U.S. patent application number 15/337563 was filed with the patent office on 2017-02-16 for antibody preparation method, and antibody and antibody library thus prepared.
This patent application is currently assigned to AbMART (SHANGHAI) CO., LTD.. The applicant listed for this patent is AbMART (SHANGHAI) CO., LTD.. Invention is credited to Zeyong CHEN, Xun MENG, GuoXing WANG, XiaoQing WANG.
Application Number | 20170044245 15/337563 |
Document ID | / |
Family ID | 46559130 |
Filed Date | 2017-02-16 |
United States Patent
Application |
20170044245 |
Kind Code |
A1 |
MENG; Xun ; et al. |
February 16, 2017 |
ANTIBODY PREPARATION METHOD, AND ANTIBODY AND ANTIBODY LIBRARY THUS
PREPARED
Abstract
Provided is a method for preparing antibodies against a protein
of interest, through which highly specific antibodies against all
proteins can be effectively and rapidly prepared with low cost, and
the epitope to which the antibody is directed can be determined, so
that a library covering epitopes on the surface of the proteins of
interest and a library of antibodies against all the epitopes can
be established. The antibodies are proved to be useful in
detection, protein function investigation and antibody
pharmaceuticals.
Inventors: |
MENG; Xun; (Shanghai,
CN) ; WANG; XiaoQing; (Shanghai, CN) ; CHEN;
Zeyong; (Shanghai, CN) ; WANG; GuoXing;
(Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbMART (SHANGHAI) CO., LTD. |
Shanghai |
|
CN |
|
|
Assignee: |
AbMART (SHANGHAI) CO., LTD.
Shanghai
CN
|
Family ID: |
46559130 |
Appl. No.: |
15/337563 |
Filed: |
October 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13982820 |
Jul 31, 2013 |
9511342 |
|
|
PCT/CN2012/070768 |
Jan 30, 2012 |
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15337563 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2803 20130101;
G01N 33/6854 20130101; B01J 19/0046 20130101; C07K 16/00 20130101;
C07K 16/40 20130101; C07K 2317/10 20130101; C07K 16/18 20130101;
C07K 16/2809 20130101; C07K 16/28 20130101; C40B 50/06
20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; G01N 33/68 20060101 G01N033/68; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 31, 2011 |
CN |
201110034648.7 |
Claims
1. An antibody library obtained by: (a) predicting and/or selecting
peptide fragment located on the surface of the protein of interest,
wherein said peptide fragment is a linear surface signature peptide
and/or a conformational surface signature domain of the protein of
interest; (b) synthesizing or expressing one or more of said
peptide fragments; (c) using the product of step (b) to immunize an
animal, optionally in combination with an adjuvant; (d) using
lymphocyte from the immunized animal of step (c) to obtain
antibodies; (e) using the peptide fragment of step (a) or said
protein of interest in native conformation thereof to screen the
antibodies obtained in step (d), so as to obtain an antibody
library against said protein of interest, wherein said peptide
fragment of step (a) is predicted or selected through the following
process: (i) determining a surface peptide by calculating a
parameter according to the sequence of the protein of interest,
said parameter is selected from: solvent accessibility, disorder
index, protein-protein interaction domain prediction, or any
combination thereof; (ii) aligning the surface peptide determined
in step (i) with the proteome of the species that the protein of
interest is originated from, so as to select a specific peptide
fragment of said protein of interest; (iii) aligning the surface
peptide determined in step (i) with homologous proteins from other
species, so as to screen a conservative sequence of said protein of
interest.
2. The antibody library of claim 1, wherein said protein of
interest is a native protein, and/or an alternative splicing
isoform thereof, and/or a mutant thereof.
3. The antibody library of claim 1, wherein said signature peptide
is a peptide, which is 6-12 amino acids in length, which is high
hydrophilic, which has high antigenicity, which is not signal
peptide, which is not in the trans-membrane region, and which is
located in disordered region.
4. The antibody library of claim 1, wherein said signature domain
is a sequence specific protein fragment which is 100-500 amino
acids in length, and which is expected to have 3 dimensional
structure.
5. The antibody library of claim 1, which is used for producing an
antibody library against all the proteins of a species.
6. The antibody library of claim 1, wherein the antibody library
produced in step (e) comprises antibodies against all the epitopes
of the protein of interest.
7. The antibody library of claim 1, wherein the antibody obtained
in step (d) is obtained through at least one process selected from:
(1) fusing lymphocyte from the immunized animal of step (c) with
amyeloma cell, so that a hybridoma is generated and then expressed
to obtain the antibody; (2) isolating antigen specific B cell from
lymphocyte of the immunized animal of step (c), and then using PCR
to clone and express the gene of the antibody so as to obtain the
antibody; (3) isolating mRNA from lymphocyte of the immunized
animal of step (c), and then obtaining the antibody through phage
display, or ribosome display, or yeast display, or bacteria
display, or Baculovirus display, or mammal cell display, or mRNA
display.
8. The antibody library of claim 1, wherein one or more of the
peptide fragments of step (b) are recombinantly expressed in the
form of a fused protein with a protein which enhances the
immunogenicity and/or increases the copy number.
9. The antibody library of claim 8, wherein the protein which
enhances the immunogenicity and/or increases the copy number is a
virus-like particle protein carrier.
