NO:
8); 14 to 33 of human or rabbit LBP-1, called BHF-1, as set forth
in FIG. 9 (SEQ ID NO: 9); a polynucleotide capable of hybridizing
to and which is at least about 80% identical, more preferably at
least about 90% identical, more preferably yet at least about 95%
identical, and most preferably at least about 98% identical to any
of the above polynucleotides, and wherein the encoded polypeptide
is capable of binding to LDL; or a biologically active fragment of
any of the above polynucleotides wherein the encoded polypeptide is
capable of binding to LDL.
[0056] This invention also includes an isolated polynucleotide
comprising a polynucleotide encoding the polypeptide having amino
acid residues 329-343 (SEQ ID NO: 19), 329-354 (SEQ ID NO: 20),
344-354 (SEQ ID NO: 21) or 529-538 (SEQ ID NO: 22) of human LBP-2
as set forth in FIG. 7A (SEQ ID NO: 43); amino acid residues 14-43
(SEQ ID NO: 23) or 38-43 (SEQ ID NO: 24) of rabbit or human LBP-1
as set forth in FIG. 1 (SEQ ID NO: 1) and FIG. 6 (SEQ ID NO: 6);
amino acid residues 338-353 (SEQ ID NO: 25), 338-365 (SEQ ID NO:
26), 354-365 (SEQ ID NO: 27) or 444-453 (SEQ ID NO: 28) of rabbit
LBP-2 as set forth in FIG. 2A (SEQ ID NO: 47); amino acid residues
96-110 (SEQ ID NO: 29) of rabbit LBP-3 as set forth in FIG. 5 (SEQ
ID NO: 5); amino acid residues 69-75 (SEQ ID NO: 41) of human LBP-3
as set forth in FIG. 8A (SEQ ID NO: 44); a polynucleotide capable
of hybridizing to and which is at least about 80% identical, more
preferably at least about 90% identical, more preferably yet at
least about 95% identical, and most preferably at least about 98%
identical to any of the above polynucleotides, and wherein the
encoded polypeptide is capable of binding to LDL; or a biologically
active fragment of any of the above polynucleotides wherein the
encoded polypeptide is capable of binding to LDL.
[0057] By a polynucleotide encoding a polypeptide is meant a
polynucleotide which includes only coding sequence for the
polypeptide, as well as a polynucleotide which includes additional
coding and/or non-coding sequences. Thus, e.g., the polynucleotides
which encode for the mature polypeptides of FIGS. 1-9 (SEQ ID NOS:
1-9, 43, 44 and 47) may include only the coding sequence for the
mature polypeptide; the coding sequence for the mature polypeptide
and additional coding sequence such as a leader or secretory
sequence or a proprotein sequence; the coding sequence for the
mature polypeptide (and optionally additional coding sequence) and
non-coding sequence, such as introns or non-coding sequences 5'
and/or 3' of the coding sequence for the mature polypeptide. The
polynucleotides of the invention are also meant to include
polynucleotides in which the coding sequence for the mature
polypeptide is fused in the same reading frame to a polynucleotide
sequence which aids in expression and/or secretion of a polypeptide
from a host cell, e.g., a leader sequence. The polynucleotides are
also meant to include polynucleotides in which the coding sequence
is fused in frame to a marker sequence which, e.g., allows for
purification of the polypeptide.
[0058] The polynucleotides of the present invention may be in the
form of RNA, DNA or PNA, e.g., cRNA, cDNA, genomic DNA, or
synthetic DNA, RNA or PNA. The DNA may be double-stranded or single
stranded, and if single stranded may be the coding strand or
non-coding (anti-sense) strand.
[0059] In preferred embodiments, the polynucleotide comprises the
nucleic acid of rabbit LBP-1 as set forth in FIG. 10 (SEQ ID NO:
10); rabbit LBP-2 as set forth in FIG. 2A (SEQ ID NO:48) or FIG. 11
(SEQ ID NO:11); nucleotide 256 to 1617 of SEQ ID NO: 11 of rabbit
LBP-2 as set forth in FIG. 12 (SEQ ID NO: 12); nucleotide 196 to
1617 of SEQ ID NO: 11 of rabbit LBP-2 as set forth in FIG. 13 (SEQ
ID NO: 13); rabbit LBP-3 as set forth in FIG. 14 (SEQ ID NO: 14);
human LBP-1 as set forth in FIG. 15 (SEQ ID NO: 15); human LBP-2 as
set forth in FIG. 7A (SEQ ID NO: 45) or FIG. 16 (SEQ ID NO: 16);
human LBP-3 as set forth in FIG. 8A (SEQ ID NO: 46) or FIG. 17 (SEQ
ID NO: 17); or nucleotide 97 to 156 of rabbit LBP-1 or nucleotide
157 to 216 of human LBP-1, (BHF-1), as set forth in FIG. 18 (SEQ ID
NO: 18).
[0060] In other preferred embodiments. the polynucleotide comprises
the nucleic acid as set forth in
1 SEQ ID NO:30 (GAAGAGGAAGAAGATGATGATGAAGATGAAGATGAAGAAGATGAT), SEQ
ID NO:31 (GAAGAGGAAGAAGATGATGATGAAGATGAAGATGAAGAAGA TGAT
GTGTCAGAGGGCTCTGAAGTGCCCGAGAGTGAC), SEQ ID NO:32
(GTGTCAGAGGGCTCTGAAGTGCCCGAGAGTGAC), SEQ ID NO:33
(GAGGATGATGACCCCGATGGCTTCTTAGGC), SEQ ID NO:34
(GTGGACGTGGATGAATATGACGAGAACAAGTTCGTGGACGAA
GAAGATGGGGGCGACGGCCAGGCCGGGCCCGACGAGGGCGAGGTGGAC), SEQ ID NO:35
(GACGAGGGCGAGGTGGAC), SEQ ID NO:36
(GAGGAGGAGGAGGAGGAGGAGGAAGACGACGAGGACGACG ACGACGAC), SEQ ID NO:37
(GAGGAGGAGGAGGAGGAGGAGGAAGACGACGAGGACGACG
ACGACGACGTCGTGTCCGAGGGCTCGGAGGTGCCCGAGAGCGAT) SEQ ID NO:38
(GTCGTGTCCGAGGGCTCGGAGGTGCCCGAGAGCGAT), SEQ ID NO:39
(CCCCCCGGGAAGCCAGCCCTCCCAGGAGCC), SEQ ID NO:40
(GAGGATGGGGTCCAGGGTGAGCCCCCTGAACCTGAAGATGCA GAG), or SEQ ID NO:42
(CGTGATGTCTCTGAGGAGCTG).
[0061] The coding sequence which encodes the mature polypeptide may
be identical to the coding sequences shown in FIGS. 2A, 7A, 8A and
10-18 (SEQ ID NOS: 10-18, 45, 46, and 48) or SEQ ID NOS: 30-40 or
42, or may be a different coding sequence which coding sequence, as
a result of the redundancy or degeneracy of the genetic code,
encodes the same mature polypeptides as the DNA of FIGS. 2A, 7A, 8A
and 10-18 (SEQ ID NOS: 10-18, 45, 46, and 48) and SEQ ID NOS: 30-40
and 42.
[0062] This invention also includes recombinant vectors comprising
the polynucleotides described above. The vector can be, e.g., a
plasmid, a viral particle or a phage. In certain embodiments, the
recombinant vector is an expression vector. The vectors may also
include various marker genes which are useful in identifying cells
containing such vectors.
[0063] This invention also includes a cell comprising such a
recombinant vector. The recombinant vectors described herein can be
introduced into a host cell, e.g., by transformation, transfection
or infection.
[0064] This invention also includes a method for producing an LBP
comprising culturing such a cell under conditions that permit
expression of the LBP.
[0065] This invention also includes an isolated polypeptide
comprising a polypeptide having the amino acid sequence as set
forth in FIG. 1 (SEQ ID NO: 1); FIG. 2A (SEQ ID NO: 47); FIG. 2B
(SEQ ID NO: 2); FIG. 3 (SEQ ID NO: 3); FIG. 4 (SEQ ID NO: 4); FIG.
5 (SEQ ID NO: 5); FIG. 6 (SEQ ID NO: 6); FIG. 7A (SEQ ID NO: 43);
FIG. 7B (SEQ ID No: 7); FIG. 8A (SEQ ID NO: 44); FIG. 8B (SEQ ID
NO: 8); or FIG. 9 (SEQ ID NO: 9); or a polypeptide which is at
least about 80% identical, more preferably at least about 90%
identical, more preferably yet at least about 95% identical, and
most preferably at least about 98% identical to the above
polypeptides, and wherein said polypeptide is capable of binding to
LDL; or a biologically active fragment of any of the above
polypeptides wherein the fragment is capable of binding to LDL.
Differences in amino acids between the rabbit and human LBP-1,
LBP-2 and LBP-3 genes are depicted in bold type in the figures.
Differences in the amino acid sequences between rabbit and human
LBP-1, LBP-2 and LBP-3 are also specifically shown in FIGS. 19, 20
and 21, respectively.
[0066] This invention also includes an isolated polypeptide
comprising a polypeptide having amino acid residues 329-343 (SEQ ID
NO: 19), 329-354 (SEQ ID NO: 20), 344-354 (SEQ ID NO: 21) or
529-538 (SEQ ID NO: 22) as set forth in FIG. 7A (SEQ ID NO: 47);
amino acid residues 14-43 (SEQ ID NO: 23) or 38-43 (SEQ ID NO: 24)
as set forth in FIG. 1 (SEQ ID NO: 1) and FIG. 6 (SEQ ID NO: 6);
amino acid residues 338-353 (SEQ ID NO: 25), 338-365 (SEQ ID NO:
26), 354-365 (SEQ ID NO: 27) or 444-453 (SEQ ID NO: 28) as set
forth in FIG. 2A (SEQ ID NO: 47); amino acid residues 96-110 (SEQ
ID NO: 29) as set forth in FIG. 5 (SEQ ID NO: 5); and amino acid
residues 69-75 (SEQ ID NO: 41) as set forth in FIG. 8A (SEQ ID NO:
8); or a polypeptide which is at least about 80% identical, more
preferably at least about 90% identical, more preferably yet at
least about 95% identical, and most preferably at least about 98%
identical to the above polypeptides, and wherein said polypeptide
is capable of binding to LDL; or a biologically active fragment of
any of the above polypeptides wherein the fragment is capable of
binding to LDL.
[0067] The polypeptides of the invention are meant to include,
e.g., a naturally purified product, a chemically synthesized
product, and a recombinantly derived product.
[0068] The polypeptides can be used, e.g., to bind to LDL, thereby
inhibiting formation of atherosclerotic plaques. The polypeptides
can also be used, e.g., in gene therapy, by expression of such
polypeptides in vivo. The polypeptides can also be used in
pharmaceutical or vaccine compositions. The polypeptides can also
be used as immunogens to produce antibodies thereto, which in turn,
can be used as antagonists to the LBP polypeptides.
[0069] Without being bound by any theory, it is believed that the
LBPs provide the mechanism by which atherosclerosis is promoted
through LDL oxidation. The LBPs are believed to be required in
order for focal, irreversible LDL binding to occur at the arterial
wall, and that such binding is a critical early event in
atherosclerosis because it allows the time necessary for LDL to be
changed from its native state to a fully oxidized state.
[0070] Since oxidized, but not native, LDL is a foreign protein,
macrophages ingest it, first becoming the foam cells of type I
lesions, and subsequently forming the fatty streaks of type II
lesions.
[0071] This invention also includes a method for determining if an
animal is at risk for atherosclerosis. An animal is provided. An
aspect of LBP metabolism or structure is evaluated in the animal.
An abnormality in the aspect of LBP metabolism or structure is
diagnostic of being at risk for atherosclerosis.
[0072] By atherosclerosis is meant a disease or condition which
comprises several stages which blend imperceptibly into each other,
including irreversible binding of LDL, LDL oxidation, macrophage
recruitment, blockage of the artery and tissue death
(infarction).
[0073] By animal is meant human as well as non-human animals.
Nonhuman animals include, e.g., mammals, birds, reptiles,
amphibians, fish, insects and protozoa. Preferably, the nonhuman
animal is a mammal, e.g., a rabbit, a rodent, e.g., a mouse, rat or
guinea pig, a primate, e.g., a monkey, or a pig. An animal also
includes transgenic non-human animals. The term transgenic animal
is meant to include an animal that has gained new genetic
information from the introduction of foreign DNA, i.e., partly or
entirely heterologous DNA, into the DNA of its cells; or
introduction of a lesion, e.g., an, in vitro induced mutation,
e.g., a deletion or other chromosomal rearrangement into the DNA of
its cells; or introduction of homologous DNA into the DNA of its
cells in such a way as to alter the genome of the cell into which
the DNA is inserted, e.g., it is inserted at a location which
differs from that of the natural gene or its insertion results in a
knockout or replacement of the homologous host gene or results in
altered and/or regulatable expression and/or metabolism of the
gene. The animal may include a transgene in all of its cells
including germ line cells, or in only one or some of its cells.
Transgenic animals of the invention can serve as a model for
studying atherosclerosis or for evaluating agents to treat
atherosclerosis.
[0074] In certain embodiments, the determination for being at risk
for atherosclerosis is done in a prenatal animal.
[0075] By LBP is meant a low density lipoprotein (LDL) binding
protein which is capable of binding LDL and methylated LDL. By
methylated LDL is meant that about 50% to about 90% of the lysine
residues of LDL have a methyl group chemically attached. Methylated
LDL is not recognized by previously reported cell surface
receptors. See, e.g., Weisgraber et al., J. Biol. Chem. 253:
9053-9062 (1978). In certain embodiments, the LBP is also capable
of binding oxidized LDL. In certain preferred embodiments, the
binding of LDL to an LBP is irreversible. In certain preferred
embodiments, the LBP does not transport the LDL to any
intracellular compartment. Examples of LBPs are LBP-1, LBP-2 and
LBP-3 described herein.
[0076] By LBP metabolism is meant any aspect of the production,
release, expression, function, action, interaction or regulation of
LBP. The metabolism of LBP includes modifications, e.g., covalent
or non-covalent modifications, of LBP polypeptide. The metabolism
of LBP includes modifications, e.g., covalent or noncovalent
modifications, that LBP induces in other substances. The metabolism
of LBP also includes changes in the distribution of LBP
polypeptide, and changes LBP induces in the distribution of other
substances.
[0077] Any aspect of LBP metabolism can be evaluated. The methods
used are standard techniques known to those skilled in the art and
can be found in standard references, e.g., Auaubel et al., ed.,
Current Protocols in Mol. Biology, New York: John Wiley & Sons,
1990; Kriegler, M., ed., Gene Transfer and Expression, Stockton
Press, New York, N.Y., 1989; pDisplay gene expression system
(Invitrogen, Carlsbad, Calif.). Preferred examples of LBP
metabolism that can be evaluated include the binding activity of
LBP polypeptide to a binding molecule, e.g., LDL; the
transactivation activity of LBP polypeptide on a target gene; the
level of LBP protein; the level of LBP mRNA; the level of LBP
modifications, e.g., phosphorylation, glycosylation or acylation;
or the effect of LBP expression on transfected mammalian cell
binding of LDL.
[0078] By binding molecule is meant any molecule to which LBP can
bind, e.g., a nucleic acid, e.g., a DNA regulatory region, a
protein, e.g., LDL, a metabolite, a peptide mimetic, a non-peptide
mimetic, an antibody, or any other type of ligand. In certain
preferred embodiments, the aspect of LBP metabolism that is
evaluated is the ability of LBP to bind to native LDL and/or
methylated LDL and/or oxidized LDL. Binding to LDL can be shown,
e.g., by antibodies against LDL, affinity chromatography, affinity
coelectrophoresis (ACE) assays, or ELISA assays. See Examples. In
other embodiments, it is the ability of LBP to bind to an arterial
extracellular matrix structural component that is evaluated.
Examples of such components include proteoglycans, e.g.,
chondroitin sulfate proteoglycans and heparin sulfate
proteoglycans; elastin; collagen; fibronectin; vitronectin;
integrins; and related extracellular matrix molecules. Binding to
arterial extracellular matrix structural components can be shown by
standard methods known to those skilled in the art, e.g., by ELISA
assays. Primary antibodies to the LBP are then added, followed by
an enzyme-conjugated secondary antibody to the primary antibody,
which produces a stable color in the presence of an appropriate
substrate, and color development on the plates is measured in a
microtiter plate reader.
[0079] Transactivation of a target gene by LBP can be determined,
e.g., in a transient transfection assay in which the promoter of
the target gene is linked to a reporter gene, e.g.,
.beta.-galactosidase or luciferase, and co-transfected with an LBP
expression vector. Such evaluations can be done in vitro or in
vivo. Levels of LBP protein, mRNA or phosphorylation, can be
measured, e.g., in a sample, e.g., a tissue sample, e.g., arterial
wall, by standard methods known to those skilled in the art.
[0080] In certain embodiments an aspect of LBP structure is
evaluated, e.g., LBP gene structure or LBP protein structure. For
example, primary, secondary or tertiary structures can be
evaluated. For example, the DNA sequence of the gene is determined
and/or the amino acid sequence of the protein is determined.
Standard cloning and sequencing methods can be used as are known to
those skilled in the art. In certain embodiments, the binding
activity of an antisense nucleic acid with the cellular LBP mRNA
and/or genomic DNA is determined using standard methods known to
those skilled in the art so as to detect the presence or absence of
the target mRNA or DNA sequences to which the antisense nucleic
acid would normally specifically bind.
[0081] The risk for atherosclerosis that is determined can be a
reduced risk or an increased risk as compared to a normal animal.
For example, an abnormality which would give a reduced risk is an
inactive LBP polypeptide. An abnormality which would give an
increased risk would be, e.g., an LBP polypeptide that has higher
activity, e.g., LDL binding activity, than native LBP
polypeptide.
[0082] The invention also includes a method for evaluating an agent
for use in treating atherosclerosis. A test cell, cell-free system
or animal is provided. An agent is provided. The agent is
administered to the test cell, cell-free system or animal in a
therapeutically effective amount. The effect of the agent on an
aspect of LBP metabolism or structure is evaluated. A change in the
aspect of LBP metabolism or structure is indicative of the
usefulness of the agent in treating atherosclerosis.
[0083] In certain embodiments, the method employs two phases for
evaluating an agent for use in treating atherosclerosis, an initial
in vitro phase and then an in vivo phase. The agent is administered
to the test cell or cell-free system in vitro, and if a change in
an aspect of LBP metabolism occurs, then the agent is further
administered to a test animal in a therapeutically effective amount
and evaluated in vivo for an effect of the agent on an aspect of
LBP metabolism.
[0084] By cell is meant a cell or a group of cells, or a cell that
is part of an animal. The cell can be a human or non-human cell.
Cell is also meant to include a transgenic cell. The cell can be
obtained, e.g., from a culture or from an animal. Animals are meant
to include, e.g., natural animals and non-human transgenic animals.
In certain embodiments, the transgenic cell or nonhuman transgenic
animal has an LBP transgene, or fragment or analog thereof. In
certain embodiments, the transgenic cell or non-human transgenic
animal has a knockout for the LBP gene.
[0085] The test cell, cell-free system or animal can have a wild
type pattern or a non-wild type pattern of LBP metabolism. A
non-wild type pattern of LBP metabolism can result, e.g., from
under-expression, over-expression, no expression, or a temporal,
site or distribution change. Such a non-wild type pattern can
result, e.g., from one or more mutations in the LBP gene, in a
binding molecule gene, a regulatory gene, or in any other gene
which directly or indirectly affects LBP metabolism. A mutation is
meant to include, e.g., an alteration, e.g., in gross or fine
structure, in a nucleic acid. Examples include single base pair
alterations, e.g., missense or nonsense mutations, frameshifts,
deletions, insertions and translocations. Mutations can be dominant
or recessive. Mutations can be homozygous or heterozygous.
Preferably, an aspect of LBP-1, LBP-2 or LBP-3 metabolism is
evaluated.
[0086] An agent is meant to include, e.g., any substance, e.g., an
anti-atherosclerosis drug. The agent of this invention preferably
can change an aspect of LBP metabolism. Such change can be the
result of any of a variety of events, including, e.g., preventing
or reducing interaction between LBP and a binding molecule, e.g.,
LDL or an arterial extracellular matrix structural component;
inactivating LBP and/or the binding molecule, e.g., by cleavage or
other modification; altering the affinity of LBP and the binding
molecule for each other; diluting out LBP and/or the binding
molecule; preventing expression of LBP and/or the binding molecule;
reducing synthesis of LBP and/or the binding molecule; synthesizing
an abnormal LBP and/or binding molecule; synthesizing an
alternatively spliced LBP and/or binding molecule; preventing or
reducing proper conformational folding of LBP and/or the binding
molecule; modulating the binding properties of LBP and/or the
binding molecule; interfering with signals that are required to
activate or deactivate LBP and/or the binding molecule; activating
or deactivating LBP and/or the binding molecule in such a way as to
prevent binding; or interfering with other receptors, ligands or
other molecules which are required for the normal synthesis or
functioning of LBP and/or the binding molecule. For example, the
agent can block the binding site on LDL for LBPs expressed focally
in the arterial wall extracellular matrix, or it could block the
binding site on an LBP for LDL, or it could be bifunctional, i.e.,
it could block both binding sites.
[0087] Examples of agents include LBP polypeptide, e.g., LBP-1,
LBP-2 or LBP-3, or a biologically active fragment or analog
thereof; a nucleic acid encoding LBP polypeptide or a biologically
active fragment or analog thereof; a nucleic acid encoding an LBP
regulatory sequence or a biologically active fragment or analog
thereof; a binding molecule for LBP polypeptide; a binding molecule
for LBP nucleic acid, the LBP nucleic acid being, e.g., a nucleic
acid comprising a regulatory region for LBP or a nucleic acid
comprising a structural region for LBP or a biologically active
fragment of LBP; an antisense nucleic acid; a mimetic of LBP or a
binding molecule; an antibody for LBP or a binding molecule; a
metabolite; or an inhibitory carbohydrate or glycoprotein. In
certain embodiments, the agent is an antagonist, agonist or super
agonist.
[0088] Knowledge of the existence of the sequence of the LBPs
allows a search for natural or artificial ligands to regulate LDL
levels in the treatment of atherosclerosis. In certain embodiments,
the agent is a natural ligand for LBP. In certain embodiments, the
agent is an artificial ligand for LBP.
[0089] By analog is meant a compound that differs from naturally
occurring LBP in amino acid sequence or in ways that do not involve
sequence, or both. Analogs of the invention generally exhibit at
least about 80% homology, preferably at least about 90% homology,
more preferably yet at least about 95% homology, and most
preferably at least about 98% homology, with substantially the
entire sequence of a naturally occurring LBP sequence, preferably
with a segment of about 100 amino acid residues, more preferably
with a segment of about 50 amino acid residues, more preferably yet
with a segment of about 30 amino acid residues, more preferably yet
with a segment of about 20 amino acid residues, more preferably yet
with a segment of about 10 amino acid residues, more preferably yet
with a segment of about 5 amino acid residues, more preferably yet
with a segment of about 4 amino acid residues, more preferably yet
with a segment of about 3 amino acid residues, and most preferably
with a segment of about 2 amino acid residues. Non-sequence
modifications include, e.g., in vivo or in vitro chemical
derivatizations of LBP. Non-sequence modifications include, e.g.,
changes in phosphorylation, acetylation, methylation,
carboxylation, or glycosylation. Methods for making such
modifications are known to those skilled in the art. For example,
phosphorylation can be modified by exposing LBP to
phosphorylation-altering enzymes, e.g., kinases or phosphatases.
Preferred analogs include LBP or biologically active fragments
thereof whose sequences differ from the wild-type sequence by one
or more conservative amino acid substitutions or by one or more
non-conservative amino acid substitutions, deletions, or insertions
which do not abolish LBP biological activity. Conservative
substitutions typically include the substitution of one amino acid
for another with similar characteristics, e.g., substitutions
within the following groups: valine, glycine; glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. Other examples of conservative
substitutions are shown in Table 1.
2TABLE 1 +HC,1CONSERVATIVE AMINO ACID SUBSTITUTIONS For Amino Acid
Code Replace with any of Alanine A D-Ala, Gly, beta-Ala, L-Cys,
D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile,
D-Met, D-Ile, Orn, D-Orn, L- NMMA, L-NAME Asparagine N D-Asn, Asp,
D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn,
Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr,
D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G
Ala, D-Ala, Pro, D-Pro, .beta.-Ala Acp Histidine H D-His Isoleucine
I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val,
D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg,
D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met,
S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe,
Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or
5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro,
L-I-thioazolidine-4-carboxylic acid, D- or
L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,
allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T
D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val,
D-Val Tryptophan W D-Trp, Phe, D-Phe, Tyr, D-Tyr Tyrosine Y D-Tyr,
Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,
D-Ile, Met, D-Met
[0090] Amino acid sequence variants of a protein can be prepared by
any of a variety of methods known to those skilled in the art. For
example, random mutagenesis of DNA which encodes a protein or a
particular domain or region of a protein can be used, e.g., PCR
mutagenesis (using, e.g., reduced Taq polymerase fidelity to
introduce random mutations into a cloned fragment of DNA; Leung et
al., BioTechnique 1: 11-15 (1989)), or saturation mutagenesis (by,
e.g., chemical treatment or irradiation of single-stranded DNA in
vitro, and synthesis of a complementary DNA strand; Mayers et al.,
Science 229: 242 (1985)). Random mutagenesis can also be
accomplished by, e.g., degenerate oligonucleotide generation
(using, e.g., an automatic DNA synthesizer to chemically synthesize
degenerate sequences; Narang, Tetrahedron 39: 3 (1983); Itakura et
al., Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules,
ed. A. G. Walton, Amsterdam: Elsevier, pp. 273-289 (1981)).
Non-random or directed mutagenesis can be used to provide specific
sequences or mutations in specific regions. These techniques can be
used to create variants which include, e.g., deletions, insertions,
or substitutions, of residues of the known amino acid sequence of a
protein. The sites for mutation can be modified individually or in
series, e.g., by (i) substituting first with conserved amino acids
and then with more radical choices depending upon results achieved,
(ii) deleting the target residue, (iii) inserting residues of the
same or a different class adjacent to the located site, or (iv)
combinations of the above. For example, analogs can be made by in
vitro DNA sequence modifications of the sequences of FIGS. 2A, 7A,
8A, 10-18 (SEQ ID NOS: 10-18, 45, 46, and 48). For example, in
vitro mutagenesis can be used to convert any of these DNA sequences
into a sequence which encodes an analog in which one or more amino
acid residues has undergone a replacement, e.g., a conservative
replacement as described in Table 1.
[0091] Methods for identifying desirable mutations include, e.g.,
alanine scanning mutagenesis (Cunningham and Wells, Science 244:
1081-1085 (1989)), oligonucleotide-mediated mutagenesis (Adelman et
al., DNA, 2: 183 (1983)); cassette mutagenesis (Wells et al., Gene
34: 315 (1985)), combinatorial mutagenesis, and phage display
libraries (Ladner et al., PCT International Appln. No. WO88/06630).
The LBP analogs can be tested, e.g., for their ability to bind to
LDL and/or to an arterial extracellular matrix component, as
described herein. Other analogs within the invention include, e.g.,
those with modifications which increase peptide stability. Such
analogs may contain, e.g., one or more non-peptide bonds (which
replace the peptide bonds) in the peptide sequence. Also included
are, e. g.: analogs that include residues other than naturally
occurring L-amino acids, e.g., D-amino acids or nonnaturally
occurring or synthetic amino acids, e.g., .beta. or .gamma. amino
acids; and cyclic analogs.
[0092] Analogs are also meant to include peptides in which
structural modifications have been introduced into the peptide
backbone so as to make the peptide non-hydrolyzable. Such peptides
are particularly useful for oral administration, as they are not
digested. Peptide backbone modifications include, e.g.,
modifications of the amide nitrogen, the .alpha.-carbon, the amide
carbonyl, or the amide bond, and modifications involving
extensions, deletions or backbone crosslinks. For example, the
backbone can be modified by substitution of a sulfoxide for the
carbonyl, by reversing the peptide bond, or by substituting a
methylene for the carbonyl group. Such modifications can be made by
standard procedures known to those skilled in the art. See, e.g.,
Spatola, A. F., "Peptide Backbone Modifications: A
Structure-Activity Analysis of Peptides Containing Amide Bond
Surrogates, Conformational Constraints, and Related Backbone
Replacements," in Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein (ed.),
Marcel Dekker, Inc., New York (1983).
[0093] An analog is also meant to include polypeptides in which one
or more of the amino acid residues include a substituent group, or
polypeptides which are fused with another compound, e.g., a
compound to increase the half-life of the polypeptide, e.g.,
polyethylene glycol.
[0094] By fragment is meant some portion of the naturally occurring
LBP polypeptide. Preferably, the fragment is at least about 100
amino acid residues, more preferably at least about 50 amino acid
residues, more preferably yet at least about 30 amino acid
residues, more preferably yet at least about 20 amino acid
residues, more preferably yet at least about 5 amino acid residues,
more preferably yet at least about 4 amino acid residues, more
preferably yet at least about 3 amino acid residues, and most
preferably at least about 2 amino acid residues in length.
Fragments include, e.g., truncated secreted forms, proteolytic
fragments, splicing fragments, other fragments, and chimeric
constructs between at least a portion of the relevant gene, e.g.,
LBP-1, LBP-2 or LBP-3, and another molecule. Fragments of LBP can
be generated by methods known to those skilled in the art. In
certain embodiments, the fragment is biologically active. The
ability of a candidate fragment to exhibit a biological activity of
LBP can be assessed by methods known to those skilled in the art.
For example, LBP fragments can be tested for their ability to bind
to LDL and/or to an arterial extracellular matrix structural
component, as described herein. Also included are LBP fragments
containing residues that are not required for biological activity
of the fragment or that result from alternative mRNA splicing or
alternative protein processing events.
[0095] Fragments of a protein can be produced by any of a variety
of methods known to those skilled in the art, e.g., recombinantly,
by proteolytic digestion, or by chemical synthesis. Internal or
terminal fragments of a polypeptide can be generated by removing
one or more nucleotides from one end (for a terminal fragment) or
both ends (for an internal fragment) of a nucleic acid which
encodes the polypeptide. Expression of the mutagenized DNA produces
polypeptide fragments. Digestion with "end-nibbling" endonucleases
can thus generate DNAs which encode an array of fragments. DNAs
which encode fragments of a protein can also be generated; e.g., by
random shearing, restriction digestion or a combination of the
above-discussed methods. For example, fragments of LBP can be made
by expressing LBP DNA which has been manipulated in vitro to encode
the desired fragment, e.g., by restriction digestion of any of the
DNA sequences of FIGS. 2A, 7A, 8A, 10-18 (SEQ ID NOS: 10-18, 45,
46, and 48).
[0096] Fragments can also be chemically synthesized using
techniques known in the art, e.g., conventional Merrifield solid
phase f-Moc or t-Boc chemistry for example, peptides of the present
invention can be arbitrarily divided into fragments of desired
length with no overlap of the fragments, or divided into
overlapping fragments of a desired length.
[0097] An LBP or a biologically active fragment or analog thereof,
or a binding molecule or a biologically active fragment or analog
thereof, can, e.g., compete with its cognate molecule for the
binding site on the complementary molecule, and thereby reduce or
eliminate binding between LBP and the cellular binding molecule.
LBP or a binding molecule can be obtained, e.g., from purification
or secretion of naturally occurring LBP or binding molecule, from
recombinant LBP or binding molecule, or from synthesized LBP or
binding molecule.
[0098] Therefore, methods for generating analogs and fragments and
testing them for activity are known to those skilled in the
art.
[0099] An agent can also be a nucleic acid used as an antisense
molecule. Antisense therapy is meant to include, e.g.,
administration or in situ generation of oligonucleotides or their
derivatives which specifically hybridize, e.g., bind, under
cellular conditions, with the cellular mRNA and/or genomic DNA
encoding an LBP polypeptide, or mutant thereof, so as to inhibit
expression of the encoded protein, e.g., by inhibiting
transcription and/or translation. The binding may be by
conventional base pair complementarity, or, for example, in the
case of binding to DNA duplexes, through specific interactions in
the major groove of the double helix.
[0100] In certain embodiments, the antisense construct binds to a
naturally-occurring sequence of an LBP gene which, e.g., is
involved in expression of the gene. These sequences include, e.g.,
promoter, start codons, stop codons, and RNA polymerase binding
sites. In other embodiments, the antisense construct binds to a
nucleotide sequence which is not present in the wild type gene. For
example, the antisense construct can bind to a region of an LBP
gene which contains an insertion of an exogenous, non-wild type
sequence. Alternatively, the antisense construct can bind to a
region of an LBP gene which has undergone a deletion, thereby
bringing two regions of the gene together which are not normally
positioned together and which, together, create a non-wild type
sequence. When administered in vivo to a subject, antisense
constructs which bind to non-wild type sequences provide the
advantage of inhibiting the expression of a mutant LBP gene,
without inhibiting expression of any wild type LBP gene.
[0101] An antisense construct of the present invention can be
delivered, e.g., as an expression plasmid which, when transcribed
in the cell, produces RNA which is complementary to at least a
unique portion of the cellular mRNA which encodes an LBP
polypeptide. An alternative is that the antisense construct is an
oligonucleotide which is generated ex vivo and which, when
introduced into the cell causes inhibition of expression by
hybridizing with the mRNA (duplexing) and/or genomic sequences
(triplexing) of an LBP gene. Such oligonucleotides are preferably
modified oligonucleotides which are resistant to endogenous
nucleases, e. g. exonucleases and/or endonucleases, and are
therefore stable in vivo. Exemplary nucleic acid molecules for use
as antisense oligonucleotides are phosphoramidate, phosphothioate,
phosphorodithioates and methylphosphonate analogs of DNA and
peptide nucleic acids (PNA). (See also U.S. Pat. Nos. 5,176,996;
5,264,564; and 5,256,775). Additionally, general approaches to
constructing oligomers useful in antisense therapy have been
reviewed. (See, e.g., Van der Krol et al., Biotechniques 6:
958-976, (1988); Stein et al., Cancer Res. 48: 2659-2668
(1988)).
[0102] By mimetic is meant a molecule which resembles in shape
and/or charge distribution LBP or a binding molecule. The mimetic
can be a peptide or a non-peptide. Mimetics can act as therapeutic
agents because they can, e.g., competitively inhibit binding of LBP
to a binding molecule. By employing, e.g., scanning mutagenesis,
e.g., alanine scanning mutagenesis, linker scanning mutagenesis or
saturation mutagenesis, to map the amino acid residues of a
particular LBP polypeptide involved in binding a binding molecule,
peptide mimetics, e.g., diazepine or isoquinoline derivatives, can
be generated which mimic those residues in binding to a binding
molecule, and which therefore can inhibit binding of the LBP to a
binding molecule and thereby interfere with the function of LBP.
Non-hydrolyzable peptide analogs of such residues can be generated
using, e.g., benzodiazepine (see, e.g., Freidinger et al., in
Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM
Publisher: Leiden, Netherlands (1988)); azepine (see, e.g., Huffman
et al., in Peptides: Chemistry and Biology, G. R. Marshall ed.,
ESCOM Publisher: Leiden, Netherlands (1988)); substituted gamma
lactam rings (see, e.g., Garvey et al., in Peptides: Chemistry and
Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands
(1988)); keto-methylene pseudopeptides (see, e.g. Ewenson et al.,
J. Med. Chem. 29: 295 (1986); Ewenson et al., in Peptides:
Structure and Function (Proceedings of the 9th American Peptide
Symposium) Pierce Chemical Co. Rockland, Ill. (1985)); .beta.-turn
dipeptide cores (see, e.g., Nagai et al., Tetrahedron Lett. 26: 647
(1985); Sato et al., J. Chem. Soc. Perkin Trans. 1: 1231 (1986));
or .beta.-aminoalcohols (see, e.g., Gordon et al., Biochem.
Biophys. Res. Commun. 126:419 (1985); Dann et al., Biochem.
Biophys. Res. Commun. 134:71 (1986)).
[0103] Antibodies are meant to include antibodies against any
moiety that directly or indirectly affects LBP metabolism. The
antibodies can be directed against, e.g., LBP or a binding
molecule, or a subunit or fragment thereof. For example, antibodies
include anti-LBP-1, LBP-2 or LBP-3 antibodies; and anti-binding
molecule antibodies. Antibody fragments are meant to include, e.g.,
Fab fragments, Fab' fragments, F(ab').sub.2 fragments, F(v)
fragments, heavy chain monomers, heavy chain dimers, heavy chain
trimers, light chain monomers, light chain dimers, light chain
trimers, dimers consisting of one heavy and one light chain, and
peptides that mimic the activity of the anti-LBP or anti-binding
molecule antibodies. For example, Fab.sub.2' fragments of the
inhibitory antibody can be generated through, e.g., enzymatic
cleavage. Both polyclonal and monoclonal antibodies can be used in
this invention. Preferably, monoclonal antibodies are used. Natural
antibodies, recombinant antibodies or chimeric-antibodies, e.g.,
humanized antibodies, are included in this invention. Preferably,
humanized antibodies are used when the subject is a human. Most
preferably, the antibodies have a constant region derived from a
human antibody and a variable region derived from an inhibitory
mouse monoclonal antibody. Production of polyclonal antibodies to
LBP is described in Example 6. Monoclonal and humanized antibodies
are generated by standard methods known to those skilled in the
art. Monoclonal antibodies can be produced, e.g., by any technique
which provides antibodies produced by continuous cell lines
cultures. Examples include the hybridoma technique (Kohler and
Milstein, Nature 256: 495-497 (1975), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today
4:72 (1983)), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., in Monoclonal Antibodies and
Cancer Therapy, A. R. Lisa, Inc., pp. 77-96 (1985)). Preferably,
humanized antibodies are raised through conventional production and
harvesting techniques (Berkower, I., Curr. Opin. Biotechnol.
7:622-628 (1996); Ramharayan and Skaletsky, Am. Biotechnol. Lab
13:26-28 (1995)). In certain preferred embodiments, the antibodies
are raised against the LBP, preferably the LDL-binding site, and
the Fab fragments produced. These antibodies, or fragments derived
therefrom, can be used, e.g., to block the LDL-binding sites on the
LBP molecules.
[0104] Agents also include inhibitors of a molecule that are
required for synthesis, post-translational modification, or
functioning of LBP. and/or a binding molecule, or activators of a
molecule that inhibits the synthesis or functioning of LBP and/or
the binding molecule. Agents include, e.g., cytokines, chemokines,
growth factors, hormones, signaling components, kinases,
phosphatases, homeobox proteins, transcription factors, editing
factors, translation factors and post-translation factors or
enzymes. Agents are also meant to include ionizing radiation,
non-ionizing radiation, ultrasound and toxic agents which can,
e.g., at least partially inactivate or destroy LBP and/or the
binding molecule.
[0105] An agent is also meant to include an agent which is not
entirely LBP specific. For example, an agent may alter other genes
or proteins related to arterial plaque formation. Such overlapping
specificity may provide additional therapeutic advantage.
[0106] The invention also includes the agent so identified as being
useful in treating atherosclerosis.
[0107] The invention also includes a method for evaluating an agent
for the ability to alter the binding of LBP polypeptide to a
binding molecule. An agent is provided. An LBP polypeptide is
provided. A binding molecule is provided. The agent, LBP
polypeptide and binding molecule are combined. The formation of a
complex comprising the LBP polypeptide and binding molecule is
detected. An alteration in the formation of the complex in the
presence of the agent as compared to in the absence of the agent is
indicative of the agent altering the binding of the LBP polypeptide
to the binding molecule.
[0108] In preferred embodiments, the LBP polypeptide is LBP-1,
LBP-2 or LBP-3. Examples of a binding molecule include native LDL,
modified LDL, e.g., methylated LDL or oxidized LDL, and arterial
extracellular matrix structural components.
[0109] Altering the binding includes, e.g., inhibiting or promoting
the binding. The efficacy of the agent can be assessed, e.g., by
generating dose response curves from data obtained using various
concentrations of the agent. Methods for determining formation of a
complex are standard and are known to those skilled in the art,
e.g., affinity coelectrophoresis (ACE) assays or ELISA assays as
described herein.
[0110] The invention also includes the agent so identified as being
able to alter the binding of an LBP polypeptide to a binding
molecule.
[0111] The invention also includes a method for evaluating an agent
for the ability to bind to an LBP polypeptide. An agent is
provided. An LBP polypeptide is provided. The agent is contacted
with the LBP polypeptide. The ability of the agent to bind to the
LBP polypeptide is evaluated. Preferably, the LBP polypeptide is
LBP-1, LBP-2 or LBP-3. Binding can be determined, e.g., by
measuring formation of a complex by standard methods known to those
skilled in the art, e.g., affinity coelectrophoresis (ACE) assays
or ELISA assays as described herein.
[0112] The invention also includes the agent so identified as being
able to bind to LBP polypeptide.
[0113] The invention also includes a method for evaluating an agent
for the ability to bind to a nucleic acid encoding an LBP
regulatory sequence. An agent is provided. A nucleic acid encoding
an LBP regulatory sequence is provided. The agent is contacted with
the nucleic acid. The ability of the agent to bind to the nucleic
acid is evaluated. Preferably, the LBP regulatory sequence is an
LBP-1, LBP-2 or LBP-3 regulatory sequence. Binding can be
determined, e.g., by measuring formation of a complex by standard
methods known to those skilled in the art, e.g., DNA mobility shift
assays, DNase I footprint analysis Molecular Biology, The invention
being able to bind sequence. (Ausubel et al., ed., Current
Protocols in John Wiley & Sons, New York, N.Y., (1989)).
[0114] The invention also includes the agent so identified as to a
nucleic acid encoding an LBP regulatory sequence.
[0115] The invention also includes a method for treating
atherosclerosis in an animal. An animal in need of treatment for
atherosclerosis is provided. An agent capable of altering an aspect
of LBP structure or metabolism is provided. The agent is
administered to the animal in a therapeutically effective amount
such that treatment of the atherosclerosis occurs.
[0116] In certain preferred embodiments, the agent is an LBP
polypeptide, e.g., LBP-1, LBP-2 or LBP-3, or a biologically active
fragment or analog thereof. The agent can be, e.g., the polypeptide
as set forth in SEQ ID NOS: 1-9, 43, 44, and 47. Preferably, the
agent is a polypeptide of no more than about 100 amino acid
residues in length, more preferably of no more than about 50 amino
acid residues, more preferably yet of no more than about 30 amino
acid residues, more preferably yet of no more than about 20 amino
acid residues, more preferably yet of no more than about 10 amino
acid residues, more preferably yet of no more than about 5 amino
acid residues, more preferably yet of no more than about 4 amino
acid residues, more preferably yet of no more than about 3 amino
acid residues, and most preferably of no more than about 2 amino
acid residues. Preferably, the polypeptide includes at least about
20% acidic amino acid residues, more preferably yet at least about
40% acidic amino acid residues, more preferably yet at least about
60% acidic amino acid residues, more preferably yet at least about
80% acidic amino acid residues, more preferably yet at least about
90% acidic amino acid residues, more preferably yet at least about
95% acidic amino acid residues, and most preferably at least about
98% acidic amino acid residues. Acidic amino acid residues include
aspartic acid and glutamic acid. An example of such an LBP
poly-peptide is BHF-1, which is a 20 amino acid length fragment of
human or rabbit LBP-1 which contains amino acid residues 14 through
33. See FIG. 9 (SEQ ID NO: 9). 45% of the amino acid residues of
BHF-1 are acidic. The invention also includes biologically active
fragments and analogs of BHF-1.
[0117] Other preferred acidic regions from the LBPs are amino acid
residues 329 through 343 (SEQ ID NO: 19), 329 through 354 (SEQ ID
NO: 20), 344 through 354 (SEQ ID NO: 21), and 529 through 538 (SEQ
ID NO: 22) of human LBP-2 as depicted in FIG. 7A (SEQ. ID NO: 43);
amino acid residues 14 through 43 (SEQ ID NO: 23)and 38 through 43
(SEQ ID NO: 24) of rabbit or human LBP-1 as depicted in FIG. 1 (SEQ
ID NO: 1) and FIG. 6 (SEQ ID NO: 6); amino acid residues 338
through 353 (SEQ ID NO: 25), 338 through 365 (SEQ ID NO: 26), 354
through 365 (SEQ ID NO: 27), and 444 through 453 (SEQ ID NO: 28) of
rabbit LBP-2 as depicted in FIG. 2A (SEQ ID NO: 47); amino acid
residues 96 through 110 (SEQ ID NO: 29) of rabbit LBP-3 as depicted
in FIG. 5 (SEQ ID NO: 5); and amino acid residues 69-75 (SEQ ID NO:
41) of human LBP-3 as depicted in FIG. 8A (SEQ ID NO: 44). The
invention is also meant to include biologically active fragments
and analogs of any of these polypeptides.
[0118] Other examples of agents include homopolymers and
heteropolymers of any amino acid or amino acid analog. In certain
preferred embodiments, the agent is a homopolymer of an acidic
amino acid or analog thereof. In certain embodiments, the agent is
a heteropolymer of one or more acidic amino acids and one or more
other amino acids, or analogs thereof. For example, agents include
poly(glu), poly(asp), poly(glu asp), poly(glu N), poly (asp N) and
poly(glu asp N). By N is meant any amino acid, or analog thereof,
other than glu or asp. By poly(glu asp) is meant all permutations
of glu and asp for a given length peptide. A preferred peptide is
poly(glu) of no more than about 10 amino acids in length,
preferably about 7 amino acids in length.
[0119] In certain preferred embodiments, the agent is an LBP
nucleic acid or a biologically active fragment or analog thereof,
e.g., a nucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide, or
a biologically active fragment or analog thereof. The agent can be,
e.g., a nucleic acid comprising a nucleotide sequence as set forth
in SEQ ID NOS: 10-18, 45, 46, and 48. In other embodiments, the
agent is an antisense molecule, e.g., one which can bind to an LBP
gene sequence.
[0120] Treating is meant to include, e.g., preventing, treating,
reducing the symptoms of, or curing the atherosclerosis.
Administration of the agent can be accomplished by any method which
allows the agent to reach the target area, e.g., a target cell or
the extracellular matrix. These methods include, e.g., injection,
deposition, implantation, suppositories, oral ingestion,
inhalation, topical administration, or any other method of
administration where access to the target area by the agent is
obtained. Injections can be, e.g., intravenous, intradermal,
subcutaneous, intramuscular or intraperitoneal. Implantation
includes inserting implantable drug delivery systems, e.g.,
microspheres, hydrogels, polymeric reservoirs, cholesterol
matrices, polymeric systems, e.g., matrix erosion and/or diffusion
systems and non-polymeric systems, e.g., compressed, fused or
partially fused pellets. Suppositories include glycerin
suppositories. Oral ingestion doses can be enterically coated.
Inhalation includes administering the agent with an aerosol in an
inhalator, either alone or attached to a carrier that can be
absorbed.
[0121] Administration of the agent can be alone or in combination
with other therapeutic agents. In certain embodiments, the agent
can be combined with a suitable carrier, incorporated into a
liposome, or incorporated into a polymer release system.
[0122] In certain embodiments of the invention, the administration
can be designed so as to result in sequential exposures to the
agent over some time period, e.g., hours, days, weeks, months or
years. This can be accomplished by repeated administrations of the
agent by one of the methods described above, or alternatively, by a
controlled release delivery system in which the agent is delivered
to the animal over a prolonged period without repeated
administrations. By a controlled release delivery system is meant
that total release of the agent does not occur immediately upon
administration, but rather is delayed for some time. Release can
occur in bursts or it can occur gradually and continuously.
Administration of such a system can be, e.g., by long acting oral
dosage forms, bolus injections, transdermal patches or subcutaneous
implants.
[0123] Examples of systems in which release occurs in bursts
include, e.g., systems in which the agent is entrapped in liposomes
which are encapsulated in a polymer matrix, the liposomes being
sensitive to a specific stimulus, e.g., temperature, pH, light,
magnetic field, or a degrading enzyme, and systems in which the
agent is encapsulated by an ionically-coated microcapsule with a
microcapsule core-degrading enzyme. Examples of systems in which
release of the agent is gradual and continuous include, e.g.,
erosional systems in which the agent is contained in a form within
a matrix, and diffusional systems in which the agent permeates at a
controlled rate, e.g., through a polymer. Such sustained release
systems can be, e.g., in the form of pellets or capsules.
[0124] The agent can be suspended in a liquid, e.g., in dissolved
form or colloidal form. The liquid can be a solvent, partial
solvent or non-solvent. In many cases water or an organic liquid
can be used.
[0125] The agent can be administered prior to or subsequent to the
appearance of atherosclerosis symptoms. In certain embodiments, the
agent is administered to patients with familial histories of
atherosclerosis, or who have phenotypes that may indicate a
predisposition to atherosclerosis, or who have been diagnosed as
having a genotype which predisposes the patient to atherosclerosis,
or who have other risk factors, e.g., hypercholesterolemia,
hypertension or smoking.
[0126] The agent is administered to the animal in a therapeutically
effective amount. By therapeutically effective amount is meant that
amount which is capable of at least partially preventing or
reversing atherosclerosis. A therapeutically effective amount can
be determined on an individual basis and will be based, at least in
part, on consideration of the species of animal, the animal's size,
the animal's age, the agent used, the type of delivery system used,
the time of administration relative to the onset of atherosclerosis
symptoms, and whether a single, multiple, or controlled release
dose regimen is employed. A therapeutically effective amount can be
determined by one of ordinary skill in the art employing such
factors and using no more than routine experimentation.
[0127] Preferably, the concentration of the agent is at a dose of
about 0.1 to about 1000 mg/kg body weight/day, more preferably at
about 0.1 to about 500 mg/kg/day, more preferably yet at about 0.1
to about 100 mg/kg/day, and most preferably at about 0.1 to about 5
mg/kg/day. The specific concentration partially depends upon the
particular agent used, as some are more effective than others. The
dosage concentration of the agent that is actually administered is
dependent at least in part upon the final concentration that is
desired at the site of action, the method of administration, the
efficacy of the particular agent, the longevity of the particular
agent, and the timing of administration relative to the onset of
the atherosclerosis symptoms. Preferably, the dosage form is such
that it does not substantially deleteriously affect the animal. The
dosage can be determined by one of ordinary skill in the art
employing such factors and using no more than routine
experimentation.
[0128] In certain embodiments, various gene constructs can be used
as part of a gene therapy protocol to deliver nucleic acids
encoding an agent, e.g., either an agonistic or antagonistic form
of an LBP polypeptide. For example, expression vectors can be used
for in vivo transfection and expression of an LBP polypeptide in
particular cell types so as to reconstitute the function of, or
alternatively, abrogate the function of, LBP polypeptide in a cell
in which non-wild type LBP is expressed. Expression constructs of
the LBP polypeptide, and mutants thereof, may be administered in
any biologically effective carrier, e.g., any formulation or
composition capable of effectively delivering the LBP gene to cells
in vivo. Approaches include, e.g., insertion of the subject gene in
viral vectors including, e.g., recombinant retroviruses,
adenovirus, adeno-associated virus, and herpes simplex virus-1, or
recombinant bacterial or eukaryotic plasmids. Viral vectors infect
or transduce cells directly; plasmid DNA can be delivered with the
help of, for example, cationic liposomes (lipofectin.TM. (Life
Technologies, Inc., Gaithersburg, Md.) or derivatized (e.g.
antibody conjugated), polylysine conjugates, gramacidin S,
artificial viral envelopes or other such intracellular carriers, as
well as direct injection of the gene construct or
Ca.sub.3,(PO.sub.4).sub.2 precipitation carried out in vivo. The
above-described methods are known to those skilled in the art and
can be performed without undue experimentation. Since transduction
of appropriate target cells represents the critical first step in
gene therapy, choice of the particular gene delivery system will
depend on such factors as the phenotype of the intended target and
the route of administration, e.g., locally or systemically.
Administration can be directed to one or more cell types, and to
one or more cells within a cell type. so as to be therapeutically
effective, by methods that are known to those skilled in the art.
In a preferred embodiment, the agent is administered to arterial
wall cells of the animal. For example, a genetically engineered LBP
gene is administered to arterial wall cells. In certain
embodiments, administration is done in a prenatal animal or
embryonic cell. It will be recognized that the particular gene
construct provided for in vivo transduction of LBP expression is
also useful for in vitro transduction of cells, such as for use in
the diagnostic assays described herein.
[0129] In certain embodiments, therapy of atherosclerosis is
performed with antisense nucleotide analogs of the genes which code
for the LBPs. Preferably, the antisense nucleotides have
non-hydrolyzable "backbones," e.g., phosphorothioates,
phosphorodithioates or methylphosphonates. The nucleoside base
sequence is complementary to the sequence of a portion of the gene
coding for, e.g., LBP-1, 2 or 3. Such a sequence might be, e.g.,
ATTGGC if the gene sequence for the LBP is TAACCG. One embodiment
of such therapy would be incorporation of an antisense analog of a
portion of one of the LBP genes in a slowrelease medium, e.g.,
polyvinyl alcohol, which is administered, e.g., by subcutaneous
injection, so as to release the antisense nucleotide analog over a
period of weeks or months. In another embodiment, the antisense
analog is incorporated into a polymeric matrix, e.g., polyvinyl
alcohol, such that the gel can be applied locally to an injured
arterial wall to inhibit LBP synthesis and prevent LDL
accumulation, e.g., after angioplasty or atherectomy.
[0130] The invention also includes a method for treating an animal
at risk for atherosclerosis. An animal at risk for atherosclerosis
is provided. An agent capable of altering an aspect of LBP
structure or metabolism is provided. The agent is administered to
the animal in a therapeutically effective amount such that
treatment of the animal occurs. Being at risk for atherosclerosis
can result from, e.g., a family history of atherosclerosis, or
phenotypic symptoms which predispose to atherosclerosis, e.g.,
having hypercholesterolemia, hypertension or smoking.
[0131] The invention also includes a method for treating a cell
having an abnormality in structure or metabolism of LBP. A cell
having an abnormality in structure or metabolism of LBP is
provided. An agent capable of altering an aspect of LBP structure
or metabolism is provided. The agent is administered to the cell in
a therapeutically effective amount such that treatment of the cell
occurs.
[0132] In certain embodiments, the cell is obtained from a cell
culture or tissue culture or an embryo fibroblast. The cell can be,
e.g., part of an animal, e.g., a natural animal or a nonhuman
transgenic animal. Preferably, the LBP is LBP-1, LBP-2 or
LBP-3.
[0133] The invention also includes a pharmaceutical composition for
treating atherosclerosis in an animal comprising a therapeutically
effective amount of an agent, the agent being capable of altering
an aspect of LBP metabolism or structure in the animal so as to
result in treatment of the atherosclerosis, and a pharmaceutically
acceptable carrier. Pharmaceutically acceptable carriers include,
e.g., saline, liposomes and lipid emulsions.
[0134] In certain preferred embodiments, the agent of the
pharmaceutical composition is an LBP polypeptide, e.g., LBP-1,
LBP-2 or LBP-3, or a biologically active fragment or analog
thereof. The agent can be, e.g., the polypeptide as set forth in
SEQ ID NOS: 1-9, 43, 44, and 47. Preferably, the agent is a
polypeptide of no more than about 100 amino acid residues in
length, more preferably of no more than about 50 amino acid
residues, more preferably yet of no more than about 30 amino acid
residues, more preferably yet of no more than about 20 amino acid
residues, more preferably yet of no more than about 10 amino acid
residues, more preferably yet of no more than about 5 amino acid
residues, more preferably yet of no more than about 4 amino acid
residues, more preferably yet of no more than about 3 amino acid
residues, and most preferably of no more than about 2 amino acid
residues. Preferably, the polypeptide includes at least about 20%
acidic amino acid residues, more preferably yet at least about 40%
acidic amino acid residues, more preferably yet at least about 60%
acidic amino acid residues, more preferably yet at least about 80%
acidic amino acid residues, more preferably yet at least about 90%
acidic amino acid residues, more preferably yet at least about 95%
acidic amino acid residues, and most preferably at least about 98%
acidic amino acid residues.
[0135] In certain preferred embodiments, the agent is an LBP
nucleic acid, e.g., a nucleic acid encoding LBP-1, LBP-2 or LBP-3
polypeptide, or a biologically active fragment or analog thereof.
The agent can be, e.g., a nucleic acid comprising a nucleotide
sequence as set forth in SEQ ID NOS: 10-18, 45, 46, and 48.
[0136] The invention also includes a vaccine composition for
treating atherosclerosis in an animal comprising a therapeutically
effective amount of an agent, the agent being capable of altering
an aspect of LBP metabolism or structure in the animal so as to
result in treatment of the atherosclerosis, and a pharmaceutically
acceptable carrier.
[0137] The invention also includes a method for diagnosing
atherosclerotic lesions in an animal. An animal is provided. A
labeled agent capable of binding to LBP present in atherosclerotic
lesions is provided. The labeled agent is administered to the
animal under conditions which allow the labeled agent to interact
with the LBP so as to result in labeled LBP. The localization or
quantification of the labeled LBP is determined by imaging so as to
diagnose the presence of atherosclerotic lesions in the animal.
[0138] Preferably, the LBP is LBP-1, LBP-2 or LBP-3. The imaging
can be performed by standard methods known to those skilled in the
art, including, e.g., magnetic resonance imaging, gamma camera
imaging, single photon emission computed tomographic (SPECT)
imaging, or positron emission tomography (PET).
[0139] Preferably, agents that bind tightly to LBPs in
atherosclerotic lesions are used for atherosclerotic imaging and
diagnosis. The agent is radiolabeled with, e.g., .sup.99mTc or
another isotope suitable for clinical imaging by gamma camera,
SPECT, PET scanning or other similar technology. Since LBPs occur
in very early lesions, such imaging is more sensitive than
angiography or ultrasound for locating very early lesions which do
not yet impinge on the arterial lumen to cause a visible bulge or
disturbed flow. In addition to locating both early and more
developed lesions, the imaging agents which bind to LBPs can also
be used to follow the progress of atherosclerosis, as a means of
evaluating the effectiveness of both dietary and pharmacological
treatments.
[0140] Thus, a diagnostic embodiment of the invention is the
adaptation of, e.g., a peptide complementary to one of the LBPs, by
radiolabeling it and using it as an injectable imaging agent for
detection of occult atherosclerosis. The peptide is selected from
those known to bind to LBPs, e.g., RRRRRRR or KKLKLXX, or any other
polycationic peptide which binds to the highly electronegative
domains of the LBPs. For extracorporeal detection with a gamma
scintillation (Anger) camera, technetium-binding ligands, e.g.,
CGC, GGCGC, or GGCGCF, can be incorporated into the peptides at the
N-terminus or C-terminus for .sup.99mTc labeling. For external
imaging by magnetic resonance imaging (MRI), e.g., the
gadolinium-binding chelator, diethylene triamine penta-acetic acid
(DTPA), is covalently bound to the N- or C-terminus of the
peptides. In yet other embodiments, the LBP-binding peptides are
covalently bound, e.g., to magnetic ion oxide particles by standard
methods known to those skilled in the art, e.g., conjugating the
peptides with activated polystyrene resin beads containing magnetic
ion oxide.
[0141] The invention also includes a method for immunizing an
animal against an LBP, e.g., LBP-1, LBP-2 or LBP-3, or fragment or
analog thereof. An animal having LDL is provided. An LBP or
fragment or analog thereof is provided. The LBP or fragment or
analog thereof is administered to the animal so as to stimulate
antibody production by the animal to the LBP or fragment or analog
thereof such that binding of the LBP to the LDL is altered, e.g.,
decreased or increased.
[0142] The invention also includes a method of making a fragment or
analog of LBP polypeptide, the fragment or analog having the
ability to bind to modified LDL and native LDL. An LBP polypeptide
is provided. The sequence of the LBP polypeptide is altered. The
altered LBP polypeptide is tested for the ability to bind to
modified LDL, e.g., methylated LDL, oxidized LDL, acetylated LDL,
cyclohexanedione-treated LDL (CHD-LDL), and to native LDL.
[0143] The fragments or analogs can be generated and tested for
their ability to bind to these modified LDLs and to native LDL, by
methods known to those skilled in the art, e.g., as described
herein. Preferably, they are tested for their ability to bind to
methylated LDL and native LDL. The binding activity of the fragment
or analog can be greater or less than the binding activity of the
native LBP. Preferably, it is greater. In preferred embodiments,
the LBP is LBP-1, LBP-2 or LBP-3.
[0144] The invention also includes a method for isolating a cDNA
encoding an LBP. A cDNA library is provided. The cDNA library is
screened for a cDNA encoding a polypeptide which binds to native
LDL and modified LDL, e.g., methylated LDL or oxidized LDL. The
cDNA which encodes this polypeptide is isolated, the cDNA encoding
an LBP.
[0145] Atherosclerosis in a hyperlipidemic subject can be reduced
following the generation of an immune response in the subject by
immunization with LBPs. Numerous immunotherapeutic products can be
used to generate antibodies that will block the binding between LDL
and LBPs.
[0146] The injection of one or more LBPs can result in the
production of anti-LBP antibodies, resulting in a reduction in
,e.g., aortic atherosclerosis. This effect is thought to be
mediated by an inhibition of LBP binding to LDL. LBP immunogens
that can be used in the invention include human LBPs, non-human
LBPs, recombinant LBPs, and proteins structurally related to the
LBPs described herein, e.g. non-naturally occurring proteins that
differ from a naturally occurring LBP at one or more amino acid
residues. In addition to full length proteins, injecting one or
more peptides that include an LBP domain can generate an effective
immune response. For example, the injection of a peptide comprising
an LBP domain having LDL-binding activity can cause an organism to
make antibodies to the LBP binding sites for LDL. These peptide
immunogens can include sequences derived from human LBPs, non-human
LBPs, recombinant LBPs, and proteins structurally related to the
LBPs described herein.
[0147] Modifications can be made to a protein or peptide immunogen
of the invention to increase its immunogenicity. The immunogen can
be conjugated or coupled with a carrier, e.g. a Cholera toxin B
chain or monoclonal antibodies. The immunogen can be precipitated
with aluminum salts or cross-linked with formaldehyde or other
aldehydes. The protein may be mixed with a physiologically
acceptable diluent such as water, phosphate buffered saline, or
saline. The composition may further include an adjuvant. In
addition to RIBI adjuvant, adjuvants such as incomplete Freund's
adjuvant, aluminum phosphate, aluminum hydroxide are all well known
in the art. Adjustments in the adjuvant of the invention can be
made to affect the immunogenicity of the peptide or protein.
Examples of such modifications include using: aluminum salts;
cytokines; MF59 (microfluidized emulsion of oil and surfactants);
SAF-1 (oil-based emulsion); saponin derivatives; polymers (such as
polyphosphazene); and bacterial toxins. Additional descriptions of
antigenic protein-adjuvant combinations are described in WO
99/54452 (herein incorporated by reference) and WO 99/49890 (herein
incorporated by reference).
[0148] In addition to delivery of the proteins and peptides
described above, numerous other delivery systems can be used to
generate the anti-atherosclerotic immunity of the invention. The
LBP immunogen can be delivered either directly as a protein antigen
or alternatively as a nucleic acid that encodes the protein
antigen. The immunotherapeutic products of the invention, either
protein or nucleic acid, can be delivered by numerous delivery
routes. These include injection, deposition, implantation,
suppositories, oral ingestion, inhalation (e.g., delivery via a
nasal spray), and topical administration (e.g., delivery via a skin
patch).
[0149] A nucleic acid encoding an immunogen of the invention can be
directly administered, for example by injection, to tissues and
expressed as a protein. The DNA or RNA can be either associated
with a delivery vehicle (e.g., viruses, bacteria, liposomes, and
gold beads) or naked (free from association with
transfection-facilitating proteins, viral particles, liposomal
formulations, charged lipids and calcium phosphate precipitating).
The nucleic acid can optionally include a promoter, e.g. a viral
promoter. The immunogen encoded by the nucleic acid is produced in
the host, resulting in the generation of an immune response.
Methods for the delivery of nucleic acid sequences encoding
therapeutic proteins and peptides are described in detail by
Felgner et al. (U.S. Pat. No. 5,580,859; herein incorporated by
reference) and Barbet et al. (U.S. Pat. No. 6,025,338:; herein
incorporated by reference). Vaccine compositions of viral liposomes
comprising a nucleic acid, e.g. an RNA, encoding a protein antigen
are described in WO 99/52503 (herein incorporated by reference).
Proteins and nucleic acids encoding peptides can also be delivered
to an individual by their encapsulation in liposomes,
microparticles, and ISCOMS, all of which are well known in the art
(see, e.g., U.S. Pat. No. 6,013,258, herein incorporated by
reference).
[0150] A nucleic acid encoding an immunogen of the invention can
also be included in the genome of a plant, so as to result in the
production of the immunogen by plant tissues. The genetically
modified plant may then consumed by an individual, resulting in the
ingestion of the immunogen and the generation of an anti-LBP immune
response. Methodology for the generation and usage of edible plant
vaccines is described in WO 99/54452 (herein incorporated by
reference).
[0151] Numerous plants may be useful for the production of an
edible vaccine, including: tobacco, tomato, potato, eggplant,
pepino, yam, soybean, pea, sugar beet, lettuce, bell pepper,
celery, carrot, asparagus, onion, grapevine, muskmelon, strawberry,
rice, sunflower, rapeseed/canola, wheat, oats, maize, cotton,
walnut, spruce/conifer, poplar and apple. The edible vaccine can
include a plant cell transformed with a nucleic acid construct
comprising a promoter and a sequence encoding an LBP. The sequence
may optionally encode a chimeric protein, comprising a cholera
toxin subunit B peptide fused to the LBP peptide. Preferred plant
promoters of the invention include CaMV 35S, patatin, mas, and
granule-bound starch synthase promoters. Additional useful
promoters and enhancers are described in WO 99/54452.
[0152] The edible vaccine of the invention can be administered to a
mammal suffering from or at risk of atherosclerosis. Preferably, an
edible vaccine is administered orally, e.g. consuming a transgenic
plant of the invention. The transgenic plant can be in the form of
a plant part, extract, juice, liquid, powder, or tablet. The edible
vaccine can also be administered via an intranasal route.
[0153] Microorganisms, e.g., attenuated viruses or bacteria, can be
used in the invention by including a nucleic acid encoding an LBP
immunogen in the genome of the microorganism. This modified vector
can then be delivered to a host, resulting in the in vivo
production of the immunogen. The immune response generated by these
vectors is expected to result in anti-atherosclerotic immunity.
Nucleic acid molecules are inserted into microorganism genomes by
standard methods known in the art (U.S. Pat. Nos. 5,866,136 and
6,025,164, both of which are herein incorporated by reference)
[0154] The anti-atherosclerotic methods of the invention are
directed to treating a subject, e.g., a human, primate, horse, dog,
cat, or goat, at risk for atherosclerosis by stimulating an
anti-LBP response in the subject by immunotherapy. The LBP proteins
and peptides of the invention may be delivered to the subject by
the numerous delivery systems described herein. The immunotherapy
may comprise an initial immunization followed by additional, e.g.
one, two, or three, boosters.
[0155] The invention also includes a method of treating a subject
at risk for atherosclerosis by (1) providing a subject at risk for
atherosclerosis and (2) administering to the subject one or more of
the following: (a) an LBP protein or fragment or analog thereof and
an adjuvant; (b) a nucleic acid encoding an LBP protein; (c) a
virus or bacteria comprising a nucleic acid encoding an LBP
protein; and (d) an edible plant comprising a nucleic acid encoding
an LBP protein. The LBP protein used in this method can be any LBP
described herein, e.g., LBP-1 , LBP-2, or LBP-3. A combination of
more than one nucleic acid or LBP protein or fragment or analog
thereof can be administered to the subject. For example,
combinations of LBP proteins, or nucleic acids encoding LBP
proteins, include: (1) LBP-1 and LBP-2; (2) LBP-1 and LBP-3;
(3)LBP-2 and LBP-3; and (4) LBP-1, LBP-2, and LBP-3. This method
optionally includes a step of diagnosing the subject as being at
risk for atherosclerosis.
[0156] Also provided by the invention is a method of treating a
subject at risk for atherosclerosis whereby a non-autologous LBP
protein or a nucleic acid encoding a non-autologous LBP protein is
delivered to the subject to generate an immune response to an
autologous LBP. Specifically, this method entails identifying one
or more autologous LBP proteins, e.g., LBP-1, LBP-2, or LBP-3,
produced by the subject. The identification can by, e.g., DNA
sequence analysis, protein sequence analysis, antibody reactivity,
hybridization analysis, or nucleic acid amplification. Next, a
non-autologous LBP protein, e.g., allogeneic, xenogeneic, or a
genetically modified, non-naturally occurring protein that differs
at one or more amino acid residues from the one or more LBP
proteins, is administered to the subject. Alternatively, a nucleic
acid encoding a non-autologous LBP protein is administered to the
subject. The anti-atherosclerotic effectiveness of this
immunotherapeutic product is determined by its ability to induce an
immune response against one or more autologous LBP proteins when
administered to the subject. It is therefore expected that
extensive differences between a non-autologous and autologous LBP
protein will not result in cross immunoreactivity. This method
optionally includes a step of diagnosing the subject as being at
risk for atherosclerosis.
[0157] Another method of the invention is a method of treating a
subject at risk for atherosclerosis by increasing the levels of one
or more LBP proteins circulating in the plasma. According to this
method, either autologous or non-autologous LBP levels may be
increased. Non-autologous LBP proteins include, e.g., allogeneic
LBP, xenogeneic LBP, and genetically modified LBP. The plasma
levels of one or more LBP proteins can be increased by the delivery
of a nucleic acid encoding an LBP protein. Because LBP generally
does not normally occur as a circulating protein, the endogenous
molecule is expected to be susceptible to immune recognition when
delivered in a soluble form. This method optionally includes a step
of diagnosing the subject as being at risk for atherosclerosis.
[0158] Also included in the invention is a pharmaceutical
composition containing one or more LBP proteins, e.g., LBP-1,
LBP-2, or LBP-3, mixed with an adjuvant, suitable for use in
humans. The pharmaceutical composition can contain a combination of
more than one LBP protein. For example, compositions can include
any of the following: (1) LBP-1 and LBP-2; (2) LBP-1 and LBP-3; (3)
LBP-2 and LBP-3; and (4) LBP-1, LBP-2, and LBP-3.
[0159] Also included in the invention is a cell therapy system,
whereby a cell expressing an LBP is delivered to a subject at risk
for atherosclerosis. This cell can be engineered to express either
an autologous or non-autologous LBP protein or peptide of the
invention. Delivery of this engineered cell to a subject results in
the in vivo production of an LBP protein and the associated
immunotherapy produced when either the protein or a nucleic acid
encoding the protein is provided to an individual. Cell therapy
methods are described in U.S. Pat. No. 5,955,095 (herein
incorporated by reference).
[0160] The following non-limiting examples further illustrate the
present invention.
EXAMPLES
Example 1
[0161] Construction of a Rabbit cDNA Library
[0162] This example illustrates the construction of a rabbit cDNA
library using mRNA from balloon-deendothelialized healing rabbit
abdominal aorta. Balloon-catheter deendothelialized rabbit aorta
has been shown to be a valid model for atherosclerosis (Minick et
al., Am. J. Pathol. 95:131-158 (1979).
[0163] The mRNA was obtained four weeks after ballooning to
maximize focal LDL binding in the ballooned rabbit aorta. First
strand cDNA synthesis was carried out in a 50 .mu.l reaction
mixture containing 4 .mu.g mRNA; 2 .mu.g oligo d(T)primer;
methylation dNTP mix (10 mM each); 10 mM DTT; 800 units superscript
II RT (Life Technologies, Gaithersburg, Md.); 1.times.first strand
cDNA synthesis buffer (50 mM Tris-HCl, pH 8.3; 75 mM KCl; 5 mM
MgCl.sub.2), which was incubated for 1 hr at 37.degree. C. The
reaction mixture was then adjusted to 250 .mu.l through the
addition of 1.times.second strand buffer (30 mM Tris-HCl, pH 7.5;
105 mM .sub.KCl; 5.2 mM MgCl.sub.2); 0.1 mM DTT; methylation dNTP
mix (10 mM each); 50 units E. coli DNA polymerase I, 3 units RNase
H; 15 units E. coli DNA ligase (all enzymes from Life
Technologies), which was incubated for an additional 2.5 hr at
15.degree. C. The resulting double-stranded cDNAs (dscDNA) were
then treated with 1.5 units T4 DNA polymerase (Novagen Inc.,
Madison, Wis.) for 20 min at 11.degree. C. to make blunt-ended
dscDNA. These were then concentrated by ethanol precipitation and
EcoRl/Hind III linkers were attached to the ends by T4 DNA ligase
(Novagen Inc.). The linker-ligated cDNAs were treated with EcoRl
and Hind111 restriction enzymes to produce EcoRl and Hind III
recognition sequences at their 5'and 3'ends, respectively. After
the removal of linker DNA by gel exclusion chromatography, the
dscDNAs were inserted into .lambda.EXlox phage arms (Novagen Inc.)
in a unidirectional manner by T4 DNA ligase and packaged into phage
particles according to the manufacturer's protocol (Novagen Inc.).
A phage library of cDNAs containing 2.times.10.sup.6 independent
clones was established from 4 .mu.g of mRNA.
Example 2
[0164] Identification of Rabbit cDNAs Encoding LDL Binding Proteins
(LBPs)
[0165] This example illustrates a method of functionally screening
a rabbit cDNA library so as to identify cDNAs encoding LBPs which
bind to both native LDL and methyl LDL. Methyl LDL is not
recognized by previously reported cell surface receptors. See,
e.g., Weisgraber et al., J. Biol. Chem. 253:9053-9062 (1978).
[0166] A fresh overnight culture of E. coli ER1647 cells (Novagen
Inc.) was infected with the cDNA phage obtained from Example 1, and
plated at a density of 2.times.10.sup.4 plaque-forming units (pfu)
in 150 mm diameter plates containing 2.times.YT agar. A total of 50
plates, equivalent to 1.times.10.sup.6 phage, were plated and
incubated at 37.degree. C. until the plaques reached 1 mm in
diameter (5-6 hr). A dry nitrocellulose membrane, which had
previously been saturated with 10 mM IPTG solution, was layered on
top of each plate to induce the production of recombinant protein,
as well as to immobilize the proteins on the membranes. The plates
were incubated at 37.degree. C. for an additional 3-4 hr, and then
overnight at 4.degree. C.
[0167] The next day, the membranes were lifted from each plate and
processed as follows. Several brief rinses in TBST solution (10 mM
Tris-HCl, pH 8.0; 150 mM NaCl, 0.05% Tween 20); two 10-min rinses
with 6M guanidine-HCl in HBB (20 mM HEPES, pH 7.5; 5 mM MgCl.sub.2,
1 mM DTT, and 5 mM KCl); two 5-min rinses in 3M guanidine-HCl in
HBB; a final brief rinse in TBSEN (TBS, 1 mM EDTA, 0.02%
NaN.sub.3).
[0168] The membranes were then blocked for 30 min at room
temperature in a solution of TBSEN with 5% non-fat dry milk,
followed by 10 min in TBSEN with 1% non-fat dry milk. Following
blocking, the membranes were incubated with native human LDL
(obtained as described in Example 11 or methylated human LDL
(meLDL) (see Weisgraber et al., J. Biol. Chem. 253:9053-9062
(1978)), at a concentration of 4 .mu.g/ml, in a solution containing
1.times.TBSEN, 1% non-fat dry milk, 1 mM PMSF, 0.5.times.protease
inhibitor solution (1 mM .epsilon.-amino caproic acid/1 mM
benzamidine). Incubation was for 4 hr at room temperature in a
glass Petri dish with gentle stirring on a stirring table, followed
by overnight at 4.degree. C. with no stirring.
[0169] Specifically bound meLDL and native LDL were detected on the
nitrocellulose membranes by antibodies against human LDL. Sheep
anti-human LDL polyclonal antibodies (Boehringer Mannheim,
Indianapolis, Ind.) were adsorbed with E. coli plys E cell extracts
to abolish background. For adsorption, E. coli plys E cells were
grown to log phase, spun down and resuspended in PBS containing 1
mM PMSF, 2 mM .epsilon.-amino caproic acid, and 1 mM benzamidine.
The cell suspension then underwent 8 freeze-thaw cycles via
immersion in liquid nitrogen and cold running tap water,
respectively. The anti LDL antibodies/cell extract solution were
incubated with gentle stirring for 1 hr at 4.degree. C. (1 ml of
antibody solution/3 mg crude cell extract). Following incubation,
the mixture was centrifuged (10,000.times.g; 10 min; 4.degree. C.)
and the supernatant was stored at 4.degree. C. in the presence of
0.02% NaN.sub.3, until use. The membranes were processed for
immunoscreening as follows: (i) three 5-min washes at room
temperature in TBSEN containing 1% gelatin; (ii) 30 min incubation
in PBS, pH 7.4 with 1% gelatin; (iii) two-hr room temperature
incubation with gentle stirring in fresh PBS/gelatin solution
containing adsorbed sheep anti-human LDL antibodies (Boehringer
Manheim, Indianapolis, Ind.) (1:1000 dilution); (iv) three brief
washes in TBS, pH 7.4; (v) one-hr room temperature incubation with
gentle stirring in PBS/gelatin solution containing donkey
anti-sheep alkaline phosphatase-conjugated antibodies (Sigma, St.
Louis, Mo.) (1:10,000 dilution); (vi) three brief washes with TBS,
PH 7.4.; and (vii) development according to the manufacturer's
instructions, using an alkaline phosphatase substrate development
kit (Novagen Inc.). Phage plaques which produced LBPs appeared as
blue-colored "donuts" on the membranes.
[0170] The phage from Example 1 containing the LBP cDNAs were
plaque-purified and converted into plasmid subclones by following a
protocol called "Autosubcloning by Cre-mediated Plasmid Excision"
provided by Novagen Inc. DNA sequences were obtained by the
dideoxynucleotide chain-termination method (Sanger et al., Proc.
Natl. Acad. Sci., USA 74: 5463-5467 (1977), and analyzed by an
Applied Biosystems automated sequencer. The open reading frame
(ORF) of each cDNA was determined from consensus sequences obtained
from both the sense and antisense strands of the cDNAs. Sequencing
confirmed that three previously unknown genes had been isolated.
Since the genes were selected by functional screening for LDL
binding, the proteins coded by these genes were termed LDL binding
proteins (LBPs), specifically, LBP-1, LBP-2 and LBP-3. The cDNA
sequences for rabbit LBP-1, LBP-2 and LBP-3 and the corresponding
proteins are set forth in SEQ ID NOS: 10-14 and 48.
[0171] Based on their respective cDNA coding sequences, the sizes
of the recombinant proteins were determined to be 16.2 kDa for
LBP-1, 40 kDa for LBP-2, and 62.7 kDa for LBP-3.
Example 3
[0172] Northern Blot Analysis of Rabbit RNA Using LBP cDNA or
cRNA
[0173] This example illustrates the size and tissue distribution of
LBP mRNAs. Total RNA was isolated from different rabbit tissues:
adrenals, thoracic aorta, abdominal aorta, ballooned and
reendothelialized abdominal aorta, heart, kidney, smooth muscle
cells, lung and liver, by Trizol reagent (Life Technologies) and
concentrated by ethanol precipitation. Gel electrophoresis of RNA
was carried out in 1.2% agarose gel containing 1.times.MOPS buffer
(0.2M MOPS, pH 7.0; 50 mM sodium acetate; 5 mM EDTA, pH 8.0) and
0.37M formaldehyde. Gels were loaded with 20 .mu.g total RNA from
each tissue examined and electrophoresed at 100 volts for 2 hr in
1.times.MOPS buffer. RNAs were blotted onto supported
nitrocellulose membranes (Schleicher & Schuell, Keene, N.H.)
and immobilized by baking at 80.degree. C. for 2 hr. Hybridization
to radiolabeled LBP-1, LBP-2 and LBP-3 cDNA or cRNA probes was
carried out by standard procedures known to those skilled in the
art (see, e.g., Ausubel et at., Current Protocols in Molecular
Biology; John Wiley & Sons (1989)); signals were detected by
autoradiography.
[0174] The results were as follows: the sizes of the mRNAs were
about 1.3 kb for LBP-1, about 2.3-2.5 kb for LBP-2, and about 4.7
kb for LBP-3. LBP-1, LBP-2 and LBP-3 mRNA were found in all tissues
tested, but the highest amount was in ballooned abdominal
aorta.
Example 4
[0175] Isolation of Human LBP cDNAs and Genomic Clones
[0176] This example illustrates isolation of human LBP cDNAs. Human
LBP cDNA clones were isolated from three cDNA libraries. A human
fetal brain cDNA library was obtained from Stratagene, LaJolla,
Calif., a human liver and a human aorta cDNA library were obtained
from Clontech, Palo Alto, Calif., and screened with a radiolabeled
cDNA probe derived from rabbit LBP-1, LBP-2 or LBP-3, according to
the method described in Law et al., Gene Expression 4:77-84 (1994).
Several strongly hybridizing clones were identified and
plaque-purified. Clones were confirmed to be human LBP-1, LBP-2 and
LBP-3, by DNA sequencing using the dideoxynucleotide
chain-termination method and analysis by an Applied Biosystems
automated sequencer. The cDNA sequences and the corresponding
proteins for human LBP-1, LBP-2 and LBP-3 are set forth in SEQ ID
NOS: 15, 16 and 17, respectively.
[0177] A human genomic library was screened with each of the LBP-1,
LBP-2, and LBP-3 clones obtained from the cDNA library screening.
Clones hybridizing to each of the three cDNAs were isolated and
sequenced. The genomic sequence for LBP-1, LBP-2, and LBP-3 are set
forth in FIGS. 22-24, respectively. The LBP-1 open reading frame
spans four exons of the LBP-1 gene (FIG. 22; SEQ ID NO:49). The
LBP-1 protein predicted by the genomic sequence is identical to
that predicted by the cDNA clone described above. The LBP-2 open
reading frame spans five exons of the LBP-2 gene (FIG. 23; SEQ ID
NO:50). The LBP-2 protein predicted by the genomic sequence differs
from that predicted by the cDNA clone in that it contains an
additional 321 amino acids at its amino terminus (the LBP-2 cDNA is
a 5' truncation). The LBP-3 open reading frame spans ten exons of
the LBP-3 gene (FIG. 24; SEQ ID NO:51). The LBP-3 protein predicted
by the genomic sequence differs from that predicted by the cDNA
clone in that it contains an additional 16 amino acids at its amino
terminus (the LBP-3 cDNA is a 5' truncation) and an Asn at amino
acid position 130 (the cDNA predicts a Tyr at this position). A
comparison between the corresponding LBP-1, LBP-2 and LBP-3 protein
sequences for rabbit and human are shown in FIGS. 19, 20 and
21.
Example 5
[0178] Isolation of Recombinant LBP-1, LBP-2 and LBP-3 Rabbit
Proteins from E. coli
[0179] LBP cDNA was isolated from the original pEXlox plasmids
obtained as described in Examples 1 and 2, and subcloned into the
pPROEX-HT vector (Life Technologies) for recombinant protein
expression. Induction of the recombinant protein by IPTG addition
to transformed E. coli DH10B cultures resulted in the expression of
recombinant protein containing a 6-histidine tag (N-terminal). This
tagged protein was then purified from whole cell proteins by
binding to Ni-NTA (nickel nitrilo-triacetic acid) as described in
the protocol provided by the manufacturer (Qiagen, Inc., Santa
Clara, Calif.). The preparation obtained after the chromatography
step was approximately 90% pure; preparative SDS-PAGE was performed
as the final purification step.
[0180] When required by the characterization procedure, iodination
of LBPs was carried out using Iodobeads (Pierce, Rockford, Ill.).
The Iodobeads were incubated with 500 .mu.Ci of Na.sup.125I
solution (17 Ci/mg) (New England Nuclear, Boston, Mass.) in a
capped microfuge tube for 5 min at room temperature. The protein
solution was added to the Iodobeads-Na .sup.125I microfuge tube and
incubated for 15 min at room temperature. At the end of this
incubation, aliquots were removed for the determination of total
soluble and TCA precipitable counts. The radiolabeled protein was
then precipitated with cold acetone (2.5 vol; -20 C.; 2.5 hr).
Following this incubation, precipitated protein was collected by
centrifugation (14,000 g; 1 hr; room temperature) and resuspended
in sample buffer (6 M urea/50 mM Tris, pH 8.0/2 mM EDTA). Integrity
of the protein preparation was assessed by SDS-PAGE.
[0181] The identities of the recombinant LBPs were confirmed using
standard protein sequencing protocols known to those skilled in the
art. (A Practical Guide for Protein and Peptide Purification for
Microsequencing, Matsudaira, ed., Academic Press, Inc., 2d edition
(1993)). Analysis was performed using an Applied Biosystems Model
477A Protein Sequencer with on-line Model 120 PTH amino acid
analyzer.
Example 6
[0182] Production of Antibodies to LBP-1, LBP-2 and LBP-3
[0183] This example illustrates the production of polyclonal
antibodies to LBP-1 , LBP-2 and LBP-3. A mixture of purified
recombinant LBP protein (0.5 ml; 200 .mu.g) and RIBI adjuvant (RIB1
ImmunoChem. Research, Inc., Hamilton, Mont.) was injected
subcutaneously into male guinea pigs (Dunkin Hartley; Hazelton
Research Products, Inc., Denver, Pa.) at 3-5 sites along the dorsal
thoracic and abdominal regions of the guinea pig. Blood was
collected by venipuncture on days 1 (pre-immune bleeding), 28, 49
and 70. Booster injections were administered on days 21 (100 .mu.g;
SC), 42 (50 .mu.g; SC), and 63 (25 .mu.g; SC). The titer of the
guinea pig antiserum was evaluated by serial dilution "dot
blotting." Preimmune antiserum was evaluated at the same time.
After the third booster of LBP protein, the titer against the
recombinant protein reached a maximal level with a detectable
calorimetric response on a dot blot assay of 156 pg.
[0184] Specificity of the polyclonal antibody for recombinant
LBP-1, LBP-2 or LBP-3 was demonstrated using Western blot analysis.