10. The antibody library of claim 9, wherein the protein which
enhances the immunogenicity and/or increases the copy number is
Hepatitis B virus nucleocapsid (HBC) protein.
11. The antibody library of claim 10, wherein said one or more
peptide fragments are inserted into loop, N-terminus, or C-terminus
of the HBC protein.
12. The antibody library of claim 11, wherein the position into
which said one or more peptide fragments are inserted is located
between the amino acid residue at position 77 and the amino acid
residue at position 82 of the HBC protein.
13. The antibody library of claim 10, wherein 2-10 of the peptide
fragments are linked by a linker and inserted into the HBC
protein.
14. The antibody library of claim 13, wherein said linker is
(GGGGS).sub.n, and wherein n=1, 2, 3 or 4.
15. The antibody library of claim 14, wherein n=1 or 2.
16. The antibody library of claim 1, wherein the one or more
peptide fragments expressed in step (b) are further coupled with an
immune-enhancing protein carrier.
17. The antibody library of claim 16, wherein said immune-enhancing
protein carrier is keyhole limpet hemocyanin (KLH).
18. The antibody library of claim 1, wherein the one or more
peptide fragments of step (b) are chemically synthesized.
19. The antibody library of claim 1, wherein the adjuvant is
selected from: Freund's complete adjuvant, aluminum, CpG, or any
combination thereof.
20. The antibody library of claim 1, wherein in said step (c) the
animal is immunized at multiple sites.
21. The antibody library of claim 20, wherein the immunization at
multiple sites is performed at at least 2 sites selected from: neck
and back, tail end, hind foot palm, hind leg inguen, front leg
armpit, hind leg muscle.
22. The antibody library of claim 20, wherein multiple
immunizations are performed, with the time interval of 2-14
days.
23. The antibody library of claim 22, wherein multiple
immunizations are performed with the time interval of 3-4 days.
24. The antibody library of claim 22, wherein the immunization
protocol used in step (C) comprises the following steps: (A) the
first immunization: the expression product of step (b) together
with Freund's complete adjuvant are used to immunize the animal at
neck and back, tail end, hind foot palm, hind leg inguen, and front
leg armpit; and the expression product of step (b) together with
the adjuvant of aluminum+CpG are used to immunize the animal at
hind leg muscle; (B) the second immunization: the expression
product of step (b) together with Freund's complete adjuvant are
used to immunize the animal at neck and back, hind leg inguen, and
front leg armpit; and the expression product of step (b) together
with the adjuvant of aluminum+CpG are used to immunize the animal
at hind leg muscle; (C) the third immunization: the expression
product of step (b) together with the adjuvant of aluminum+CpG are
used to immunize the animal at hind leg muscle, tail end, and front
leg armpit; (D) the fourth immunization: the expression product of
step (b) together with the adjuvant of aluminum+CpG are used to
immunize the animal at hind leg muscle, tail end, and front leg
armpit.
25. The antibody library of claim 24, wherein the first
immunization is performed on the first day, the second immunization
is performed on the fifth day, the third immunization is performed
on the eighth day, the fourth immunization is performed on the
eleventh day.
26. The antibody library of claim 21, wherein multiple
immunizations are performed, with the time interval of 2-14
days.
27. The antibody library of claim 26, wherein multiple
immunizations are performed with the time interval of 3-4 days.
28. The antibody library of claim 26, wherein the immunization
protocol used in step (C) comprises the following steps: (A) the
first immunization: the expression product of step (b) together
with Freund's complete adjuvant are used to immunize the animal at
neck and back, tail end, hind foot palm, hind leg inguen, and front
leg armpit; and the expression product of step (b) together with
the adjuvant of aluminum+CpG are used to immunize the animal at
hind leg muscle; (B) the second immunization: the expression
product of step (b) together with Freund's complete adjuvant are
used to immunize the animal at neck and back, hind leg inguen, and
front leg armpit; and the expression product of step (b) together
with the adjuvant of aluminum+CpG are used to immunize the animal
at hind leg muscle; (C) the third immunization: the expression
product of step (b) together with the adjuvant of aluminum+CpG are
used to immunize the animal at hind leg muscle, tail end, and front
leg armpit; (D) the fourth immunization: the expression product of
step (b) together with the adjuvant of aluminum+CpG are used to
immunize the animal at hind leg muscle, tail end, and front leg
armpit.
29. The antibody library of claim 28, wherein the first
immunization is performed on the first day, the second immunization
is performed on the fifth day, the third immunization is performed
on the eighth day, the fourth immunization is performed on the
eleventh day.
30. The antibody library of claim 1, wherein the produced antibody
is singly IgG subtype.
31. The antibody library of claim 30, wherein the produced antibody
is monoclonal antibody.
32. The antibody library of claim 30, wherein the produced antibody
is polyclonal antibody.
33. The antibody library of claim 1, wherein the antibodies
produced in step (d) is screened through affinity sorting in said
step (e).
34. The antibody library of claim 1, further comprising a step (f)
of screening functional antibody and detection antibody.
35. The antibody library of claim 34, wherein said detection
antibody is screened through Western blotting, IP, IF, IHC, flow
cytometry, ELISA, or any combination thereof.
36. The antibody library of claim 34, wherein said functional
antibody is screened through blocking or neutralizing assay.