(Towbin et al., Proc. Natl. Acad. Sci. USA 76: 4350 (1979)). The
protein-antibody complex was visualized immunochemically with
alkaline phosphatase-conjugated goat antiguinea pig IgG, followed
by staining with nitro blue tetrazolium (BioRad Laboratories,
Hercules, Calif.). Non-specific binding was blocked using 3%
non-fat dry milk in Tris buffered saline (100 mM Tris; 0.9% NaCl,
pH 7.4).
Example 7
[0185] Immunohistochemical Characterization
[0186] This example illustrates the presence of LBPs in or on
endothelial cells covering plaques, in or on adjacent smooth muscle
cells, and in the extracellular matrix. In addition,
co-localization of LDL and LBPs was demonstrated. These results
were obtained by examining ballooned rabbit arterial lesions and
human atherosclerotic plaques by immunohistochemical methods.
[0187] Ballooned deendothelialized aorta was obtained from rabbits
which had received a bolus injection of human LDL (3 mg; i.v.) 24
hr prior to tissue collection. Human aortas containing
atherosclerotic plaques were obtained from routine autopsy
specimens. Tissues were fixed in 10% buffered formalin (<24 hr)
and imbedded in paraffin using an automated tissue-imbedding
machine. Tissue sections were cut (5-7 1.mu.) and mounted onto
glass slides by incubating for 1 hr at 60.degree. C. Sections were
deparaffinized. After a final wash with deionized H.sub.2O,
endogenous peroxidase activity was eliminated by incubating the
sections with 1% H.sub.2O.sub.2/H.sub.2O buffer for 5 min at room
temperature. Sections were rinsed with phosphate buffered saline
(PBS) for 5 min at room temperature and nonspecific binding was
blocked with 5% normal goat serum or 5% normal rabbit serum
depending on the source of the secondary antibody (Sigma, St.
Louis, Mo.) (1 hr; room temperature). Sections were then incubated
with a 1:50 dilution (in 5% normal goat serum/PBS) of a guinea pig
polyclonal antibody against the rabbit form of recombinant LBP-1,
LBP-2 or LBP-3. Controls included preimmune serum as well as
specific antisera to LBP-1, LBP-2, or LBP-3 in which the primary
antibody was completely adsorbed and removed by incubation with
recombinant LBP-1, LBP-2 or LBP-3 followed by centrifugation prior
to incubation with the tissue sections. An affinity purified rabbit
polyclonal antibody against human apolipoprotein B (Polysciences
Inc.; Warrington, Pa.) was used at a dilution of 1:100 (in 5%
normal rabbit serum/PBS). Sections were incubated for 2 hr at room
temperature in a humidified chamber. At the end of incubation,
sections were rinsed with PBS and incubated with a 1:200 dilution
(in 5% normal goat serum/PBS) of goat anti-guinea pig biotinylated
IgG conjugate (Vector Laboratories, Burlingame, Calif.) or a 1:250
dilution (in 5% normal rabbit serum/PBS)of rabbit anti-goat
biotinylated IgG conjugate (Vector Laboratories, Burlingame,
Calif.) for 1 hr at room temperature in a humidified chamber.
Sections were then rinsed with PBS and antigen-antibody signal
amplified using avidinibiotin HRP conjugate (Vectastain ABC kit;
Vector Laboratories, Burlingame, Calif.). Sections were developed
using DAB substrate (4-6 min; room temperature) and counterstained
with hematoxylin. In the ballooned rabbit artery,
immunohistochemistry with the anti-LBP-1, LBP-2 and LBP-3
antibodies showed that LBP-1, LBP-2 and LBP-3 were located in or on
functionally modified endothelial cells at the edges of
regenerating endothelial islands, the same location in which
irreversible LDL binding has been demonstrated (Chang et al.,
Arteriosclerosis and Thrombosis 12:1088-1098 (1992)). LBP-1, LBP-2
and LBP-3 were also found in or on intimal smooth muscle cells
underneath the functionally modified endothelial cells, and to a
lesser extent, in extracellular matrix. No LBP-1, LBP-2 or LBP-3
was detected in still deendothelialized areas, where LDL binding
had been shown to be reversible (Chang et al., Arteriosclerosis and
Thrombosis 12:1088-1098 (1992)). Immunohistochemistry of ballooned
rabbit aorta with anti-human apolipoprotein B antibodies showed the
presence of LDL at the same locations as that found for LBP-1,
LBP-2 and LBP-3.
[0188] In the human atherosclerotic plaques taken at routine
autopsies, immunohistochemistry with the anti-LBP-1,. anti-LBP-2
and anti-LBP-3 antibodies showed that LBP-1, LBP-2, and LBP-3 were
also found in or on endothelial cells covering plaques and in or on
adjacent smooth muscle cells. In the human tissue, there was
greater evidence of LBP-1, LBP-2 and LBP-3 in extracellular
matrix.
[0189] The results obtained with paraffin sections were identical
to those of frozen sections.
Example 8
[0190] Affinity Coelectrophoresis (ACE) Assays of LBPs and LDL or
HDL
[0191] This example illustrates that binding occurs between LBP-1,
LBP-2 or LBP-3 and LDL, and that this binding is specific, as
illustrated by the fact that binding does not occur between LBP-1,
LBP-2 or LBP-3 and HDL (high density lipoprotein). Analysis of the
affinity and specificity of recombinant rabbit LBP-1, LBP-2 or
LBP-3 binding to LDL was carried out using the principle of
affinity electrophoresis (Lee and Lander, Proc. Natl. Acad. Sci.
USA 88:2768-2772 (1991)). Melted agarose (1%; 65.degree. C.) was
prepared in 50 mM sodium MOPS, pH 7.0; 125 mM sodium acetate, 0.5%
CHAPS. A teflon comb consisting of nine parallel bars
(45.times.4.times.4 mm/3 mm spacing between bars) was placed onto
GelBond film (FMC Bioproducts, Rockland, Me.) fitted to a
plexiglass casting tray with the long axis of the bars parallel to
the long axis of the casting tray. A teflon strip
(66.times.1.times.1 mm) was placed on edge with the long axis
parallel to the short axis of the casting tray, at a distance of 4
mm from the edge of the teflon comb. Melted agarose (>65.degree.
C.) was then poured to achieve a height of approximately 4 mm.
Removal of the comb and strip resulted in a gel containing nine
45.times.4.times.4 mm rectangular wells adjacent to a 66.times.1 mm
slot. LDL or HDL samples were prepared in gel buffer (50 mM sodium
MOPS, pH 7.0, 125 mM sodium acetate) at twice the desired
concentration. Samples were then mixed with an equal volume of
melted agarose (in 50 mM MOPS, pH 7.0; 125 mM sodium acetate;
50.degree. C.), pipetted into the appropriate rectangular wells and
allowed to gel. The binding affinity and specificity of LBP-1 and
LBP-3 was tested using several concentrations of LDL (540 to 14 nM)
and HDL (2840177 nM). A constant amount (0.003 nM -0.016 nM) of
.sup.125I-labeled LBP1, LBP-2 or LBP-3 (suspended in 50 mM sodium
MOPS, pH 7.0; 125 mM sodium acetate; 0.5% bromphenol blue; 6%
(wt/vol) sucrose) was loaded into the slot. Gels were
electrophoresed at 70 v/2 hr/20.degree. C. At the end of the run,
the gels were air dried and retardation profiles were visualized by
exposure of X-ray films to the gels overnight at -70.degree. C.,
with intensifying screens.
[0192] LDL retarded LBP-1, LBP-2 and LBP-3 migration through the
gel in a concentration-dependent, saturable manner, indicating that
LBP-1, LBP-2 and LBP-3 binding to LDL was highly specific. This
conclusion is supported by the fact that HDL did not retard LBP-1,
LBP-2 or LBP-3. A binding curve generated from the affinity
coelectrophoresis assay indicated that LBP-1 binds to LDL with a
K.sub.d of 25.6 nM, that LBP-2 (rabbit clone 26) binds to LDL with
a K.sub.d of 100 nM, and that LBP-3 (80 kDa fragment) binds to LDL
with a K.sub.d of 333 nM.
[0193] In addition to testing affinity and specificity of LBP-1,
LBP-2 and LBP-3 binding to LDL, the ability of "cold" (i.e.,
non-radiolabeled) LBP-1, LBP-2 or LBP-3 to competitively inhibit
radiolabeled LBP-1, LBP-2 or LBP-3 binding to LDL, respectively,
was tested. Competition studies were carried out using fixed
concentrations of cold LDL and radiolabeled LBP-1 and increasing
amounts of cold recombinant LBP-1 (6-31 .mu.M). The ACE assay
samples and gel were prepared as described herein. Cold LBP-1
inhibited binding of radiolabeled LBP-1 to LDL in a
concentration-dependent manner, cold LBP-2 inhibited binding of
radiolabeled LBP-2 to LDL in a concentration-dependent manner, and
cold LBP-3 inhibited binding of radiolabeled LBP-3 to LDL in a
concentration-dependent manner.
[0194] Rabbit and human LBP-2 contain a long stretch of acidic
amino acids at the amino terminal (rabbit LBP-2 amino acid residues
338 through 365 and human LBP-2 amino acid residues 329 through
354). The possibility that this segment of LBP-2 was the LDL
binding domain was tested by subcloning two rabbit LBP-2 clones
which differ from each other by the presence or absence of this
acidic region (clone 26 and clone 45, respectively) into expression
vectors, by standard methods known to those skilled in the art. ACE
assays were then conducted in order to assess the affinity and
specificity of the binding of these two clones to LDL. LDL retarded
clone 26 derived radiolabeled LBP-2 migration through the gel in a
concentration-dependent, saturable, manner while clone 45 derived
radiolabeled LBP-2 migration was not retarded.
[0195] Competition studies using fixed concentrations of cold LDL
and clone 26 derived radiolabeled LBP-2 and increasing
concentrations of cold recombinant LBP-2/clone 26 and LBP-a/clone
45 were carried out. Cold clone 26 derived LBP-2 inhibited binding
of clone 26 derived radiolabeled LBP-2 to LDL in a
concentration-dependent manner. Clone 45 derived LBP-2, on the
other hand, did not affect the binding of clone 26 derived
radiolabeled LBP-2 to LDL. These results indicate that the long
stretch of acidic amino acids contain a binding domain of LBP-2 to
LDL.
Example 9
[0196] Affinity Coelectrophoreses (ACE) Assays of LBP-1 or LBP-2
and LDL in the Presence of Inhibitors
[0197] This example illustrates that binding between LBP-1 or LBP-2
and LDL is inhibited by polyglutamic acid or BHF-1. The ability of
a third compound to inhibit binding between two proteins previously
shown to interact was tested by a modification of the ACE assays
described in Example 8. The third compound was added to the top or
wells together with the radiolabeled protein. If the third compound
inhibited binding, the radiolabeled protein would run through the
gel. If the third compound did not inhibit binding, migration of
the radiolabeled protein was retarded by the protein cast into the
gel.
[0198] Inhibition of LBP-1/LDL or LBP-2/LDL binding by polyglutamic
acid (average MW about 7500, corresponding to about 7 monomers) was
shown by casting a constant amount of LDL (148 nM) in all the
rectangular lanes. A constant amount (1 .mu.l) of .sup.125I-labeled
LBP-1 or LBP-2 (0.003 nM-0.016 nM) was loaded in the wells at the
top of the gel, together with increasing concentrations of
polyglutamic acid (obtained from Sigma) (0-0.4 nM). The gel was
electrophoresed at 70 volts for 2 hr, dried and placed on X-ray
film, with intensifying screens, overnight at -70.degree. C. before
the film was developed to determine the retardation profile of
LBP-1 and LBP-2. As the concentration of polyglutamic acid
increased, retardation of radiolabeled LBP-1 and LBP-2 migration by
LDL decreased in a concentration-dependent manner, which showed
that polyglutamic acid inhibited binding between LBP-1, LBP-2 and
LDL.
[0199] Inhibition of LBP-1/LDL binding by BHF-1 was shown by
casting a constant amount of LDL (148 nM) in all the rectangular
lanes. A constant amount of .sup.125I-labeled LBP-1 (0.003 nM-0.016
nM) was loaded in the wells at the top of the gel, together with
increasing concentrations of BHF-1 (0-10 nM), obtained as described
in Example 15. The gel was electrophoresed at 70 volts for 2 hr,
dried and placed on X-ray film, with intensifying screens,
overnight at -70.degree. C. The film was then developed to
determine the retardation profile of .sup.125I-LBP-1. As the
concentration of BHF-1 increased, retardation of LBP-1 by LDL
decreased in a concentration-dependent manner, which demonstrated
that BHF-1 inhibited binding between LBP-1 and LDL.
Example 10
[0200] Affinity Coelectrophoreses (ACE) Assays for Identifying
Fragments, Analogs and Mimetics of LBPs which Bind to LDL
[0201] This example illustrates a method for identifying fragments,
analogs or mimetics of LBPs which bind to LDL, and which thus can
be used as inhibitors of LDL binding to LBP in the arterial walls,
by occupying binding sites on LDL molecules, thereby rendering
these sites unavailable for binding to LBP in the arterial
wall.
[0202] Fragments of LBPs are generated by chemical cleavage or
synthesized from the known amino acid sequences. Samples of these
fragments are individually added (cold) to radiolabeled LBP as
described in Example 8, to assess the inhibitory potency of the
various fragments. By iterative application of this procedure on
progressively smaller portions of fragments identified as
inhibitory, the smallest active polypeptide fragment or fragments
are identified. In a similar manner, analogs of the LBPs are tested
to identify analogs which can act as inhibitors by binding to LDL.
And, similarly, mimetics of LBP (molecules which resemble the
conformation and/or charge distributions of the LDL-binding sites
on LBP molecules) are tested in a similar fashion to identify
molecules exhibiting affinities for the LDL-binding sites on
LBP.
[0203] The affinities of the inhibitors so identified are at least
as strong as the affinity of LDL itself for the LDL-binding sites
on LBP. The inhibitors bind at least competitively, and some
irreversibly and preferentially as well, to the LDL-binding sites,
thereby rendering such sites unavailable for binding to humoral
LDL.
Example 11
[0204] ELISA Assays
[0205] This example illustrates the use of ELISA plate assays for
the quantification of a test compound's capacity to inhibit the
binding of LDL to a specific LBP.
[0206] In one example, the ELISA assay was carried out as follows:
LDL was diluted in 50 mM Na.sub.2HCO.sub.3, pH 9.6/0.02% NaN.sub.3
and added to the wells of a 96-well plate (ImmunoWare 96-Well
Reacti-Bind EIA Polystyrene Plates; Pierce (Rockford, Ill.)) to
achieve a final concentration ranging from 0.1 to 1 .mu.g/well. The
plates were incubated for 6 hr at room temperature. At the end of
the incubation period, the wells were washed 3 times with
Tris-buffered saline, pH 7.4 (TBS), and blocked overnight with 200
.mu.l of 1% bovine serum albumin (BSA) in TBS/0.02% NaN.sub.3
(Sigma; St. Louis Mo.) at room temperature. The wells were then
incubated with 200 .mu.l of LBP protein (5-10 .mu.g/well) in TBS
and varying concentrations of the test compound. Plates were
incubated for 1 hr at room temperature. The wells were then washed
three times with TBS and blocked for 2 hr with 200 .mu.l of 1% BSA
in TBS/0.02% NaN.sub.3 at room temperature. At the end of the
incubation period, the wells were washed 3 times with TBS and a
1:1000 dilution (in TBS/0.05% Tween 20) of the appropriate guinea
pig anti-LBP protein polyclonal antibody was added to the wells and
incubated for 1 hr at room temperature. The wells were then washed
3 times with TBS/0.05% Tween 20; a 1:30,000 dilution of goat
anti-guinea pig IgG alkaline phophatase conjugate (Sigma) was added
to each well. Plates were incubated for 1 hr at room temperature.
The wells were washed 3 times with TBS/0.05% Tween 20 and a
calorimetric reaction was carried out by adding 200 ml of
p-nitrophenyl phosphate substrate (Sigma; St. Louis Mo.) to the
wells. The reaction was allowed to proceed for 30 min at room
temperature and stopped with 50 .mu.l of 3N NaOH. The absorbance
was determined at 405 nm using an ELISA plate reader. The test
compound's effectiveness in blocking the binding of LDL to the
recombinant protein was assessed by comparing the absorbance values
of control and treated groups.
[0207] In a second example, the ELISA assay was carried out as
follows: LDL was diluted in Tris-buffered saline, pH 7.4 (TBS) and
added to the wells of a 96-well plate (Immuno Ware 96-Well
Reacti-Bind EIA Polystyrene Plates; Pierce (Rockford Ill.)) to give
a plate-saturating concentration of 0.2 .mu.g/well. The plate was
incubated for 1 hr at room temperature, after which the wells were
washed three times with TBS, before being blocked for 1 hr at room
temperature with 1% bovine serum albumin (BSA in TBS). The wells
were then washed twice with TBS before LBP-1 or LBP-2 (0.025
.mu.g/well), or LBP-3 (0.01 .mu.g/well) were added, without and
with varying concentrations of the test inhibitor compound. Each
condition was set up in quadruplicate. The plate was incubated for
1 hr at room temperature, then washed three times with TBS/0.02%
Tween 20 (TBS/Tween). An appropriate dilution of guinea pig
anti-LBP polyclonal antibody (1:750 to 1:1500, depending on the
antibody) was added to three wells for each condition and incubated
for 1 hr. Anti-LBP antibody was replaced by buffer for the fourth
well of each condition, as a negative control. After 1 hr. the
plate was again washed three times with TBS/Tween before a 1:10,000
dilution (in TBS/Tween) of goat anti-guinea pig IgG alkaline
phosphatase-conjugated antibody (Sigma) was added to each well. The
plate was incubated for 1 hr at room temperature, then washed three
times with TBS/Tween. A fresh solution of substrate was prepared
from an Alkaline Phosphatase Substrate Kit (Bio-Rad, Hercules,
Calif.) as follows: Mix 1 ml 5.times.concentrated diethanolamine
buffer with 4 ml distilled water. Add one tablet of
p-nitrophenylphosphate (5 mg) and vortex until tablet is completely
dissolved. Subtrate solution was added to wells immediately.
Increasing concentrations of diluted alkaline
phosphatase-conjugated goat anti-guinea pig IgG (1:100,000 dilution
in TBS/Tween) were added to five empty wells, followed by
substrate, as a positive control. Following addition of substrate,
the plate was immediately placed in an ELISA plate reader, allowed
to stand at 37.degree. C., generally for 75 min, before absorbance
was measured at 405 nm. Incubation in the ELISA reader at
37.degree. C. was sometimes adjusted to optimize absorbance (60-90
min). The effectiveness of the test inhibitor was determined, after
subtracting absorbance of negative controls, by comparing
absorbance in wells where an LBP was mixed with test inhibitor to
absorbance in wells containing LBP with no inhibitor.
[0208] Alternatively, LBPs, rather than LDL, were bound to the
plate. Recombinant LBP protein binding to LDL and the effect of
varying concentration of the inhibitor on LBP-LDL binding was
determined through the use of antibodies against LDL. This
interaction was visualized through the use of a secondary antibody
conjugated to a reporter enzyme (e.g. alkaline phosphatase).
[0209] ELISA plate assays were used to screen agents which can
affect the binding of LBP proteins to LDL. For example, peptides
derived from LBP-1 and human LBP-3 protein sequences (BHF-1 and
BHF-2, respectively) were synthesized and have been shown to reduce
the binding of LDL to recombinant LBP-1 and LBP-2 in this format.
These results were in agreement with those obtained with the ACE
assays.
Example 12
[0210] Administration of Humanized Antibodies Against LBPs so as to
Block LDL-Binding Sites on the LBPs
[0211] This example illustrates administration to patients of
humanized antibodies against LBP-1, LBP-2 or LBP-3 so as to block
LDL-binding sites on arterial LBP molecules. Mouse monoclonal
antibodies are humanized by recombinant DNA techniques and produced
by standard procedures known to those skilled in the art (Berkower,
I., Curr. Opin. Biotechnol. 7:622-628 (1996); Ramharayan and
Skaletsky, Am. Biotechnol. Lab 13: 26-28 (1995)) against LBPs
and/or the LDL-binding sites on the LBPs. The corresponding Fab
fragments are also produced, as described in Goding, J. W.,
Monoclonal Antibodies: Principles and Practice, Academic Press, New
York, N.Y. (1986). These antibodies are administered parenterally
in sufficient quantity so as to block LDL-binding sites on the LBP
molecules, i.e., 1-10 mg/kg daily. This prevents the irreversible
arterial uptake of LDL that is required to facilitate oxidation of
the LDL.
Example 13
[0212] Preparation of LDL
[0213] This example illustrates the preparation of LDL. LDL was
prepared from the plasma of normolipemic donors (Chang et al.,
Arterioscler. Thromb. 12:1088-1098 (1992)). 100 ml of whole blood
was placed into tubes containing 100 mM disodium EDTA. Plasma was
separated from red blood cells by low-speed centrifugation (2,000
g; 30 min; 4.degree. C.). Plasma density was adjusted to 1.025
gm/ml with a solution of KBr and centrifuged for 18-20 hr,
100,000.times.g, 12.degree. C. Very low density lipoproteins (VLDL)
were removed from the tops of the centrifuge tubes with a Pasteur
pipette. The density of the infranate was raised to 1.050 gm/ml
with KBr solution and centrifuged for 22-24 hr, 100,000.times.g,
12.degree. C. LDL was removed from the tops of the centrifuge tubes
with a drawn out Pasteur pipette tip. Purity of the LDL preparation
was checked by Ouchterlony double immunodiffusion against
antibodies to human LDL, human HDL, human immunoglobulins, and
human albumin. KBr was removed from the LDL solution by dialysis (1
L,.times.2, approximately 16 hr) against 0.9% saline, pH 9.0,
containing 1 mM EDTA and 10 .mu.M butylated hydroxytoluene (BHT),
the latter to prevent oxidation of LDL. Following dialysis, LDL
protein was measured by the method of Lowry (Lowry et al., J. Biol.
Chem. 193:265-275 (1951)), and the LDL was stored at 4.degree. C.
until use. LDL preparations were kept for no more than 4-6
weeks.
Example 14
[0214] Preparation of HDL
[0215] This example illustrates the preparation of HDL. HDL was
prepared from plasma of normolipemic donors. 100 ml of whole blood
was placed into tubes containing 100 mM disodium EDTA and plasma
was collected by centrifugation (2000 g; 30 min; 4.degree. C.).
Apolipoprotein B containing lipoproteins present in plasma were
then precipitated by the sequential addition of sodium heparin
(5,000 units/ml) and MnCl.sub.2 (1M) to achieve a final
concentration of 200 units/ml and 0.46 M, respectively (Warnick and
Albers, J. Lipid Res. 19:65-76 (1978)). Samples were then
centrifuged, (2000 g; 1 hr; 4.degree. C.). The supernatant was
collected and density adjusted to 1.21 g/ml by the slow addition of
solid KBr. HDL was separated by ultracentrifugation (100,000
g;>46 hr; 12.degree. C.). Purity of the HDL preparation was
assessed via Ouchterlony double immunodiffusion test using
antibodies against human HDL, human LDL, human immunoglobulins, and
human albumin. HDL samples were dialyzed against saline pH 9.0/1 mM
EDTA/10 .mu.M BHT (4 L; 24 hr/4.degree. C.) and total protein was
determined by the Lowry protein assay (Lowry et al., J. Biol. Chem.
193:265-275 (1951)). HDL was stored at 4.degree. C. until use. HDL
preparations were kept for no longer than 2 weeks.
Example 15
[0216] Synthesis of BHF-1
[0217] This example illustrates the synthesis of BHF-1, a fragment
of human or rabbit LBP-1 which contains amino acid residues 14
through 33. BHF-1 was synthesized using an Applied Biosystems Model
430A peptide synthesizer with standard T-Boc NMP chemistry cycles.
The sequence of BHF-1 is as follows:
[0218] val
-asp-val-asp-glu-tyr-asp-glu-asn-lys-phe-val-asp-glu-glu-asp-gl-
y-gly-asp-gly (SEQ ID NO: 9).
[0219] After synthesis, the peptide was cleaved with hydrofluoric
acid/anisole (10/1 v/v) for 30 min at -10.degree. C. and then
incubated for 30 min at 0.degree. C. BHF-1 was then precipitated
and washed three times with cold diethyl ether. Amino acid coupling
was monitored with the ninhydrin test (>99%).
[0220] The BHF-1 peptide was purified to homogeneity by high
performance liquid chromatography on a reverse phase Vydac C.sub.4
column (2.24.times.25 cm) using a linear gradient separation (2-98%
B in 60 min) with a flow rate of 9 ml/min. Buffer A consisted of
0.1% trifluoroacetic acid (TFA)/Milli Q water and Buffer B
consisted of 0.085% TFA/80% acetonitrile. The gradient was run at
room temperature and absorbance monitored at 210 and 277 nm.
[0221] Fast atom bombardment-mass spectrometry gave a protonated
molecular ion peak (M+H).sup.+ at m/z=2290.2, in good agreement
with the calculated value. On amino acid analysis, experimental
values for the relative abundance of each amino acid in the peptide
were in good agreement with theoretical values. The lyophilized
peptide was stored at -20.degree. C.
Example 16
[0222] In Vitro Screening for Agents which Inhibit Binding Between
LDL and LBPs
[0223] This example illustrates in vitro screening for agents which
inhibit binding between LDL and LBPs.
[0224] A candidate polypeptide for being an agent is chosen, e.g.,
LBP-1, LBP-2, LBP-3, BHF-1 or any other polypeptide. The shortest
fragment of the polypeptide that inhibits LDL binding to LBPs in
vitro is determined. Peptides are synthesized by standard
techniques described herein. Inhibition assays are performed using
standard ELISA techniques for screening, and affinity
coelectrophoresis (ACE) assays to confirm the ELISA results, as
described herein. Additional assays that can be used in this
screening method include, e .g., fluorescence polarization and
pulsed ultra-filtration electrospray mass spectrometry. Short
peptides ranging, e.g., from dimers to 20-mers are constructed
across sequences of the candidate polypeptide whose chemical
characteristics make them likely LDL binding sites, e.g., acidic
regions. The ability of shorter and shorter lengths of the peptides
to inhibit LDL binding in vitro and to mammalian cells in culture
is tested. For example, the effect of the peptide on inhibiting LDL
binding in mammalian cells transfected to express an LBP gene is
tested. Each of the peptides so identified as an inhibitor is
tested with each of LBP-1, LBP-2 and LBP-3, to determine whether a
single inhibitor works against all three LBPs.
[0225] Once the minimum active sequence is determined, the peptide
backbone is modified so as to inhibit proteolysis, as discussed
herein. For example, modification is accomplished by substitution
of a sulfoxide for the carbonyl, by reversing the peptide bond, by
substituting a methylene for the carbonyl group, or other similar
standard methodology. See Spatola, A. F., "Peptide Backbone
Modifications: A Structure-Activity Analysis of Peptides Containing
Amide Bond Surrogates, Conformational Constraints, and Related
Backbone Replacements," in Chemistry and Biochemistry of Amino
Acids, Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein
(ed.), Marcel Dekker, Inc., New York (1983). The ability of these
analogs to inhibit LDL binding to the LBPs in vitro is tested in a
similar manner as for the natural peptides described above, e.g.,
by ELISA, ACE, fluorescence polarization, and/or pulsed
ultra-filtration electrospray mass spectrometry.
Example 17
[0226] In Vitro Screening with Cultured Mammalian Cells for Agents
which Inhibit Binding Between LDL and LBPs
[0227] This example illustrates cell-based in vitro screening of
agents which have been shown by in vitro tests such as ACE assay
and ELISA to be potential inhibitors of binding between LDL and
LBPs.
[0228] Mammalian cells, such as 293 cells, which are commonly used
for expression of recombinant gene constructs, are used to develop
cell lines which express LBPs on the cell surface. This is done by
subcloning LBP open reading frames (ORFS) into a mammalian
expression plasmid vector, pdisplay (Invitrogen, Carlsbad, Calif.),
which is designed to express the gene of interest on the cell
surface. The use of mammalian cells to produce LBPs allows for
their expression in a functionally active, native conformation.
Therefore, stably transfected mammalian cell lines with surface
expression of LBPs individually, or in combination, are
particularly suitable for assaying and screening inhibitors that
block LDL binding in cell culture, as well as to evaluate the
cytotoxicity of these compounds.
[0229] Specifically, LBP ORFs are amplified by PCR (Perkin Elmer,
Foster City, Calif.) from cDNA templates using Taq polymerase
(Perkin Elmer) and appropriate primers. The amplified LBP ORFs are
purified by agarose gel electrophoresis and extracted from gel
slices with the Bio-Rad DNA Purification kit (Bio-Rad, Hercules,
Calif.). The purified DNAs are then cut with the restriction
enzymes Bgl II and Sal I (New England Biolabs, Beverly, Mass.) to
generate cohesive ends, and purified again by agarose gel
electrophoresis and DNA extraction as described above. The LBP ORFs
are then subcloned into the Bgl II/Sal I sites in the mammalian
expression vector, pDisplay (Invitrogen) by ligation. Recombinant
plasmids are established by transformation in E. coli strains TOP10
(Invitrogen) or DH5.alpha. (Life Technologies, Grand Island, N.Y.).
Recombinant pDisplay/LBP plasmid DNA is isolated from overnight E.
coli cultures with the Bio-Rad Plasmid Miniprep kit, cut with Bgl
II/Sal I, and analyzed by agarose gel electrophoresis. LBP ORFs in
successfully transformed clones are verified by automated dideoxy
DNA sequencing. To transfect human kidney 293 cells, 1-2 .mu.g of
DNA is mixed with 6 .mu.l lipofectamine reagent (Life Technologies)
and incubated with the cells as described in the Life Technologies
protocol. LBP expression in transfected cells is confirmed by
Western blot analysis of cell extracts obtained 48 hr after
transfection. To select for stably transfected 293 cells, the
antibiotic G418 (Life Technologies) is added to the growth medium
at a concentration of 800 .mu.g/ml. Colonies resistant to G418 are
tested for recombinant LBP expression by Western blot, and
recombinant clones expressing LBPs are expanded, assayed for LDL
binding and used to test compounds for their ability to inhibit LDL
binding.
Example 18
[0230] In Vivo Screening for Agents which Inhibit Binding Between
LDL and LBPs
[0231] This example illustrates in vivo screening of agents which
have been shown by in vitro tests to be promising candidate
inhibitors of binding between LDL and LBPs.
[0232] In vivo inhibitory activity is first tested in the healing
balloon-catheter deendothelialized rabbit aorta model of arterial
injury (Roberts et al., J. Lipid Res. 24:1160-1167 (1983); Chang et
al., Arterioscler. Thromb. 12:1088-1098 (1992)). This model was
shown to be an excellent analog for human atherosclerotic lesions.
Other useful animal models for human atherosclerosis include Apo E
knockout mice and LDL receptor knockout mice. Both of these mouse
models are characterized by high levels of plasma cholesterol and
the development of naturally-occurring atherosclerotic-like
lesions.
[0233] Each candidate inhibitor is tested in five to ten ballooned
rabbits, while an equal number of rabbits receive a control
peptide, or placebo. Four weeks following aortic
deendothelialization, when reendothelialization (healing) is
partially complete, daily parenteral (intravenous or subcutaneous)
or intragastric administration of the peptides and the analogs
begins at an initial concentration of 10 mg/kg body weight, which
is varied down, or up to 100 mg/kg depending on results. 30 min
later, a bolus of intravenously injected .sup.125I(or .sup.99mTc-)
labeled LDL is given to test the candidate inhibitor's ability in
short term studies to inhibit LDL sequestration in healing arterial
lesions. If .sup.125I-LDL is used, the animals are sacrificed 8-24
hr later, the aortas excised, washed and subjected to quantitative
autoradiography of excised aortas, as previously described (Roberts
et al., J. Lipid Res. 24:1160-1167 (1983); Chang et al.,
Arterioscler. Thromb. 12:1088-1098 (1992)). If .sup.99mTc-LDL is
used, analysis is by external gamma camera imaging of the live
anesthetized animal at 2-24 hr, as previously described (Lees and
Lees, Syndromes of Atherosclerosis, in Fuster, ed., Futura
Publishing Co., Armonk, N.Y., pp. 385-401 (1996)), followed by
sacrifice, excision and imaging of the excised aorta. Immediately
before the end of testing, the animals have standard toxicity
tests, including CBC, liver enzymes, and urinalysis.
[0234] The compounds which are most effective and least toxic are
then tested in short term studies of rabbits fed a 2% cholesterol
diet (Schwenke and Carew, Arteriosclerosis 9:895-907 (1989)). Each
candidate inhibitor is tested in five to ten rabbits, while an
equal number of rabbits receive a control peptide, or placebo.
Animals receive one or more doses per day of the candidate
inhibitor, or placebo, for up to two weeks. Daily frequency of
doses is determined by route of administration. If active drug or
placebo are administered parenterally, they are given 1-3 times
daily and the 2% cholesterol diet is continued. If drug or placebo
are given orally, they are mixed with the 2% cholesterol diet.
Schwenke and Carew (Arteriosclerosis 9:895-907 (1989)) have shown
that the LDL concentration in lesion-prone areas of the rabbit
aorta is increased 22-fold above normal in rabbits fed a 2%
cholesterol diet for 16 days, and that the increased LDL content
precedes the histological evidence of early atherosclerosis.
Therefore, analysis of the effect of the candidate inhibitors is
tested two weeks after the start of cholesterol feeding by
injecting .sup.125I-LDL, allowing it to circulate for 8-24 hr, and
then performing quantitative autoradiography on the excised aortas
of both test and control animals. If appropriate, quantitation of
aortic cholesterol content is also carried out (Schwenke and Carew,
Arteriosclerosis 9:895-907 (1989); Schwenke and Carew,
Arteriosclerosis 9:908-918 (1989).
[0235] The above procedures identify the most promising candidate
inhibitors, as well as the best route and frequency of their
administration. Inhibitors so identified are then tested in
long-term studies of cholesterol-fed rabbits. These tests are
carried out in the same way as the short-term cholesterol feeding
studies, except that inhibitor effectiveness is tested by injection
of .sup.125I-LDL at longer intervals following the initiation of
cholesterol feeding, and lesion-prone areas of the aorta are
examined histologically for evidence of atherosclerosis. Testing
times are at two, four, and six months. Major arteries are examined
grossly and histologically or evidence and extent of
atherosclerosis. If necessary, other accepted animal models, such
as atherosclerosis-susceptible primates (Williams et al.,
Arterioscler. Thromb. Vast. Biol. 15:827-836 (1995)), genetically
altered mice, and/or Watanabe rabbits are tested with short- and
long-term cholesterol feeding.
Example 19
[0236] In Vivo Inhibition of Radiolabeled LDL Accumulation in the
Ballooned Deendothelialized Rabbit Aorta via Induction of Active
Immunity Against LBP Protein
[0237] This example illustrates the effect that induction of
immunity against LBP protein has on the accumulation of
radiolabeled LDL in the ballooned deendothelialized rabbit aorta
model of atherosclerosis.
[0238] Immunity was induced in male New Zealand White rabbits
(Hazelton Research Products, Denver, Pa.) as follows: A mixture of
purified human recombinant LBP-2 or BHF-1 peptide (1 ml; 1 mg) and
RIBI adjuvant (RIBI ImmunoChem Research, Inc., Hamilton, Mont.) was
injected subcutaneously at 2-5 sites along the dorsal thoracic and
abdominal regions of the rabbits. Blood was collected by
venipuncture on days 1 (preimmune bleeding), 35, 63, and 91.
Booster injections were administered on days 28 (500 .mu.g; SC), 56
(250 .mu.g; SC), and 84 (125 .mu.g; SC).
[0239] The titer of the rabbits was evaluated by serial dilution
using an ELISA plate format. Preimmune serum was evaluated at the
same time. After the third booster of LBP protein or peptide, the
titer reached a maximal level with a detectable calorimetric
response on an ELISA plate of 156 pg. Titer is defined as the
maximum dilution of antibody which generates an absorbance reading
of 0.5 above control in 30 min. Specificity of the polyclonal
antibodies was demonstrated using Western blot analysis as
described in Example 6.
[0240] On day 93, the abdominal aorta of immunized and control
rabbits was deendothelialized using a Fogarty number 4 embolectomy
catheter (Chang et al., Arteriosclerosis and Thrombosis
12:1088-1098 (1992)). Four weeks after ballooning, rabbits received
a bolus injection of .sup.125I-labeled LDL (1 ml; i.v.). Blood
samples were collected at 1 hr intervals for 8 hr, and 24 hr post
injection. Blood samples were centrifuged for 30 min at 2000 rpm
(40.degree. C.)and total activity present in the serum was
determined using a Gamma counter. Total TCA precipitable counts
were determined by addition of TCA to the serum to a final
concentration of 10% followed by incubation for 10 min at 4.degree.
C. Serum samples were then centrifuged (2000 rpm; 30 min;
40.degree. C.) and total activity present in the supernate was
determined. TCA precipitable counts were calculated by substration:
total soluble counts minus counts present in the supernate after
TCA precipitation. Blood samples for the determination of antibody
titers were collected prior to the injection of the radiolabeled
LDL.
[0241] After 24 hr, the rabbits were injected intravenously with 5%
Evan's blue dye which was allowed to circulate for 15 min. Areas of
the aorta in which the endothelial covering is absent stain blue
while those areas covered by endothelium remain unstained. At the
end of the incubation period, the rabbits were euthanized and the
abdominal and thoracic aorta were dissected out, rinsed, and fixed
overnight in 10% TCA at room temperature. The aortas were then
rinsed exhaustively with physiological saline, weighed, counted,
blotted dry and placed onto X-ray film in order to visualize the
pattern of radiolabeled LDL accumulation in the deendothelialized
rabbit abdominal aorta.
[0242] Immunization of rabbits against recombinant human LBP-2 or
BHF-1 peptide altered the pattern of radiolabeled LDL accumulation
in the ballooned deendothelialized abdominal aorta. When corrected
for dosage, and percent reendothelialization, immunized-ballooned
rabbits had lower accumulation of radiolabeled LDL compared to
nonimmune-ballooned rabbits. These results indicate that active
immunization against LBP provides an effective means by which the
accumulation of LDL in the injured arterial wall can be
modified.
Example 20
[0243] Screening Agents in Humans which Inhibit Binding Between LDL
and LBPs
[0244] Human studies are carried out according to standard FDA
protocols for testing of new drugs for safety (Phase I), efficacy
(Phase II), and efficacy compared to other treatments (Phase III).
Subjects, who are enrolled into studies after giving informed
consent, are between the ages of 18 and 70. Women who are pregnant,
or likely to become pregnant, or subjects with diseases other than
primary atherosclerosis, such as cancer, liver disease, or
diabetes, are excluded. Subjects selected for study in FDA Phase II
and Phase III trials have atherosclerotic disease previously
documented by standard techniques, such as ultrasound and/or
angiography, or are known to be at high risk of atherosclerosis by
virtue of having at least one first degree relative with documented
atherosclerosis. Subjects themselves have normal or abnormal plasma
lipids. Initial testing includes 20-50 subjects on active drug and
20-50 subjects, matched for age, sex, and atherosclerotic status,
on placebo. The number of subjects is pre-determined by the number
needed for statistical significance. Endpoints for inhibitor
efficacy includes ultrasound measurements of carotid artery
thickness in high risk subjects, as well as in subjects with known
carotid or coronary disease; atherosclerotic events;
atherosclerotic deaths; and all-cause deaths in all subjects.
Non-invasive analysis (carotid artery thickness by ultrasound) as
per Stadler (Med. and Biol. 22:25-34 (1996)) are carried out at 6-
to 12-month intervals for 3 years. Atherosclerotic events and
deaths, as well as all-cause deaths are tabulated at 3 years.
[0245] Oral dosage of drug in FDA Phase I trials ranges from 0.01
to 10 gm/day, and is determined by results of animal studies,
extrapolated on a per kg basis. Based on data obtained from Phase I
studies, the dose range and frequency are narrowed in Phase II and
III trials. If parenteral administration of drug is determined by
animal studies to be the only effective method, parenteral
administration in human subjects is tested by injection, as well as
by the transdermal and nasal insufflation routes. Testing of
parenteral drug follows the same outline as that for oral
administration.
[0246] The optimal treatment schedule and dosage for humans is thus
established.
Example 21
[0247] Treating an Individual Having Atherosclerosis with BHF-1
[0248] This example illustrates a method for treating an individual
having atherosclerosis with an LBP fragment, e.g., BHF-1, so as to
decrease the levels of arterially bound LDL in the individual.
BHF-1 is obtained as described herein. The BHF-1 is administered to
the mammal intravenously as a bolus or as an injection at a
concentration of 0.5-10 mg/kg body weight. Such administrations are
repeated indefinitely in order to prevent the development or
progression of symptomatic atherosclerosis, just as is done
currently with cholesterol lowering drugs. Stable subjects are
examined twice yearly to evaluate the extent of any atherosclerotic
disease by physical exam and non-invasive studies, such as carotid
artery thickness, ultrasound, and/or gamma camera imaging of the
major arteries, to determine if atherosclerotic lesions are
present, and, if previously present, have regressed or progressed.
Such a regimen results in treatment of the atherosclerosis.
Example 22
[0249] In Vivo Reduction of Atherosclerosis in Apo E Knockout Mice
by Immunization with LBPs
[0250] Separate immunization experiments were performed with each
of LBP-1, LBP-2, and LBP-3. Immunity was induced by injecting apo E
knockout mice with the LBP protein (LBP-1, LBP-2, or LBP-3)
together with an RIBI adjuvant (RIBI ImmunoChem Research, Inc.,
Hamilton, Mont.). Apo E knockout mice (Jackson Laboratories, Bar
Harbor, Me.) are hyperlipidemic and thus a model for human
atherosclerosis. Apo E knockout mice have high levels of plasma
cholesterol and develop naturally-occurring atherosclerotic-like
lesions.
[0251] Four week old apo E knockout mice (Jackson Laboratories, Bar
Harbor, Me.) were ear tagged, randomly assigned to different cages
and weighed. Body weights were determined weekly. Animals were
allowed to habituate for 1 week. Normal rodent chow was provided ad
libitum and animals were maintained in a 12:12 light:dark cycle.
The following four groups of mice were treated with either
recombinant LBP proteins (40 .mu.g of recombinant protein/mouse)
plus RIBI adjuvant or RIBI adjuvant alone (control group).
[0252] LBP-1: Immunized with rabbit recombinant LBP-1 (6-His
tag).
[0253] LBP-2: Immunized with rabbit recombinant LBP-2 clone 26
(6-His tag).
[0254] LBP-3: Immunized with rabbit recombinant LBP-3 (6-His
tag).
[0255] Control: Received adjuvant.
[0256] Blood samples (pre-immune serum) were collected prior to the
initial injection of recombinant protein and RIBI adjuvant (as
described in the manufacturer's manual). After 21 days, mice
received a booster injection (half-initial dose) and were then bled
seven days later. Titer was defined as the maximum dilution of
serum that yielded a change in absorbance equivalent to
2.times.that of control serum (60 min; 37.degree. C.). The amount
of recombinant protein per well was 100 ng.
[0257] Booster injections took place at 21 day intervals until an
average titer value of 1:10,000 was reached. At this time, mice
were switched to western type diet (Harland Teklad, Madison, Wis.)
and fed ad libitum. Blood samples were collected at this time
(retro-orbital sinus bleeding technique) and monthly
thereafter.
[0258] Blood samples were analyzed for total cholesterol, HDL
cholesterol, and triglyceride concentration with a commercially
available total cholesterol and triglycerides assay kits (Sigma;
St. Louis Mo.) using an ELISA format. HDL concentration was
determined after Apo B containing lipoproteins were precipitated
using heparin/MnCl.sub.2.
[0259] Apo E knockout mice were sacrificed at 26 weeks of age. The
mice were anesthetized with methoxyfluorane and exanguinated via
cardiac puncture. A midline thoracotomy was performed, a cannula
inserted into the right ventricle and perfusate allowed
unrestricted flow via an incision into the right atrium. The mice
were perfused with saline, followed by 10% phosphate buffered
formalin until fasciculations stopped. At this time, the aorta was
exposed and adventitial fat removed in situ. The aorta was then
removed from the heart down to the iliac bifurcation and placed in
10% phosphate buffered formalin overnight.
[0260] The aorta was stained as follows: after a brief 70% ethanol
rinse, it was immersed in a filtered solution of 0.5%
(weight/volume) Sudan IV in 35% ethanol/50% acetone with continuous
shaking for 10 minutes at room temperature. Unbound dye was removed
by incubating the aorta in an 80% ethanol solution with shaking
until the background color was clear. The vessel was then rinsed in
distilled water, placed in physiological saline and opened
longitudinally from the aortic arch down to the iliac bifurcation.
The vessel was pinned out and photographed. Photographs were then
digitized using an Astra 1200S scanner (UMAX Technologies Inc.,
Freemont, Calif.) and a commercially available graphics program
(Canvas; Deneba Software, Miami Fla.). Total and lesion areas were
determined using the signal processing toolbox of MATLAB (The
Mathworks Inc., Natick, Mass.). Percent involvement was calculated
by dividing lesion area by total area.
[0261] A second analysis was done to measure aortic atherosclerosis
by a cholesterol extraction method whereby cholesterol is
determined as a unit weight of artery. This method may be more
accurate in measuring lesion size than attempting to measure the
thickness of many sections. Specifically, the weight of an artery
was measured, then the cholesterol was extracted. Aortic
cholesterol content was then measured by gas-liquid chromatography.
The amount of cholesterol per unit weight of artery was then
determined.
[0262] After the first booster injection, some of the apo E
knockout mice immunized against LBP-1 had relatively high
anti-LBP-1 titers (<1:5000) while others in the same group
exhibited moderate levels (>1:500 to <1:1000). LBP-2/26
titers were low in the apoE knockout mice (<1:500) at this time.
LBP-3 titers ranged from moderate to low (>1:500 to <1:1000)
to low (<1:500) in the apoE knockout mice.
[0263] After the second booster injection, Apo E knockout mice
immunized against LBP-1 had moderate to high titers (>1:1000 to
<1:8000). Apo E knockout mice immunized against LBP-2/26 had
moderate titer levels (>1:2000). LBP-3 titers range from
moderate to high (>1:1000 to >1:8000) in the Apo knockout
mice.
[0264] After the third booster injection, most of the mice
immunized against LBP-1 had relatively high titers (>1:10,000)
while others had moderate to high titers (>1000 to
<1:10,000). Some of the Apo E knockout mice had moderate
(<1:5000) to low (<1:1000) titers. LBP-3titers ranged from
high(>1:5000to <1:10,000)to moderate(>1:1000to
<1:5000).
[0265] Data were analyzed using T-tests and Wilcoxons. Immunization
against LBP-1, LBP-2/26 or LBP-3 did not have a significant effect
(P>0.05) on body weight of Apo E knockout mice. Due to the small
sample size and the large variability present in the Apo E knockout
mice, it was not possible to determine whether immunization against
LBP-1, LBP-2/26 or LBP-3 had an effect on total cholesterol, HDL
cholesterol or triglycerides concentration, but it did not appear
to.
[0266] Immunization against LBP-1 or LBP-3 did not have a
significant effect (P>0.05) on lesions of the apo E knockout
mice or LDL receptor negative knockout mice. However, immunization
of the apo E knockout mice against LBP-2 had a significant effect
on lesion area (Table 2), and, once outliers were deleted, a
significant effect on arterial wall cholesterol content (Table 3).
The LBP-2 immunized apo E knockout mice had significantly reduced
aortic atherosclerosis as compared to the control, non-immunized
mice. Without being bound to any particular theory, the circulating
antibodies generated against LBP-2 proteins are thought to block
LDL binding to the artery wall.
3TABLE 2 Lesion Area in LBP-Immunized Apo E Mice Lesion Area
Treated Area P-Value Apo E Mice % Coverage Change Wilcoxon Control
9.40 LBP-1 6.05 -0.36% 0.07 LBP-2 6.01 -0.36% 0.01 LBP-3 7.14
-0.24% 0.36
[0267]
4TABLE 3 Arterial Cholesterol Content in LBP-Immunized Apo E Mice
Arterial Wall Treated Area Cholesterol (ug Cholesterol P-Value Apo
E Mice cholesterol/mg aorta) Change Wilcoxon Control 6.33 LBP-1
3.82 -0.40% 0.14 LBP-2 3.28 -0.48% 0.07 LBP-2 (outliers 1.83 -0.71%
0.01 deleted) LBP-3 4.48 -0.29% 0.20
Example 23
[0268] In Vivo Reduction of Atherosclerosis in LDL Receptor
Knockout Mice by Immunization with BHF-1
[0269] An immunization experiment was performed with the BHF-1
peptide. LDL receptor (LDLR) knockout mice (B6,
129S-Ldlr.sup.tmlHer, Jackson Laboratories, Bar Harbor, Me.) were
injected with the BHF-1 peptide (see Example 15 for methods of
synthesizing the BHF-1 peptide) together with an RIBI adjuvant
(RIBI ImmunoChem Research, Inc., Hamilton, Mont.). LDLR knockout
mice are hyperlipidemic and thus a model for human atherosclerosis.
LDLR knockout mice have high levels of plasma cholesterol and
develop naturally-occurring atherosclerotic-like lesions.
[0270] Four week old LDLR knockout mice were ear tagged, randomly
assigned to different cages and weighed. Body weights were
subsequently determined weekly. Animals were allowed to habituate
for one week prior to experimentation. Normal rodent chow was
provided ad libitum and animals were maintained in a 12:12
light:dark cycle. Animals were divided into experimental and
control groups, as follows: (1) experimental, 16 mice were
immunized with the BHF-1.20.L peptide; (2) control, 8 mice were
immunized against bovine serum albumin.
[0271] Mice in the experimental group received subcutaneous
injections (9.99 .mu.g/g body weight; 200 .mu.l final volume) of
the BHF-1.20.L peptide daily for 2 weeks, from 5 to 7 weeks of age,
prior to the initial injection with the peptide and adjuvant. Blood
samples (pre-immune serum) were collected prior to the initial
injection of BHF-1.20.L and RIBI adjuvant (50 .mu.g of
peptide/mouse) (as described in the manufacturer's manual) at 7
weeks of age. After 21 days, mice received a booster injection
(half-initial dose) and were then bled 7 days later. Titer was
defined as the maximum dilution of serum that yielded a change in
absorbance equivalent to 2.times. that of control serum (60 min;
37.degree. C.). The amount of peptide per well was 100 ng. Booster
injections took place at 21 days interval.
[0272] Blood samples were analyzed for total cholesterol, HDL
cholesterol, and triglyceride concentration, using commercially
available total cholesterol and triglycerides assay kits (Sigma;
St. Louis Mo.) (ELISA). HDL concentration was determined after Apo
B containing lipoproteins were precipitated using
heparin/MnCl.sub.2.
[0273] When fed a normal rodent chow, total serum cholesterol
concentration in LDLR knockout mice remains relatively low. A high
fat diet, on the other hand, results in an increase in total serum
cholesterol concentration in these mice. The animals were thus
switched at 16 weeks of age to a modified "Western Type" diet (0.1%
cholesterol content) (Harland Teklad, Madison, Wis.) and fed ad
libitum. This diet was expected to increase the total serum
cholesterol concentration to a range of 600-800 mg/dl, thereby
increasing the rate of lesion formation. Blood samples were
collected at 18 weeks of age (retro-orbital sinus bleeding
technique) and monthly thereafter.
[0274] At 30 weeks of age, the mice were sacrificed and aorta were
removed as described in Example 22. Aortic atherosclerosis was
measured by the cholesterol extraction method described in Example
22, whereby cholesterol is determined as a unit weight of
artery.
[0275] Immunization against BHF-1.20.L had no effect on body weight
of LDLR knockout mice. Consumption of the modified "Western Type"
diet for 12 weeks significantly (P<0.05) increased total serum
cholesterol, HDL cholesterol and triglycerides in both experimental
and control animals. Levels of total serum cholesterol, HDL serum
cholesterol and serum triglyceride concentration were not
significantly different (P>0.05) between experimental and
control animals.
[0276] Mice that were immunized with the BHF-1 peptide had 24% less
aortic cholesterol content (P>0.037) as compared to the control,
non-immunized mice. Without being bound to any particular theory,
the immunization is thought to generate circulating antibodies
against the BHF-1 peptide. These antibodies are thought to block
LDL binding to the artery wall, thereby reducing aortic cholesterol
content.
[0277] Those skilled in the art will be able to ascertain using no
more than routine experimentation, many equivalents of the specific
embodiments of the invention described herein. These and all other
equivalents are intended to be encompassed by the following claims.
Sequence CWU 1
1
53 1 151 PRT Oryctolagus cuniculus 1 Met Ser Lys Asn Thr Val Ser
Ser Ala Arg Phe Arg Lys Val Asp Val 1 5 10 15 Asp Glu Tyr Asp Glu
Asn Lys Phe Val Asp Glu Glu Asp Gly Gly Asp 20 25 30 Gly Gln Ala
Gly Pro Asp Glu Gly Glu Val Asp Ser Cys Leu Arg Gln 35 40 45 Gly
Asn Met Thr Ala Ala Leu Gln Ala Ala Leu Lys Asn Pro Pro Ile 50 55
60 Asn Thr Arg Ser Gln Ala Val Lys Asp Arg Ala Gly Ser Ile Val Leu
65 70 75 80 Lys Val Leu Ile Ser Phe Lys Ala Gly Asp Ile Glu Lys Ala
Val Gln 85 90 95 Ser Leu Asp Arg Asn Gly Val Asp Leu Leu Met Lys
Tyr Ile Tyr Lys 100 105 110 Gly Phe Glu Ser Pro Ser Asp Asn Ser Ser
Ala Val Leu Leu Gln Trp 115 120 125 His Glu Lys Ala Leu Ala Ala Gly
Gly Val Gly Ser Ile Val Arg Val 130 135 140 Leu Thr Ala Arg Lys Thr
Val 145 150 2 317 PRT Oryctolagus cuniculus VARIANT (1)...(317) Xaa
= Any Amino Acid 2 Asp Cys Arg Ser Ser Ser Asn Asn Arg Xaa Pro Lys
Gly Gly Ala Ala 1 5 10 15 Arg Ala Gly Gly Pro Ala Arg Pro Val Ser
Leu Arg Glu Val Val Arg 20 25 30 Tyr Leu Gly Gly Ser Ser Gly Ala
Gly Gly Arg Leu Thr Arg Gly Arg 35 40 45 Val Gln Gly Leu Leu Glu
Glu Glu Ala Ala Ala Arg Gly Arg Leu Glu 50 55 60 Arg Thr Arg Leu
Gly Ala Leu Ala Leu Pro Arg Gly Asp Arg Pro Gly 65 70 75 80 Arg Ala
Pro Pro Ala Ala Ser Ala Arg Ala Ala Arg Asn Lys Arg Ala 85 90 95
Gly Glu Glu Arg Val Leu Glu Lys Glu Glu Glu Glu Glu Glu Glu Glu 100
105 110 Asp Asp Glu Asp Asp Asp Asp Asp Val Val Ser Glu Gly Ser Glu
Val 115 120 125 Pro Glu Ser Asp Arg Pro Ala Gly Ala Gln His His Gln
Leu Asn Gly 130 135 140 Gly Glu Arg Gly Pro Gln Thr Ala Lys Glu Arg
Ala Lys Glu Trp Ser 145 150 155 160 Leu Cys Gly Pro His Pro Gly Gln
Glu Glu Gly Arg Gly Pro Ala Ala 165 170 175 Gly Ser Gly Thr Arg Gln
Val Phe Ser Met Ala Ala Leu Ser Lys Glu 180 185 190 Gly Gly Ser Ala
Ser Ser Thr Thr Gly Pro Asp Ser Pro Ser Pro Val 195 200 205 Pro Leu
Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly Thr Pro 210 215 220
Phe Gly Cys Pro Ala Gly Arg Lys Glu Lys Pro Ala Asp Pro Val Glu 225
230 235 240 Trp Thr Val Met Asp Val Val Glu Tyr Phe Thr Glu Ala Gly
Phe Pro 245 250 255 Glu Gln Ala Thr Ala Phe Gln Glu Gln Glu Ile Asp
Gly Lys Ser Leu 260 265 270 Leu Leu Met Gln Arg Thr Asp Val Leu Thr
Gly Leu Ser Ile Arg Leu 275 280 285 Gly Pro Ala Leu Lys Ile Tyr Glu
His His Ile Lys Val Leu Gln Gln 290 295 300 Gly His Phe Glu Asp Asp
Asp Pro Glu Gly Phe Leu Gly 305 310 315 3 232 PRT Oryctolagus
cuniculus 3 Ala Ser Ala Arg Ala Ala Arg Asn Lys Arg Ala Gly Glu Glu
Arg Val 1 5 10 15 Leu Glu Lys Glu Glu Glu Glu Glu Glu Glu Glu Asp
Asp Glu Asp Asp 20 25 30 Asp Asp Asp Val Val Ser Glu Gly Ser Glu
Val Pro Glu Ser Asp Arg 35 40 45 Pro Ala Gly Ala Gln His His Gln
Leu Asn Gly Gly Glu Arg Gly Pro 50 55 60 Gln Thr Ala Lys Glu Arg
Ala Lys Glu Trp Ser Leu Cys Gly Pro His 65 70 75 80 Pro Gly Gln Glu
Glu Gly Arg Gly Pro Ala Ala Gly Ser Gly Thr Arg 85 90 95 Gln Val
Phe Ser Met Ala Ala Leu Ser Lys Glu Gly Gly Ser Ala Ser 100 105 110
Ser Thr Thr Gly Pro Asp Ser Pro Ser Pro Val Pro Leu Pro Pro Gly 115
120 125 Lys Pro Ala Leu Pro Gly Ala Asp Gly Thr Pro Phe Gly Cys Pro
Ala 130 135 140 Gly Arg Lys Glu Lys Pro Ala Asp Pro Val Glu Trp Thr
Val Met Asp 145 150 155 160 Val Val Glu Tyr Phe Thr Glu Ala Gly Phe
Pro Glu Gln Ala Thr Ala 165 170 175 Phe Gln Glu Gln Glu Ile Asp Gly
Lys Ser Leu Leu Leu Met Gln Arg 180 185 190 Thr Asp Val Leu Thr Gly
Leu Ser Ile Arg Leu Gly Pro Ala Leu Lys 195 200 205 Ile Tyr Glu His
His Ile Lys Val Leu Gln Gln Gly His Phe Glu Asp 210 215 220 Asp Asp
Pro Glu Gly Phe Leu Gly 225 230 4 252 PRT Oryctolagus cuniculus 4
Thr Arg Leu Gly Ala Leu Ala Leu Pro Arg Gly Asp Arg Pro Gly Arg 1 5
10 15 Ala Pro Pro Ala Ala Ser Ala Arg Ala Ala Arg Asn Lys Arg Ala
Gly 20 25 30 Glu Glu Arg Val Leu Glu Lys Glu Glu Glu Glu Glu Glu
Glu Glu Asp 35 40 45 Asp Glu Asp Asp Asp Asp Asp Val Val Ser Glu
Gly Ser Glu Val Pro 50 55 60 Glu Ser Asp Arg Pro Ala Gly Ala Gln
His His Gln Leu Asn Gly Gly 65 70 75 80 Glu Arg Gly Pro Gln Thr Ala
Lys Glu Arg Ala Lys Glu Trp Ser Leu 85 90 95 Cys Gly Pro His Pro
Gly Gln Glu Glu Gly Arg Gly Pro Ala Ala Gly 100 105 110 Ser Gly Thr
Arg Gln Val Phe Ser Met Ala Ala Leu Ser Lys Glu Gly 115 120 125 Gly
Ser Ala Ser Ser Thr Thr Gly Pro Asp Ser Pro Ser Pro Val Pro 130 135
140 Leu Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly Thr Pro Phe
145 150 155 160 Gly Cys Pro Ala Gly Arg Lys Glu Lys Pro Ala Asp Pro
Val Glu Trp 165 170 175 Thr Val Met Asp Val Val Glu Tyr Phe Thr Glu
Ala Gly Phe Pro Glu 180 185 190 Gln Ala Thr Ala Phe Gln Glu Gln Glu
Ile Asp Gly Lys Ser Leu Leu 195 200 205 Leu Met Gln Arg Thr Asp Val
Leu Thr Gly Leu Ser Ile Arg Leu Gly 210 215 220 Pro Ala Leu Lys Ile
Tyr Glu His His Ile Lys Val Leu Gln Gln Gly 225 230 235 240 His Phe
Glu Asp Asp Asp Pro Glu Gly Phe Leu Gly 245 250 5 557 PRT
Oryctolagus cuniculus 5 Met Lys Asn Gln Asp Lys Lys Asn Gly Ala Ala
Lys Gln Pro Asn Pro 1 5 10 15 Lys Ser Ser Pro Gly Gln Pro Glu Ala
Gly Ala Glu Gly Ala Gln Gly 20 25 30 Arg Pro Gly Arg Pro Ala Pro
Ala Arg Glu Ala Glu Gly Ala Ser Ser 35 40 45 Gln Ala Pro Gly Arg
Pro Glu Gly Ala Gln Ala Lys Thr Ala Gln Pro 50 55 60 Gly Ala Leu
Cys Asp Val Ser Glu Glu Leu Ser Arg Gln Leu Glu Asp 65 70 75 80 Ile
Leu Ser Thr Tyr Cys Val Asp Asn Asn Gln Gly Ala Pro Gly Glu 85 90
95 Asp Gly Val Gln Gly Glu Pro Pro Glu Pro Glu Asp Ala Glu Lys Ser
100 105 110 Arg Ala Tyr Val Ala Arg Asn Gly Glu Pro Glu Pro Gly Thr
Pro Val 115 120 125 Val Asn Gly Glu Lys Glu Thr Ser Lys Ala Glu Pro
Gly Thr Glu Glu 130 135 140 Ile Arg Thr Ser Asp Glu Val Gly Asp Arg
Asp His Arg Arg Pro Gln 145 150 155 160 Glu Lys Lys Lys Ala Lys Gly
Leu Gly Lys Glu Ile Thr Leu Leu Met 165 170 175 Gln Thr Leu Asn Thr
Leu Ser Thr Pro Glu Glu Lys Leu Ala Ala Leu 180 185 190 Cys Lys Lys
Tyr Ala Glu Leu Leu Glu Glu His Arg Asn Ser Gln Lys 195 200 205 Gln
Met Lys Leu Leu Gln Lys Lys Gln Ser Gln Leu Val Gln Glu Lys 210 215
220 Asp His Leu Arg Gly Glu His Ser Lys Ala Ile Leu Ala Arg Ser Lys
225 230 235 240 Leu Glu Ser Leu Cys Arg Glu Leu Gln Arg His Asn Arg
Ser Leu Lys 245 250 255 Glu Glu Gly Val Gln Arg Ala Arg Glu Glu Glu
Glu Lys Arg Lys Glu 260 265 270 Val Thr Ser His Phe Gln Met Thr Leu
Asn Asp Ile Gln Leu Gln Met 275 280 285 Glu Gln His Asn Glu Arg Asn
Ser Lys Leu Arg Gln Glu Asn Met Glu 290 295 300 Leu Ala Glu Arg Leu
Lys Lys Leu Ile Glu Gln Tyr Glu Leu Arg Glu 305 310 315 320 Glu His
Ile Asp Lys Val Phe Lys His Lys Asp Leu Gln Gln Gln Leu 325 330 335
Val Asp Ala Lys Leu Gln Gln Ala Gln Glu Met Leu Lys Glu Ala Glu 340
345 350 Glu Arg His Gln Arg Glu Lys Asp Phe Leu Leu Lys Glu Ala Val
Glu 355 360 365 Ser Gln Arg Met Cys Glu Leu Met Lys Gln Gln Glu Thr
His Leu Lys 370 375 380 Gln Gln Leu Ala Leu Tyr Thr Glu Lys Phe Glu
Glu Phe Gln Asn Thr 385 390 395 400 Leu Ser Lys Ser Ser Glu Val Phe
Thr Thr Phe Lys Gln Glu Met Glu 405 410 415 Lys Met Thr Lys Lys Ile
Lys Lys Leu Glu Lys Glu Thr Thr Met Tyr 420 425 430 Arg Ser Arg Trp
Glu Ser Ser Asn Lys Ala Leu Leu Glu Met Ala Glu 435 440 445 Glu Lys
Thr Leu Arg Asp Lys Glu Leu Glu Gly Leu Gln Val Lys Ile 450 455 460
Gln Arg Leu Glu Lys Leu Cys Arg Ala Leu Gln Thr Glu Arg Asn Asp 465
470 475 480 Leu Asn Lys Arg Val Gln Asp Leu Ser Ala Gly Gly Gln Gly
Pro Val 485 490 495 Ser Asp Ser Gly Pro Glu Arg Arg Pro Glu Pro Ala
Thr Thr Ser Lys 500 505 510 Glu Gln Gly Val Glu Gly Pro Gly Ala Gln
Val Pro Asn Ser Pro Arg 515 520 525 Ala Thr Asp Ala Ser Cys Cys Ala
Gly Ala Pro Ser Thr Glu Ala Ser 530 535 540 Gly Gln Thr Gly Pro Gln
Glu Pro Thr Thr Ala Thr Ala 545 550 555 6 151 PRT Homo sapiens 6
Met Ser Lys Asn Thr Val Ser Ser Ala Arg Phe Arg Lys Val Asp Val 1 5
10 15 Asp Glu Tyr Asp Glu Asn Lys Phe Val Asp Glu Glu Asp Gly Gly
Asp 20 25 30 Gly Gln Ala Gly Pro Asp Glu Gly Glu Val Asp Ser Cys
Leu Arg Gln 35 40 45 Gly Asn Met Thr Ala Ala Leu Gln Ala Ala Leu
Lys Asn Pro Pro Ile 50 55 60 Asn Thr Lys Ser Gln Ala Val Lys Asp
Arg Ala Gly Ser Ile Val Leu 65 70 75 80 Lys Val Leu Ile Ser Phe Lys
Ala Asn Asp Ile Glu Lys Ala Val Gln 85 90 95 Ser Leu Asp Lys Asn
Gly Val Asp Leu Leu Met Lys Tyr Ile Tyr Lys 100 105 110 Gly Phe Glu
Ser Pro Ser Asp Asn Ser Ser Ala Met Leu Leu Gln Trp 115 120 125 His
Glu Lys Ala Leu Ala Ala Gly Gly Val Gly Ser Ile Val Arg Val 130 135
140 Leu Thr Ala Arg Lys Thr Val 145 150 7 217 PRT Homo sapiens 7
Glu Glu Arg Val Leu Glu Lys Glu Glu Glu Glu Asp Asp Asp Glu Asp 1 5
10 15 Glu Asp Glu Glu Asp Asp Val Ser Glu Gly Ser Glu Val Pro Glu
Ser 20 25 30 Asp Arg Pro Ala Gly Ala Gln His His Gln Leu Asn Gly
Glu Arg Gly 35 40 45 Pro Gln Ser Ala Lys Glu Arg Val Lys Glu Trp
Thr Pro Cys Gly Pro 50 55 60 His Gln Gly Gln Asp Glu Gly Arg Gly
Pro Ala Pro Gly Ser Gly Thr 65 70 75 80 Arg Gln Val Phe Ser Met Ala
Ala Met Asn Lys Glu Gly Gly Thr Ala 85 90 95 Ser Val Ala Thr Gly
Pro Asp Ser Pro Ser Pro Val Pro Leu Pro Pro 100 105 110 Gly Lys Pro
Ala Leu Pro Gly Ala Asp Gly Thr Pro Phe Gly Cys Pro 115 120 125 Pro
Gly Arg Lys Glu Lys Pro Ser Asp Pro Val Glu Trp Thr Val Met 130 135
140 Asp Val Val Glu Tyr Phe Thr Glu Ala Gly Phe Pro Glu Gln Ala Thr
145 150 155 160 Ala Phe Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu
Leu Met Gln 165 170 175 Arg Thr Asp Val Leu Thr Gly Leu Ser Ile Arg
Leu Gly Pro Ala Leu 180 185 190 Lys Ile Tyr Glu His His Ile Lys Val
Leu Gln Gln Gly His Phe Glu 195 200 205 Asp Asp Asp Pro Asp Gly Phe
Leu Gly 210 215 8 530 PRT Homo sapiens 8 Lys Ser Ser Pro Gly Gln
Pro Glu Ala Gly Pro Glu Gly Ala Gln Glu 1 5 10 15 Arg Pro Ser Gln
Ala Ala Pro Ala Val Glu Ala Glu Gly Pro Gly Ser 20 25 30 Ser Gln
Ala Pro Arg Lys Pro Glu Gly Ala Gln Ala Arg Thr Ala Gln 35 40 45
Ser Gly Ala Leu Arg Asp Val Ser Glu Glu Leu Ser Arg Gln Leu Glu 50
55 60 Asp Ile Leu Ser Thr Tyr Cys Val Asp Asn Asn Gln Gly Gly Pro
Gly 65 70 75 80 Glu Asp Gly Ala Gln Gly Glu Pro Ala Glu Pro Glu Asp
Ala Glu Lys 85 90 95 Ser Arg Thr Tyr Val Ala Arg Asn Gly Glu Pro
Glu Pro Thr Pro Val 100 105 110 Val Tyr Gly Glu Lys Glu Pro Ser Lys
Gly Asp Pro Asn Thr Glu Glu 115 120 125 Ile Arg Gln Ser Asp Glu Val
Gly Asp Arg Asp His Arg Arg Pro Gln 130 135 140 Glu Lys Lys Lys Ala
Lys Gly Leu Gly Lys Glu Ile Thr Leu Leu Met 145 150 155 160 Gln Thr
Leu Asn Thr Leu Ser Thr Pro Glu Glu Lys Leu Ala Ala Leu 165 170 175
Cys Lys Lys Tyr Ala Glu Leu Leu Glu Glu His Arg Asn Ser Gln Lys 180
185 190 Gln Met Lys Leu Leu Gln Lys Lys Gln Ser Gln Leu Val Gln Glu
Lys 195 200 205 Asp His Leu Arg Gly Glu His Ser Lys Ala Val Leu Ala
Arg Ser Lys 210 215 220 Leu Glu Ser Leu Cys Arg Glu Leu Gln Arg His
Asn Arg Ser Leu Lys 225 230 235 240 Glu Glu Gly Val Gln Arg Ala Arg
Glu Glu Glu Glu Lys Arg Lys Glu 245 250 255 Val Thr Ser His Phe Gln
Val Thr Leu Asn Asp Ile Gln Leu Gln Met 260 265 270 Glu Gln His Asn
Glu Arg Asn Ser Lys Leu Arg Gln Glu Asn Met Glu 275 280 285 Leu Ala
Glu Arg Leu Lys Lys Leu Ile Glu Gln Tyr Glu Leu Arg Glu 290 295 300
Glu His Ile Asp Lys Val Phe Lys His Lys Asp Leu Gln Gln Gln Leu 305
310 315 320 Val Asp Ala Lys Leu Gln Gln Ala Gln Glu Met Leu Lys Glu
Ala Glu 325 330 335 Glu Arg His Gln Arg Glu Lys Asp Phe Leu Leu Lys
Glu Ala Val Glu 340 345 350 Ser Gln Arg Met Cys Glu Leu Met Lys Gln
Gln Glu Thr His Leu Lys 355 360 365 Gln Gln Leu Ala Leu Tyr Thr Glu
Lys Phe Glu Glu Phe Gln Asn Thr 370 375 380 Leu Ser Lys Ser Ser Glu
Val Phe Thr Thr Phe Lys Gln Glu Met Glu 385 390 395 400 Lys Met Thr
Lys Lys Ile Lys Lys Leu Glu Lys Glu Thr Thr Met Tyr 405 410 415 Arg
Ser Arg Trp Glu Ser Ser Asn Lys Ala Leu Leu Glu Met Ala Glu 420 425
430 Glu Lys Thr Val Arg Asp Lys Glu Leu Glu Gly Leu Gln Val Lys Ile
435 440 445 Gln Arg Leu Glu Lys Leu Cys Arg Ala Leu Gln Thr Glu Arg
Asn Asp 450 455 460 Leu Asn Lys Arg Val Gln Asp Leu Ser Ala Gly Gly
Gln Gly Ser Leu 465 470 475 480 Thr Asp Ser Gly Pro Glu Arg Arg Pro
Glu Gly Pro Gly Ala Gln Ala 485 490 495 Pro Ser Ser Pro Arg Val Thr
Glu Ala Pro Cys Tyr Pro Gly Ala Pro 500 505 510 Ser Thr Glu Ala Ser
Gly Gln Thr Gly Pro Gln Glu Pro Thr Ser Ala 515 520 525 Arg Ala 530
9 20 PRT Homo sapiens 9 Val Asp Val Asp Glu Tyr Asp Glu Asn Lys Phe
Val Asp Glu Glu Asp 1
5 10 15 Gly Gly Asp Gly 20 10 1404 DNA Oryctolagus cuniculus CDS
(58)...(510) 10 aagcctcgca gcggtcgggg cggcgccgcg gaggctcgag
ggcggcgggc ggcggcg atg 60 Met 1 tcg aag aac acg gtg tcg tcg gcg cgg
ttc cgg aag gtg gac gtg gat 108 Ser Lys Asn Thr Val Ser Ser Ala Arg
Phe Arg Lys Val Asp Val Asp 5 10 15 gag tac gac gag aac aag ttc gtg
gac gag gaa gac ggc ggc gac ggc 156 Glu Tyr Asp Glu Asn Lys Phe Val
Asp Glu Glu Asp Gly Gly Asp Gly 20 25 30 cag gcg ggg ccg gac gag
ggc gag gtg gac tcg tgc ctg cgg caa ggg 204 Gln Ala Gly Pro Asp Glu
Gly Glu Val Asp Ser Cys Leu Arg Gln Gly 35 40 45 aac atg aca gcc
gcc ctg cag gcg gcg ctg aag aac cct ccc atc aac 252 Asn Met Thr Ala
Ala Leu Gln Ala Ala Leu Lys Asn Pro Pro Ile Asn 50 55 60 65 acc agg
agc cag gcg gtg aag gac cgg gca ggc agc atc gtg ctg aag 300 Thr Arg
Ser Gln Ala Val Lys Asp Arg Ala Gly Ser Ile Val Leu Lys 70 75 80
gtg ctc atc tcc ttc aag gcc ggc gac ata gaa aag gcc gtg cag tcc 348
Val Leu Ile Ser Phe Lys Ala Gly Asp Ile Glu Lys Ala Val Gln Ser 85
90 95 ctg gac agg aac ggc gtg gac ctg ctc atg aag tac atc tac aag
ggc 396 Leu Asp Arg Asn Gly Val Asp Leu Leu Met Lys Tyr Ile Tyr Lys
Gly 100 105 110 ttc gag agc ccc tcc gac aac agc agc gcc gtg ctc ctg
cag tgg cac 444 Phe Glu Ser Pro Ser Asp Asn Ser Ser Ala Val Leu Leu
Gln Trp His 115 120 125 gag aag gcg ctg gct gca gga gga gtg ggc tcc
atc gtc cgt gtc ctg 492 Glu Lys Ala Leu Ala Ala Gly Gly Val Gly Ser
Ile Val Arg Val Leu 130 135 140 145 act gca agg aaa acc gtg
tagcctggca ggaacgggtg cctgccgggg 540 Thr Ala Arg Lys Thr Val 150
agcgggagct gccggtacaa agaccaaaac gcccagatgc cgccgctgcc ctgtgggcgg
600 cgtctgttcc cagcttcgct ttttcccttt cccgtgtctg tcaggattac
ataaggtttc 660 ccttcgtgag aatcggagtg gcgcagaggg tcctgttcat
acgcgccgtg cgtccggctg 720 tgtaagaccc ctgccttcag tgtccttgag
caacggtagc gtgtcgccgg ctgggtttgg 780 ttttgtcgtg gagggatctg
gtcagaattt gaggccagtt tcctaactca ttgctggtca 840 ggaaatgatc
ttcatttaaa aaaaaaaaaa agactggcag ctattatgca aaactggacc 900
ctcttccctt atttaagcag agtgagtttc tggaaccagt ggtgcccccc cccccgcccc
960 ggccgccgtc ctgctcaagg gaagcctccc tgcagagcag cagagcccct
gggcaggagc 1020 gccgcgtccc gctcccagga gacagcatgc gcggtcacgc
ggcacttcct gtgcctccca 1080 gccccagtgc cccggagttc ttcagggcga
cagggacctc agaagactgg atccgatcca 1140 gacagacgcc cattcttggt
tcagctcagt gttttcaaaa ggaacgtgct accgtgggta 1200 gagcacactg
gttctcagaa cacggccggc gcttgacggt tgtcacagct ccagaacaaa 1260
tcctgggaga caggcgagcg cgagtcgccg ggcaggaatt ccacacactc gtgctgtttt
1320 tgatacctgc tttttgtttt gttttgtaaa aatgatgcac ttgagaaaat
aaaacgtcag 1380 tgttgacaaa aaaaaaaaaa aaaa 1404 11 1617 DNA
Oryctolagus cuniculus CDS (1)...(951) 11 gac tgc cgc agc agc agc
aac aac cgc tag ccg aag ggt ggc gcg gcg 48 Asp Cys Arg Ser Ser Ser
Asn Asn Arg * Pro Lys Gly Gly Ala Ala 1 5 10 15 cgg gcc ggc ggc ccg
gcg cgg ccc gtg agc ctg cgg gaa gtc gtg cgc 96 Arg Ala Gly Gly Pro
Ala Arg Pro Val Ser Leu Arg Glu Val Val Arg 20 25 30 tac ctc ggg
ggt agc agc ggc gct ggc ggc cgc ctg acc cgc ggc cgc 144 Tyr Leu Gly
Gly Ser Ser Gly Ala Gly Gly Arg Leu Thr Arg Gly Arg 35 40 45 gtg
cag ggt ctg ctg gaa gag gag gcg gcg gcg cgg ggc cgc ctg gag 192 Val
Gln Gly Leu Leu Glu Glu Glu Ala Ala Ala Arg Gly Arg Leu Glu 50 55
60 cgc acc cgt ctc gga gcg ctt gcg ctg ccc cgc ggg gac agg ccc gga
240 Arg Thr Arg Leu Gly Ala Leu Ala Leu Pro Arg Gly Asp Arg Pro Gly
65 70 75 cgg gcg cca ccg gcc gcc agc gcc cgc gcg gcg cgg aac aag
aga gct 288 Arg Ala Pro Pro Ala Ala Ser Ala Arg Ala Ala Arg Asn Lys
Arg Ala 80 85 90 95 ggc gag gag cga gtg ctt gaa aag gag gag gag gag
gag gag gag gaa 336 Gly Glu Glu Arg Val Leu Glu Lys Glu Glu Glu Glu
Glu Glu Glu Glu 100 105 110 gac gac gag gac gac gac gac gac gtc gtg
tcc gag ggc tcg gag gtg 384 Asp Asp Glu Asp Asp Asp Asp Asp Val Val
Ser Glu Gly Ser Glu Val 115 120 125 ccc gag agc gat cgt ccc gcg ggt
gcg cag cat cac cag ctg aat ggc 432 Pro Glu Ser Asp Arg Pro Ala Gly
Ala Gln His His Gln Leu Asn Gly 130 135 140 ggc gag cgc ggc ccg cag
acc gcc aag gag cgg gcc aag gag tgg tcg 480 Gly Glu Arg Gly Pro Gln
Thr Ala Lys Glu Arg Ala Lys Glu Trp Ser 145 150 155 ctg tgt ggc ccc
cac cct ggc cag gag gaa ggg cgg ggg ccg gcc gcg 528 Leu Cys Gly Pro
His Pro Gly Gln Glu Glu Gly Arg Gly Pro Ala Ala 160 165 170 175 ggc
agt ggc acc cgc cag gtg ttc tcc atg gcg gcc ttg agt aag gag 576 Gly
Ser Gly Thr Arg Gln Val Phe Ser Met Ala Ala Leu Ser Lys Glu 180 185
190 ggg gga tca gcc tct tcg acc acc ggg cct gac tcc ccg tcc ccg gtg
624 Gly Gly Ser Ala Ser Ser Thr Thr Gly Pro Asp Ser Pro Ser Pro Val
195 200 205 cct ttg ccc ccc ggg aag cca gcc ctc cca gga gcc gat ggg
acc ccc 672 Pro Leu Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly
Thr Pro 210 215 220 ttt ggc tgc cct gcc ggg cgc aaa gag aag ccg gca
gac ccc gtg gag 720 Phe Gly Cys Pro Ala Gly Arg Lys Glu Lys Pro Ala
Asp Pro Val Glu 225 230 235 tgg aca gtc atg gac gtc gtg gag tac ttc
acc gag gcg ggc ttc cct 768 Trp Thr Val Met Asp Val Val Glu Tyr Phe
Thr Glu Ala Gly Phe Pro 240 245 250 255 gag caa gcc acg gct ttc cag
gag cag gag atc gac ggc aag tcc ctg 816 Glu Gln Ala Thr Ala Phe Gln
Glu Gln Glu Ile Asp Gly Lys Ser Leu 260 265 270 ctg ctc atg cag cgc
acc gat gtc ctc acc ggc ctg tcc atc cgc ctg 864 Leu Leu Met Gln Arg
Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu 275 280 285 ggg cca gcg
ttg aaa atc tat gag cac cat atc aag gtg ctg cag cag 912 Gly Pro Ala
Leu Lys Ile Tyr Glu His His Ile Lys Val Leu Gln Gln 290 295 300 ggt
cac ttc gag gac gat gac ccg gaa ggc ttc ctg gga tgagcacaga 961 Gly
His Phe Glu Asp Asp Asp Pro Glu Gly Phe Leu Gly 305 310 315
gccgccgcgc cccttgtccc cacccccacc ccgcctggac ccattcctgc ctccatgtca
1021 cccaaggtgt cccagaggcc aggagctgga ctgggcaggc gaggggtgcg
gacctaccct 1081 gattctggta gggggcgggg ccttgctgtg ctcattgcta
cccccccacc ccgtgtgtgt 1141 ctctgcacct gcccccagca cacccctccc
ggagcctgga tgtcgcctgg gactctggcc 1201 tgctcatttt gcccccagat
cagccccctc cctccctcct gtcccaggac attttttaaa 1261 agaaaaaaag
gaaaaaaaaa aattggggag ggggctggga aggtgcccca agatcctcct 1321
cggcccaacc aggtgtttat tcctatatat atatatatat gttttgttct gcctgttttt
1381 cgttttttgg tgcgtggcct ttcttccctc ccaccaccac tcatggcccc
agccctgctc 1441 gccctgtcgg cgggagcagc tgggaatggg aggagggtgg
gaccttgggt ctgtctccca 1501 ccctctctcc cgttggttct gttgtcgctc
cagctggctg tattgctttt taatattgca 1561 ccgaagggtt gttttttttt
ttttaaataa aattttaaaa aaaggaaaaa aaaaaa 1617 12 1362 DNA
Oryctolagus cuniculus CDS (1)...(696) 12 gcc agc gcc cgc gcg gcg
cgg aac aag aga gct ggc gag gag cga gtg 48 Ala Ser Ala Arg Ala Ala
Arg Asn Lys Arg Ala Gly Glu Glu Arg Val 1 5 10 15 ctt gaa aag gag
gag gag gag gag gag gag gaa gac gac gag gac gac 96 Leu Glu Lys Glu