37. The antibody library of claim 1, wherein said method can be
used to produce detection antibody and/or functional antibody
against more than 90% of proteins of interest.
38. The antibody library of claim 1, which comprises antibodies
against all the proteins of a specie.
39. The antibody library of claim 1, which comprises antibodies
against all the epitopes on the surface of one protein of
interest.
40. The antibody library of claim 1, wherein the antibodies are
monoclonal antibodies.
41. The antibody library of claim 1, wherein said library comprises
antibodies used for detecting the protein of interest in Western
blotting, IP, ELISA, IHC, IF or flow cytometry, and/or antibodies
used for blocking and/or neutralizing the function of the protein
of interest.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a division of U.S. patent
application Ser. No. 13/982,820 filed 31 Jul. 2013, which in turn
is a 35 U.S.C. .sctn.371 National Phase Entry Application of
PCT/CN2012/070768, filed 30 Jan. 2012, designating the United
States, which in turn claims priority to Chinese Patent Application
No. 201110034648.7, filed on 31 Jan. 2011. Each application is
incorporated herein by reference.
SEQUENCE SUBMISSION
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is entitled
2398-114SequenceListing.txt, created on 5 Oct. 2016 and is 68 kb in
size. The information in the electronic format of the Sequence
Listing is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates to a method for the
preparation and/or screening of a specific antibody for a protein
of interest. The present invention specifically relates to a
high-throughput, low-cost method with high success rate, which can
be used for predicting epitopes of a protein of interest at
proteome level, preparing specific antibodies thereof, and
screening and examining the thus produced antibodies.
TECHNICAL BACKGROUND
[0004] The developments of genomic techniques provide a great deal
of basic information at gene level for the investigation of
biological functions, diagnosis and treatment of disease, and
development of medicaments etc. But for most of the biological
systems, the gene information alone is not sufficient for
illustrating the function mechanism thereof. Furthermore, if these
information as well as the function mechanism are to be used for
the purposes of applications, such as identification and analysis
for pathological conditions, and verification and determination for
the function mechanism of medicaments, then it will be necessary to
further obtain more information at the protein level, such as
analysis and investigation for various protein propertied like the
structure, the function, the expression, the localization, the
modification, and the interaction etc.
[0005] The identification for various properties of a protein will
need many technical means, such as MS, chromatography,
electrophoresis, chip, and various labeling techniques. Many of
these technical means will need to be carried out based on
antigen-antibody interactions. Further, antigen-antibody
interaction per se is also an important technical means, which can
be widely used in various fields like scientific researches,
medical treatment, and medicament development, such as the
development of therapeutic antibody medicament etc. With the
development of protein researches, the demand for various
antibodies as well as antibody libraries is increasing. For
example, it could be necessary to prepare a specific antibody
library against all the proteins in the proteome of a specie, or to
prepare specific antibodies against a particular type of proteins
like kinases or G-protein receptors. However, only limited amount
of antibodies against several thousands of proteins are known in
the art, and the specificities thereof are not sufficient for the
requirements of many technical means. Therefore, it would be
important to rapidly and effectively prepare large amount of
antibodies against any protein of interest.
[0006] Currently, the commonly used methods for obtaining
antibodies include hybridoma techniques, recombinant antibody
techniques, various molecular display techniques, and the
combination of these techniques with high-throughput
techniques.
[0007] For the preparation of antibodies, generally a native or
recombinant protein or fragment thereof is used to immunize an
animal, so that an antibody that can specifically recognize and
bind the protein is produced in the animal. Then various technical
means can be used based on corresponding requirements to obtain
antibody from the animal, such as monoclonal antibody or polyclonal
antibody. The production of monoclonal antibody will typically rely
on hybridoma techniques. In such techniques, after immunizing the
animal, the cells of the animal will be taken and fused to generate
an antibody-producing hybridoma, which will then be cloned to
construct a strain for producing antibody, and subsequently the
antibody will be purified and identified. The antigen's epitope for
the antibody can also be further determined according to the
requirements. Such hybridoma techniques for producing monoclonal
antibodies were first applied in mouse model (Kohler and Milstein,
Nature vol. 256, 1975). Currently they are widely used in various
animal models, and the detailed procedure thereof can be seen in
various textbooks and operation manuals (such as, Bazin, "Rat
hybridomas and rat monoclonal antibodies", CRC Press, 1990; Goding,
"Monoclonal antibodies: principles and practice", 3.sup.rd edition,
Academic Press, 1996; Shepherd and Dean "Monoclonal antibodies"
Oxford University Press, 2000 etc.). Although these methods
currently are widely used in the preparations of antibodies, they
also have many disadvantages, such as very long preparation
periods, very complicated preparation techniques, incomplete
recognition of epitopes, and high cost etc. Further, such methods
cannot be used for all the proteins, e.g. for many antigens with
low immunogenicity or antigens with toxicity, such methods would be
inappropriate (Sinclair N R (et al, 2004) B cell/antibody tolerance
to our own antigens. Front Biosci 9: 3019-3028).
[0008] Furthermore, in order to obtain monoclonal antibodies with
specificity, generally, a chemical synthesized peptide fragment is
coupled to a carrier protein, which is then used to immunize a
mouse. Such a method can generate an antibody against a single
epitope of one protein. But due to the differences in the
immunogenicity of different fragments, the overall success rate of
such a strategy is relatively low, and especially for proteins with
high homology, the fragments of which have poor immunogenicity and
can hardly stimulate the mouse to produce potent immune responses.