Glu Glu Glu Glu Glu Glu Glu Asp Asp Glu Asp Asp 20 25 30 gac gac
gac gtc gtg tcc gag ggc tcg gag gtg ccc gag agc gat cgt 144 Asp Asp
Asp Val Val Ser Glu Gly Ser Glu Val Pro Glu Ser Asp Arg 35 40 45
ccc gcg ggt gcg cag cat cac cag ctg aat ggc ggc gag cgc ggc ccg 192
Pro Ala Gly Ala Gln His His Gln Leu Asn Gly Gly Glu Arg Gly Pro 50
55 60 cag acc gcc aag gag cgg gcc aag gag tgg tcg ctg tgt ggc ccc
cac 240 Gln Thr Ala Lys Glu Arg Ala Lys Glu Trp Ser Leu Cys Gly Pro
His 65 70 75 80 cct ggc cag gag gaa ggg cgg ggg ccg gcc gcg ggc agt
ggc acc cgc 288 Pro Gly Gln Glu Glu Gly Arg Gly Pro Ala Ala Gly Ser
Gly Thr Arg 85 90 95 cag gtg ttc tcc atg gcg gcc ttg agt aag gag
ggg gga tca gcc tct 336 Gln Val Phe Ser Met Ala Ala Leu Ser Lys Glu
Gly Gly Ser Ala Ser 100 105 110 tcg acc acc ggg cct gac tcc ccg tcc
ccg gtg cct ttg ccc ccc ggg 384 Ser Thr Thr Gly Pro Asp Ser Pro Ser
Pro Val Pro Leu Pro Pro Gly 115 120 125 aag cca gcc ctc cca gga gcc
gat ggg acc ccc ttt ggc tgc cct gcc 432 Lys Pro Ala Leu Pro Gly Ala
Asp Gly Thr Pro Phe Gly Cys Pro Ala 130 135 140 ggg cgc aaa gag aag
ccg gca gac ccc gtg gag tgg aca gtc atg gac 480 Gly Arg Lys Glu Lys
Pro Ala Asp Pro Val Glu Trp Thr Val Met Asp 145 150 155 160 gtc gtg
gag tac ttc acc gag gcg ggc ttc cct gag caa gcc acg gct 528 Val Val
Glu Tyr Phe Thr Glu Ala Gly Phe Pro Glu Gln Ala Thr Ala 165 170 175
ttc cag gag cag gag atc gac ggc aag tcc ctg ctg ctc atg cag cgc 576
Phe Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu Leu Met Gln Arg 180
185 190 acc gat gtc ctc acc ggc ctg tcc atc cgc ctg ggg cca gcg ttg
aaa 624 Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly Pro Ala Leu
Lys 195 200 205 atc tat gag cac cat atc aag gtg ctg cag cag ggt cac
ttc gag gac 672 Ile Tyr Glu His His Ile Lys Val Leu Gln Gln Gly His
Phe Glu Asp 210 215 220 gat gac ccg gaa ggc ttc ctg gga tgagcacaga
gccgccgcgc cccttgtccc 726 Asp Asp Pro Glu Gly Phe Leu Gly 225 230
cacccccacc ccgcctggac ccattcctgc ctccatgtca cccaaggtgt cccagaggcc
786 aggagctgga ctgggcaggc gaggggtgcg gacctaccct gattctggta
gggggcgggg 846 ccttgctgtg ctcattgcta cccccccacc ccgtgtgtgt
ctctgcacct gcccccagca 906 cacccctccc ggagcctgga tgtcgcctgg
gactctggcc tgctcatttt gcccccagat 966 cagccccctc cctccctcct
gtcccaggac attttttaaa agaaaaaaag gaaaaaaaaa 1026 aattggggag
ggggctggga aggtgcccca agatcctcct cggcccaacc aggtgtttat 1086
tcctatatat atatatatat gttttgttct gcctgttttt cgttttttgg tgcgtggcct
1146 ttcttccctc ccaccaccac tcatggcccc agccctgctc gccctgtcgg
cgggagcagc 1206 tgggaatggg aggagggtgg gaccttgggt ctgtctccca
ccctctctcc cgttggttct 1266 gttgtcgctc cagctggctg tattgctttt
taatattgca ccgaagggtt gttttttttt 1326 ttttaaataa aattttaaaa
aaaggaaaaa aaaaaa 1362 13 1422 DNA Oryctolagus cuniculus CDS
(1)...(756) 13 acc cgt ctc gga gcg ctt gcg ctg ccc cgc ggg gac agg
ccc gga cgg 48 Thr Arg Leu Gly Ala Leu Ala Leu Pro Arg Gly Asp Arg
Pro Gly Arg 1 5 10 15 gcg cca ccg gcc gcc agc gcc cgc gcg gcg cgg
aac aag aga gct ggc 96 Ala Pro Pro Ala Ala Ser Ala Arg Ala Ala Arg
Asn Lys Arg Ala Gly 20 25 30 gag gag cga gtg ctt gaa aag gag gag
gag gag gag gag gag gaa gac 144 Glu Glu Arg Val Leu Glu Lys Glu Glu
Glu Glu Glu Glu Glu Glu Asp 35 40 45 gac gag gac gac gac gac gac
gtc gtg tcc gag ggc tcg gag gtg ccc 192 Asp Glu Asp Asp Asp Asp Asp
Val Val Ser Glu Gly Ser Glu Val Pro 50 55 60 gag agc gat cgt ccc
gcg ggt gcg cag cat cac cag ctg aat ggc ggc 240 Glu Ser Asp Arg Pro
Ala Gly Ala Gln His His Gln Leu Asn Gly Gly 65 70 75 80 gag cgc ggc
ccg cag acc gcc aag gag cgg gcc aag gag tgg tcg ctg 288 Glu Arg Gly
Pro Gln Thr Ala Lys Glu Arg Ala Lys Glu Trp Ser Leu 85 90 95 tgt
ggc ccc cac cct ggc cag gag gaa ggg cgg ggg ccg gcc gcg ggc 336 Cys
Gly Pro His Pro Gly Gln Glu Glu Gly Arg Gly Pro Ala Ala Gly 100 105
110 agt ggc acc cgc cag gtg ttc tcc atg gcg gcc ttg agt aag gag ggg
384 Ser Gly Thr Arg Gln Val Phe Ser Met Ala Ala Leu Ser Lys Glu Gly
115 120 125 gga tca gcc tct tcg acc acc ggg cct gac tcc ccg tcc ccg
gtg cct 432 Gly Ser Ala Ser Ser Thr Thr Gly Pro Asp Ser Pro Ser Pro
Val Pro 130 135 140 ttg ccc ccc ggg aag cca gcc ctc cca gga gcc gat
ggg acc ccc ttt 480 Leu Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp
Gly Thr Pro Phe 145 150 155 160 ggc tgc cct gcc ggg cgc aaa gag aag
ccg gca gac ccc gtg gag tgg 528 Gly Cys Pro Ala Gly Arg Lys Glu Lys
Pro Ala Asp Pro Val Glu Trp 165 170 175 aca gtc atg gac gtc gtg gag
tac ttc acc gag gcg ggc ttc cct gag 576 Thr Val Met Asp Val Val Glu
Tyr Phe Thr Glu Ala Gly Phe Pro Glu 180 185 190 caa gcc acg gct ttc
cag gag cag gag atc gac ggc aag tcc ctg ctg 624 Gln Ala Thr Ala Phe
Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu 195 200 205 ctc atg cag
cgc acc gat gtc ctc acc ggc ctg tcc atc cgc ctg ggg 672 Leu Met Gln
Arg Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly 210 215 220 cca
gcg ttg aaa atc tat gag cac cat atc aag gtg ctg cag cag ggt 720 Pro
Ala Leu Lys Ile Tyr Glu His His Ile Lys Val Leu Gln Gln Gly 225 230
235 240 cac ttc gag gac gat gac ccg gaa ggc ttc ctg gga tgagcacaga
766 His Phe Glu Asp Asp Asp Pro Glu Gly Phe Leu Gly 245 250
gccgccgcgc cccttgtccc cacccccacc ccgcctggac ccattcctgc ctccatgtca
826 cccaaggtgt cccagaggcc aggagctgga ctgggcaggc gaggggtgcg
gacctaccct 886 gattctggta gggggcgggg ccttgctgtg ctcattgcta
cccccccacc ccgtgtgtgt 946 ctctgcacct gcccccagca cacccctccc
ggagcctgga tgtcgcctgg gactctggcc 1006 tgctcatttt gcccccagat
cagccccctc cctccctcct gtcccaggac attttttaaa 1066 agaaaaaaag
gaaaaaaaaa aattggggag ggggctggga aggtgcccca agatcctcct 1126
cggcccaacc aggtgtttat tcctatatat atatatatat gttttgttct gcctgttttt
1186 cgttttttgg tgcgtggcct ttcttccctc ccaccaccac tcatggcccc
agccctgctc 1246 gccctgtcgg cgggagcagc tgggaatggg aggagggtgg
gaccttgggt ctgtctccca 1306 ccctctctcc cgttggttct gttgtcgctc
cagctggctg tattgctttt taatattgca 1366 ccgaagggtt gttttttttt
ttttaaataa aattttaaaa aaaggaaaaa aaaaaa 1422 14 4722 DNA
Oryctolagus cuniculus CDS (61)...(1731) 14 gtggaaaata gcaactgtgt
ttctcaagga tccaatccca acctaaggtg gcagcgcaca 60 atg aag aat caa gac
aaa aag aac ggg gct gcc aaa cag ccc aac ccc 108 Met Lys Asn Gln Asp
Lys Lys Asn Gly Ala Ala Lys Gln Pro Asn Pro 1 5 10 15 aaa agc agc
ccg gga cag ccg gaa gca gga gcg gag gga gcc cag ggg 156 Lys Ser Ser
Pro Gly Gln Pro Glu Ala Gly Ala Glu Gly Ala Gln Gly 20 25 30 cgg
ccc ggc cgg ccg gcc ccc gcc cga gaa gcc gaa ggt gcc agc agc 204 Arg
Pro Gly Arg Pro Ala Pro Ala Arg Glu Ala Glu Gly Ala Ser Ser 35 40
45 cag gct ccc ggg agg ccg gag ggg gct caa gcc aaa act gct cag cct
252 Gln Ala Pro Gly Arg Pro Glu Gly Ala Gln Ala Lys Thr Ala Gln Pro
50 55 60 ggg gcg ctc tgt gat gtc tct gag gag ctg agc cgc cag ttg
gaa gac 300 Gly Ala Leu Cys Asp Val Ser Glu Glu Leu Ser Arg Gln Leu
Glu Asp 65 70 75 80 ata ctc agt aca tac tgt gtg gac aac aac cag ggg
gcc ccg ggt gag 348 Ile Leu Ser Thr Tyr Cys Val Asp Asn Asn Gln Gly
Ala Pro Gly Glu 85 90 95 gat ggg gtc cag ggt gag ccc cct gaa cct
gaa gat gca gag aag tct 396 Asp Gly Val Gln Gly Glu Pro Pro Glu Pro
Glu Asp Ala Glu Lys Ser 100 105 110 cgc gcc tat gtg gca agg aat ggg
gag ccg gag ccg ggc acc cca gta 444 Arg Ala Tyr Val Ala Arg Asn Gly
Glu Pro Glu Pro Gly Thr Pro Val 115 120 125 gtc aat ggc gag aag gag
acc tcc aag gca gag ccg ggc acg gaa gag 492 Val Asn Gly Glu Lys Glu
Thr Ser Lys Ala Glu Pro Gly Thr Glu Glu 130 135 140 atc cgg acg agc
gat gag gtc gga gac cga gac cac cgg agg cca cag 540 Ile Arg Thr Ser
Asp Glu Val Gly Asp Arg Asp His Arg Arg Pro Gln 145 150 155 160 gaa
aag aag aag gcc aag ggt ctg gga aag gag atc acg ctg ctg atg 588 Glu
Lys Lys Lys Ala Lys Gly Leu Gly Lys Glu Ile Thr Leu Leu Met 165 170
175 cag aca ctg aac acg ctg agc acc cca gag gag aag ctg gcg gct ctg
636 Gln Thr Leu Asn Thr Leu Ser Thr Pro Glu Glu Lys Leu Ala Ala Leu
180 185 190 tgc aag aag tat gcg gaa ctg ctc gag gag cac cgg aac tcg
cag aag 684 Cys Lys Lys Tyr Ala Glu Leu Leu Glu Glu His Arg Asn Ser
Gln Lys 195 200 205 cag atg aag ctg ctg cag aag aag cag agc cag ctg
gtg cag gag aag 732 Gln Met Lys
Leu Leu Gln Lys Lys Gln Ser Gln Leu Val Gln Glu Lys 210 215 220 gac
cac ctg cgt ggc gag cac agc aag gcc atc ctg gcc cgc agc aag 780 Asp
His Leu Arg Gly Glu His Ser Lys Ala Ile Leu Ala Arg Ser Lys 225 230
235 240 ctc gag agc ctg tgc cgg gag ctg cag cgg cac aac cgc tcg ctc
aag 828 Leu Glu Ser Leu Cys Arg Glu Leu Gln Arg His Asn Arg Ser Leu
Lys 245 250 255 gaa gaa ggt gtg cag cga gcc cga gag gag gag gag aag
cgc aag gag 876 Glu Glu Gly Val Gln Arg Ala Arg Glu Glu Glu Glu Lys
Arg Lys Glu 260 265 270 gtg acg tca cac ttc cag atg acg ctc aac gac
att cag ctg cag atg 924 Val Thr Ser His Phe Gln Met Thr Leu Asn Asp
Ile Gln Leu Gln Met 275 280 285 gag cag cac aac gag cgc aac tcc aag
ctg cgc cag gag aac atg gag 972 Glu Gln His Asn Glu Arg Asn Ser Lys
Leu Arg Gln Glu Asn Met Glu 290 295 300 ctg gcc gag cgg ctc aag aag
ctg att gag cag tac gag ctg cga gaa 1020 Leu Ala Glu Arg Leu Lys
Lys Leu Ile Glu Gln Tyr Glu Leu Arg Glu 305 310 315 320 gag cac atc
gac aaa gtc ttc aaa cac aag gat ctg cag cag cag ctg 1068 Glu His
Ile Asp Lys Val Phe Lys His Lys Asp Leu Gln Gln Gln Leu 325 330 335
gtg gac gcc aag ctc cag cag gcc cag gag atg ctg aag gag gca gag
1116 Val Asp Ala Lys Leu Gln Gln Ala Gln Glu Met Leu Lys Glu Ala
Glu 340 345 350 gag cgg cac cag cgg gag aag gac ttt ctc ctg aag gag
gcc gtg gag 1164 Glu Arg His Gln Arg Glu Lys Asp Phe Leu Leu Lys
Glu Ala Val Glu 355 360 365 tcc cag agg atg tgc gag ctg atg aag caa
cag gag acc cac ctg aag 1212 Ser Gln Arg Met Cys Glu Leu Met Lys
Gln Gln Glu Thr His Leu Lys 370 375 380 cag cag ctt gcc cta tac aca
gag aag ttt gag gag ttc cag aac act 1260 Gln Gln Leu Ala Leu Tyr
Thr Glu Lys Phe Glu Glu Phe Gln Asn Thr 385 390 395 400 ctt tcc aaa
agc agc gag gtg ttc acc aca ttc aaa cag gaa atg gaa 1308 Leu Ser
Lys Ser Ser Glu Val Phe Thr Thr Phe Lys Gln Glu Met Glu 405 410 415
aag atg aca aag aag atc aag aag ctg gag aaa gag acc acc atg tac
1356 Lys Met Thr Lys Lys Ile Lys Lys Leu Glu Lys Glu Thr Thr Met
Tyr 420 425 430 cgt tcc cgg tgg gag agc agc aac aag gcc ctg ctt gag
atg gct gag 1404 Arg Ser Arg Trp Glu Ser Ser Asn Lys Ala Leu Leu
Glu Met Ala Glu 435 440 445 gag aaa aca ctc cgg gac aaa gag ctg gaa
ggc ctg cag gtg aaa atc 1452 Glu Lys Thr Leu Arg Asp Lys Glu Leu
Glu Gly Leu Gln Val Lys Ile 450 455 460 cag cgg ctg gag aag ctg tgc
cgg gca ctg cag aca gag cgc aat gac 1500 Gln Arg Leu Glu Lys Leu
Cys Arg Ala Leu Gln Thr Glu Arg Asn Asp 465 470 475 480 ctg aac aag
agg gtg cag gac ctg agt gcc ggt ggc cag ggc ccc gtc 1548 Leu Asn
Lys Arg Val Gln Asp Leu Ser Ala Gly Gly Gln Gly Pro Val 485 490 495
tcc gac agc ggt cct gag cgg agg cca gag ccc gcc acc acc tcc aag
1596 Ser Asp Ser Gly Pro Glu Arg Arg Pro Glu Pro Ala Thr Thr Ser
Lys 500 505 510 gag cag ggt gtc gag ggc ccc ggg gct caa gta ccc aac
tct cca agg 1644 Glu Gln Gly Val Glu Gly Pro Gly Ala Gln Val Pro
Asn Ser Pro Arg 515 520 525 gcc aca gac gct tcc tgc tgc gca ggt gca
ccc agc aca gag gca tca 1692 Ala Thr Asp Ala Ser Cys Cys Ala Gly
Ala Pro Ser Thr Glu Ala Ser 530 535 540 ggc cag aca ggg ccc cag gag
ccc acc act gcc act gcc tagagagctt 1741 Gly Gln Thr Gly Pro Gln Glu
Pro Thr Thr Ala Thr Ala 545 550 555 ggtgctgggg tgtgccagga
agggagcagg cagcccagcc aggcctggcc cagcccaggc 1801 tcccatgcta
agcagtccgg tgctgaggcc aggatgttct gacctggctg gcacctgacc 1861
ctctgcagtc ttggattttg tgggtcagtt ttacatgcat atggcacaca tgcaaggcct
1921 cacacatttg tgtctctaag tgtactgtgg gcttgcatcg ggggtgacga
tggacagatg 1981 aagccagcgg ctcccttgtg agctgaagtc ttacggagga
gacggcgtct gcactgccat 2041 cgcagtgacc tgcaggacga gttccttgag
ctttccctgc ctgctttgag gctgagaccc 2101 ctcccggccc ttcagagctc
ctgacaggtg atacacaccc agccttgacc gcacttctct 2161 tgggtagctg
ggctctccta gcctccccca gaggcgccat tgcttctctt gacttggaga 2221
ggggatgccc aggcgtggcc ttggcaggca ctgggagcta gtgattgggc tgctctcctg
2281 cctcgagcag gggcaggagt gtttctggtg ggatgatgcg ctcgctggtc
aggagccccg 2341 tgggcgctgc ttcccccgcc ctctggtgat gccaggacca
ggccagtgat gcttctcagt 2401 agccttacca ttcacaggtg cctctccagc
ccgcacagtg agtgacaaga tcatccaaag 2461 gattccttct gaaggtgttc
gtttcgtttt gttttgttgc acgtgacggt ttgtattgag 2521 gaccctctga
ggaagagggg tgctgtagca gtggtccctg cgtgcctggc tccagtgtcc 2581
tgccctcccc cccctcgcca tggctcctcg gccgccttgg tgctgaggtt tctgtttggt
2641 gagatcaggt tgtctgttca gagagaagag gcgtctgatg gctttgccgc
cagcttgcct 2701 gcgggcctca atcccgggag gccgcccggt tcccgtcact
gttgtccccg tgcagtgcgt 2761 tgctggtccc caggaccagc tgctcgtttg
ctgtatgggt cagtttctgc ttcctgcccc 2821 ccactccacc taactgcaat
ccttggggtt tccctggttc tcgtccctgg tacctctgtg 2881 cccaagaagt
agccttcttt gggattcttg ttctgcccat gcgggagctg ctgctgtctg 2941
acaggtgagg cctgagactc agcggctgac agagctgcag agctctgcac ggtggctccc
3001 ggggcggcct ctgtgtgctg cacaccgctg ctctgctggc actggccagt
ctgtgcagag 3061 catttgagta ctggctcagg agggagggct ctgctggcct
cgagggacag cgccacgtct 3121 ccagctgggc tcagggagag ccccagactg
gctgcgtagg gtgcttgggg tttgcttctt 3181 gcagtatttc ttggaagctg
ttttgttgtc ctgctattcc ttcatcttcc acagtccacg 3241 ctcagccttt
aacttggatc cctcacataa cagggttcat gagacccgca agtacgccca 3301
agctacgtat ggctgaggcc agctggcagg tgaatggcac gccattgctg ctgctaatcc
3361 ctggcatatc tttagttcac ctcgaaatgc ccccgccaca gtgcaagcag
tgagtccacg 3421 tgccaccctg ggctgaatcc caccccctgt gagtgttgcc
cgagattgtg tctcttctga 3481 atgccttcac tgggaatggc ctctgccgcc
tcctgctcag ggaggctttc cccttccctc 3541 agcccctgtg ccagactgag
gtacaagaac cgccaagccc atgcaaggtg tggctaggcg 3601 ccagggtgca
ggaaggaggc aggtagctgc ctgcaccctt gaaagccaag aggcctacgg 3661
tggcctccat cctggcttgc ctcacttcag ctacctcgca tagcccaggg gtggggctat
3721 tggattccag ggtgggggga tgggaagctg cagggggcag gtggctctca
ctaggcttcc 3781 cagctcagga atgtgggcct caggtagggg agagcctttg
ctccactcca cccatttgca 3841 ggcatctagg ccagtctaga tggcgacccc
ttctcttcct ctccattgac caaatcgtac 3901 ctgtctctcc agctgctcgc
ttgctctgct ttccaaagtc agcccaggta cccaggtgcc 3961 gcccacattg
gcctggaacc tggaccagag gcaagggagg tggcctatcc ttgagtgata 4021
gccagtgcct tcctcacccg gtggcttcca tgcctgtgac ctcagattta ggaccaagag
4081 ctgtgttggt ttcttacgtt gtgagctttc cctccagggg accacagcag
gtgaggctcg 4141 gagcccagag cccttggcgc cgccagcagt aacttgtgtc
cggaccttgt ccagctgagc 4201 gcttcgtgta tgactcagct tcgtgtgtga
gtccagcgga gtgcgtcacg tgacctagac 4261 tcagcggtgt cagccgcact
ttgatttgtt tgttttccat gaggtttttg gaccatgggc 4321 ttagctcagg
caacttttct gtaaggagaa tgttaacttt ctgtaaagat gcttatttaa 4381
ctaacgcctg cttcccccac tcccaaccag gtggccaccg agagctcacc aggaggccaa
4441 tagagctgct ccagctctcc catcttgcac cgcacaaagg tggccgcccc
agggacagcc 4501 aggcacctgc ctgggggagg ggcttctctt ccttatggcc
tggccatcta gattgtttaa 4561 agttgtgctg acagcttttt ttggtttttt
ggtttttgtt tttgtttttg tttttgtttt 4621 tgtctacttt tggtattcac
aacagccagg gacttgattt tgatgtattt taagccacat 4681 taaataaaga
gtctgttgcc ttaaaaaaaa aaaaaaaaaa a 4722 15 1925 DNA Homo sapiens
CDS (118)...(570) 15 gacgcctcag agcggaacag ggaagtgaat caggcgccgg
gtagtgggtt gctgggctgg 60 gcttgctgag gtagaggcag cgccaagaag
aggcctttgc cgctggtcgg gattggg atg 120 Met 1 tcg aag aac aca gtg tcg
tcg gcc cgc ttc cgg aag gtg gac gtg gat 168 Ser Lys Asn Thr Val Ser
Ser Ala Arg Phe Arg Lys Val Asp Val Asp 5 10 15 gaa tat gac gag aac
aag ttc gtg gac gaa gaa gat ggg ggc gac ggc 216 Glu Tyr Asp Glu Asn
Lys Phe Val Asp Glu Glu Asp Gly Gly Asp Gly 20 25 30 cag gcc ggg
ccc gac gag ggc gag gtg gac tcc tgc ctg cgg caa gga 264 Gln Ala Gly
Pro Asp Glu Gly Glu Val Asp Ser Cys Leu Arg Gln Gly 35 40 45 aac
atg aca gct gcc cta cag gca gct ctg aag aac ccc cct atc aac 312 Asn
Met Thr Ala Ala Leu Gln Ala Ala Leu Lys Asn Pro Pro Ile Asn 50 55
60 65 acc aag agt cag gca gtg aag gac cgg gca ggc agc att gtc ttg
aag 360 Thr Lys Ser Gln Ala Val Lys Asp Arg Ala Gly Ser Ile Val Leu
Lys 70 75 80 gtg ctc atc tct ttt aaa gct aat gat ata gaa aag gca
gtt caa tct 408 Val Leu Ile Ser Phe Lys Ala Asn Asp Ile Glu Lys Ala
Val Gln Ser 85 90 95 ctg gac aag aat ggt gtg gat ctc cta atg aag
tat att tat aaa gga 456 Leu Asp Lys Asn Gly Val Asp Leu Leu Met Lys
Tyr Ile Tyr Lys Gly 100 105 110 ttt gag agc ccg tct gac aat agc agt
gct atg tta ctg caa tgg cat 504 Phe Glu Ser Pro Ser Asp Asn Ser Ser
Ala Met Leu Leu Gln Trp His 115 120 125 gaa aag gca ctt gct gct gga
gga gta ggg tcc att gtt cgt gtc ttg 552 Glu Lys Ala Leu Ala Ala Gly
Gly Val Gly Ser Ile Val Arg Val Leu 130 135 140 145 act gca aga aaa
act gtg tagtctggca ggaagtggat tatctgcctc 600 Thr Ala Arg Lys Thr
Val 150 gggagtggga attgctggta caaagaccaa aacaaccaaa tgccaccgct
gccctgtggg 660 tagcatctgt ttctctcagc tttgccttct tgctttttca
tatctgtaaa gaaaaaaatt 720 acatatcagt tgtcccttta atgaaaattg
ggataatata gaagaaattg tgttaaaata 780 gaagtgtttc atcctttcaa
aaccatttca gtgatgttta taccaatctg tatatagtat 840 aatttacatt
caagttttaa ttgtgcaact tttaaccctg ttggctggtt tttggttctg 900
tttggttttg tattattttt aactaatact gaaaaatttg gtcagaattt gaggccagtt
960 tcctagctca ttgctagtca ggaaatgata tttataaaaa atatgagaga
ctggcagcta 1020 ttaacattgc aaaactggac catatttccc ttatttaata
agcaaaatat gtttttggaa 1080 taagtggtgg gtgaatacca ctgctaagtt
atagctttgt ttttgcttgc ctcctcatta 1140 tctgtactgt gggtttaagt
atgctacttt ctctcagcat ccaataatca tggcccctca 1200 atttatttgt
ggtcacgcag ggttcagagc aagaagtctt gctttataca aatgtatcca 1260
taaaatatca gagcttgttg ggcatgaaca tcaaactttt gttccactaa tatggctctg
1320 tttggaaaaa actgcaaatc agaaagaatg atttgcagaa agaaagaaaa
actatggtgt 1380 aatttaaact ctgggcagcc tctgaatgaa atgctacttt
ctttagaaat ataatagctg 1440 ccttagacat tatgaggtat acaactagta
tttaagatac catttaatat gccccgtaaa 1500 tgtcttcagt gttcttcagg
gtagttggga tctcaaaaga tttggttcag atccaaacaa 1560 atacacattc
tgtgttttag ctcagtgttt tctaaaaaaa gaaactgcca cacagcaaaa 1620
aattgtttac tttgttggac aaaccaaatc agttctcaaa aaatgaccgg tgcttataaa
1680 aagttataaa tatcgagtag ctctaaaaca aaccacctga ccaagaggga
agtgagcttg 1740 tgcttagtat ttacattgga tgccagtttt gtaatcactg
acttatgtgc aaactggtgc 1800 agaaattcta taaactcttt gctgtttttg
atacctgctt tttgtttcat tttgttttgt 1860 tttgtaaaaa tgataaaact
tcagaaaata aaatgtcagt gttgaataat taaaaaaaaa 1920 aaaaa 1925 16 1208
DNA Homo sapiens CDS (1)...(651) 16 gaa gag cga gta ctt gag aaa gaa
gag gaa gaa gat gat gat gaa gat 48 Glu Glu Arg Val Leu Glu Lys Glu
Glu Glu Glu Asp Asp Asp Glu Asp 1 5 10 15 gaa gat gaa gaa gat gat
gtg tca gag ggc tct gaa gtg ccc gag agt 96 Glu Asp Glu Glu Asp Asp
Val Ser Glu Gly Ser Glu Val Pro Glu Ser 20 25 30 gac cgt cct gca
ggt gcc cag cac cac cag ctt aac ggc gag cgg gga 144 Asp Arg Pro Ala
Gly Ala Gln His His Gln Leu Asn Gly Glu Arg Gly 35 40 45 cct cag
agt gcc aag gag agg gtc aag gag tgg acc ccc tgc gga ccg 192 Pro Gln
Ser Ala Lys Glu Arg Val Lys Glu Trp Thr Pro Cys Gly Pro 50 55 60
cac cag ggc cag gat gaa ggg cgg ggg cca gcc ccg ggc agc ggc acc 240
His Gln Gly Gln Asp Glu Gly Arg Gly Pro Ala Pro Gly Ser Gly Thr 65
70 75 80 cgc cag gtg ttc tcc atg gca gcc atg aac aag gaa ggg gga
aca gct 288 Arg Gln Val Phe Ser Met Ala Ala Met Asn Lys Glu Gly Gly
Thr Ala 85 90 95 tct gtt gcc acc ggg cca gac tcc ccg tcc ccc gtg
cct ttg ccc cca 336 Ser Val Ala Thr Gly Pro Asp Ser Pro Ser Pro Val
Pro Leu Pro Pro 100 105 110 ggc aaa cca gcc cta cct ggg gcc gac ggg
acc ccc ttt ggc tgt cct 384 Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly
Thr Pro Phe Gly Cys Pro 115 120 125 ccc ggg cgc aaa gag aag cca tct
gat ccc gtc gag tgg acc gtg atg 432 Pro Gly Arg Lys Glu Lys Pro Ser
Asp Pro Val Glu Trp Thr Val Met 130 135 140 gat gtc gtc gaa tat ttt
act gag gct gga ttc ccg gag cag gcg aca 480 Asp Val Val Glu Tyr Phe
Thr Glu Ala Gly Phe Pro Glu Gln Ala Thr 145 150 155 160 gct ttc caa
gag cag gaa att gat ggc aaa tct ttg ctg ctc atg cag 528 Ala Phe Gln
Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu Leu Met Gln 165 170 175 cgc
aca gat gtg ctc acc ggc ctg tcc atc cgc ctc ggg cca gcc ctg 576 Arg
Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly Pro Ala Leu 180 185
190 aaa atc tac gag cac cac atc aag gtg ctt cag caa ggc cac ttt gag
624 Lys Ile Tyr Glu His His Ile Lys Val Leu Gln Gln Gly His Phe Glu
195 200 205 gat gat gac ccc gat ggc ttc tta ggc tgagcgccca
gcctcacccc 671 Asp Asp Asp Pro Asp Gly Phe Leu Gly 210 215
tgccccagcc cattccggcc cccatctcac ccaagatccc ccagagtcca ggagctggac
731 ggggacaccc tcagccctca taacagattc caaggagagg gcaccctctt
gtccttatct 791 ttgccccttg tgtctgtctc acacacatct gctcctcagc
acgtcggtgt ggggagggga 851 ttgctcctta aaccccaggt ggctgaccct
ccccacccag tccaggacat tttaggaaaa 911 aaaaaatgaa atgtgggggg
cttctcatct ccccaagatc ctcttccgtt cagccagatg 971 tttcctgtat
aaatgtttgg atctgcctgt ttattttggt gggtggtctt tcctccctcc 1031
cctaccaccc atgcccccct tctcagtctg cccctggcct ccagccccta ggggactagc
1091 tgggttgggg ttcctcgggc cttttctctc ctccctcttt tctttctgtt
gattgtcgct 1151 ccagctggct gtattgcttt ttaatattgc accgaaggtt
ttttaaataa aatttta 1208 17 4697 DNA Homo sapiens CDS (3)...(1592)
17 ca aaa agc agc cca gga caa ccg gaa gca gga ccc gag gga gcc cag
47 Lys Ser Ser Pro Gly Gln Pro Glu Ala Gly Pro Glu Gly Ala Gln 1 5
10 15 gag cgg ccc agc cag gcg gct cct gca gta gaa gca gaa ggt ccc
ggc 95 Glu Arg Pro Ser Gln Ala Ala Pro Ala Val Glu Ala Glu Gly Pro
Gly 20 25 30 agc agc cag gct cct cgg aag ccg gag ggg gct caa gcc
aga acg gct 143 Ser Ser Gln Ala Pro Arg Lys Pro Glu Gly Ala Gln Ala
Arg Thr Ala 35 40 45 cag tct ggg gcc ctt cgt gat gtc tct gag gag
ctg agc cgc caa ctg 191 Gln Ser Gly Ala Leu Arg Asp Val Ser Glu Glu
Leu Ser Arg Gln Leu 50 55 60 gaa gac ata ctg agc aca tac tgt gtg
gac aat aac cag ggg ggc ccc 239 Glu Asp Ile Leu Ser Thr Tyr Cys Val
Asp Asn Asn Gln Gly Gly Pro 65 70 75 ggc gag gat ggg gca cag ggt
gag ccg gct gaa ccc gaa gat gca gag 287 Gly Glu Asp Gly Ala Gln Gly
Glu Pro Ala Glu Pro Glu Asp Ala Glu 80 85 90 95 aag tcc cgg acc tat
gtg gca agg aat ggg gag cct gaa cca act cca 335 Lys Ser Arg Thr Tyr
Val Ala Arg Asn Gly Glu Pro Glu Pro Thr Pro 100 105 110 gta gtc tat
gga gag aag gaa ccc tcc aag ggg gat cca aac aca gaa 383 Val Val Tyr
Gly Glu Lys Glu Pro Ser Lys Gly Asp Pro Asn Thr Glu 115 120 125 gag
atc cgg cag agt gac gag gtc gga gac cga gac cat cga agg cca 431 Glu
Ile Arg Gln Ser Asp Glu Val Gly Asp Arg Asp His Arg Arg Pro 130 135
140 cag gag aag aaa aaa gcc aag ggt ttg ggg aag gag atc acg ttg ctg
479 Gln Glu Lys Lys Lys Ala Lys Gly Leu Gly Lys Glu Ile Thr Leu Leu
145 150 155 atg cag aca ttg aat act ctg agt acc cca gag gag aag ctg
gct gct 527 Met Gln Thr Leu Asn Thr Leu Ser Thr Pro Glu Glu Lys Leu
Ala Ala 160 165 170 175 ctg tgc aag aag tat gct gaa ctg ctg gag gag
cac cgg aat tca cag 575 Leu Cys Lys Lys Tyr Ala Glu Leu Leu Glu Glu
His Arg Asn Ser Gln 180 185 190 aag cag atg aag ctc cta cag aaa aag
cag agc cag ctg gtg caa gag 623 Lys Gln Met Lys Leu Leu Gln Lys Lys
Gln Ser Gln Leu Val Gln Glu 195 200 205 aag gac cac ctg cgc ggt gag
cac agc aag gcc gtc ctg gcc cgc agc 671 Lys Asp His Leu Arg Gly Glu
His Ser Lys Ala Val Leu Ala Arg Ser 210 215 220 aag ctt gag agc cta
tgc cgt gag ctg cag cgg cac aac cgc tcc ctc 719 Lys Leu Glu Ser Leu
Cys Arg Glu Leu Gln Arg His Asn Arg Ser Leu 225 230 235 aag gaa gaa
ggt gtg cag cgg gcc cgg gag gag gag gag aag cgc aag 767 Lys Glu Glu
Gly Val Gln Arg Ala Arg Glu Glu Glu Glu Lys Arg Lys 240 245 250 255
gag gtg acc tcg cac ttc cag gtg aca ctg aat gac att cag ctg cag 815
Glu Val Thr Ser His Phe Gln Val Thr Leu Asn Asp Ile Gln Leu Gln 260
265 270 atg gaa cag cac aat gag cgc
aac tcc aag ctg cgc caa gag aac atg 863 Met Glu Gln His Asn Glu Arg
Asn Ser Lys Leu Arg Gln Glu Asn Met 275 280 285 gag ctg gct gag agg
ctc aag aag ctg att gag cag tat gag ctg cgc 911 Glu Leu Ala Glu Arg
Leu Lys Lys Leu Ile Glu Gln Tyr Glu Leu Arg 290 295 300 gag gag cat
atc gac aaa gtc ttc aaa cac aag gac cta caa cag cag 959 Glu Glu His
Ile Asp Lys Val Phe Lys His Lys Asp Leu Gln Gln Gln 305 310 315 ctg
gtg gat gcc aag ctc cag cag gcc cag gag atg cta aag gag gca 1007
Leu Val Asp Ala Lys Leu Gln Gln Ala Gln Glu Met Leu Lys Glu Ala 320
325 330 335 gaa gag cgg cac cag cgg gag aag gat ttt ctc ctg aaa gag
gca gta 1055 Glu Glu Arg His Gln Arg Glu Lys Asp Phe Leu Leu Lys
Glu Ala Val 340 345 350 gag tcc cag agg atg tgt gag ctg atg aag cag
caa gag acc cac ctg 1103 Glu Ser Gln Arg Met Cys Glu Leu Met Lys
Gln Gln Glu Thr His Leu 355 360 365 aag caa cag ctt gcc cta tac aca
gag aag ttt gag gag ttc cag aac 1151 Lys Gln Gln Leu Ala Leu Tyr
Thr Glu Lys Phe Glu Glu Phe Gln Asn 370 375 380 aca ctt tcc aaa agc
agc gag gta ttc acc aca ttc aag cag gag atg 1199 Thr Leu Ser Lys
Ser Ser Glu Val Phe Thr Thr Phe Lys Gln Glu Met 385 390 395 gaa aag
atg act aag aag atc aag aag ctg gag aaa gaa acc acc atg 1247 Glu
Lys Met Thr Lys Lys Ile Lys Lys Leu Glu Lys Glu Thr Thr Met 400 405
410 415 tac cgg tcc cgg tgg gag agc agc aac aag gcc ctg ctt gag atg
gct 1295 Tyr Arg Ser Arg Trp Glu Ser Ser Asn Lys Ala Leu Leu Glu
Met Ala 420 425 430 gag gag aaa aca gtc cgg gat aaa gaa ctg gag ggc
ctg cag gta aaa 1343 Glu Glu Lys Thr Val Arg Asp Lys Glu Leu Glu
Gly Leu Gln Val Lys 435 440 445 atc caa cgg ctg gag aag ctg tgc cgg
gca ctg cag aca gag cgc aat 1391 Ile Gln Arg Leu Glu Lys Leu Cys
Arg Ala Leu Gln Thr Glu Arg Asn 450 455 460 gac ctg aac aag agg gta
cag gac ctg agt gct ggt ggc cag ggc tcc 1439 Asp Leu Asn Lys Arg
Val Gln Asp Leu Ser Ala Gly Gly Gln Gly Ser 465 470 475 ctc act gac
agt ggc cct gag agg agg cca gag ggg cct ggg gct caa 1487 Leu Thr
Asp Ser Gly Pro Glu Arg Arg Pro Glu Gly Pro Gly Ala Gln 480 485 490
495 gca ccc agc tcc ccc agg gtc aca gaa gcg cct tgc tac cca gga gca
1535 Ala Pro Ser Ser Pro Arg Val Thr Glu Ala Pro Cys Tyr Pro Gly
Ala 500 505 510 ccg agc aca gaa gca tca ggc cag act ggg cct caa gag
ccc acc tcc 1583 Pro Ser Thr Glu Ala Ser Gly Gln Thr Gly Pro Gln
Glu Pro Thr Ser 515 520 525 gcc agg gcc tagagagcct ggtgttgggt
catgctggga agggagcggc 1632 Ala Arg Ala 530 agcccagcca ggcctggccc
ataaaaggct cccatgctga gcagcccatt gctgaagcca 1692 ggatgttctt
gacctggctg gcatctggca cttgcaattt tggattttgt gggtcagttt 1752
tacgtacata gggcattttg caaggccttg caaatgcatt tatacctgta agtgtacagt
1812 gggcttgcat tggggatggg ggtgtgtaca gatgaagtca gtggcttgtc
tgtgagctga 1872 agagtcttga gaggggctgt catctgtagc tgccatcaca
gtgagttggc agaagtgact 1932 tgagcatttc tctgtctgat ttgaggctca
gacccctccc tgccctttca gagctcaaaa 1992 caagtaatac accaaggtct
tgactgcatt tgtcttgtga gcagggcttg cttggtcagc 2052 tcaggccctc
ctagctgctt ggaggctcct ttgattctct agacctggaa aaggtgtccc 2112
taggcagagc cctggcaggg cgctcagagc tgggatttcc tgcctggaac aagggacctg
2172 gagaatgttt ttgcgtggga tgatgtgctg gtcaggagcc ccttgggcat
cgcttcccct 2232 gccctttggt agtgccagga ccaggccaat gatgcttctc
agtagcctta tcattcacag 2292 gtgcctctct agcctgcaca aatgattgac
aagagatcac ccaaaggatt atttctgaag 2352 gtgttttttt ctttatttct
ttttcttttt ttttttttct ttttcttttt tttttgcaca 2412 tgacagtgtt
tgtattgagg accttccaag gaaaagggat gctgtaccag tggtgcctgg 2472
gtgcctggcc tccagtgtcc cacctccttc accaccccac ttggctcctt tgccatcttg
2532 atgctgaggt ttcctgtttg gtgagatcag gttgtttgtg gtaaaagaaa
ggaaagggct 2592 tctgatggct ttgccacaag cttacctgtg ggtttcagtc
ctgagaggcc accaccagtt 2652 cccatcagca ctgtctccat gcagcagttg
ctgggtccca tgtccagctg cctctttggc 2712 ttcatgggtt tttctgcttc
ctgcccccac ccccacatgt gcaatcctca agatttgtcc 2772 tgattctatt
tcctggcacc tccctgcctg tccttgggga ttctacttct tcctgtgtgg 2832
ggcccatagc tgttgtctaa caggtaagaa atgaaattga actattgact gggccccaga
2892 aatccataaa atggctgcag acagttgttt ctgtgtcctg ttctaccccc
actccagtac 2952 ataactacta tgtactgtgt agagccattc tatatgctga
atgttctgct gttgcaaact 3012 tgccagggta ttagccagtg tttgtgccaa
gcagttttcg gggacaacag aatgactcag 3072 accaagatgg ataggatggt
tagggctttg cttcttgctg tttttctttg aactagtcat 3132 tgtcctgcag
gtcccttcat cttccatacc tagcccactc ttttagccct taccttaaat 3192
ctctcagata agttggttca caaagaatgt taagtactga atcatgtgtg actgagacca
3252 gagatggcaa atgaatggca caccatttct ccttctcctg ccccagggca
ggtaccactg 3312 atctgcatca gagttgcctg ctattctctg gtgtatcctt
cacatctagg tgccctcaag 3372 cagctgtgtg agtgttgaga tctctgccat
ctctggctga gatactgctg tcctgtgaag 3432 tgtttcccat gacctttttc
ttcccctttg aatccctctt gtctggagta gtccttgcct 3492 tcttcttgct
ccagtaggcc ttttccttac cccagccctt gtgccaggct aagctggtac 3552
aagagctgcc aactcacaga gttttgctag gcgagagagg tgcagggaag aggcagaggt
3612 atgcaccttc ccccttgaag agaggggaaa ggcctacagt ggcccacata
attgcctgac 3672 tcacacttca gctacctctt aatgcctgtg gagggactgg
agctgctgga tcccagtgtg 3732 gtggtgtagg aggccacagt gagcaggtgg
ccccagctgg gtttcccagg tcaggaatgt 3792 gggccccagg caaggtgcag
cctttgctca cagctccatc catgtctaga ccttcaggcc 3852 agtctgcaga
tgaggttccc tacctttttc ttctcttcat tgaccaaatc aaccaatcac 3912