Another commonly used strategy is to produce the immunogen with
full length protein or protein fragment, which can partially solve
the above problem; but there still exists an disadvantage of poor
overall success rate for protein expression (30-70% for commonly
used expression and purification systems)(Thorsten Kohl, Christian
Schmidt, Stefan Wiemann, Annemarie Poustka and Ulrike Korf. Drew,
2003). For protein fragments with high homology with the proteins
of the animal used as model, the immune responses in the animal are
generally very weak, and thus the success rate for preparing
monoclonal antibody is quite low (Sinclair N R et al, 2004,
Automated production of recombinant human proteins as resource for
proteome research Proteome Science 2008, 6:4; Sinclair N R (2004) B
cell/antibody tolerance to our own antigens. Front Biosci 9:
3019-3028).
[0009] The techniques of recombinant antibody can be various
molecular display techniques, so as to produce antibodies (with
high affinity to the target) against several antigens, and the
antigen epitopes can also be simultaneously determined. Therefore,
they are commonly used in the development of medicaments (Christine
Rothe, Stefanie Urlinger, Makiko Yamashita et al. The Human
Combinatorial Antibody Library HuCAL GOLD Combines Diversification
of All Six CDRs According to the Natural Immune System with a Novel
Display Method for Efficient Selection of High-Affinity Antibodies.
J. Mol. Biol. (2008) 376, 1182-1200, 2007). However, the operation
of the techniques of recombinant antibody is complicated, the cost
thereof is high, and the yield thereof is relatively low. Further,
there often exists non-specific binding. Therefore the large scale
application of such techniques is limited. (Levitan, B. Stochastic
modeling and optimization of phage display. J. Mol. Biol. 277,
893-916 (1998). Bradbury et al, 2004)
[0010] In order to increase the efficiency of immunization and
screening, the above techniques can be combined with
high-throughput methods, such as the high-throughput strategy in
which several immunogens are simultaneously used for the
immunization and chip techniques are used for the screening, e.g.
as described in CN200510026873.0. However, such immunization
strategy will require a great amount of immunogens with high
immunogenicity. This can hardly be accomplished for proteins that
are difficult to be expressed, or for proteins with very low
immunogenicity.
[0011] Furthermore, in conventional immunization methods using
several immunogens, the epitope that the produced antibody is
directed to can only be passively determined based on the
requirements using particular techniques such as epitope mapping
after the antibody is produced (see, Glenn E. Morris, "Methods in
Molecular Biology: Epitope Mapping Protocols", Humana Press, 1996).
Sometimes the epitope is not unique for the protein of interest,
and can present in many other proteins, such that the specificity
of the produced antibody is relatively low.
[0012] In order to solve the various problems mentioned above, new
methods for preparing and screening antibodies are desired, so as
to effectively and rapidly prepare and screen high specific
antibodies against all the proteins with low cost.
SUMMARY OF THE INVENTION
[0013] The present invention provides a method for preparing an
antibody against a protein of interest, the method comprises: (a)
predicting and/or selecting peptide fragment located on the surface
of the protein of interest; (b) synthesizing or expressing one or
more of said peptide fragments; (c) using the product of step (b)
to immunize an animal, optionally in combination with an adjuvant;
(d) using lymphocyte from the immunized animal of step (c) to
obtain antibodies; (e) using the peptide fragment of step (a) or
said protein of interest in native conformation thereof to screen
the antibodies obtained in step (d), so as to obtain an antibody
library against said protein of interest.
[0014] In one embodiment, in the method of the invention, said
peptide fragment of step (a) is predicted or selected through the
following process: (i) determining a surface peptide by calculating
a parameter according to the sequence of the protein of interest,
said parameter is selected from: solvent accessibility, disorder
index, protein-protein interaction domain prediction, or any
combination thereof; (ii) aligning the surface peptide determined
in step (i) with the proteome of the specie that the protein of
interest is originated from, so as to select a specific peptide
fragment of said protein of interest; and (iii) aligning the
surface peptide determined in step (i) with homologous proteins
from other species, so as to screen a conservative sequence of said
protein of interest.
[0015] In one embodiment, said peptide fragment of step (a) in the
method of the invention is a linear surface signature peptide
and/or a conformational surface signature domain of the protein of
interest. In one embodiment, said signature peptide is
characterized in that: it is 6-12 amino acids in length, it is high
hydrophilic, it has high antigenicity, it is not a signal peptide,
it is not located in trans-membrane region but rather in disordered
region. In another embodiment, said signature domain is a sequence
specific protein fragment which is 100-500 amino acids in length,
and which is expected to have 3 dimensional structure.
[0016] In one embodiment, one or more of the peptide fragments of
step (b) in the method of the invention are recombinantly expressed
in the form of a fused protein with a protein which enhances the
immunogenicity and/or increases the copy number. In one embodiment,
the protein which enhances the immunogenicity and/or increases the
copy number is a virus-like particle protein carrier. In one
embodiment, said virus-like particle protein carrier is Hepatitis B
virus nucleocapsid (HBc) protein. In one embodiment, said one or
more peptide fragments are inserted into loop, N-terminus, or
C-terminus of the HBC protein, e.g. the inserted site can be
located between the amino acid residue at position 77 and the amino
acid residue at position 82 of the HBC protein. In one embodiment,
2-10 of the peptide fragments linked by linker are inserted into
the HBC protein. Said linker can be (GGGGS).sub.n, (SEQ ID
NO:158).sub.n, wherein n is any integer, such as 1, 2, 3, or 4.