tacagctgct ctgcttctgc tttccaaagt agcccaggtc ctgggccaga tgcaggggag
3972 gtgcctatcc atgagtgaag gccagtgtct tcctcacctg ggtggtccca
cacttgtgac 4032 cctcagtttt aggacccaag atctgtgttg gtttcttaga
ttgctagctt ttcctccagg 4092 ggaccacagc aggtgaagct caagagcgca
tggctctgct aatagtaaat tgttttcagg 4152 gccttgtcca gctgagagct
tcatgtccac cagattctga gaggtgtcag cagcactttt 4212 tttttttatt
tgttgtttgt tttccatgag gttatcggac catgggctga gctcaggcac 4272
tttctgtagg agactgttat ttctgtaaag atggttattt aaccctcctc caccccatca
4332 cggtggccct gagggctgac ccggaggcca gtggagctgc ctggtgtcca
cgggggaggg 4392 ccaaggcctg ctgagctgat tctccagctg ctgccccagc
ctttccgcct tgcacagcac 4452 agaggtggtc accccaggga cagccaggca
cctgctcctc ttgcccttcc tgggggaaag 4512 gagctgcctt ctgtccctgt
aactgctttc cttatggccc aacccggcca ctcagacttg 4572 tttgaagctg
cactggcagc ttttttgtct cctttgggta ttcacaacag ccagggactt 4632
gattttgatg tattttaaac cacattaaat aaagagtctg ttgccttaaa aaaaaaaaaa
4692 aaaaa 4697 18 60 DNA Homo sapiens CDS (1)...(60) 18 gtg gac
gtg gat gag tac gac gag aac aag ttc gtg gac gag gaa gac 48 Val Asp
Val Asp Glu Tyr Asp Glu Asn Lys Phe Val Asp Glu Glu Asp 1 5 10 15
ggc ggc gac ggc 60 Gly Gly Asp Gly 20 19 15 PRT Homo sapiens 19 Glu
Glu Glu Glu Asp Asp Asp Glu Asp Glu Asp Glu Glu Asp Asp 1 5 10 15
20 26 PRT Homo sapiens 20 Glu Glu Glu Glu Asp Asp Asp Glu Asp Glu
Asp Glu Glu Asp Asp Val 1 5 10 15 Ser Glu Gly Ser Glu Val Pro Glu
Ser Asp 20 25 21 11 PRT Homo sapiens 21 Val Ser Glu Gly Ser Glu Val
Pro Glu Ser Asp 1 5 10 22 10 PRT Homo sapiens 22 Glu Asp Asp Asp
Pro Asp Gly Phe Leu Gly 1 5 10 23 30 PRT Oryctolagus cuniculus 23
Val Asp Val Asp Glu Tyr Asp Glu Asn Lys Phe Val Asp Glu Glu Asp 1 5
10 15 Gly Gly Asp Gly Gln Ala Gly Pro Asp Glu Gly Glu Val Asp 20 25
30 24 6 PRT Oryctolagus cuniculus 24 Asp Glu Gly Glu Val Asp 1 5 25
16 PRT Oryctolagus cuniculus 25 Glu Glu Glu Glu Glu Glu Glu Glu Asp
Asp Glu Asp Asp Asp Asp Asp 1 5 10 15 26 28 PRT Oryctolagus
cuniculus 26 27 12 PRT Oryctolagus cuniculus 27 Val Val Ser Glu Gly
Ser Glu Val Pro Glu Ser Asp 1 5 10 28 10 PRT Oryctolagus cuniculus
28 Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala 1 5 10 29 15 PRT
Oryctolagus cuniculus 29 Glu Asp Gly Val Gln Gly Glu Pro Pro Glu
Pro Glu Asp Ala Glu 1 5 10 15 30 45 DNA Homo sapiens 30 gaagaggaag
aagatgatga tgaagatgaa gatgaagaag atgat 45 31 78 DNA Homo sapiens 31
gaagaggaag aagatgatga tgaagatgaa gatgaagaag atgatgtgtc agagggctct
60 gaagtgcccg agagtgac 78 32 33 DNA Homo sapiens 32 gtgtcagagg
gctctgaagt gcccgagagt gac 33 33 30 DNA Homo sapiens 33 gaggatgatg
accccgatgg cttcttaggc 30 34 90 DNA Homo sapiens 34 gtggacgtgg
atgaatatga cgagaacaag ttcgtggacg aagaagatgg gggcgacggc 60
caggccgggc ccgacgaggg cgaggtggac 90 35 18 DNA Homo sapiens 35
gacgagggcg aggtggac 18 36 48 DNA Homo sapiens 36 gaggaggagg
aggaggagga ggaagacgac gaggacgacg acgacgac 48 37 84 DNA Homo sapiens
37 gaggaggagg aggaggagga ggaagacgac gaggacgacg acgacgacgt
cgtgtccgag 60 ggctcggagg tgcccgagag cgat 84 38 36 DNA Homo sapiens
38 gtcgtgtccg agggctcgga ggtgcccgag agcgat 36 39 30 DNA Homo
sapiens 39 ccccccggga agccagccct cccaggagcc 30 40 45 DNA Homo
sapiens 40 gaggatgggg tccagggtga gccccctgaa cctgaagatg cagag 45 41
7 PRT Homo sapiens 41 Arg Asp Val Ser Glu Glu Leu 1 5 42 21 DNA
Homo sapiens 42 cgtgatgtct ctgaggagct g 21 43 538 PRT Homo sapiens
43 Met Ala Gly Pro Pro Ala Leu Pro Pro Pro Glu Thr Ala Ala Ala Ala
1 5 10 15 Thr Thr Ala Ala Ala Ala Ser Ser Ser Ala Ala Ser Pro His
Tyr Gln 20 25 30 Glu Trp Ile Leu Asp Thr Ile Asp Ser Leu Arg Ser
Arg Lys Ala Arg 35 40 45 Pro Asp Leu Glu Arg Ile Cys Arg Met Val
Arg Arg Arg His Gly Pro 50 55 60 Glu Pro Glu Arg Thr Arg Ala Glu
Leu Glu Lys Leu Ile Gln Gln Arg 65 70 75 80 Ala Val Leu Arg Val Ser
Tyr Lys Gly Ser Ile Ser Tyr Arg Asn Ala 85 90 95 Ala Arg Val Gln
Pro Pro Arg Arg Gly Ala Thr Pro Pro Ala Pro Pro 100 105 110 Arg Ala
Pro Arg Gly Ala Pro Ala Ala Ala Ala Ala Ala Ala Pro Pro 115 120 125
Pro Thr Pro Ala Pro Pro Pro Pro Pro Ala Pro Val Ala Ala Ala Ala 130
135 140 Pro Ala Arg Ala Pro Arg Ala Ala Ala Ala Ala Ala Thr Ala Pro
Pro 145 150 155 160 Ser Pro Gly Pro Ala Gln Pro Gly Pro Arg Ala Gln
Arg Ala Ala Pro 165 170 175 Leu Ala Ala Pro Pro Pro Ala Pro Ala Ala
Pro Pro Ala Val Ala Pro 180 185 190 Pro Ala Gly Pro Arg Arg Ala Pro
Pro Pro Ala Val Ala Ala Arg Glu 195 200 205 Pro Pro Leu Pro Pro Pro
Pro Gln Pro Pro Ala Pro Pro Gln Gln Gln 210 215 220 Gln Pro Pro Pro
Pro Gln Pro Gln Pro Pro Pro Glu Gly Gly Ala Val 225 230 235 240 Arg
Ala Gly Gly Ala Ala Arg Pro Val Ser Leu Arg Glu Val Val Arg 245 250
255 Tyr Leu Gly Gly Ser Gly Gly Ala Gly Gly Arg Leu Thr Arg Gly Arg
260 265 270 Val Gln Gly Leu Leu Glu Glu Glu Ala Ala Ala Arg Gly Arg
Leu Glu 275 280 285 Arg Thr Arg Leu Gly Ala Leu Ala Leu Pro Arg Gly
Asp Arg Pro Gly 290 295 300 Arg Ala Pro Pro Ala Ala Ser Ala Arg Pro
Ser Arg Ser Lys Arg Gly 305 310 315 320 Gly Glu Glu Arg Val Leu Glu
Lys Glu Glu Glu Glu Asp Asp Asp Glu 325 330 335 Asp Glu Asp Glu Glu
Asp Asp Val Ser Glu Gly Ser Glu Val Pro Glu 340 345 350 Ser Asp Arg
Pro Ala Gly Ala Gln His His Gln Leu Asn Gly Glu Arg 355 360 365 Gly
Pro Gln Ser Ala Lys Glu Arg Val Lys Glu Trp Thr Pro Cys Gly 370 375
380 Pro His Gln Gly Gln Asp Glu Gly Arg Gly Pro Ala Pro Gly Ser Gly
385 390 395 400 Thr Arg Gln Val Phe Ser Met Ala Ala Met Asn Lys Glu
Gly Gly Thr 405 410 415 Ala Ser Val Ala Thr Gly Pro Asp Ser Pro Ser
Pro Val Pro Leu Pro 420 425 430 Pro Gly Lys Pro Ala Leu Pro Gly Ala
Asp Gly Thr Pro Phe Gly Cys 435 440 445 Pro Pro Gly Arg Lys Glu Lys
Pro Ser Asp Pro Val Glu Trp Thr Val 450 455 460 Met Asp Val Val Glu
Tyr Phe Thr Glu Ala Gly Phe Pro Glu Gln Ala 465 470 475 480 Thr Ala
Phe Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu Leu Met 485 490 495
Gln Arg Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly Pro Ala 500
505 510 Leu Lys Ile Tyr Glu His His Ile Lys Val Leu Gln Gln Gly His
Phe 515 520 525 Glu Asp Asp Asp Pro Asp Gly Phe Leu Gly 530 535 44
546 PRT Homo sapiens 44 Met Lys Asn Gln Asp Lys Lys Asn Gly Ala Ala
Lys Gln Ser Asn Pro 1 5 10 15 Lys Ser Ser Pro Gly Gln Pro Glu Ala
Gly Pro Glu Gly Ala Gln Glu 20 25 30 Arg Pro Ser Gln Ala Ala Pro
Ala Val Glu Ala Glu Gly Pro Gly Ser 35 40 45 Ser Gln Ala Pro Arg
Lys Pro Glu Gly Ala Gln Ala Arg Thr Ala Gln 50 55 60 Ser Gly Ala
Leu Arg Asp Val Ser Glu Glu Leu Ser Arg Gln Leu Glu 65 70 75 80 Asp
Ile Leu Ser Thr Tyr Cys Val Asp Asn Asn Gln Gly Gly Pro Gly 85 90
95 Glu Asp Gly Ala Gln Gly Glu Pro Ala Glu Pro Glu Asp Ala Glu Lys
100 105 110 Ser Arg Thr Tyr Val Ala Arg Asn Gly Glu Pro Glu Pro Thr
Pro Val 115 120 125 Val Asn Gly Glu Lys Glu Pro Ser Lys Gly Asp Pro
Asn Thr Glu Glu 130 135 140 Ile Arg Gln Ser Asp Glu Val Gly Asp Arg
Asp His Arg Arg Pro Gln 145 150 155 160 Glu Lys Lys Lys Ala Lys Gly
Leu Gly Lys Glu Ile Thr Leu Leu Met 165 170 175 Gln Thr Leu Asn Thr
Leu Ser Thr Pro Glu Glu Lys Leu Ala Ala Leu 180 185 190 Cys Lys Lys
Tyr Ala Glu Leu Leu Glu Glu His Arg Asn Ser Gln Lys 195 200 205 Gln
Met Lys Leu Leu Gln Lys Lys Gln Ser Gln Leu Val Gln Glu Lys 210 215
220 Asp His Leu Arg Gly Glu His Ser Lys Ala Val Leu Ala Arg Ser Lys
225 230 235 240 Leu Glu Ser Leu Cys Arg Glu Leu Gln Arg His Asn Arg
Ser Leu Lys 245 250 255 Glu Glu Gly Val Gln Arg Ala Arg Glu Glu Glu
Glu Lys Arg Lys Glu 260 265 270 Val Thr Ser His Phe Gln Val Thr Leu
Asn Asp Ile Gln Leu Gln Met 275 280 285 Glu Gln His Asn Glu Arg Asn
Ser Lys Leu Arg Gln Glu Asn Met Glu 290 295 300 Leu Ala Glu Arg Leu
Lys Lys Leu Ile Glu Gln Tyr Glu Leu Arg Glu 305 310 315 320 Glu His
Ile Asp Lys Val Phe Lys His Lys Asp Leu Gln Gln Gln Leu 325 330 335
Val Asp Ala Lys Leu Gln Gln Ala Gln Glu Met Leu Lys Glu Ala Glu 340
345 350 Glu Arg His Gln Arg Glu Lys Asp Phe Leu Leu Lys Glu Ala Val
Glu 355 360 365 Ser Gln Arg Met Cys Glu Leu Met Lys Gln Gln Glu Thr
His Leu Lys 370 375 380 Gln Gln Leu Ala Leu Tyr Thr Glu Lys Phe Glu
Glu Phe Gln Asn Thr 385 390 395 400 Leu Ser Lys Ser Ser Glu Val Phe
Thr Thr Phe Lys Gln Glu Met Glu 405 410 415 Lys Met Thr Lys Lys Ile
Lys Lys Leu Glu Lys Glu Thr Thr Met Tyr 420 425 430 Arg Ser Arg Trp
Glu Ser Ser Asn Lys Ala Leu Leu Glu Met Ala Glu 435 440 445 Glu Lys
Thr Val Arg Asp Lys Glu Leu Glu Gly Leu Gln Val Lys Ile 450 455 460
Gln Arg Leu Glu Lys Leu Cys Arg Ala Leu Gln Thr Glu Arg Asn Asp 465
470 475 480 Leu Asn Lys Arg Val Gln Asp Leu Ser Ala Gly Gly Gln Gly
Ser Leu 485 490 495 Thr Asp Ser Gly Pro Glu Arg Arg Pro Glu Gly Pro
Gly Ala Gln Ala 500 505 510 Pro Ser Ser Pro Arg Val Thr Glu Ala Pro
Cys Tyr Pro Gly
Ala Pro 515 520 525 Ser Thr Glu Ala Ser Gly Gln Thr Gly Pro Gln Glu
Pro Thr Ser Ala 530 535 540 Arg Ala 545 45 1614 DNA Homo sapiens
CDS (1)...(1614) 45 atg gcg ggg ccc ccg gcc cta ccc ccg ccg gag acg
gcg gcg gcc gcc 48 Met Ala Gly Pro Pro Ala Leu Pro Pro Pro Glu Thr
Ala Ala Ala Ala 1 5 10 15 acc acg gcg gcc gcc gcc tcg tcg tcc gcc
gct tcc ccg cac tac caa 96 Thr Thr Ala Ala Ala Ala Ser Ser Ser Ala
Ala Ser Pro His Tyr Gln 20 25 30 gag tgg atc ctg gac acc atc gac
tcg ctg cgc tcg cgc aag gcg cgg 144 Glu Trp Ile Leu Asp Thr Ile Asp
Ser Leu Arg Ser Arg Lys Ala Arg 35 40 45 ccg gac ctg gag cgc atc
tgc cgg atg gtg cgg cgg cgg cac ggc ccg 192 Pro Asp Leu Glu Arg Ile
Cys Arg Met Val Arg Arg Arg His Gly Pro 50 55 60 gag ccg gag cgc
acg cgc gcc gag ctc gag aaa ctg atc cag cag cgc 240 Glu Pro Glu Arg
Thr Arg Ala Glu Leu Glu Lys Leu Ile Gln Gln Arg 65 70 75 80 gcc gtg
ctc cgg gtc agc tac aag ggg agc atc tcg tac cgc aac gcg 288 Ala Val
Leu Arg Val Ser Tyr Lys Gly Ser Ile Ser Tyr Arg Asn Ala 85 90 95
gcg cgc gtc cag ccg ccc cgg cgc gga gcc acc ccg ccg gcc ccg ccg 336
Ala Arg Val Gln Pro Pro Arg Arg Gly Ala Thr Pro Pro Ala Pro Pro 100
105 110 cgc gcc ccc cgc ggg gcc ccc gcc gcc gcc gcc gcc gcc gcg ccg
ccg 384 Arg Ala Pro Arg Gly Ala Pro Ala Ala Ala Ala Ala Ala Ala Pro
Pro 115 120 125 ccc acg ccc gcc ccg ccg cca ccg ccc gcg ccc gtc gcc
gcc gcc gcc 432 Pro Thr Pro Ala Pro Pro Pro Pro Pro Ala Pro Val Ala
Ala Ala Ala 130 135 140 ccg gcc cgg gcg ccc cgc gcg gcc gcc gcc gcc
gcc aca gcg ccc ccc 480 Pro Ala Arg Ala Pro Arg Ala Ala Ala Ala Ala
Ala Thr Ala Pro Pro 145 150 155 160 tcg cct ggc ccc gcg cag ccg ggc
ccc cgc gcg cag cgg gcc gcg ccc 528 Ser Pro Gly Pro Ala Gln Pro Gly
Pro Arg Ala Gln Arg Ala Ala Pro 165 170 175 ctg gcc gcg ccg ccg ccc
gcg cca gcc gct ccc ccg gcg gtg gcg ccc 576 Leu Ala Ala Pro Pro Pro
Ala Pro Ala Ala Pro Pro Ala Val Ala Pro 180 185 190 ccg gcc ggc ccg
cgc cgc gcc ccc ccg ccc gcc gtc gcc gcc cgg gag 624 Pro Ala Gly Pro
Arg Arg Ala Pro Pro Pro Ala Val Ala Ala Arg Glu 195 200 205 ccg ccg
ctg ccg ccg ccg cca cag ccg ccg gcg ccg cca cag cag cag 672 Pro Pro
Leu Pro Pro Pro Pro Gln Pro Pro Ala Pro Pro Gln Gln Gln 210 215 220
cag ccg ccg ccg ccg cag cca cag ccg ccg ccg gag ggg ggc gcg gtg 720
Gln Pro Pro Pro Pro Gln Pro Gln Pro Pro Pro Glu Gly Gly Ala Val 225
230 235 240 cgg gcc ggc ggc gcg gcg cgg ccc gtg agc ctg cgg gaa gtc
gtg cgc 768 Arg Ala Gly Gly Ala Ala Arg Pro Val Ser Leu Arg Glu Val
Val Arg 245 250 255 tac ctc ggg ggc agc ggc ggc gcc ggc ggt cgc cta
acc cgc ggc cgc 816 Tyr Leu Gly Gly Ser Gly Gly Ala Gly Gly Arg Leu
Thr Arg Gly Arg 260 265 270 gtg cag ggg ctg ctg gag gag gag gcg gcg
gct cga ggc cgt ctg gag 864 Val Gln Gly Leu Leu Glu Glu Glu Ala Ala
Ala Arg Gly Arg Leu Glu 275 280 285 cgc acc cgt ctc gga gcg ctt gcg
ctg ccc cgc ggg gac agg ccc gga 912 Arg Thr Arg Leu Gly Ala Leu Ala
Leu Pro Arg Gly Asp Arg Pro Gly 290 295 300 cgg gcg ccg ccg gcc gcc
agc gcc cgc ccg tct cgc agc aag aga ggt 960 Arg Ala Pro Pro Ala Ala
Ser Ala Arg Pro Ser Arg Ser Lys Arg Gly 305 310 315 320 gga gaa gag
cga gta ctt gag aaa gaa gag gaa gaa gat gat gat gaa 1008 Gly Glu
Glu Arg Val Leu Glu Lys Glu Glu Glu Glu Asp Asp Asp Glu 325 330 335
gat gaa gat gaa gaa gat gat gtg tca gag ggc tct gaa gtg ccc gag
1056 Asp Glu Asp Glu Glu Asp Asp Val Ser Glu Gly Ser Glu Val Pro
Glu 340 345 350 agt gac cgt cct gca ggt gcc cag cac cac cag ctt aac
ggc gag cgg 1104 Ser Asp Arg Pro Ala Gly Ala Gln His His Gln Leu
Asn Gly Glu Arg 355 360 365 gga cct cag agt gcc aag gag agg gtc aag
gag tgg acc ccc tgc gga 1152 Gly Pro Gln Ser Ala Lys Glu Arg Val
Lys Glu Trp Thr Pro Cys Gly 370 375 380 ccg cac cag ggc cag gat gaa
ggg cgg ggg cca gcc ccg ggc agc ggc 1200 Pro His Gln Gly Gln Asp
Glu Gly Arg Gly Pro Ala Pro Gly Ser Gly 385 390 395 400 acc cgc cag
gtg ttc tcc atg gca gcc atg aac aag gaa ggg gga aca 1248 Thr Arg
Gln Val Phe Ser Met Ala Ala Met Asn Lys Glu Gly Gly Thr 405 410 415
gct tct gtt gcc acc ggg cca gac tcc ccg tcc ccc gtg cct ttg ccc
1296 Ala Ser Val Ala Thr Gly Pro Asp Ser Pro Ser Pro Val Pro Leu
Pro 420 425 430 cca ggc aaa cca gcc cta cct ggg gcc gac ggg acc ccc
ttt ggc tgt 1344 Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly Thr
Pro Phe Gly Cys 435 440 445 ccg ccc ggg cgc aaa gag aag cca tct gat
ccc gtc gag tgg acc gtg 1392 Pro Pro Gly Arg Lys Glu Lys Pro Ser
Asp Pro Val Glu Trp Thr Val 450 455 460 atg gat gtc gtc gaa tat ttt
act gag gct gga ttc ccg gag cag gcg 1440 Met Asp Val Val Glu Tyr
Phe Thr Glu Ala Gly Phe Pro Glu Gln Ala 465 470 475 480 aca gct ttc
caa gag cag gaa att gat ggc aaa tct ttg ctg ctc atg 1488 Thr Ala
Phe Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu Leu Met 485 490 495
cag cgc aca gat gtg ctc acc ggc ctg tcc atc cgc ctc ggg cca gcc
1536 Gln Arg Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly Pro
Ala 500 505 510 ctg aaa atc tac gag cac cac atc aag gtg ctt cag caa
ggc cac ttt 1584 Leu Lys Ile Tyr Glu His His Ile Lys Val Leu Gln
Gln Gly His Phe 515 520 525 gag gat gat gac ccc gat ggc ttc tta ggc
1614 Glu Asp Asp Asp Pro Asp Gly Phe Leu Gly 530 535 46 1638 DNA
Homo sapiens CDS (1)...(1638) 46 atg aag aac caa gac aaa aag aac
ggg gct gcc aaa caa tcc aat cca 48 Met Lys Asn Gln Asp Lys Lys Asn
Gly Ala Ala Lys Gln Ser Asn Pro 1 5 10 15 aaa agc agc cca gga caa
ccg gaa gca gga ccc gag gga gcc cag gag 96 Lys Ser Ser Pro Gly Gln
Pro Glu Ala Gly Pro Glu Gly Ala Gln Glu 20 25 30 cgg ccc agc cag
gcg gct cct gca gta gaa gca gaa ggt ccc ggc agc 144 Arg Pro Ser Gln
Ala Ala Pro Ala Val Glu Ala Glu Gly Pro Gly Ser 35 40 45 agc cag
gct cct cgg aag ccg gag ggt gct caa gcc aga acg gct cag 192 Ser Gln
Ala Pro Arg Lys Pro Glu Gly Ala Gln Ala Arg Thr Ala Gln 50 55 60
tct ggg gcc ctt cgt gat gtc tct gag gag ctg agc cgc caa ctg gaa 240
Ser Gly Ala Leu Arg Asp Val Ser Glu Glu Leu Ser Arg Gln Leu Glu 65
70 75 80 gac ata ctg agc aca tac tgt gtg gac aat aac cag ggg ggc
ccc ggc 288 Asp Ile Leu Ser Thr Tyr Cys Val Asp Asn Asn Gln Gly Gly
Pro Gly 85 90 95 gag gat ggg gca cag ggt gag ccg gct gaa ccc gaa
gat gca gag aag 336 Glu Asp Gly Ala Gln Gly Glu Pro Ala Glu Pro Glu
Asp Ala Glu Lys 100 105 110 tcc cgg acc tat gtg gca agg aat ggg gag
cct gaa cca act cca gta 384 Ser Arg Thr Tyr Val Ala Arg Asn Gly Glu
Pro Glu Pro Thr Pro Val 115 120 125 gtc aat gga gag aag gaa ccc tcc
aag ggg gat cca aac aca gaa gag 432 Val Asn Gly Glu Lys Glu Pro Ser
Lys Gly Asp Pro Asn Thr Glu Glu 130 135 140 atc cgg cag agt gac gag
gtc gga gac cga gac cat cga agg cca cag 480 Ile Arg Gln Ser Asp Glu
Val Gly Asp Arg Asp His Arg Arg Pro Gln 145 150 155 160 gag aag aaa
aaa gcc aag ggt ttg ggt aag gag atc acg ttg ctg atg 528 Glu Lys Lys
Lys Ala Lys Gly Leu Gly Lys Glu Ile Thr Leu Leu Met 165 170 175 cag
aca ttg aat act ctg agt acc cca gag gag aag ctg gct gct ctg 576 Gln
Thr Leu Asn Thr Leu Ser Thr Pro Glu Glu Lys Leu Ala Ala Leu 180 185
190 tgc aag aag tat gct gaa ctg ctg gag gag cac cgg aat tca cag aag
624 Cys Lys Lys Tyr Ala Glu Leu Leu Glu Glu His Arg Asn Ser Gln Lys
195 200 205 cag atg aag ctc cta cag aaa aag cag agc cag ctg gtg caa
gag aag 672 Gln Met Lys Leu Leu Gln Lys Lys Gln Ser Gln Leu Val Gln
Glu Lys 210 215 220 gac cac ctg cgc ggt gag cac agc aag gcc gtc ctg
gcc cgc agc aag 720 Asp His Leu Arg Gly Glu His Ser Lys Ala Val Leu
Ala Arg Ser Lys 225 230 235 240 ctt gag agc cta tgc cgt gag ctg cag
cgg cac aac cgc tcc ctc aag 768 Leu Glu Ser Leu Cys Arg Glu Leu Gln
Arg His Asn Arg Ser Leu Lys 245 250 255 gaa gaa ggt gtg cag cgg gcc
cgg gag gag gag gag aag cgc aag gag 816 Glu Glu Gly Val Gln Arg Ala
Arg Glu Glu Glu Glu Lys Arg Lys Glu 260 265 270 gtg acc tcg cac ttc
cag gtg aca ctg aat gac att cag ctg cag atg 864 Val Thr Ser His Phe
Gln Val Thr Leu Asn Asp Ile Gln Leu Gln Met 275 280 285 gaa cag cac
aat gag cgc aac tcc aag ctg cgc caa gag aac atg gag 912 Glu Gln His
Asn Glu Arg Asn Ser Lys Leu Arg Gln Glu Asn Met Glu 290 295 300 ctg
gct gag agg ctc aag aag ctg att gag cag tat gag ctg cgc gag 960 Leu
Ala Glu Arg Leu Lys Lys Leu Ile Glu Gln Tyr Glu Leu Arg Glu 305 310
315 320 gag cat atc gac aaa gtc ttc aaa cac aag gac cta caa cag cag
ctg 1008 Glu His Ile Asp Lys Val Phe Lys His Lys Asp Leu Gln Gln
Gln Leu 325 330 335 gtg gat gcc aag ctc cag cag gcc cag gag atg cta
aag gag gca gaa 1056 Val Asp Ala Lys Leu Gln Gln Ala Gln Glu Met
Leu Lys Glu Ala Glu 340 345 350 gag cgg cac cag cgg gag aag gat ttt
ctc ctg aaa gag gca gta gag 1104 Glu Arg His Gln Arg Glu Lys Asp
Phe Leu Leu Lys Glu Ala Val Glu 355 360 365 tcc cag agg atg tgt gag
ctg atg aag cag caa gag acc cac ctg aag 1152 Ser Gln Arg Met Cys
Glu Leu Met Lys Gln Gln Glu Thr His Leu Lys 370 375 380 caa cag ctt
gcc cta tac aca gag aag ttt gag gag ttc cag aac aca 1200 Gln Gln
Leu Ala Leu Tyr Thr Glu Lys Phe Glu Glu Phe Gln Asn Thr 385 390 395
400 ctt tcc aaa agc agc gag gta ttc acc aca ttc aag cag gag atg gaa
1248 Leu Ser Lys Ser Ser Glu Val Phe Thr Thr Phe Lys Gln Glu Met
Glu 405 410 415 aag atg act aag aag atc aag aag ctg gag aaa gaa acc
acc atg tac 1296 Lys Met Thr Lys Lys Ile Lys Lys Leu Glu Lys Glu
Thr Thr Met Tyr 420 425 430 cgg tcc cgg tgg gag agc agc aac aag gcc
ctg ctt gag atg gct gag 1344 Arg Ser Arg Trp Glu Ser Ser Asn Lys
Ala Leu Leu Glu Met Ala Glu 435 440 445 gag aaa aca gtc cgg gat aaa
gaa ctg gag ggc ctg cag gta aaa atc 1392 Glu Lys Thr Val Arg Asp
Lys Glu Leu Glu Gly Leu Gln Val Lys Ile 450 455 460 caa cgg ctg gag
aag ctg tgc cgg gca ctg cag aca gag cgc aat gac 1440 Gln Arg Leu
Glu Lys Leu Cys Arg Ala Leu Gln Thr Glu Arg Asn Asp 465 470 475 480
ctg aac aag agg gta cag gac ctg agt gct ggt ggc cag ggc tcc ctc
1488 Leu Asn Lys Arg Val Gln Asp Leu Ser Ala Gly Gly Gln Gly Ser
Leu 485 490 495 act gac agt ggc cct gag agg agg cca gag ggg cct ggg
gct caa gca 1536 Thr Asp Ser Gly Pro Glu Arg Arg Pro Glu Gly Pro
Gly Ala Gln Ala 500 505 510 ccc agc tcc ccc agg gtc aca gaa gcg cct
tgc tac cca gga gca ccg 1584 Pro Ser Ser Pro Arg Val Thr Glu Ala
Pro Cys Tyr Pro Gly Ala Pro 515 520 525 agc aca gaa gca tca ggc cag
act ggg cct caa gag ccc acc tcc gcc 1632 Ser Thr Glu Ala Ser Gly
Gln Thr Gly Pro Gln Glu Pro Thr Ser Ala 530 535 540 agg gcc 1638
Arg Ala 545 47 550 PRT Oryctolagus cuniculus 47 Met Ala Gly Pro Pro
Ala Leu Pro Pro Pro Glu Thr Ala Ala Ala Ala 1 5 10 15 Thr Thr Ala
Ala Ala Ala Ala Ser Ser Ser Ala Ala Ser Pro His Tyr 20 25 30 Gln
Glu Trp Ile Leu Asp Thr Ile Asp Ser Leu Arg Ser Arg Lys Ala 35 40
45 Arg Pro Asp Leu Glu Arg Ile Cys Arg Met Val Arg Arg Arg His Gly
50 55 60 Pro Glu Pro Glu Arg Thr Arg Ala Glu Leu Glu Lys Leu Ile
Gln Gln 65 70 75 80 Arg Ala Val Leu Arg Val Ser Tyr Lys Gly Ser Ile
Ser Tyr Arg Asn 85 90 95 Ala Ala Arg Val Gln Pro Pro Arg Arg Gly
Ala Thr Pro Pro Ala Pro 100 105 110 Pro Arg Ala Pro Arg Gly Gly Pro
Ala Ala Ala Ala Ala Pro Pro Pro 115 120 125 Thr Pro Ala Pro Pro Pro
Pro Pro Ala Pro Val Ala Ala Ala Ala Ala 130 135 140 Pro Ala Arg Ala
Pro Arg Ala Ala Ala Ala Ala Ala Ala Ala Thr Ala 145 150 155 160 Pro
Pro Ser Pro Gly Pro Ala Gln Pro Gly Pro Arg Ala Gln Arg Ala 165 170
175 Ala Pro Leu Ala Ala Pro Pro Pro Ala Pro Ala Ala Pro Pro Ala Ala
180 185 190 Ala Pro Pro Ala Gly Pro Arg Arg Ala Pro Pro Pro Ala Ala
Ala Val 195 200 205 Ala Ala Arg Glu Ser Pro Leu Pro Pro Pro Pro Gln
Pro Pro Ala Pro 210 215 220 Pro Gln Gln Gln Gln Gln Pro Pro Pro Pro
Pro Pro Pro Gln Gln Pro 225 230 235 240 Gln Pro Pro Pro Glu Gly Gly
Ala Ala Arg Ala Gly Gly Pro Ala Arg 245 250 255 Pro Val Ser Leu Arg
Glu Val Val Arg Tyr Leu Gly Gly Ser Ser Gly 260 265 270 Ala Gly Gly
Arg Leu Thr Arg Gly Arg Val Gln Gly Leu Leu Glu Glu 275 280 285 Glu
Ala Ala Ala Arg Gly Arg Leu Glu Arg Thr Arg Leu Gly Ala Leu 290 295
300 Ala Leu Pro Arg Gly Asp Arg Pro Gly Arg Ala Pro Pro Ala Ala Ser
305 310 315 320 Ala Arg Ala Ala Arg Asn Lys Arg Ala Gly Glu Glu Arg
Val Leu Glu 325 330 335 Lys Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp
Glu Asp Asp Asp Asp 340 345 350 Asp Val Val Ser Glu Gly Ser Glu Val
Pro Glu Ser Asp Arg Pro Ala 355 360 365 Gly Ala Gln His His Gln Leu
Asn Gly Gly Glu Arg Gly Pro Gln Thr 370 375 380 Ala Lys Glu Arg Ala
Lys Glu Trp Ser Leu Cys Gly Pro His Pro Gly 385 390 395 400 Gln Glu
Glu Gly Arg Gly Pro Ala Ala Gly Ser Gly Thr Arg Gln Val 405 410 415
Phe Ser Met Ala Ala Leu Ser Lys Glu Gly Gly Ser Ala Ser Ser Thr 420
425 430 Thr Gly Pro Asp Ser Pro Ser Pro Val Pro Leu Pro Pro Gly Lys
Pro 435 440 445 Ala Leu Pro Gly Ala Asp Gly Thr Pro Phe Gly Cys Pro
Ala Gly Arg 450 455 460 Lys Glu Lys Pro Ala Asp Pro Val Glu Trp Thr
Val Met Asp Val Val 465 470 475 480 Glu Tyr Phe Thr Glu Ala Gly Phe
Pro Glu Gln Ala Thr Ala Phe Gln 485 490 495 Glu Gln Glu Ile Asp Gly
Lys Ser Leu Leu Leu Met Gln Arg Thr Asp 500 505 510 Val Leu Thr Gly
Leu Ser Ile Arg Leu Gly Pro Ala Leu Lys Ile Tyr 515 520 525 Glu His
His Ile Lys Val Leu Gln Gln Gly His Phe Glu Asp Asp Asp 530 535 540
Pro Glu Gly Phe Leu Gly 545 550 48 2561 DNA Oryctolagus cuniculus
CDS (246)...(1895) 48 ggtctgtgtg tgcgtgcgtg cgagtgagtg agtgtgtgca
tatttttttt tctcttttct 60 ttctctctct tttttttttt tttgcaaaga
aacagcagcg ccgccgccgc tccgccgagg 120 cgctgcgccc cccggggggg
ggaggcggag gaggcgggca gcggcggagg gaggggagcc 180 ggggaggggg
gcgccgcgct gggagggagg cagcgcgcac ggtgcagccg ggccgggcgg 240 gaggc
atg gcg ggg ccc ccg gcc cta ccc ccg ccg gag acg gcg gcg gcc 290 Met
Ala Gly Pro Pro Ala Leu Pro Pro Pro Glu Thr Ala Ala Ala 1 5 10 15
gcc acc acg gcc gcg gcc gcc gcc tcg tcg tcc gcc gct tcc ccg cac 338
Ala Thr Thr Ala Ala Ala Ala Ala Ser Ser Ser Ala Ala Ser Pro His 20
25 30 tac caa gag tgg att
ctg gac acc atc gac tcg ctg cgc tcg cgc aag 386 Tyr Gln Glu Trp Ile
Leu Asp Thr Ile Asp Ser Leu Arg Ser Arg Lys 35 40 45 gcg cgg ccg
gac ctg gag cgc atc tgc cgg atg gtg cgg cgg cgg cac 434 Ala Arg Pro
Asp Leu Glu Arg Ile Cys Arg Met Val Arg Arg Arg His 50 55 60 ggc
ccg gag ccg gag cgc acg cgc gcc gag ctc gag aaa ctg atc cag 482 Gly
Pro Glu Pro Glu Arg Thr Arg Ala Glu Leu Glu Lys Leu Ile Gln 65 70
75 cag cgc gcc gtg ctc cgg gtc agc tac aag ggg agc atc tcg tac cgc
530 Gln Arg Ala Val Leu Arg Val Ser Tyr Lys Gly Ser Ile Ser Tyr Arg
80 85 90 95 aac gcg gcg cgc gtc cag ccg ccc cgg cgc gga gcc acc ccg
ccg gcc 578 Asn Ala Ala Arg Val Gln Pro Pro Arg Arg Gly Ala Thr Pro
Pro Ala 100 105 110 ccg ccg cgc gcc ccc cgc ggg ggc ccc gcc gcc gcc
gcc gcg ccg ccg 626 Pro Pro Arg Ala Pro Arg Gly Gly Pro Ala Ala Ala
Ala Ala Pro Pro 115 120 125 ccc acg ccc gcc ccg ccg ccg ccg ccc gcg
ccc gtc gcc gcc gcc gcc 674 Pro Thr Pro Ala Pro Pro Pro Pro Pro Ala
Pro Val Ala Ala Ala Ala 130 135 140 gcc ccg gcc cgg gcg ccc cgc gcg
gcc gcc gcc gcc gct gcc gcc aca 722 Ala Pro Ala Arg Ala Pro Arg Ala
Ala Ala Ala Ala Ala Ala Ala Thr 145 150 155 gcg ccc ccc tcg ccc ggc
ccc gcg cag ccg ggc ccc cgc gcg cag cgg 770 Ala Pro Pro Ser Pro Gly
Pro Ala Gln Pro Gly Pro Arg Ala Gln Arg 160 165 170 175 gcc gcg ccc
ctg gcc gcg ccg ccg ccc gcg ccc gcc gct ccc ccg gcg 818 Ala Ala Pro
Leu Ala Ala Pro Pro Pro Ala Pro Ala Ala Pro Pro Ala 180 185 190 gcg
gcg ccc ccg gcc ggc ccg cgc cgc gcc ccc ccg ccc gcc gcc gcc 866 Ala
Ala Pro Pro Ala Gly Pro Arg Arg Ala Pro Pro Pro Ala Ala Ala 195 200
205 gtc gcc gcc cgg gag tcg ccg ctg ccg ccg ccg cca cag ccg ccg gcg
914 Val Ala Ala Arg Glu Ser Pro Leu Pro Pro Pro Pro Gln Pro Pro Ala
210 215 220 ccg cca cag cag cag cag cag ccg ccg ccg cca ccg ccg ccg
cag cag 962 Pro Pro Gln Gln Gln Gln Gln Pro Pro Pro Pro Pro Pro Pro
Gln Gln 225 230 235 cca cag ccg ccg ccg gag ggg ggc gcg gcg cgg gcc
ggc ggc ccg gcg 1010 Pro Gln Pro Pro Pro Glu Gly Gly Ala Ala Arg
Ala Gly Gly Pro Ala 240 245 250 255 cgg ccc gtg agc ctg cgg gaa gtc
gtg cgc tac ctc ggg ggt agc agc 1058 Arg Pro Val Ser Leu Arg Glu
Val Val Arg Tyr Leu Gly Gly Ser Ser 260 265 270 ggc gct ggc ggc cgc
ctg acc cgc ggc cgc gtg cag ggt ctg ctg gaa 1106 Gly Ala Gly Gly
Arg Leu Thr Arg Gly Arg Val Gln Gly Leu Leu Glu 275 280 285 gag gag
gcg gcg gcg cgg ggc cgc ctg gag cgc acc cgt ctc gga gcg 1154 Glu
Glu Ala Ala Ala Arg Gly Arg Leu Glu Arg Thr Arg Leu Gly Ala 290 295
300 ctt gcg ctg ccc cgc ggg gac agg ccc gga cgg gcg cca ccg gcc gcc
1202 Leu Ala Leu Pro Arg Gly Asp Arg Pro Gly Arg Ala Pro Pro Ala
Ala 305 310 315 agc gcc cgc gcg gcg cgg aac aag aga gct ggc gag gag
cga gtg ctt 1250 Ser Ala Arg Ala Ala Arg Asn Lys Arg Ala Gly Glu
Glu Arg Val Leu 320 325 330 335 gaa aag gag gag gag gag gag gag gag
gaa gac gac gag gac gac gac 1298 Glu Lys Glu Glu Glu Glu Glu Glu
Glu Glu Asp Asp Glu Asp Asp Asp 340 345 350 gac gac gtc gtg tcc gag
ggc tcg gag gtg ccc gag agc gat cgt ccc 1346 Asp Asp Val Val Ser
Glu Gly Ser Glu Val Pro Glu Ser Asp Arg Pro 355 360 365 gcg ggt gcg
cag cat cac cag ctg aat ggc ggc gag cgc ggc ccg cag 1394 Ala Gly
Ala Gln His His Gln Leu Asn Gly Gly Glu Arg Gly Pro Gln 370 375 380
acc gcc aag gag cgg gcc aag gag tgg tcg ctg tgt ggc ccc cac cct
1442 Thr Ala Lys Glu Arg Ala Lys Glu Trp Ser Leu Cys Gly Pro His
Pro 385 390 395 ggc cag gag gaa ggg cgg ggg ccg gcc gcg ggc agt ggc
acc cgc cag 1490 Gly Gln Glu Glu Gly Arg Gly Pro Ala Ala Gly Ser
Gly Thr Arg Gln 400 405 410 415 gtg ttc tcc atg gcg gcc ttg agt aag
gag ggg gga tca gcc tct tcg 1538 Val Phe Ser Met Ala Ala Leu Ser
Lys Glu Gly Gly Ser Ala Ser Ser 420 425 430 acc acc ggg cct gac tcc
ccg tcc ccg gtg cct ttg ccc ccc ggg aag 1586 Thr Thr Gly Pro Asp
Ser Pro Ser Pro Val Pro Leu Pro Pro Gly Lys 435 440 445 cca gcc ctc
cca gga gcc gat ggg acc ccc ttt ggc tgc cct gcc ggg 1634 Pro Ala
Leu Pro Gly Ala Asp Gly Thr Pro Phe Gly Cys Pro Ala Gly 450 455 460
cgc aaa gag aag ccg gca gac ccc gtg gag tgg aca gtc atg gac gtc
1682 Arg Lys Glu Lys Pro Ala Asp Pro Val Glu Trp Thr Val Met Asp
Val 465 470 475 gtg gag tac ttc acc gag gcg ggc ttc cct gag caa gcc
acg gct ttc 1730 Val Glu Tyr Phe Thr Glu Ala Gly Phe Pro Glu Gln
Ala Thr Ala Phe 480 485 490 495 cag gag cag gag atc gac ggc aag tcc
ctg ctg ctc atg cag cgc acc 1778 Gln Glu Gln Glu Ile Asp Gly Lys
Ser Leu Leu Leu Met Gln Arg Thr 500 505 510 gat gtc ctc acc ggc ctg
tcc atc cgc ctg ggg cca gcg ttg aaa atc 1826 Asp Val Leu Thr Gly
Leu Ser Ile Arg Leu Gly Pro Ala Leu Lys Ile 515 520 525 tat gag cac
cat atc aag gtg ctg cag cag ggt cac ttc gag gac gat 1874 Tyr Glu
His His Ile Lys Val Leu Gln Gln Gly His Phe Glu Asp Asp 530 535 540
gac ccg gaa ggc ttc ctg gga tgagcacaga gccgccgcgc cccttgtccc 1925
Asp Pro Glu Gly Phe Leu Gly 545 550 cacccccacc ccgcctggac
ccattcctgc ctccatgtca cccaaggtgt cccagaggcc 1985 aggagctgga
ctgggcaggc gaggggtgcg gacctaccct gattctggta gggggcgggg 2045
ccttgctgtg ctcattgcta cccccccacc ccgtgtgtgt ctctgcacct gcccccagca
2105 cacccctccc ggagcctgga tgtcgcctgg gactctggcc tgctcatttt
gcccccagat 2165 cagccccctc cctccctcct gtcccaggac attttttaaa
agaaaaaaag gaaaaaaaaa 2225 aattggggag ggggctggga aggtgcccca
agatcctcct cggcccaacc aggtgtttat 2285 tcctatatat atatatatat
gttttgttct gcctgttttt cgttttttgg tgcgtggcct 2345 ttcttccctc
ccaccaccac tcatggcccc agccctgctc gccctgtcgg cgggagcagc 2405
tgggaatggg aggagggtgg gaccttgggt ctgtctccca ccctctctcc cgttggttct
2465 gttgtcgctc cagctggctg tattgctttt taatattgca ccgaagggtt
gttttttttt 2525 ttttaaataa aattttaaaa aaaggaaaaa aaaaaa 2561 49
12619 DNA Homo sapiens 49 aagctttata aagatttaac tacctaataa
ggtagagaag taatttatgt gcccactaaa 60 aaatactcaa tttctgaatg
ttcgtccaaa attaacttgt cagatcatta aatcattgac 120 tagaaacacg
ttgagtacct attatgtact aggcacttag atcattgtga gacaataaaa 180
aatactgcat tagaaaagga catttttcac atcttaaatg caataagcat tatttggctg
240 gcagttaatt acatttaaca cattaaacat atagagcaaa attctgagca
atcaaaataa 300 ttataccctt gagcaatcga ttatttaaat ttctttcact
attcccttaa gctgatttct 360 actctgggat tctttcatag ttctcaaata
agaaaataaa aaatttccta aataaggcaa 420 tacaaaagaa tagaaatgta
agagaagaga tatattagct cttgaatccc tgtttccatt 480 tgctgtcaat
agtgcctcta atgttcgatt ttctcttcaa agaaaaatct tgatttaaaa 540
ggaagaaaaa gtacaatcac ctttaacagc taaagtatac tgattagcat ctactaaagt