[0017] In one embodiment, the one or more peptide fragments
expressed in step (b) are further coupled with an immune-enhancing
protein carrier. In one embodiment, said immune-enhancing protein
carrier is keyhole limpet hemocyanin (KLH).
[0018] In one embodiment, the one or more peptide fragments of step
(b) are chemically synthesized.
[0019] In one embodiment, the immunization of step (c) in the
method of the invention is optionally performed in combination with
an adjuvant, e.g. an adjuvant selected from: Freund's complete
adjuvant, aluminum, CpG, or any combination thereof.
[0020] In one embodiment, in said step (c) of the method of in
invention, the animal is immunized at multiple sites, e.g. at least
2 sites selected from: neck and back, tail end, hind foot palm,
hind leg inguen, front leg armpit, hind leg muscle.
[0021] In one embodiment, multiple immunizations are performed,
with the time interval of 2-14 days, such as 3-4 days.
[0022] In one embodiment, the immunization protocol used in step
(c) of the method of the invention comprises the following
steps:
[0023] (A) the first immunization: the expression product of step
(b) together with Freund's complete adjuvant are used to immunize
the animal at neck and back, tail end, hind foot palm, hind leg
inguen, and front leg armpit; and the expression product of step
(b) together with the adjuvant of aluminum+CpG are used to immunize
the animal at hind leg muscle;
[0024] (B) the second immunization: the expression product of step
(b) together with Freund's complete adjuvant are used to immunize
the animal at neck and back, hind leg inguen, and front leg armpit;
and the expression product of step (b) together with the adjuvant
of aluminum+CpG are used to immunize the animal at hind leg
muscle;
[0025] (C) the third immunization: the expression product of step
(b) together with the adjuvant of aluminum+CpG are used to immunize
the animal at hind leg muscle, tail end, and front leg armpit;
and
[0026] (D) the fourth immunization: the expression product of step
(b) together with the adjuvant of aluminum+CpG are used to immunize
the animal at hind leg muscle, tail end, and front leg armpit.
[0027] In one embodiment, the first immunization is performed on
the first day, the second immunization is performed on the fifth
day, the third immunization is performed on the eighth day, and the
fourth immunization is performed on the eleventh day.
[0028] The antibody obtained in step (d) of the method of the
invention is obtained through at least one process selected from:
(1) fusing lymphocyte from the immunized animal of step (c) with
amyeloma cell, so that a hybridoma is generated and then expressed
to obtain the antibody; (2) isolating antigen specific B cell from
lymphocyte of the immunized animal of step (c), and then using PCR
to clone and express the gene of the antibody so as to obtain the
antibody; or (3) isolating mRNA from lymphocyte of the immunized
animal of step (c), and then obtaining the antibody through phage
display, or ribosome display, or yeast display, or bacteria
display, or baculovirus display, or mammal cell display, or mRNA
display.
[0029] In one embodiment, the antibody produced in the method of
the invention is singly IgG subtype.
[0030] In one embodiment, the antibody library produced in step (e)
of the method of the invention is an antibody library against all
the proteins of a specie. In another embodiment, the antibody
library produced in step (e) of the method of the invention
comprises antibodies against all the epitopes of the protein of
interest.
[0031] In one embodiment, the antibodies produced in step (d) are
screened through affinity sorting in step (e) of the method of the
invention.
[0032] The method of the invention can also comprise a further step
(f) of screening functional antibody and detection antibody. For
example, said detection antibody is screened through Western
blotting, IP, IF, IHC, flow cytometry, ELISA, or any combination
thereof; said functional antibody is screened through blocking or
neutralizing assay.
[0033] In another aspect, the invention provides a method for
determining epitopes of a protein of interest, and said method
comprises the step of using the peptide fragment predicted and/or
screened in above mentioned step (a) to construct a detection
antigen, which can be used to screen the produced antibodies so as
to determine the epitopes.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1: the design diagram and nucleotide sequence of the
multiple cloning site. The upper strand sequence is set forth in SQ
ID NO:159, and the lower strand is set forth in SEQ ID NO:160.
[0035] FIG. 2: schematic diagram of artificial HBc. The
.beta.-gal(8) sequence is set forth in SEQ ID NO:161. The L-N
linker is set forth in SEQ ID NO:162. The L-C linker is set forth
in SEQ ID NO:163.
[0036] FIG. 3: the structure of H vector.
[0037] FIG. 4: Western result for the verification of 4A1 antibody.
C2C12 cells were lysed with RIPA buffer (50 mM Tris pH7.4, 150 mM
NaCl, 1% Triton-X-100, 1% sodium deoxycholate, 0.1% SDS lysis
buffer) containing protease inhibitor (Roche), electrophoresed with
SDS-PAGE, and then transferred to PVDF membrane; 1:5 diluted
supernatant of monoclonal antibody cell strain culture was added,
ECL Plus (Amersham) was used for the examination. WB: Western
blotting.
[0038] FIG. 5: IF verification data for 4A1 antibody.
5.times.10.sup.3 BHK cells were inoculated on a cell slide and
cultured at 37.degree. C. overnight, after being fixed with chilled
methanol (-20.degree. C.) for 15 min, washed with 1.times.PBS for
2.times.3 min, and blocked with 1% BSA at room temperature for 1 h,
they were then incubated at room temperature for 1 hour with the
addition of 1:1 supernatant of monoclonal antibody cell strain
culture, and washed; they were incubated at room temperature for 1
hour with the addition of secondary antibody anti-mouse Dylight 549
(Abcam) at 1:400 and DAPI at 1:4000, washed, and then examined with
fluorescence microscope (Olympus) with a picture being taken.
[0039] FIG. 6: Western result for the verification of the antibody
1A6 produced against GPR116 protein. WB: Western blotting.
[0040] FIG. 7: Western result for the verification of the antibody
2F1 produced against Aof1 protein. WB: Western blotting
DETAILED DESCRIPTION OF THE INVENTION
[0041] Unless otherwise specified, all the technical and scientific
terms have common meanings known to a person skilled in the art.
The patents, patent applications, publications, GenBank sequences,
websites and other published material are all incorporated herein
as references in their entirety, unless otherwise specified.
[0042] The present invention provides a method for producing an
antibody of a protein of interest, which can be used to efficiently
and rapidly prepare highly specific antibodies of the protein with
low-cost, so as to construct a epitope library encompassing the
surface epitopes of a protein of interest and an antibody library
against all the epitopes. The method also provides a method for
indicating the specific epitope that an antibody is directed
to.
[0043] The present invention provides a method for preparing an
antibody against a protein of interest, the method comprises: (a)
predicting and/or selecting peptide fragment located on the surface
of the protein of interest; (b) synthesizing or expressing one or
more of said peptide fragments; (c) using the product of step (b)
to immunize an animal, optionally in combination with an adjuvant;
(d) using lymphocyte from the immunized animal of step (c) to
obtain antibodies; (e) using the peptide fragment of step (a) or
said protein of interest in native conformation thereof to screen
the antibodies obtained in step (d), so as to obtain an antibody
library against said protein of interest.
[0044] As used herein, the term "a protein of interest" refers to
any native protein, or an isoform of a native protein obtained
through alternative splicing, or a mutant of a native protein, or
any combination of the various proteins mentioned above.
"Alternative splicing" as used herein refers to a process of
producing different mRNA splicing isomers from a same mRNA
precursor through different splicing manners (i.e. combining exons
through different splicing sites). The protein products obtained
through alternative splicing are isoforms to each other, which may
exhibit different functional and structural properties, or may
result in different phenotypes due to different expression levels
in a same cell. "Mutant" as used herein refers to a mutated protein
obtained through substitution, deletion, or addition of one or more
amino acids in a native protein.
[0045] The term "peptide fragment" as used herein refers to a
continuous or incontinuous amino acids sequence in the protein of
interest, which can be a continuous linear polypeptide, and which
can also be a combination of incontinuous polypeptides constituting
the domain conformation.
[0046] In one embodiment of the invention, said peptide fragment of
step (a) is predicted or selected through the following process:
(i) determining a surface peptide by calculating a parameter
according to the sequence of the protein of interest, said
parameter is selected from: solvent accessibility, disorder index,
protein-protein interaction domain prediction, or any combination
thereof; (ii) aligning the surface peptide determined in step (i)
with the proteome of the specie that the protein of interest is
originated from, so as to select a specific peptide fragment of
said protein of interest; and (iii) aligning the surface peptide
determined in step (i) with homologous proteins from other species,
so as to screen a conservative sequence of said protein of
interest. In one embodiment, said peptide fragment in step (a) is
linear surface signature peptide and/or a conformational surface
signature domain of the protein of interest.
[0047] The term "surface peptide" as used herein refers to linear
peptide fragment located on the surface of the protein of interest,
and/or combination of incontinuous polypeptides located on the
surface of the protein of interest that constitute the domain
conformation. The term "signature peptide" as used herein refers to
a unique linear continuous peptide sequence in the protein of
interest when compared to other protein sequences in the proteome
of the same specie. The term "signature domain" as used herein
refers to a unique domain in the protein of interest when compared
to the domains of other protein in the proteome of the same specie.
The term "surface signature peptide" and "surface signature domain"
respectively refer to "signature peptide" and "signature domain"
located on the surface of the native conformation of a protein.
[0048] The term "solvent accessibility" refers to an indicator
representing the degree that an amino acids within a protein
exposes to the conformational surface of the protein (see, Bent
Petersen, Thomas Nordahl Petersen, Pernille Andersen Morten Nielsen
and Claus Lundegaard. A generic method for assignment of
reliability scores applied to solvent accessibility predictions.
BMC Structural Biology 2009, 9:51).
[0049] The term "disorder index" used herein is a parameter
representing the complexity of amino acids (see, Predrag Radivojac,
Lilia M. Iakoucheva, Christopher J. Oldfield, Zoran Obradovic,
Vladimir N. Uversky, and A. Keith Dunker. Intrinsic Disorder and
Functional Proteomics. Biophysical Journal Volume 92, 1439-1456
(2007)).