600 tagcaaagac tgaaactgaa aaaaaattgt aaaatcttta ttctaagtta
tataacgcca 660 ttcaccatag taatgatttt atactttggt atatggcttt
ttaaaataaa tattgccaac 720 aggtaaaaat ttttcctttg ctgtcttaag
gcattcctaa gagaattttt accagtgtgt 780 gttcataact tgaatgttaa
tttaaacaat gttacttcta tcacctaaat gatatactta 840 tagaagagtg
gtttaattgg gaacagaaaa acaccacatt gcttcttccc aagaaaaagg 900
gatgtattcc attctcgagg tctctctccc actctctatt tatatataat atactgcata
960 gataaatata cacacattat atatgtattt ttttgaactt aaagaagact
ggacatatgt 1020 atttacatgt atatatccaa caaatattta attttgagat
ctctctccct cttctgattt 1080 attattctca gtatgaattc tcaaactgta
cggtctttca catttcattc attcatcaag 1140 catgtatcga gtcccttctg
catgcttagc tttttgtcat atggaaggaa gatacaaaag 1200 aaaaactgtt
tctgcccttc agaatctttc catctcttct aggaaggaga taaaacacca 1260
tatatcatta agaaatttat aagactagtc ccaaaaccaa tggtacaagc aacatgcatt
1320 ttacatttat gtagaatttt agagcttgga aacactttcg tgatatataa
tcctaagaac 1380 aatcttgtaa agtgcacatt attagctcca tttcagtgat
gaggaatctg agacagaatt 1440 ttaagtgaca tgtctcgttc aaacattatg
agtggaagag tcaacactta agcctgagtt 1500 ttctgattct aagcctagtg
ctcttttcaa cacagcactg gaaaccaaag attgtggtac 1560 acaacaaggc
aacagccagt cttcttgctc gaggtccaac taaactggac ccataccgag 1620
cagtgtccag ccaaatgtcc aaattaattt tatcctgcaa atatttgttc ttcagtgtaa
1680 tacacacagc acaactacca tttccttcgt cttagtgcct ttatctccta
cattccagaa 1740 atggggatgt caaatatttt tttaaatctg gcctagatgg
aatcatataa atctcaaatc 1800 ataatataaa tcttaaaggt ctggtttcca
ccaatccttc cacattttgt tttcccccag 1860 cactagagag cctaacctac
cctcacccct ttcgagcatt cttgctccaa acgaccacct 1920 attttaagat
gtcaatgacc ctttcccaaa ttctacaaat tcaccccagt tttgccaccc 1980
gaccccagcg cctgcccgga cacgttcccc tccctcccaa tagatttgat accgagttca
2040 ggttctgcag atcccgttgc gatgctgtca cacagcactg acagataaga
tttgaccttt 2100 cgactccgtc cttggggact tcccgctggc caagaagggt
agttccaatc ccaggaaacg 2160 ggcttcctgc tcaggaacgc agcctctagc
agcgcacagt ctgaggcaat gtctccggca 2220 attagaacga tgctgggcgc
ccgggtgtgc atcactctgc ctcatactcc taccaactgc 2280 agggcactcg
gtccggcagc cagtccatcc cacccacacc caagtcccag ccagccggac 2340
cttacgcagg accccgatga taggtcgttg acggctgcag caaaagccaa ggccacctgc
2400 cgctgctgcc catccccgcc aatctgagac cccctagact ggaccgcaga
aaagcgtttc 2460 tatgggaacc cccccaccga gaatcacgtg acgcaatcgg
acgaccaatc gcttcttacc 2520 tctgcccgcg gtccagcttt tggccctccc
tctcgccccc gcctccttcg cccagccccg 2580 ccccttgcct gcggagagcc
cgcgcctgcg cgctgtgtcc tgcgcgctcc ttccctcgcg 2640 cgcgctctcc
gtggaagagc aggggcagcg tgggaggcgc caagggagcg cgaacctgag 2700
gaggaagaaa cggggctagc gcgcaggccc agaacggtcc gagccgcggc agtcggcgac
2760 gcctcagagc ggaagaggga agtgaatcag gcgccgggta gtgggttgct
gggctgggct 2820 tgctgaggta gaggcagcgc caagaagagg cctttgccgc
tggtcgggat tgggatgtcg 2880 aagaacacag tgtcgtcggc ccgcttccgg
aaggtggacg tggatgaata tgacgagaac 2940 aagttcgtgg acgaagaaga
tgggggcgac ggccaggccg ggcccgacga gggcgaggtg 3000 gactcctgcc
tgcggcaata tccttgcatt caccgccctc cccaccccag cccagcccag 3060
cccgcccttc tcctgggacc cgggagcctg caggatccgc ggggcaccgg cgcggagctg
3120 cctctcaacc tgcggcttaa cctgtctctt tgggatcgcc cgctctgaga
gggcaagggg 3180 gaagcccccg tttcctaccc agtcggcagg agacgcgagg
gtcccactct tggaagcctg 3240 ccctaccccg cgcgccttcc acgcccccag
attcctcagg ttgcacccga gtgcctgcct 3300 gcctcgggaa ctggtcccgc
cgcccgcgcc ctcgcggcgc tggggaaggc ggccccggct 3360 ggtggggaag
gctggtgccg accgccttag tttttcttcc tagaactctg atttcctggg 3420
gtcacattag ctccagaaat ttctgattgt ggggaacctg catctttcct tagtggtttt
3480 gttttttggt tgtgtttttg ttattggtag cgttaaggta gtttattgct
taccgggggg 3540 ccgggggaga tgggactgtt cgaaaattga gggtccctgt
gctttcagcc cattggcctt 3600 tttaaaaaaa aaaaaaaaag aagaagaaga
aggggatttg gcaaaatata cattgtacag 3660 aatttgttaa ctgggggagg
ggaatgaata caaaaaatac aaaactccta gaaggaagct 3720 tggagccttt
tacctgctaa gaaaaggaca atagaaaaaa caacggggaa tgcgtgtgga 3780
gaatccttgg aaatatttaa aataaacccc aatgaataag atagaagatg agtcattcgt
3840 ataaagcaga atcatttttg taatcctaaa attgtttcca ttttagttaa
aatatggcag 3900 tcagttcccg gtttctgttt ttgcatattt gaatattcat
aactttggct tcgcatttgc 3960 attacatctt ttttagaaaa atgtaaatgt
tgcaaaaaaa ccgaagctgt agttttagaa 4020 aatctcagac actgaatttg
tatgcatttc taattcttgg gtgtattcat aaggaagact 4080 ctcaacaatg
tcctgttata gtggggaaat atgagagtga aaatatttaa tggcaacaat 4140
atcctttttt aaaggcacct aaatagagca ttagacattt atcaatatat agatagtgct
4200 ttgcccaact ttcacaatta attagctgtt gctcttttgc attatttaaa
tacttaagtg 4260 cttggagtta taaaaaatga gctaatctac atcaggcatg
cttctctaga aatccctgca 4320 gccttgaaaa taacagcttg tcaaccagag
attttgtgta agaacttttt ctttagaaaa 4380 taaatggtga acatgcttcc
taaaaacatt atttgtgatg ggataagatg gtgttttatg 4440 aaaccccagt
gtattttagg taatttgtgg tgacttttaa aaggtactgc tgtatccata 4500
tcagtggatc tgctttttga tcagttcatc ttaaaatata aagatactgt ctcttcttac
4560 cgttacatac agccaggaaa gacagcccta gtggtggggt actagagttg
gaggaacaag 4620 tgaactctgt ggttttcctt ttaggggaat gtttgtacat
tctgacagtc tgattggcct 4680 tctgtttctc atgcttgcta actcactagt
gctttcaaag agagcctgaa tttaataggt 4740 atggtctaac acagtttgaa
taacctttgt gaaatatgag agaaaatatc taaagcaaaa 4800 aattaagctg
ccacctaagg gacatatgaa ttattacatc ttctgtgatg cctcttttca 4860
tcaatattga gagattgcta atgtgtatca ttcagattgc taatctgcca gcatgttcta
4920 ccagcatttc agataataca gaatatggtt ctagcaaaag tttggtcttt
attttttcaa 4980 ttagaatcac aggaaaagac atattttggt tgataatagg
ttatttcatt tgggggacta 5040 ataattctga tatatatttt aggatttctt
taacaccact ctaggtaatg tttgcatatg 5100 tatctcactg ggaaatgaaa
gactatcaag gtgttcactt gatagttaga accaagggtg 5160 aaacagtctt
tgctttatta aaaaaaagtc taatgttcta ttttgctttt gatattttgc 5220
ctttgattaa catcctggaa accaacacat tgaatttcca gtattgaaca tagtgaccaa
5280 agtaattttc tttttatatg taaatcaagt cataaagaac cagtggttat
aatgctttct 5340 gggggccatc ctttgctgtt acacccttaa cttccatcac
aggaaacatg acagctgccc 5400 tacaggcagc tctgaagaac ccccctatca
acaccaagag tcaggcagtg aaggtgagtc 5460 gcagactaca acacagtgat
ctctgctgat atcttattct tagtaaaatc cttgcagtgc 5520 aaaaaaaaat
caatatttta actgtttgct atctttgaca agaagagttt ataatgtagt 5580
ttgataggta aaaatttcac gtgaaaaaat agccctataa tgtagttatg ataatgctgc
5640 atggtaagat acagtaagtt caaacgatag tgaaatcatt tgtgtgtgtt
tttagaggag 5700 accactcagg ctgaatttga gcaaaggttt gaaaaataag
ttaaaccttt acaaaaataa 5760 acagattgta attgcttttt aaagattttt
taaaaccata caaatactaa atacttatta 5820 tagaaagctc agacatatga
gaaggttaaa aagatagtgg tttgtggtcc cagcacccag 5880 agataacagt
tactactttg gggccttgct gtattgttac agagttccct tttgtttttt 5940
taagaatgaa tttttaaaac gggctttttc agctatatgc aatggtacat gagctttcct
6000 tccccaataa gttaatagcc ttttttaaca cttgtatatg gataagctcc
agtgtataca 6060 taactaatct tttgtttata tttagactga cttttttttt
cctattgtaa accactgaaa 6120 tcaatatttt ttggtaaatt tttaattgtt
ctctttgagt aaattgctag cagtgaatta 6180 ctggatcaaa gaatgcactt
ttttttaagg cttttggtat gcagtattgc caaattgccc 6240 ttcagaacag
ttgtgcaact tacattctct gcagtctttt actaattctt aacctattta 6300
cgtatttatt taaaatgatg cccatagcat caaccccgtt gtccatagct attcatacat
6360 cctaggagct tcaagaatct caattgaata gtagtaagta ataacttagg
taaatgcata 6420 ataattatct aggtaacata attttttatt ggggaaaatt
tctttggttt ttacaagttg 6480 taaagattgt cgttgaaatt tcatttttac
cgtggatgca aagatatttt tctaaatctg 6540 gtaattgcag tctttaaacc
aaagataaca gtaggtggta gaaacattct gtgaaatcct 6600 gaccagtagg
aatgctggag gtatcacttt gtgttgaatg gaaggagaaa cgaattgttg 6660
aaaaggtcag ttaagtgttt cctttgcttg gccggatggg taagaaaata actgcttttg
6720 aagcaggctt ttgccaaaga aaaaagatca ttattaatga acatcactat
atttcatatc 6780 tacagtcaat tcatataaat tacagtcaat tttcttttaa
gacagcttgg tttattaaaa 6840 tttttaaata aaaaagtttt taagaaaaaa
ttacttctga aggataattc aaggtgaaac 6900 tgcaaatctg cctccttgtt
ttgttgggaa tttttttttt tttttttttt ttttgagacg 6960 gagtctcact
ctatcaccca ggttggagtg cagtggtgca atctcaactc actgcaccct 7020
ccgcctcccg ggtttaagca atcctcctgc ttcagcctcc cgagtagctg ggatcacagg
7080 cacacaccac catgcctgga taatttctgt atttttagaa gaaaacaggg
ttttaccatt 7140 ttggccaggc tggtctcgaa ctcctgacct caggtgatct
gcccatctcg gcctcccaaa 7200 gtgctgggat tacagctgtg ggccaccaca
cccggccgtt ttgttgggat tttttttttt 7260 taagatcaag acataaattt
aaatgttgtt ttaataaatt gttaaattat cacattgatc 7320 tgttagcaaa
tcctctcagc tctgccttca attatgttaa tagtctgtca agtttcttac 7380
cacctccact gctactatgc ttaccacatc cagcctgtat tattgcaatt gcctcctaat
7440 tgctctccct gcttctacct tatcccctac tcccacagct tattttctgt
aacatagatg 7500 ccaaagcaat cctgttaaaa tgtgagtcag attatggcac
tgctcttaaa accttccaat 7560 gtcttctcat ttctctcagt aaaagccaaa
ctccttacaa tgcctgtagg ccttacacga 7620 tctgtcctcc cataacctct
gacttactca cgtgcttttc tcccaccaat ccactccaac 7680 cacattgggt
ttttttctgt tcctggaaca cactgaacac acactaatag cactgttctt 7740
tcctctgtct gaaacacttt cctcagttat cccaagcctt ctttcacgtc cttcaggtcc
7800 ttactcaaat gtcacattca tagtgtagac tttctgaaat tctaaaccct
cctcatacag 7860 atatgtctaa atgttctgtt atttattgac ccaccaggac
cgggcaggca gcattgtctt 7920 gaaggtgctc atctctttta aagctaatga
tatagaaaag gcagttcaat ctctggacaa 7980 gaatggtgtg gatctcctaa
tgaagtatat ttataaagga tttgagagcc cgtctgacaa 8040 tagcagtgct
atgttactgc aatggcatga aaaggtaagt tatgaattat aaatctatat 8100
gactggttct tttacaatag ggaatgacaa tgacaacctc tctcacctaa ataaccattt
8160 tgatttgttg tacatttttg ttattacaaa taaaatgcat gaaaaggata
gttcatattt 8220 atgtttacta gccttggtct taagagattc tgattccaac
acttgtgttt attcaacaat 8280 gattattagt aattaaacat aatcttgaac
tctgaattaa atcaaaactt tgtaaaagaa 8340 aataagcaat acaaatcaag
aattctttca cagtgaccaa aaggtgaaaa caacacaagg 8400 atcgaatatg
attcaaccat taaaaggaat gacattctga cacatgctat aacattaata 8460
aaccttgaaa acataccaag tgaaatgagc caaacacaaa agaactaata ttttataatt
8520 ttacttatat gaaataatct aggataggca aacacaaagg gacagaaagt
ccttagaggt 8580 tactaggaag tagggaaagc aaggaatagg gagttagtgc
ttaataggta cagagttcct 8640 ccttggagtg gtaaaaaagt tttggaaaca
gatagtggtg atggctacag tacattgtga 8700 atataattaa tgccaatgga
ttttacactt aaagatggtt aaaatggcaa attttgtgtt 8760 agatatttta
caactttttt aaagaattag gagtttggag gatcaagaat tcttaaatca 8820
tgtttttcta ttttcatgtg tatattttgc aatgtaagta gatgctggta catcatctgt
8880 caaaagagta taagtgattt tgagctttgg gtaaaaaact ggataacatg
taaatagaac 8940 cagtcataaa aatattgagt gtttgaagtg tatctgagtg
aaaacacaaa cataagaaaa 9000 aagcacatag taaaacaata gttccccctt
ttactctaaa atgcaccaat ttgggtagta 9060 atttatatgg caccctattc
atggaacact ttctgttgcc aggtaccata ctattaatgt 9120 tttatttaac
ctttacaaca accctgtgga agtatataaa tatctttatc atcctcaatt 9180
tacagatgaa aagctagctt taaaacccaa gccagcgtag
ttctagcata gcctcaagat 9240 tgcagtgaac attgattact tattatattc
cacatattct tcaaaggact ttataaatat 9300 taactcattt aatcctcata
aaaatggagg gaaatgcttg ctattattcc tcttttgtca 9360 ctgaggaaac
tgaggcatgt gtgaagtctt catttcttcc aaatgtcagt caccagtttt 9420
taccaatctt cgaagtattt ctgaaatcta tctgttcaag cgtatctaat gcagctgttc
9480 acagcatctc tcccagtctg ttgccatagc ttcctgactg gtttcccagt
taacagtttt 9540 gcctccttca aatctgttct ccacccagcc atcaaaatga
tatctttaaa atcaaaattg 9600 cccttgtcag tcacctgcag ggataaagtc
aaagttccca agtctagctt catcttccat 9660 gtcattcttc ccctcaggct
atagcaatgc cagccttttt cctgaatgca ccatattgtt 9720 tcacacctcc
atacatttgc tcatgatttt ctggtgttag cctgtcacct actcattctt 9780
ttaatgtgtc atttcctcca tgaagcctta gctgaaacat tcctctatac tgttaatctg
9840 ggtataagcc tctccctggt gctttaatag cacctgcagc acaactctca
tttcatacat 9900 tagattaaaa ttacctgttt atatgtctgt ctcctcatgc
tagaccagaa aatgctgtat 9960 ttgttcactt ttgtatcccc agcatctagc
acagtactca gtatacaaag gtattccata 10020 aatatttttt gaacagaaag
aaaccagagc tcagattcct aatacttgat cattactctc 10080 tatttttcaa
attagagtca gagttaaagt ttctaagttc ttagctatta aacaatacct 10140
tctttctttg ggagaaaaaa aatctgacaa aggctgacta atcgaagtgg aagttgggat
10200 ggttgatccc agtttgaatt ttcttctgac tatgtggtga gaatgagaaa
tgcagaatgt 10260 ccacctgttt tgagcaggaa cactatgctg cagatttttt
tttttttttt tttttttttt 10320 ttttgagacg gagtcttgct ctgtcgccca
ggctggagtg cagtggcgca atctcggctc 10380 actgcaagct ccgcctcctg
ggttcacacc attgtcctgc ctcagcctcc cgagtagctg 10440 ggactacagg
cacccgccac cacgcccggc taattttttg tatttttagt agagacgggg 10500
tttcaccatg ttagccagga tggtcttgat ctcctgacct cgtgatccgc cggcctcggc
10560 ctcccaaagt gctgggatta caggcgtgag ccaccgcgcc cggcctatgc
tgcagatttt 10620 ttaaaacatt atttagaatt aatgtactaa aatgtaaact
agtatctcac tagaatgtaa 10680 cttcatgagg gcagggactt tcaaggtttt
gtttattact gtaacctcag tgccaagaac 10740 agtacctggt gcataattgg
tgctcaagaa tttattattt gttaactaat aaattcaggg 10800 tctatagcag
tgcccattcc ttctttaaga aaaatgtttt accaaatatg agaattgacc 10860
ttttattatt ctgtcaacat ttacatcctg gtttgttttt aggcacttgc tgctggagga
10920 gtagggtcca ttgttcgtgt cttgactgca agaaaaactg tgtagtctgg
caggaagtgg 10980 attatctgcc tcgggagtgg gaattgctgg tacaaagacc
aaaacaacca aatgccaccg 11040 ctgccctgtg ggtagcatct gtttctctca
gctttgcctt cttgcttttt catatctgta 11100 aagaaaaaaa ttacatatca
gttgtccttt aatgaaaatt gggataatat agaagaaatt 11160 gtgttaaaat
agaagtgttt catcctttca aaaccatttc agtgatgttt ataccaatct 11220
gtatatagta taatttacat tcaagtttaa ttgtgcaact tttaacccct gttggctggt
11280 tttttgttct gttttgtttt gtattatttt taactaatac tgagagattt
ggtcagaatt 11340 tgaggccagt ttcctagctc attgctagtc agggaaatga
tatttataaa aaatatgaga 11400 gactggcagc tattaacatt gcaaaactgg
accatatttc ccttatttaa taagcaaaat 11460 atgtttttgg aataagtggt
gggtgaatac cactgctaag ttatagcttt gtttttgctt 11520 gcctcctgat
tatctgtact gtgggtttaa gtatgctact ttctctcagc atccaataat 11580
catggcccct caatttattt gtggtcaccc agggttcaga gcaagaagtc ttgctttata
11640 caaatgtatc cataaaatat cagagcttgt tgggcatgaa catcaaactt
ttgttccact 11700 aatatggctc tgtttggaaa aaactgcaaa tcagaaagaa
tgatttgcag aaagaaagaa 11760 aaactatggt gtaatttaaa ctctgggcag
cctctgaatg aaatgctact ttctttagaa 11820 atataatagc tgccttagac
attatgaggt atacaactag tatttaagat accatttaat 11880 atgccccgta
aatgtcttca gtgttcttca gggtagttgg gatctcaaaa gatttggttc 11940
agatccaaac aaatacacat tctgtgtttt agctcagtgt tttctaaaaa aagaaactgc
12000 cacacagcaa aaaattgttt actttgttgg acaaaccaaa tcagttctca
aaaaatgacc 12060 ggtgcttata aaaagttata aatatcgagt agctctaaaa
caaaccacct gaccaagagg 12120 gaagtgagct tgtgcttagt atttacattg
gatgccagtt ttgtaatcac tgacttatgt 12180 gcaaactggt gcagaaattc
tataaactct ttgctgtttt tgatacctgc tttttgtttc 12240 attttgtttt
gttttgtaaa aatgataaaa cttcagaaaa taaaatgtca gtgttgaata 12300
atttattttt ctctgacact ttaacaatta tgaatgtatg gttaattaag aggaaaggtt
12360 ttctgcttct accaccaagt actgtactct taacaagaac agtttggtag
ggtttttata 12420 agactatata gatataagat gatagagaag agagtcatga
atgatgtcag agcactactg 12480 aagcctttgg agtgattcca tagccttctg
gatggcagct gaatacctat atgtagtatc 12540 actgcccaaa gacctagact
agaaagtgca aagtagctta gcagctgcag tcattcactc 12600 ccagcctcca
aaattctct 12619 50 12425 DNA Homo sapiens 50 gatccctctc caggtggaag
ctcccttcat accaaagttt aaaggccctg gggatacgag 60 taactttgac
gactatgagg aagaagaaat ccgggtctcc atcaatgaga agtgtggcaa 120
ggagttttct gagttttagg ggcatgcctg tgcccccatg ggttttcttt tttctttttt
180 cttttttttg gtcggggggg tgggagggtt ggattgaaca gccagagggc
cccagagttc 240 cttgcatcta atttcacccc caccccaccc tccagggtta
gggggagcag gaagcccaga 300 taatcagagg gacagaaaca ccagctgctc
cccctcatcc ccttcaccct cctgccccct 360 ctcccacttt tcccttcctc
tttccccaca gccccccagc ccctcagccc tcccagccca 420 cttctgcctg
ttttaaacga gtttctcaac tccagtcaga ccaggtcttg ctggtgtatc 480
cagggacagg gtatggaaag aggggctcac gcttaactcc agcccccacc cacaccccca
540 tcccacccaa ccacaggccc cacttgctaa gggcaaatga acgaagcgcc
aaccttcctt 600 tcggagtaat cctgcctggg aaggagagat ttttagtgac
atgttcagtg ggttgcttgc 660 tagaattttt ttaaaaaaac aacaatttaa
aatcttattt aagttccacc agtgcctccc 720 tccctccttc ctctactccc
acccctccca tgtcccccca ttcctcaaat ccattttaaa 780 gagaagcaga
ctgactttgg aaagggaggc gctggggttt gaacctcccc gctgctaatc 840
tcccctgggc ccctccccgg ggaatcctct ctgccaatcc tgcgagggtc taggcccctt
900 taggaagcct ccgctctctt tttccccaac agacctgtct tcacccttgg
gctttgaaag 960 ccagacaaag cagctgcccc tctccctgcc aaagaggagt
catcccccaa aaagacagag 1020 ggggagcccc aagcccaagt ctttcctccc
agcagcgttt ccccccaact ccttaatttt 1080 attctccgct agattttaac
gtccagcctt ccctcagctg agtggggagg gcatccctgc 1140 aaaagggaac
agaagaggcc aagtcccccc aagccacggc ccggggttca aggctagagc 1200
tgctggggag gggctgcctg ttttactcac ccaccagctt ccgcctcccc catcctgggc
1260 gcccctcctc cagcttagct gtcagctgtc catcacctct cccccacttt
ctcatttgtg 1320 cttttttctc tcgtaataga aaagtgggga gccgctgggg
agccacccca ttcatccccg 1380 tatttccccc tctcataact tctccccatc
ccaggaggag ttctcaggcc tggggtgggg 1440 ccccgggtgg gtgcgggggc
gattcaacct gtgtgctgcg aaggacgaga cttcctcttg 1500 aacagtgtgc
tgttgtaaac atatttgaaa actattacca ataaagtttt gtttaaaaaa 1560
aaagtgtcgc tggtgttctc gacttcgatc acccacccac acacccccag ggggttggaa
1620 agggaatttc ggaccccagc gtgcaggccg atcaggtcct ggcttgaagt
ccttgtaacc 1680 agggtttagc tgaaattccg gcactccttc ggccccgcag
gagaaacgag cgtcaaactg 1740 ccctttgacc ccagattcgg ggtccccaaa
tctgcggcgc gccccctcgg cgtccagccc 1800 gggaccgaga gggcgctcta
gggaggcgct ggggctggcg cgccaggagg ccgagcggcg 1860 gcgggggcgg
ccctggcagg gggagtagaa gggggagagg gtgcgcgccc cccttcccgc 1920
atcctcagcg ccgggccagg cgcgcctgag ggacgcgggg gcggcggcag caggagggtc
1980 cccgcagcac cctgcgagcg cggcagcccc ggcccgcggg cggcgagttc
ccggtaagtg 2040 cggtcccgag agcggagcgc gctggagagg cgtggagagg
ggggctgggc gccggggacg 2100 tctgggtccc gcgcccaatg gctggagggc
ggccgagcgc cgcccgcccg ccctgcccgc 2160 cccctctccc ctccccccgg
cactcccctc cccctccccc gcccgccgct ttcccccgcc 2220 cccgccccgg
cgccaactcc gcggcgcctc cttaaaaagc gcgcgggagt tgtaaggggg 2280
ggccggagcg agccggagtg agcgagagcg cagggtaaag ggggcgggcg gggggcccgg
2340 gctccacctt aaaagcgggc gcgtgggggt gggagggagg aaggcgggcg
gcggggagga 2400 gggagggagg gaaggaaggg gggccggagt gtcccgggcg
cagggcgcgc gtgcggcggc 2460 ggcggcggcg gggaggggcc ggccgcgccg
cgctcccctc ctccccctcg catccccggc 2520 cccgcgcgcg cccagcagaa
gcgggtctgt gtgtgcgtgc gtgcgagtga gtgagtgtgt 2580 gcatattttt
ttctctcttt tctttctctc tcactgtttt ttcctctctc tctctctccc 2640
tctctctctc tttttttttt tttttttttt gcaaagaaac agcagcgccg ccgccgctcc
2700 gccgaggcgc tgcgcccccc ggggggggag gcggaggagg cgggcagcgg
cggagggagg 2760 ggagccgggg aggggggcgc cgcgctggga gggaggcagc
gcgcacggtg cagccgggcc 2820 gggcgggagg catggcgggg cccccggccc
tacccccgcc ggagacggcg gcggccgcca 2880 ccacggcggc cgccgcctcg
tcgtccgccg cttccccgca ctaccaagag tggatcctgg 2940 acaccatcga
ctcgctgcgc tcgcgcaagg cgcggccgga cctggagcgc atctgccgga 3000
tggtgcggcg gcggcacggc ccggagccgg agcgcacgcg cgccgagctc gagaaactga
3060 tccagcagcg cgccgtgctc cgggtcagct acaaggggag catctcgtac
cgcaacgcgg 3120 cgcgcgtcca gccgccccgg cgcggagcca ccccgccggc
cccgccgcgc gccccccgcg 3180 gggcccccgc cgccgccgcc gccgccgcgc
cgccgcccac gcccgccccg ccgccaccgc 3240 ccgcgcccgt cgccgccgcc
gccccggccc gggcgccccg cgcggccgcc gccgccgcca 3300 cagcgccccc
ctcgcctggc cccgcgcagc cgggcccccg cgcgcagcgg gccgcgcccc 3360
tggccgcgcc gccgcccgcg ccagccgctc ccccggcggt ggcgcccccg gccggcccgc
3420 gccgcgcccc cccgcccgcc gtcgccgccc gggagccgcc gctgccgccg
ccgccacagc 3480 cgccggcgcc gccacagcag cagcagccgc cgccgccgca
gccacagccg ccgccggagg 3540 ggggcgcggt gcgggccggc ggcgcggcgc
ggcccgtgag cctgcgggaa gtcgtgcgct 3600 acctcggggg cagcggcggc
gccggcggtc gcctaacccg cggccgcgtg caggggctgc 3660 tggaggagga
ggcggcggct cgaggccgtc tggagcgcac ccgtctcgga gcgcttgcgc 3720
tgccccgcgg ggacaggccc ggacgggcgc cgccggccgc cagcgcccgc ccgtctcgca
3780 gcaaggtgag cgcgccgggg agcgggggcg ccgcgcggtg ggcaggtgcg
ggcgaagttg 3840 gtggcggggg cgcgagtccc gggaggaact gggtggcggg
tggctggggc tttgcgcgcg 3900 tttcctgcgg gctcggtgcg tggtgacctt
ggcaagtgat tgaatctccc ggagcctcag 3960 tttcctccgc tgtaaacgcg
gtttaataac agtagcgacc ccttggggtt gttgagcgag 4020 tttagtaaga
tttggttgtc gagggcttta gttaacacag agcctggcac ggagtgaatg 4080
cgtaaaagtt agtccgtatt gttcttaaag gtggaatcgg ttcctcctcc ccaccgcccg
4140 gacgccacag tcagggtctg ggattagaac agctactaat tttgcatgct
tctctcctcg 4200 gctccagaga ggtggagaag agcgagtact tgagaaagaa
gaggaagaag atgatgatga 4260 agatgaagat gaagaagatg atgtgtcaga
gggctctgaa gtgcccgaga gtgaccgtcc 4320 tgcaggtgcc cagcaccacc
agcttaacgg cgagcgggga cctcagagtg ccaaggagag 4380 ggtcaaggag
tggaccccct gcggaccgca ccagggccag gatgaagggc gggggccagc 4440
cccgggcagc ggcacccgcc aggtgttctc catggcagcc atgaacaagg aagggggaac
4500 aggtaaggat ccctctgggt ggggaagagt gctaggtgga gaggaactca
gcccgaagac 4560 aaagccaaag acaggtgttt ttttccttcc cagcttctgt
tgccaccggg ccagactccc 4620 cgtcccccgt gcctttgccc ccaggcaaac
cagccctacc tggggccgac gggaccccct 4680 ttggctgtcc gtaagttggg
gtattggaga catgggggtg ctgctcaggt gtgtggtaca 4740 gccagagaga
catccgtgtt cactggtgtc tgtttgtttt gatgcagtcc cgggcgcaaa 4800
gagaagccat ctgatcccgt cgagtggacc gtgatggatg tcgtcgaata ttttactgag
4860 gctggattcc cggagcaggc gacagctttc caagagcagg tgagtttcca
gcccaggact 4920 acacactgac agacacagag ggcctccctg ggatgtgccc
tgatcccggc tttctctgtt 4980 cctgtcccac ccaggaaatt gatggcaaat
ctttgctgct catgcagcgc acagatgtgc 5040 tcaccggcct gtccatccgc
ctcgggccag ccctgaaaat ctacgagcac cacatcaagg 5100 tgcttcagca
aggccacttt gaggatgatg accccgatgg cttcttaggc tgagcgccca 5160
gcctcacccc tgccccagcc cattccggcc cccatctcac ccaagatccc ccagagtcca
5220 ggagctggac ggggacaccc tcagccctca taacagattc caaggagagg
gcaccctctt 5280 gtccttatct ttgccccttg tgtctgtctc acacacatct
gctcctcagc acgtcggtgt 5340 ggggagggga ttgctcctta aaccccaggt
ggctgaccct ccccacccag tccaggacat 5400 tttaggaaaa aaaaaatgaa
atgtgggggg cttctcatct ccccaagatc ctcttccgtt 5460 cagccagatg
tttcctgtat aaatgtttgg atctgcctgt ttattttggt gggtggtctt 5520
tcctccctcc cctaccaccc atgcccccct tctcagtctg cccctggcct ccagccccta
5580 ggggactagc tgggttgggg ttcctcgggc cttttctctc ctcccttttt
ctttctgttg 5640 attgtcgctc cagctggctg tattgctttt taatattgca
ccgaaggttt tttaaataaa 5700 attttaaaaa aagaaaaagg gaaaaaaaag
ccacggagtc cattttatga atggggtggg 5760 gagagggcac taaagagcct
cctaagagag cctcaggtta ggacagaatt gtttggggag 5820 ggagaaaaac
agaaacaatg aattatagct gcctcacagc catgtataac aataattgct 5880
ccaggaaggt gggaatattt gctttttttt cttctgtaat ctcaccgtgt ccgtgtccag
5940 aacagagcta ggcacacagc aggtgctcaa tttttgtttt tcgtttagac
aggtttcatt 6000 ctttcaccca ggctggagtg cagtggtgct atcatagctc
attgtagcct caaactcctg 6060 ggctgaagtg atcctcccac ctcagcctcc
tgagtagctg ggactacagg tgcactctgc 6120 catgccgggc taacttttaa
aaatttttgt ccgggcacag tggctcatgc ctgtaatccc 6180 agcactttgg
gaggccgagg tgggtggatc atgaggtcag gagttcaaga tcagcctggc 6240
caagatgatg aaaccctgtc tctactaaaa atataaaaaa aaattagctg ggcgtggtgg
6300 tgggtgcctg taatcctagc tattcaggag gctgaggcag aggattgctt
acacctggga 6360 ggcggagggt gcagtgagcc aagatcgtgc cactgcactc
cagcctgggt gacaaagtga 6420 gactctgtct caaaaaaaaa tctttgtgtg
tgtgtggaga tgagggtatg cactttgttg 6480 gccaggttgg cctcgaactc
ccagccaagc aattctgcct gggattacaa gcgtgagcca 6540 ccatgcctgg
cctcaaatat tgttgaatgg ctagcagtta agtccttggg tttataagca 6600
tttcctcaac tgtcctccca agtccccata agacaaaaaa ctcataaaat cccaccttac
6660 agaagaggca gctggcccgg cacagagatg ctgtctgccc cgggtcacac
agggtggcat 6720 ctgacaccct gtctgagttc ttcactcaga gtctttaaat
ataattagcg tatttgacat 6780 aatgtacatt aaaaactata aacctgtcag
cctttgtcta ctgcaaagaa tccactacaa 6840 atattggggc agggatctgt
tcttggacca tagtagtgtc tccagacctc atggtcctct 6900 tcattaaaac
aacagaaaat tccttctggg ccatcagatg agaccatgag atagaagatt 6960
tccaagtgaa gattttgttt caagacagag tcttgctctg tcactcaggc tagagtgtac
7020 tggtgcaatc ataactgtgg tgacagcctc gaacttttgg gtacaagtga
ttctcatgcc 7080 tcagacaaca cccaactaat attttggttt ttgtatagac
agggtcttgc tatgtggctt 7140 aggctggtct tgaactcctg gcctcaagca
gtcctcccgc ttcagcctcc taaagtgtca 7200 ggattacaga catgagccac
caagtccagc ctgaagattt ttaaaaatta ttgttagtag 7260 tagtcgccag
agttactaca tccaaagtcc ctactaagtt ctaagtagtc cctactaagt 7320
tctaaggcag tttctcaact cattagagtt gttttttgtt tttaaagaaa aaaagaggct
7380 gggcacttta ggagaccgac acgggaggat cgcttgagtc caggagtttg
agaccaacct 7440 gggcaacatg ggcccccatc tctaaaaatt ttaaattaaa
aaaatgtttt aacaacaaaa 7500 agcgttctgg gagtgagggg ctggggcctg
ggcggcctca ttccatatac ctgtgccggg 7560 ttgaggggtt ggagacacgt
ttagagaccc ctccactcta ggaatccacc tcgagagata 7620 aaggtcccgg
ccctagccac acccccagga cacggccaga ggccacctcc ctaggcgggt 7680
ccctccccac cgccaggttc ctggagcgcg tgcggcgcgt gtgcaggggt agggggccgc
7740 aggcgcgcgg actggagagg cgcgcccctc ccgcgtgttg aaattcaaaa
gaggcgaacg 7800 gcccccggcg cggcggcgcg gctccggtgg agaggtcaag
gcaggggcca gtcggaggct 7860 cccggggcgg ggtcgaaccc gcggccaacc
tgagcagcag cggaagctta aagagctcag 7920 gttcccgccc cccggcccta
ccatggctac agagcagtgg ttcgaggggt cgctccccct 7980 ggaccctgga
gaaacaccgc ctccagacgc cttggaacct gggacgccgc cctgcggaga 8040
cccctccagg tcgacgcccc ctggcaggcc tgggaaccca tctgagccgg atcctgaaga
8100 tgccgagggg cggctggctg aggcccgggc ctccacgtct tcccccaaac
ctctggtccc 8160 ccggcctggg ccagcacctc cccgcctatc cctggacact
ttgttcagcc ccatcaccca 8220 acagctgcgc tacctactga agaaggcaga
tgatttccag agctacttgc tctacaggtg 8280 atgctggaca gggtcccagg
tccccatggg taaggagact tggaggggag gcgacaggat 8340 gggtgacaca
caccagggtc gcaaaattac aagcgctagg agccagaggg agacagtgga 8400
agaagctagc atattagaat ccagtttaag agaatgagga agactgtaga attgcgggta
8460 ggggatggct gctattactg tcgtggcagg gtgggcctgg ggttgtcaag
tctctaggac 8520 tttttctccc agtttttaag tgctgtctta cattttgagc
cctgtgctgg ctaaacaaga 8580 cccacctgag ccaaacttgg cctgcaggac
atcagtttga gactccaaag gataatgtga 8640 ttcccagacc aggtttccct
gtgactctca atttcagtgt ccattggaat ttcctaggag 8700 gctgggttgg
gtttgtttgc gtgtttgttt ttgagatgga gtctcactct gtcgcccagg 8760
ctggagtgca gtggtgcaat ctcagctcac tgcaacctcc gcctcccgga ttgaagcaat
8820 tctctgcctc agcctcccga gtagctggga ttacaggcgc ccaccaacat
gtgttgcccg 8880 gctaattttt ttcttttctt agtagagaca gagtttcacc
atcttggcca gactggtctt 8940 gagctcctga cctcatgatc cacccgcctt
ggcctcccaa agtgctggaa ttacagacgt 9000 gagccaccgc gcctacccga
ggctgggttt ttttgttttg ttttgttgtt atgtgttttt 9060 ttgaaatgga
gtcttgctct gtcacctagg ctggagtgca gtggggcgaa ctcagctcac 9120
tgcaacctcc gcctcccagg ttcgagggat tctcatgagg ctgttttttt ttttttaatg
9180 agacagggtc tcgctctgtc acccaagctg gagtgcaagt ggggcagtca
tagctcactg 9240 caccctcgaa ctcctggtct caagcaatct tccacctccc
ctcctgggta actgggacta 9300 caggtgccac catgcccagc taattatttt
tgtgtagaga tgggttcttg ctatgttgcc 9360 taggcttgtc tggaactcct
ggcctcaagc aatcctccag cctcagcctc ccaaaactct 9420 aggattgcag
gcgtgagcca ctgtgcccag accctgcagg aagctctggg tcctaagtgt 9480
tgtgacactc aggtgtcagc actttaacaa gtgttccaaa tgggtttgat gcaggtaaac
9540 cagaaagatg ttcagaaaag acctgaaact gggggctttt ctaatgggtc
aaagccaggg 9600 atacaggttg ggattgagta gaatggggaa aactgcgggg
tggggagggg ttgtgaggga 9660 ttccaggcaa aggccccctt cttccttcag
cagagaccaa gtacagaagg agcagctggc 9720 caaggccatg cccaccttct
tacagatgtg tgagccctac ttcctgtacc tggaggcagc 9780 cgcgagaagc
atacccccca tctatggacc cctgcaggag ctggtccgaa agggggtgtg 9840
tggaggtttc ttagacccca cgcccctttc ttctcgcagc tctgagcctg tggggatggt
9900 ggagggggag gcccactcct cgcaggccag ctgatctcac tgtacccccc
tcttgtatgc 9960 agctgttaga gatctcccaa cagctgaccc tgcgcctgga
acagctggtc ctcatgtacg 10020 cttcctttgg gttcgtggac ctggaggaga
tgaaccccct taggtaaaat ggtaggagac 10080 tcagatgggg ggatgaagga
gtccaaggcc cagcctcacc cctccattct ctcatgtctc 10140 gccagcatct
cctgtttctt ttgcgggagg ttctccatca gcctgtccca tgaggtctcc 10200
atcttcagat actgtgcccc aaccgcctac actgccagcc gcttcccccg ctacctctat
10260 aagaagatgc gctggcacct ggaagccacc ccagaggccc ctggtcgggg
acaagattcc 10320 cttgtggatt agtaagtcct cttacccaaa tcaaagtcct
cccctttcta tgatgaatgc 10380 caatatgacc ctccaaaccg tcaccagcaa
agtgaaaagt gagccagggc ccgaggcagt 10440 ggctcacgcc tgtaatccca
acactttggg aggccgaggc aggaggatca cttgagctca 10500 agagtttgag
atcagcctgg gcaagatggc aagaccctgt ctcaacaaca aagaaattcg 10560
ccaggcgtga tggctggcac ctgtagtccc agctacttgg gaggcttagg caggaggagc
10620 acttgagccc aggaatcaag gctacggtga gctgtgattg tgccactgca
ctccaccctg 10680 agtggaagca ataatctgtc tcttaaaaaa aaaaaaaagt
gaaccaggaa actaaaggct 10740 tttgaaaggc tacctctatt ttcttaaaac
ccaccctccc accaaaataa aagttctcat 10800 cttaaaagta ggctggcagg
gagaaaaggc cttggagtca cattcctacc tgagaacttc 10860 agggcaactt
ctgatgagtt cccacctcaa ctccaaaatt aaagccctca acagaagtag 10920
ctaggaagct gatcacttct aattacagct ccctcccctc ctagctactt tctgtgctat
10980 cgagatactt gggaagacac aggccagagt ccagccaatt cgtgcccaca
gatccagaag 11040 ctgtggtcca tcggccgatg ggtgccccta ggaccagccg
aggatgacct ttattcatgg 11100 taggagctag ggcaatagca acgtgggcct
gggagctgga gggggaggca gaaccccacc 11160 aaagacaatc caccttccca
aacactttgc ttcccttagt agtgatagca ttttattgtg 11220 ccctgaaaag
cacttcatgc agaccccagt aacaacccat ggagatctat gctattggcc 11280
ccatttaaca aagaaaacag ggtgctcaga gaagttgtta cctgcccaag gacacacagc
11340 tagcagagcg aatggacagg tcaggaccag ttattcagcc tctaggagcc
attactaagt 11400 ctctgatcaa caaggaaaca agtttccccc gggggttttt
cccacccgca gctgaaacaa 11460 agcctctttc acctgagcct ctcactcaaa