[0050] As used herein, "protein-protein interaction domain
prediction" means that, in protein-protein interaction, some
domains play a key role in the interaction, and binding manner of
two proteins can be predicted by analyzing the amino acid
composition of the proteins. Typical software such as Autodock can
be used (see, Bin Liu, Xiaolong Wang, Lei Lin, Buzhou Tang, Qiwen
Dong and Xuan Wang. Prediction of protein binding sites in protein
structures using hidden Markov support vector machine. BMC
Bioinformatics 2009, 10:381).
[0051] As used herein, the term "proteome" refers to the collection
of all the proteins expressed by the genome of a certain specie, or
by a certain cell or tissue or cell.
[0052] The sequence alignment in the invention can be performed
using various methods known in the art, such as using various
conventional softwares or online services, e.g. BLASTP as provided
by NCBI.
[0053] The prediction and/or selection in the invention mean that:
based on bioinformatics methods, through secondary structure
prediction, comparison in the same genome, structural prediction of
homologous proteins, signature peptides and/or signature domains
are first defined for a certain protein as potential epitopes,
wherein said signature peptides and/or signature domains have
protein specificity and can cover relatively large portion of the
protein.
[0054] Such bioinformatics methods are widely used for the
prediction of protein structures, functions and interactions. For
example, Berglund L. et al. (see, Protein Science, 17:606-613,
2008) introduced a method for screening specific epitopes at
proteome level against the entire human proteome, wherein based on
sequence alignment for sliding windows of 8, 10, 12 amino acid
residues, heuristic processes are used to predict the sequence
identity of every human protein against the entire human proteome.
Based on such method, at least one specific epitop can be found for
90% of human protein. Anderson H P et al. (see, Protein Science,
15:2558-2567, 2006) introduced method DiscoTope for predicting
incontinuous epitopes using 3D structural data. This method
assesses amino acids statistics, spatial information, and surface
accessibility based on incontinuous epitopes determined in X-ray
structure of antigen-antibody protein complex. Yan C H et al. (see,
BMC Bioinformatics, 7:262, 2006) introduces a method for predicting
potential DNA-biding sites through amino acid sequence, which uses
Naive Bayes classifier to predict whether a certain amino acid
sequence is a DNA-binding site.
[0055] In one embodiment, the signature peptide of the invention is
6-12 amino acids in length, it is high hydrophilic, it has high
antigenicity, it is not a signal peptide, and it is not located in
trans-membrane region but rather in disordered region. In one
embodiment, the signature domain of the invention is a sequence
specific protein fragment which is 100-500 amino acids in length,
and which is expected to have 3 dimensional structure.
[0056] In one embodiment, in step (a) of the method of the
invention, several peptide fragments located on the surface of the
protein of interest are predicted and/or selected, e.g. 2, 3, 4, 5,
6, 7, 8, 9, 10 peptide fragments, until all the peptide fragments
located on the surface of the protein of interest that can be used
as potential epitopes are predicted and/or selected.
[0057] In one embodiment, said several peptide fragments are
designed as one tandem polypeptide of immunization antigens. For
example, said tandem polypeptide of immunizing antigens can be
designed as follows: predicting immunizing parameters of the
protein of interest, such as antigenicity, hydrophilicity,
accessibility, isoelectric point, predicting the signaling, and
predicting the trans-membrane region; the setting rules for
parameters like specificity can be seen in Kolaskar A S et al. FEBS
Lett. 276(1-2):172-4, 1990; Parker J M et al., Biochemistry.
25(19):5425-32, 1986; Emini E A et al., J Virol. 55(3): 836-9,
1985. For any protein of interest, the prediction results for
isoelectric point and signal-peptide are first used to exclude
these regions in the protein. For example, a sliding window with
the length of 5-20 amino acids can be set, and then sequentially
moved for one amino acid along the protein. The sequences of the
sliding windows are added into a collection. As an example, for a
protein with the length of n amino acids (suppose it has no
signal-peptide or trans-membrane region), all together n-9 short
peptide sequences can be generated (the length of the sliding
window is 10 amino acids). For every short peptide, the isoelectric
point, accessibility, immunogenicity, hydrophilicity, and specie
specificity according to BLAST methods can be calculated (see,
Kolaskar A S et al., FEBS Lett. 276(1-2):172-4, 1990; Parker J M et
al., Biochemistry. 25(19):5425-32, 1986; Emini E A et al., J Virol.
55(3): 836-9, 1985). When the isoelectric point is larger than 3.5,
the weighted average value of the above parameters are calculated
for every short peptide
(0.2*immunogenicity+0.1*accessibility+0.2*hydrophilicity+0.5*specie
specificity). These short peptides are sorted according to the
weighted average value, and the short peptides having the highest
score and with an inter-peptide overlap <3 are selected, e.g. 7
short peptides are selected.
[0058] The peptide fragment of the invention can be obtained by any
known techniques in the art, e.g. by recombinant expression or by
chemical synthesis. That is, the peptide fragment of the invention
can be expressed in prokaryotic or eukaryotic expression systems
using recombinant expression methods known in the art, or the
peptide fragment can be synthesized using conventional chemical
methods.
[0059] In one example, e.g. for a protein the expression and
purification of which are difficult, or for a protein which has
very low immunogenicity, in order to prepare specific antibodies,
expression vectors with high immunogenicity and soluble protein
expression systems with high success rate can be used to facilitate
the production of these proteins as potential antigens. Virus-like
particle protein carrier Hepatitis B virus nucleocapsid (HBc)
protein can be mentioned as an example.