gggagggact cccgaggggc agggggcact 11520 caagtccagg cctgtctatc
cctggccccc ccaccccagg attttgtgcc cgcaccgctt 11580 ggggactacc
agcagctgct gaccatcggc ttcgaggagc ccacgcccac gctggccacc 11640
gacctgctgg tgcagatcct cacgggccag gcaggccagg cccggcctcc gagcgcagcc
11700 gggcctgcgg ggtgggcagc gcaggggtct tgaacctggg gaagagggta
ggagctggaa 11760 cttgacagtt ccaaactcca gaataggggg caggggaggg
gctcactcgt tctcgcagtg 11820 cagccgggcc tcgccttcca aagggccagg
ccgagctgac ctgtctgcac cgagtccggc 11880 ttggccgtgg ggccctgaat
gcggacacgt cagttttgtg ttaaataaaa gaaagaaaga 11940 ggtcacaggc
tcagcgtccg ctgcgaatgc cgcgcccctc ccccggggga ttgccccacc 12000
cactcgcgtg gccttctggg aaatgtagtc ttttgaaaga agcctggaat tcgccaatag
12060 gcggacgaga gtttggcgca tgcgcatagg cgcacatgaa gcaaaaaggg
aagtggtgcc 12120 cgtcaacacc ggaacccaga aaactgcaag tttagggtac
cggggaaatt caacgtccac 12180 tggaggaaga gacttaaggc tacgcccact
cccatatttt gacccggaag ttatttattt 12240 tagcgtagaa gactactttt
cccgacgcgc cccaggaaag tgccctcgat cagtttccta 12300 agggcccgag
ttagactttt tttttctctt ccagcttttg ggacttgggg gccggacagg 12360
tcgtcgtctt tcttggggta tccggggtgc ggacaaggtg ggagagccct acggtatcca
12420 agctt 12425 51 22255 DNA Homo sapiens 51 caacatgctt
gggaccagaa gtgtttccaa tttgggattt tctcaaattt taccggttga 60
gcttccccaa tctgaaaatc tgaaatccaa catgcacggc tctgaagtct ttcactgagc
120 ctttggggga aatatttaac atcctaacag ccctaaacca acgctcaatt
agcacaacag 180 tttacaatct tctctaccca cagcctgatg cgaggctctg
ggactagact atttagccaa 240 cagttcttgc aaaattaact gacttataag
taaatagtaa tttcaacacc tcactgctaa 300 tgctgtaaca actctgcaga
cctagggagc aagtacggtt tgcagagcac tgggaaggct 360 ctgaagtgac
ctttgaactg ggcctcaaaa aattttgggt ttggcaaaag tcaaatctct 420
taggcttcaa attccaggca caaggattgt tgggtttgat ttcattatcc agaagcaatg
480 gggatacaga attgtgatct catgtgtagg gaactgtggg ggttttttct
actttaaccc 540 cagtgagact ttgtagagtg tggggtagag aaaaggctca
tgaatatgcc tgaagcctaa 600 ctcagcacct ttctgaggaa ctgactgcca
aaatggtaat ggagagggga aaatatgacc 660 tactttcaca agttaccttg
actgcctcag ggaaacctgc tgtggtagtg tttcttctgg 720 gtgaaagacc
aggtaattac ctgggtgctg gtctcagact taccagtttt gaatccctgt 780
tttaaccact cactatcgat atgaccttgg ataagttacc taacctttct cttactgtcc
840 ttttccgtaa aatggggata acagatagta gttatttcta tgagtggtta
tgagaaccaa 900 gctattagat agcgggaaag cacacagtaa gcgttcaagg
aactgctatt gttattaaaa 960 gcctcctttg gaagaaggac attgaggccc
agagagagaa cagaacgtcc agccacacag 1020 caaatccgtg atgaagttgg
gactggagta tgggtctcct gagtctcagc ccaggactct 1080 atccctcttc
ccgagtcctc ggagttcccg gatggagtca catttgttca cggccaggga 1140
ggaaggtttg atggaggcct gcaggaaaca acagccaggc gcaaggcttt gggagttgaa
1200 gcatagcttc tgcgagatag aaacaaggtt gacatgggca ctcgtgcaga
atgacgggct 1260 ccttttggac tcccaggact acagtccctt atgcaccttg
ggatctgcgg ctagcccctg 1320 cgtaaagagg gacgcgtagt cttttccctg
ccccgccctg ccggggcgcc cgcctccgag 1380 gccgccctcg cttcgtcctt
cccagcaagc tccgcgccgg cgccggctat tgattggctg 1440 aggcgggagc
aggcggctgg ccggcagcag ttactcgggg tttccggtgc gaggccagag 1500
gtggggaagc catcggacgt cggcggtgag gtacgtgcag cggcggccgg tgggcgagac
1560 tatttgagag tgtgcgggcc gggatgttct cggcctgtgg ggaaatcacg
ccaactcccc 1620 gcgtgggccg ggggctgtct ggggatatgc gcatgcgcgg
gcgtgcctcg cggcttgagg 1680 gcgcgcgggg cgtgggtggc tgcgcgcgcg
gggggcgcac gtggggcctg aggggcgggg 1740 gcggtgccgg gagtcccgcc
acgtcagtct ccggccctga gccaatcccg cgcccggcct 1800 gccgcgaggg
ggccggttgt gccgggaagt ggctccaggg agaagaggcc tcttccctca 1860
cccgctgtgg gagctgcgcc ccgaaagcct gccccggcac gtcgggctct cctgacccgc
1920 caagaccaga gagccgttgg cgccctccgc ccgggcctgc cggtccgttt
attttaagaa 1980 gctttgtgcg cctgctgtgg ggatttctga tccaggctgc
gaagaatttc gaagtctgga 2040 aaatagcaac tgtgtttgtt tctaaaggat
cttctcctga cccagcatcg ctcatcacaa 2100 tgaagaacca agacaaaaag
aacggggctg ccaaacaatc caatccaaaa agcagcccag 2160 gacaaccgga
agcaggaccc gagggagccc aggagcggcc cagccaggcg gctcctgcag 2220
tagaagcaga aggtcccggc agcagccagg ctcctcggaa gccggagggt gtgtgccagc
2280 tctgcgttgc cagcgggcag ggggaggagc tgtggggtcg gcctcgcttc
tggacttaca 2340 ggccgaggcc aggttgtccg ggaggaggag atgtagaatg
agaggacagt gctgggggcc 2400 gcggtccccc ctgcgctctg gcgagttggc
ggagctgccc cctctaagca caggaacaga 2460 gttctggaga gaagctccga
cgggattaag tcaggtggca gccaaacgag gcacccagtc 2520 aggaaatcca
ggtcccgtta gaaacacctc agccaccagc agctaactgc ccttcctgtt 2580
tgaggcattt ctagaatgat ctgaatggca agaaatgggt tttgtggggg ggaaggagat
2640 ggactagaag ttgctccgtg ccatccctgt gtgctgatgc tttacatact
tttatgatct 2700 aacaaatatg ttcgggtggt agtgagaaat agttgtgtca
ttttacaagt aaacagactt 2760 aaagaagtta ggcaacgatt actataattt
cttgatttaa aagatgtttc gaatctaaat 2820 tctgacagga actagatttg
ctgaatgata ctccattctt gcttctcagt ttccataaaa 2880 aaaaaagtta
ggcaacattt aactcaaact gatgagtttg gctgggcctg aaaaatccca 2940
accagtggta taatcgtctt ctttctcact ctacccctca tcctctcctg ctgtaggggc
3000 tcaagccaga acggctcagt ctggggccct tcgtgatgtc tctgaggagc
tgagccgcca 3060 actggaagac atactgagca catactgtgt ggacaataac
caggggggcc ccggcgagga 3120 tggggcacag ggtgagccgg ctgaacccga
agatgcagag aagtcccgga cctatgtggc 3180 aaggaatggg gagcctgaac
caactccagt agtcaatgga gagaaggaac cctccaaggg 3240 ggatccaaac
acagaagaga tccggcagag tgacgaggtc ggagaccgag accatcgaag 3300
gccacaggag aagaaaaaag ccaagggttt gggtgagcag agggcggctc tttgtgaagc
3360 tggtgaggag agggagtttg gacttgacgt tctctgggcc agtctgttct
gccaggattc 3420 aaaggaaaac ggtacttctc agagcagcaa gtcactctag
tctaatcaaa gccagggatg 3480 tgggggccac ggcatagaga gatgcaggag
ttaccagcac aaagccttct gggttttgga 3540 gcaactggag cttggcatgg
gacctgttct ctctttgaga aaatggagac gggaggctag 3600 ggtaggctcc
tgtgccagcc agtactacct gctgtgtgac cttgggtgtg tcccttctcc 3660
tctctgggtc ttagtttata tttctcttta cagtaagaaa attagactag gccagagttg
3720 aaaacccaaa tatctgcata agctgggctt ggccatgggg ccacctgaag
atggaggctt 3780 tactgcttcc ctgattagtt gctctcacta gccaactgag
agcaggcaaa actacaggct 3840 gggtgcagtc aggctttttt tttttttttt
tttttttaaa taaagaaaag ccagaaatct 3900 agagttatgt gagaactcta
gattttttca tagttagcag ctaaaatggt aagagccaaa 3960 caaaacccat
ccgtgggttg gatttggcac acatgcctgc gaattgcagt ctccatgctg 4020
atctcttggg cccttctggg gaggcagagg gaaggctccc tgactcagtc acaggcaatg
4080 gggaataggc agtgacagtc attttacagc agggtatgta tgtttaagag
tctaggccgg 4140 ggtgtggtgg ctcacgcctg taattgcagc actttgggag
gccgaggcgg gtggatcacc 4200 tgagggtcag gagttcgaga acagcctggc
caacatgatg aaatcccgtc tctactaaaa 4260 atacaaaaat tagctggaca
tgctggcaca cgcctgtaat cccagctact tgggaggctg 4320 aggcaggaga
atggcttgaa cccgggaggc agaggttgca gtgaactgag attgtgccac 4380
tacatccagc ctgggtgaca agagtgaaac tctgtctcaa aaaaaaaaaa aaagaatcta
4440 gaatctaagt cgagtgtcat tatatccatg ttttattcct attccctttt
ccccttatgt 4500 atcctcttac tttaaagagg aactttaaaa aatcttaggg
acgactaggc agagtggctc 4560 acacctgtaa ctccagcact ttgggaggcc
aaggcaggca gattatgagg tcaggagttc 4620 gagaccagcc tggccaacat
ggtgaaaccc cagttctact aaagatacaa aaaatcagcc 4680 gggcgtggtg
gcacgtgcct ataatcccag atactcggga ggctgaggca ggagaatcac 4740
ttgaacccgt gaggcaaagt tttcagtgag ctgagatcat gccattgcac tccacctggg
4800 tgacagggtg agactccatc tcaaaaaaag aaaaaggaaa aaatcttaac
gtcacataca 4860 tggaaagatc atctttttca ccccccaccc ccaactgaga
tggagttttg ctcttgtcac 4920 ccaagctgga gtgcactggc gcgatctagc
tccctgcaag ctccgcctcc cgggttcaca 4980 ccattctccc tgcctcagcc
tcccgagtag ctgggactac aggctcctgc taccatgccc 5040 ggctaatttt
tttgtatttt ttttagtaga gacggggttt catctgtgtt agccaggatg 5100
gttttgatct cctgacctcg tgatccgccc gcctcagcct cccaaagtgc tgggattaca
5160 ggcgtaagcc actgcacccc gccttttttt tttaattaat taattttttt
agacagagtc 5220 tcgctctgtc ccaagctgga gtgcagtggc gcgatctggg
ctcactgcaa cctccgcctc 5280 ctgggttcac ggcgattctc ctgcctcagc
ctcccgagta gctgggacta caggctcctg 5340 ctaccatgcc cggctaattt
ttttgtattt tttttagtag agacggggtt tcactgtgtt 5400 agccaggatg
gttttgatct cctgacctcg tgatccgccc gcctcagcct cccaaagtcc 5460
gcctcagcct cccaaagtgc tgggattaca ggcgtaagcc actgtaccct gccttttttt
5520 tttaattaat taattttttt agacagagtc tcgctctgtc accaagctgg
agtgcagtgg 5580 cgcgatttgg gctcactgca acctccgctt cttgggttca
agcgattttc ctacctcagc 5640 ctccggagta actgggacta caggcgcgtg
ccaccacacc aagctaattt ttttgtgtat 5700 gtctttagta gagatggggt
ttcaccatgt taggatggtc tcgatctctt gacctcgtga 5760 tccgcctgcc
tcggcctccc aaagtgctgg gattacaggc atgagccacc ttgcctggcc 5820
gaaagtatct tcattttaaa gttcactgtt tggctactct gttgacaaga gtttagtatt
5880 tctcaaggag gctaagatac ctattccttt ttggatccta cctctatcag
gagggtgggc 5940 cttccttgca ttgaaacagt atgaaaacag tagccctgaa
ttcataagtg ggacaccttt 6000 cttctattgg tagagcaggc agtttttttc
tcctgccaat ggtgcctact aaggagattt 6060 cactagggta cagtcgttca
tttgataagc atttgttgag catatcctct gtgatggtac 6120 tatggacagt
actggggcta tagtgagggc aggattgagt tggtccttat ggcaaggaag 6180
gcagctaatc aacaagcaaa atataaagta tgatggggag ggctgtcttc agcactcatg
6240 agtgtgagcc caggcctgga ggggacacct ggagaagagg gtgcatgtct
ttgctcctgt 6300 gcttttcagg gaaggagatc acgttgctga tgcagacatt
gaatactctg agtaccccag 6360 aggagaagct ggctgctctg tgcaagaagt
atgctgaact ggtcagttcc cccctccgcg 6420 ggcaccttcc ctgcgttggg
aaaatcagca tgccacctgg tgtaaggttg ggggtgcaga 6480 gtcaagtagg
tggcttaatt cctgttcagc ttttctctga actatctgtt aaatggggaa 6540
tcacttccag ccagcctctt cagggctgtg cagcaagagg agaaactgca tattccttga
6600 aagaaatttc tcaaagaatg attccaaggt ggtagagccc ttgttcctgg
cctgagtcca 6660 agacaccttg tgatcttgat gcttcttcct caaatacaga
tgcatagagc cattatcaca 6720 gttaataaaa ctaacactag tcacttgata
ctttttcctt ttactccaga gcagtcttct 6780 tgtcactgcc tcctcatatt
ccccatgaca ttgactttta acagaaacta gactagctgt 6840 cttgtaggat
gcccccttct agctttgtca tctctgtggt atcattttac ttctttacct 6900
cctggtacat gtaagtgaag tagaagttag ctctaaagct tgatccaatt cagcttcaac
6960 tttttgacaa gaattcttca taagtacttc atgttccatc acaataaatg
caaagcatgc 7020 tcttcccact ttgttgtaac attgttcagt gggttggggg
tggggcagcc agattcttcc 7080 atcatcaggt cccttgtcag aatttgaact
aacagattta tccattgatg gtcacagcct 7140 gtgtatgtat gtatgtatgt
atgtatgtat gtatttattt atttatttat tttttgagac 7200 ggggtcttgc
tctgtcgccc aggctggggt gcagtggcac gatctcggct cgctgcaagc 7260
tccgccttct gggttcatgc cattctcctg cctcagcctc ccgagtagct gggtctacag
7320 gcgcccgcca ccatgctagg ctattttttt tttttttttt ttttttagta
gagacggggt 7380 ttcaccgtgt tagccaggat ggtctcgatc tcttgacctc
gtgatccgcc cgcctcggcc 7440 tcccaaagtg ctgggattac aggcttgagc
caccacgcct ggcctattta tttatttatt 7500 cagagtcaga gtctcgctct
gtcaccaggc tggagtgcag tggcgcgatc tcggctcatt 7560 gcaacctcca
cctcccaggt tcaagcgagt ctcctgcctc agcctcccga gtagctggga 7620
ttacaggtgc atgtcaccat gcctggctaa attttgtatg ttttagtaga gacagagttt
7680 cagtatgttg gccaggatgg tcttgatctc ttggcctcgt gatccgcccg
tctcagcctc 7740 ccaaagtgct gggattacag gtgtgagcca ctgtgcctgg
cctctaagta tttattttaa 7800 aattaattca ttccacacac atttattaat
attttcctgt aaggaacttt actcatcttt 7860 aaaatgggga atgtcatacc
tgcctaatga cattcttgta aggattaaat aaaaggtata 7920 aggaagataa
gcaccctttt ggagtgatcc agccagggga aaattgctga tgcaagagag 7980
gaaatgagtt gctagagtgg tgttgtgagt agaggagggg agctgaggcc tgcccaagaa
8040 gggggcttgg ctgtggtaac cacatggcta ggtctgtgtg actggaggag
aggacggggc 8100 aggtggactg gtagatgtgc agcttgtgcc cctgattctc
tagtttcttc tgtgttttga 8160 gatttgatga gaacgatgaa atagttgtct
ggaaggagag gagtgtgaat agcatatgca 8220 ttgtattggg attgctggtc
ttcctgaaat tggtggccat gaatttaaag tgagactctt 8280 caagtagggt
tgttatagta ctggtgtaaa gcaggaaggt gctttactag ggttgcagta 8340
ctactgggga agggccaaga gagttgaggg tgtaagaaat ccaagccagg taatgtagtt
8400 attttaaagg agagtggaag gatggttgag tcaatggatt ggaggtccta
tagggtaaga 8460 gactttctga ggatcacaga tactgattgg aatgagctaa
aaagataggt gatggtagtc 8520 ctggactggg atgctggaaa ttgagatagt
gggtgtgctc tctggtagtg acaaatctag 8580 atctgcgctg tccaagataa
attcgtctct agctaattga catgtggcca gtttgaattt 8640 gaacatgcta
taaatgtaag atacacatca gcttttgaag acttaagcaa aaacaaagaa 8700
tataaaacat ctttttgtga gagagtgtct cagtcaccca ggctggagtg cagtggcgtg
8760 atgtcctgct tccaggttca aacgattctc ctgcctcaca gcctcctgga
gtaactgaga 8820 ttacaggcgc atgccaccaa actggctact tttttgtatt
ttttttttag tagaaacggt 8880 ttcaccatgt tggccaggct ggtcttgaac
tcctgacctc aagtgatctg cctgcctcag 8940 cctcccaaag tgctgggatt
acaggcatga gccaccactc ccggcctcac ttttttacat 9000 tgattccgtg
ttgaaattgt aatgttttgg atattaggtt aaatacatat attactaaaa 9060
ttaatttcac ctgtttttta cttttttagt gcggccagta gaatattttt aattacttat
9120 gtggtttgca ttatatttct gttgtacagg cctggatagg gtcatgggag
gggaactgag 9180 ctggggaaag gagtgggttt gtggaagagg tgatggactg
tgaggccagg gagttagaag 9240 gattatctgt tgatactgaa gtggccacaa
atgagaaaag taattgtgtt ggggagagcg 9300 ctgatgaacg cagcgctaac
gttttgaagg aatgcgaggg agcgatgggg gtctgtctgt 9360 taataggcac
aaggtacggt agcaggtggt ctcatcctcg ggcatgagtg tccagcaagt 9420
tggggaaatg caacagcttg aagtggctct agtggcccag agtcagagct ggaataggaa
9480 ttggcatctg ctggctgtgt ggcccctgct tgccctagtg agttaccatt
tctctgtccc 9540 tacggtggag cctttggggt tattgtgagt tcatgggagg
agcgtgtaag caccggcaca 9600 gcatcagccc atgagagtgc tcctggcctg
agagggtaag ggtcagggca gctcaggaga 9660 ccctagacct gcatagtgat
ccccccacca ggaaggcccc acaagatgct cacctgccct 9720 ccctatccct
gtccccagct ggaggagcac cggaattcac agaagcagat gaagctccta 9780
cagaaaaagc agagccagct ggtgcaagag aaggaccacc tgcgcggtga gcacagcaag
9840 gccgtcctgg cccgcagcaa gcttgagagc ctatgccgtg agctgcagcg
gcacaaccgc 9900 tccctcaagg taggcctggg ccccctggaa caggtgactc
tggtttcctt gacttccact 9960 taatgtttct ttcatgggct ttcctcttaa
aaagtagtgc aggctagggc caggcgcagt 10020 ggcacacata agtgattaaa
aatcttctgg ccactaaaaa acagaaatta attttagtaa 10080 tatacttaac
ccaatatcca aaacattaca atttcaacat gaaatcagtg taaaaaagca 10140
aggctgggtg tggtggctca cacctgtaat cccaacactt tgggaggctg aggtggatgg
10200 atcacttgag gccaggagtt tgagaccaac ctggtcaacg cagtgaaacc
ccattctact 10260 aaaaatacaa aaattagccg agtgtgctgg caaatgccta
taatcccagc tactcaggtg 10320 gctcaggcat gagaattgct tgcacctggg
aggctgaggt tgcagtgagc cgagattgca 10380 tcactgcatt acagcctggg
caacagagtg agactcagtg tccaaaaaaa aaaaaaagta 10440 gtgcaggctt
gtggcataga aatacacttt ctcaataatg ccttacgtta agagagtact 10500
gcttgtaatc atttgacatg tattagataa ggtgaaggat aaagtactaa gagaatccat
10560 aatgcactgg cgttagtatt tctcaatgaa atgacagtcc cctggtaagc
ggaggcctgg 10620 ctctgacaag cagctcttgt cccagacgtt ggtcagtcag
gaacctgggt ccttcccatg 10680 ttctgctgct tctatggtga ggtcagtctg
tggttacacc aagtttaaat acagcctttt 10740 aactttcttt tttatatgta
aaatcttaca tgtagttttt agaatgaaat tattatacat 10800 gtaccatttc
atatcctgtg cctttttttc actttacata acatttttcc ctatcagtat 10860
gtgtagggct atcttctcat tatatggata tattatatca gtgccctagt taaagcattt
10920 tgggggttgt ttacaatttt tcattattac atatagaact atagtgaaaa
ttcttgttat 10980 atttatcact ggtcagttat atagaactta tctgtaggat
aagtcatgga attgaaatgg 11040 ctaggtcaca gtatatgcag atttttcatt
ttaatagatt ttgctggatt gccttccagt 11100 gagggggcag tgtgccttcc
ccatcaaaag tgttgagtgc ctaattctgc acaactttgc 11160 aaaccctggg
tgttactaaa ttttaacagc ttggtctctg ggggtacaga ggggacaaat 11220
gcacattaat ctgaaatctg gaagaatagg ccttaggaga tccgacttgc ttcagaatgg
11280 cacttagcac ttacatgtgt gcatgtgtgc ctgcattttt tcttcctttt
tttttttttg 11340 gggacggagt cttgctctgt ggcccatcgc ccaggctgga
gtgcagtggc gcgatcatag 11400 ctcaccacaa cctccgcctc ccaggttcaa
atgactcctc tgcctcagcc tcccaagcag 11460 ctgggaccac aggtgcacac
catcacgccg gctaattttt gtattttagt agaaacgggg 11520 tttcaccata
ttggccaggc tggtctcaaa ctcctgacct cgtgatccgc ccacctcagc 11580
ctcccaaagt gctgggatta caggcgtgag ccaccgcgcc tgccatgtgc ctgcattttt
11640 ctagggggag aatctcactt gatgtcacct gatatacaga ggggcccatt
ggaacccgca 11700 ttgcacaaca tcctggagtc tggctactcc acgctttggg
agcagggagg gctgttggca 11760 gagaccatct gtggactagc tgggggaccc
ttgtgaggta gcagtggatg atggctctcg 11820 ggctgacttc tttgcccagg
aagaaggtgt gcagcgggcc cgggaggagg aggagaagcg 11880 caaggaggtg
acctcgcact tccaggtgac actgaatgac attcagctgc agatggaaca 11940
gcacaatgag cgcaactcca agctgcgcca agagaacatg gagctggctg agaggctcaa
12000 gaagctgatt gagcagtatg agctgcgcga ggaggtaagg gtatcacgga
cagcagtcat 12060 ggcccagaaa ttgtgaggtt ttgagtgtgt gctaggcact
gggacagtac cttttcaggc 12120 ttcatcccat tctccctttc ttcctcctcc
tcctccttgg gaggagagta atgttattcc 12180 tcatagataa aaaacaggtg
tggagaagag actcacttac agccacacag ccccaggtcc 12240 acagtgcctt
gtcccaaatg actgggccag gcatcttttg gaattagaac tatccacatt 12300
ttagaatgga ggtacatgta tggactgtgt gttatatagc accctcagca gggccttggg
12360 gaagccagac acattaatgt atttatgcag tagaacttcc aaatactcac
ctacattatg 12420 ggcttacaat gatgcaggtc aagtctggct gccagcttat
gacaatttcc attttcagaa 12480 ctttgtagaa tttggaattg caggggaggg
gtgtacctgt gatcagtgat ggactccaga 12540 gactgtgtcc actgattcct
tgctgctcct gccactcaaa aggcagaatt tatcaggctg 12600 ggcgtggtgg
ctcatgcctg taatcccaac actttgggag gccaaagcgg gcggatcacc 12660
tgaggtcagg agttcaagac cagcctggcc aacatggtga aaccctgtct ctactaaaaa
12720 tacaaaaaat tagccaggtg tggtggtgca cggctgtagt cccagctact
caggaggctg 12780 aggcaggaga attgcttgaa cccaggaggc agaggttgca
atgagccaag attgtgctac 12840 tgcactctag cctgggtgat ataccgagac
tccatctcaa aaaaaaaaaa aaaaaaaagc 12900 aggatgtcac tccctttgtc
actgcgttgg ctgccacccc aggcacttga atctttggat 12960 cttccctgcc
agtcacctgg ctgttctggg cgcgttctca tcatgagaag ggagacctgc 13020
agccccctta cagggctggc agaggacctg ctctggatta ggccctttcc tagcccctgg
13080 ggtgtggcag tgggtgagac cgggaagatc tgccctctta ggttcatagg
ccaaagtgat 13140 gatcgtgtgt gcaggaccta gagggcgctc ccctgaccca
cccctttcct tgccatactt 13200 catcctctgg gaacaaagct gcttgtttgg
tttgagggga gttggtttgg ttcttatccc 13260 tcagcgctga gacatagagg
cttcctgggc cactacagtg agacacgaac ttcaagaatc 13320 tgaatacccc
cgttttctct ccccgccaag gcaaaaaagg acttagtact acctgtggag 13380
aaggaggtgc aggactacca ggccctgctg ctttgcattt acagccctcc ccagacagac
13440 acaggcaccc tcatcatacc caaactggac ttacctgcta ggcaccttcc
cttccccatc 13500 caaaaaaatg gagttatttt cccttatttc agcaagtcca
gttgatttta cctttgaagt 13560 agcacctgag tccttcacct tctctccatc
ccttctctct cacctgacac aggtctgcag 13620 cgctcctcta gtaggcagga
cagccattcc ttggggatgc acatgtctag tctttgccta 13680 gatatggcaa
gtctttgcca actgagctag gctgttatgt tcttagaggc attgtttttg 13740
cccattcttc ccatttacaa gagaatcagg gacacagaag tgagggcttc cagccccata
13800 ggtgatcaat cctggggtca gagatttgag tgtgtttatt gcttgccttc
ttgggagcag 13860 attccatcca taaaccatgt gcttaccaag gtctgactca
ctgggagaga aacgacgtga 13920 ggttggaaag ctgaccttcc agagacttgg
ggcccatgtt gtgtggtaca catgggagtc 13980 catcatatca gattgagatg
gggggctggg caaagtgccc tggtctgtgg ctgtggggct 14040 accctgagaa
agggagcgcc tgacaagccg actgctccca ccatctttgt tgcagcatat 14100
cgacaaagtc ttcaaacaca aggacctaca acagcagctg gtggatgcca
agctccagca 14160 ggcccaggag atgctaaagg aggcagaaga gcggcaccag
cgggagaagg attttgtgag 14220 gctcaggccc cagggttggg gtgggggtgt
gggaggagac aggctgggct ctggctcagc 14280 tcatagccgg gttatatggg
agaagtctgg ccagaccagg cacagattcc ttgagtacca 14340 gtctgagagc
aggaagcctc agtgggtctg gtgcttgtgg ctaaaaacca aacatagccc 14400
ctgggggctt ctgacaggat ctggggttct gtcttggaaa tagctcctga aagaggcagt
14460 agagtcccag aggatgtgtg agctgatgaa gcagcaagag acccacctga
agcaacaggt 14520 gagagcatat aacctgaccc tgtgccttca agtttccctc
actgggcccc atcctggggg 14580 tagtgaaatg ggaccctcat tctaggactg
gctgtgtcct ggctgctatg acgccttggt 14640 tgagcttagg tgggctcaga
ggacttcatt tgtagctcag aaatgtattg cttttgagga 14700 ggtaggaaca
gaagagtttg aaaatcaaca taaaggcaaa ataaaagtca ccctaagtct 14760
cctactttcc aggcttagca ttttggatta tatccttcca aatatatagc tttgctttgt
14820 tttaaggaaa aatagtatct caatagaatt actggtcaga gagtcaagga
cgggtctgag 14880 tgtgttgacc agagtgcctc ccagagaaac ccagtcttat
ctgtgggctg ctttctcccc 14940 acagcttgcc ctatacacag agaagtttga
ggagttccag aacacacttt ccaaaagcag 15000 cgaggtattc accacattca
agcaggagat ggaaaaggta actgtggtcc aggccaggca 15060 tggctgctgg
ggcataagct gcttcattca aaattgttgg gcctgccttc aggaagctcc 15120
catctggggt gtctcaaggg cagggctgtt aggaaggttc acagcctttc ccctcttgag
15180 gcagtatcag tggtatgtat acactccagg ttgtcccagg gaatggggca
gtcttttctg 15240 tttgtttggt ttttttgggg ggtttgttgt tgttgttgtt
gttgttgttg ttgtttgaga 15300 tggagactca cctattgccc aggctggagt
gcagtggcat gatctcagct cattgcagcc 15360 tttgcccccc gggttcaagt
gattctcctg cctcagcctc ctgactagct ggaattacag 15420 gcgcgtgcca
ccatgcctgg ctaatttttt ctttcttttt tttttgtatt tttagtagag 15480
acggggtttc accatgttgg ccaggctggt ctcgaactct tggcctcaag tgatctgccc
15540 gccttggcct cccaaagtgc tgggattata ggcgtgagcc accatgcctg
gccccttacc 15600 attccttgtt attggtggtg gacacctctg acttcctggt
ggtgaggtgg cacagagggc 15660 attgactgca tcctgtaatg ccttgcgcct
tgggatcaat cattccccac cttggagaca 15720 caggtgcagt ccccaccttg
gagacacaga ccttggagag gccagctctg accatttcct 15780 tctgtctgtc
acataaccta gatgactaag aagatcaaga agctggagaa agaaaccacc 15840
atgtaccggt cccggtggga gagcagcaac aaggccctgc ttgagatggc tgaggaggtg
15900 ggctgtctgt gatctgcagc cagggtgggg gtgtgcactt agcgcatatc
aggccctttc 15960 ctgtatgttc tacccatcag tgacacagct agcatgaggt
agaggtgaga tttgcacaca 16020 atgtccaagt ccaaagttaa tgctgttctc
tccccatggg aggtggtgag cccagtggta 16080 ggtctccagt gggagtgaag
ggagcaaatg gaagaaagga ataaaagagc agaaaaaaac 16140 gggtgccagt
gatgtgcctg gtttacatgt aaagcagccc aggtagtttg tgatttcaca 16200
gcttgtaatg tagaagaaag gaactaacga tggagcagca actgcaagcc agaccttgct
16260 gaaagttttt gggttttttt tgtctttttt gctgctgaat gtttttaggt
acgttgttca 16320 ttgaaccttc tcttgagctc tgaggatggt attagtagtc
ctgttttata gatgagacag 16380 gctcaaaagt caagtccttt gccaaggtca
cgtggtagat aaatggagga atacgttatc 16440 tccaagccgt gccccttttc
tgcaccatgc tgccccacct gacagcctag tcatggcttc 16500 aactaggact
gtttcctaaa gggggccagc tttggactcg gtctgctctc agccttgtta 16560
aagtgtttgc cgccaagtgg tgatggtaag tgggaggttg atggggcacg gcactgaagg
16620 tctcatttct ttccctagaa aacagtccgg gataaagaac tggagggcct
gcaggtaaaa 16680 atccaacggc tggagaagct gtgccgggca ctgcagacag
agcgcaatga cctgaacaag 16740 agggtacagg acctgagtgc tggtggccag
ggctccctca ctgacagtgg ccctgagagg 16800 aggccagagg ggcctggggc
tcaagcaccc agctccccca gggtcacaga agcgccttgc 16860 tacccaggag
caccgagcac agaagcatca ggccagactg ggcctcaaga gcccacctcc 16920
gccagggcct agagagcctg gtgttgggtc atgctgggaa gggagcggca gcccagccag
16980 gcctggccca taaaaggctc ccatgctgag cagcccattg ctgaagccag
gatgttctga 17040 cctggctggc atctggcact tgcaattttg gattttgtgg
gtcagtttta cgtacatagg 17100 gcattttgca aggccttgca aatgcattta
tacctgtaag tgtacagtgg gcttgcattg 17160 gggatggggg tgtgtacaga
tgaagtcagt ggcttgtctg tgagctgaag agtcttgaga 17220 ggggctgtca
tctgtagctg ccatcacagt gagttggcag aagtgacttg agcatttctc 17280
tgtctgattt gaggctcaga cccctccctg cccttcagag ctcaagacaa gtaatacacc
17340 caggtcttga ctgcatttgt cttgtgagca gggcttgctt ggtcagctca
ggccctccta 17400 gctgctctgg aggctccttt gattctctag acctggaaaa
ggtgtcccta ggcagagccc 17460 tggcagggcg ctcagagctg gggatttgct
gcctggaaca agggacctgg agaatgtttt 17520 tgcgtgggat gatgtgctgg
tcaggagccc cttgggcatc gcttcccctg ccctttggta 17580 gtgccaggac
caggccaatg atgcttctca gtagccttat cattcacagg tgcctctcta 17640
gcctgcacaa atgattgaca agagatcacc caaaggatta tttctgaagg tgtttttttc
17700 tttatttctt tttctttttt tttttttttc tttttctttt ttttttgcac
atgacagtgt 17760 ttgtattgag gaccttccaa ggaagaggga tgctgtagca
gtggtgcctg ggtgcctggc 17820 ctccagtgtc ccacctcctt caccacccca
cttggctcct ttgccatctt gatgctgagg 17880 tttcctgttt ggtgagatca
ggttgtttgt ggtaaaagaa aggaaagggc ttctgatggc 17940 tttgccacaa
gcttacctgt gggtttcagt cctgagaggc caccaccagt tcccatcagc 18000
actgtctcca tgcagcagtt gctgggtccc atgtccagct gcctctttgg cttcatgggt
18060 ttttctgctt cctgccccca cccccacatg tgcaatcctc aagatttgtc
ctgattctat 18120 ttcctggcac ctccctgcct gtccttgggg attctacttc
ttcctgtgtg ggagcccata 18180 gctgttgtct aacaggtaag aaatgaaatt
gaactattga ctgggcccca gaaatccata 18240 aaatggctgc agacagttgt
ttctgtgtcc tgttctaccc ccactccagt acataactac 18300 tatgtactgt
gtagagccat tctatatgct gaatgttctg ctgttgcaaa cttgccaggg 18360
tattagccag tgtttgtgcc aagcagtttt ctgggacaac agaatgactc agaccaagat
18420 ggataggatg gttagggctt tgcttcttgc tgtttttctt tgaagctagt
tcattgtcct 18480 gcaggtccct tcatcttcca tacctagccc actcttttag
cccttacctt aaatctctca 18540 gataagttgg ttcacaaaga atgttaagta
ctgaatcatg tgtgactgag accagagatg 18600 gcaaatgaat ggcacaccat
ttctccttct cctgccccag ggcaggtacc actgatctgc 18660 atcagagttg
cctgctattc tctggtgtat ccttcacatc taggtgccct caagcagctg 18720
tgtgagtgtt gagatctctg ccatctctgg ctgagatact gctgtcctgt gaagtgtttc
18780 ccatgacctt tttcttcccc tttgaatccc tctgtctgga gtagtccttg
cctcttcctg 18840 ctccagtagg gccttttccc taccccagcc cctgtgccag
gctaagctgg tacaagagct 18900 gccaacctca cagagtgttt gctaggcgag
agaggtgcag ggaagaggca gaggtatgca 18960 ccttccccct tgaagagagg
ggaaaggcct acagtggccc acataattgc ctgactcaca 19020 cttcagctac
ctcttaatgc ctgtggaggg actggagctg ctggatccca gtgtggtggt 19080
gtaggaggcc acagtgagca ggtggcccca gctgggtttc ccaggtcagg aatgtgggcc
19140 ccaggcaagg tgcagccttt gctcacagct ccatccatgt ctagaccttc
aggccagtct 19200 gcagatgagg ttccctacct ttttcttctc ttcattgacc
aaatcaacca atcactacag 19260 ctgctctgct tctgctttcc aaagtagccc
aggtcctggg ccagatgcag gggaggtgcc 19320 tatccatgag tgaaggccag
tgtcttcctc acctgggtgg gtcccacact tgtgacctca 19380 gttttaggac
caagatctgt gttggtttct tagattgcta gcttttcctc caggggacca 19440
cagcaggtga agctcaagag cgcatggctc tgctaatagt aaattgtttt cagggccttg
19500 tccagctgag agcttcatgt ccaccagatt ctgagaggtg tcagcagcac
tttttttttt 19560 tatttgttgt ttgttttcca tgaggttatc ggaccatggg
ctgagctcag gcactttctg 19620 taggagactg ttatttctgt aaagatggtt
atttaaccct tctcacccca tcacggtggc 19680 cctgagggct gacccggagg
ccagtggagc tgcctggtgt ccacggggga gggccaaggc 19740 ctgctgagct
gattctccag ctgctgcccc agcctttccg ccttgcacag cacagaggtg 19800
gtcaccccag ggacagccag gcacctgctc ctcttgccct tcctggggga agggagctgc
19860 cttctgtccc tgtaactgct ttccttatgg cccagcccgg ccactcagac
ttgtttgaag 19920 ctgcactggc agcttttttg tctcctttgg gtattcacaa
cagccaggga cttgattttg 19980 atgtatttta aaccacatta aataaagagt
ctgttgcctt acttgtttct ctcctgacct 20040 gtgtattcct ttgtttctgg
atctgatcca ttcagcccct tccatcatca ctgacttgtt 20100 caggtctgct
gcagagcgcc catggtggtt ccctggtatc ttacatattc cacagtgtct 20160
ttgagcagtc gccacagcct caggatgctg gcatattcac ttgagctgcc tgagtggagc
20220 ccttggcaaa gttggcaaga cccttgcctc agagaggatc acacacacac
aaaaaagttt 20280 tccctgacct gggggctcac aggctagtga agggaaaagg
tacttttagc tatagacagg 20340 tcaatggtgc tgagagcaga gaggaggccc
ctgccccctt cagcaaggtg agggggtgat 20400 acctggaatg gccttctgaa
ccacagggca ggtagaagat gaacgtcatt tagtgattaa 20460 atggtacagc
tgggaagcag gtccatggga ctgggagagg gggtgaggct gggcccagag 20520
tctgggtacc aggttaagga atgtgggcta gatccagagg gcaggggggg caactgaagg
20580 tgtttcaata ggaaattgat aggctccagc agtaaggcaa aaggcatgga
gccaggcata 20640 ggccatttga ggcccaggtt aagaggggtg gacactcatc
actgctattt gggtctgagc 20700 tgtgggtagg ctcctatagc cctggcctgc
ccaagggaat tcacaggggc ctctaattgt 20760 atgcattcct taaggagagc
acattctctg ttcagttttt acacccccca tttacccacc 20820 tcaagcatgg
gactcctata tgggagacat gctgctggtg gcctcaccca gcaccctgtt 20880
ctctctgggt cctgggttgg tcaggcacaa aggatgatat gtgctgaatg cccaggaaat
20940 ggcagagaca acccacctgc ccttccctcc aggcctccac aaatagatgt
gcccacaatg 21000 actgtgacag tcccagcaga gcctctgacc cttctagctg
ggtcctgata catgttttcc 21060 atgctggcca tgttatttct agtcgcagat
cctctggagg gtgtgggggg ggtgccgccc 21120 caactcttgg agattccaag
caaagcagct ctgagaataa tgaggtttct gaccccccag 21180 tgaagcagct
gaggatggga accacagggg tgctccctct gtcagcagca ttaccactgt 21240
ctactctagc agctccggtg gggaaggaga gggatttctg ttgtccccag tctgggcccc
21300 tggttattga aaaagttcgg aattactctt tacccttgtg gagtgttctg
agtgttggaa 21360 gtacccagga agaagccctg agcaggtgcc ctcaggagca
gtgcccatgg ctccccacat 21420 cagccaagag gcccaacccc aggaagccac
tcctgcccgg ggatggggaa ggtgggctgg 21480 gtggctgtgt gcactgccct
gggccagctc acttgagcct gctgagccgc ctggccaaac 21540 atgagcctct
ctcctgttgt atcagatgct gttctgggga cctgcgccag gagcctctgc 21600
cagggcttta aatagctgcc cccattgatc tggctgcagg cagcagcagt cacactgggt
21660 cagcctccat caggtgctca ggtttccctg aggactggag tcaggtgcca
gggaatcgcg 21720 tggtctacct tatgacctgg tgctccccac acctgtctcc
taggcctggg gggtggggag 21780 gactcctgtc acttcatctg cggcaaaata
cagcccccac cacttaccag agaaaactgt 21840 ctggcattgt agagagaggg
gttttgccct caaaagactg ttgcttactt tcagtagaat 21900 ggggaatgac
actggtatct tccttaaggg ttgttatggg gatgaaatgt atgtaaagtg 21960
ctcaataggg cactggactc actccattga tggctgtctt tgctcgaagt gtcttcctga
22020 tgctgctgct gttgctgctt gtgcttcttc tgtgcttaca ttctctctct
ctcactcact 22080 cactctgtct ctcctctccc ccgccccacc ccctttctga
caaagccacc accattttgt 22140 aaggaactgt agcttctctc tgaaactgcc
gggaaaggga aaatcttttt aaaatagaca 22200 tcacacaacc aacagggtcc
cctaggttca ggcggggagg tgaggtcgag tgaga 22255 52 7 PRT Artificial
Sequence Polycatonic peptide 52 Arg Arg Arg Arg Arg Arg Arg 1 5 53
7 PRT Artificial Sequence Polycatonic peptide 53 Lys Lys Leu Lys
Leu Xaa Xaa 1 5
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