[0060] HBC protein is a non-infectious virus-like particle carrier
that can carry peptides of various sources. It can be used to
combine target peptide so as to generate high-titer antibody
response, and thus is widely used in vaccine preparation. HBC
protein can carry foreign amino acids at particular sites such as
loops, N-terminus or C-terminus, and a foreign sequence of up to
238amino acids can be displayed on the surface of the protein so as
to promote the generation of immune-response. Detailed description
of HBC carrier can be seen in: Good M F et al., Science,
235:1059-1063, 1987; Pumpens P and Grens E, Intervirology,
44:98-114, 2001; Clarke B E et al., Nature 330:381-384, 1987;
Francis M J et al., Nature 330:168-170, 1987; Whitacre D C et al.,
Expert Rev. Vaccines 8:11 1565-1573, 2009.
[0061] In one embodiment, the one or more peptide fragments in step
(b) of the method of the invention are inserted into loop,
N-terminus, or C-terminus of the HBC protein, e.g. into a position
between the amino acid residue at position 77 and the amino acid
residue at position 82 of the HBC protein. 2-10 of the peptide
fragments of the invention can also be linked by linker and then
inserted into the HBC protein. Said linker can be any linker in the
art, e.g. (GGGGS).sub.n, wherein n is any integer, such as 1, 2, 3,
or 4.
[0062] For instance, in one example, N (5.ltoreq.N.ltoreq.20)
peptide fragments with relatively high immunogenicity and
specificity are selected, and the lengths thereof may be M
(5.ltoreq.M.ltoreq.20) amino acids. In order to avoid the
generation of new antigen epitope between different peptide
fragments, one or more linkers GGGGS with low immunogenicity are
inserted between different peptide fragments, so as to connect said
peptide fragments in tandem. For different lengths of proteins,
different lengths (5.ltoreq.M.ltoreq.20) of the peptide fragment
can be selected. The number of the inserted fragments can be
between 5-20, the length of the peptide fragment can be, e.g. 6-12
amino acids, and the number of the fragments can be, e.g. less than
10.
[0063] In an embodiment of the invention, the one or more peptide
fragments expressed in step (b) can also be further coupled with an
immune-enhancing protein carrier, and said immune-enhancing protein
carrier can be, e.g. keyhole limpet hemocyanin (KLH). KLH protein
is an oxygen-carrying metalloprotein originated from Megathura
crenulata, which is commonly used as carrier protein for generating
antibodies. The many epitopes contained therein and its differences
with mammal-derived protein make KLH a good carrier protein for
generating antibodies. The applications of KLH protein can be seen
in Harris J R et al., Micron. 30(6):597-623, 1999 and WO
2001/047552.
[0064] The immunization of animal in step (c) can be conducted
using any methods known in the art. The animal used for
immunization in the invention can be conventional animals in the
art, e.g. mouse, rat, rabbit, sheep, goat, horse, cattle etc.
[0065] In one embodiment, an appropriate adjuvant can be used, so
as to rapidly and effectively obtain antibodies of a single subtype
and with high titer. In one embodiment, the antibodies obtained
through the method of the invention can be antibodies mainly
comprising single IgG subtype. The adjuvant used in the invention
can be selected from: Freund's complete adjuvant, aluminum, CpG, or
any combination thereof.
[0066] In one embodiment, the method of the invention comprises
multiple immunizations in an animal. The specific protocol of using
multiple sites immunization strategy for rapid production of
antibodies with high affinity can be seen in Kilpatrick K E et al.,
(Hybridoma 16:(4) 381-389, 1997). The protocol for adjusting the
affinity and subtype of antibodies can be seen in Karagouni L et
al., Scand. J. Immunol. 31:745-754, 1990, and Petty R E et al.,
Immunology 32:49-55, 1977.
[0067] The multiple immunizations in an animal of the invention can
be conducted at at least 2 sites selected from: neck and back, tail
end, hind foot palm, hind leg inguen, front leg armpit, hind leg
muscle.
[0068] In one embodiment of the invention, multiple immunizations
are conducted in an animal. The time interval between multiple
immunizations can be determined based on common technical knowledge
in the art, e.g. 2-14 days, such as 3 or 4 days.
[0069] In one embodiment, the immunization in step (c) of the
method of the invention comprises the following steps:
[0070] (A) the first immunization: the expression product of step
(b) together with Freund's complete adjuvant are used to immunize
the animal at neck and back, tail end, hind foot palm, hind leg
inguen, and front leg armpit; and the expression product of step
(b) together with the adjuvant of aluminum+CpG are used to immunize
the animal at hind leg muscle;
[0071] (B) the second immunization: the expression product of step
(b) together with Freund's complete adjuvant are used to immunize
the animal at neck and back, hind leg inguen, and front leg armpit;
and the expression product of step (b) together with the adjuvant
of aluminum+CpG are used to immunize the animal at hind leg
muscle;
[0072] (C) the third immunization: the expression product of step
(b) together with the adjuvant of aluminum+CpG are used to immunize
the animal at hind leg muscle, tail end, and front leg armpit;
and
[0073] (D) the fourth immunization: the expression product of step
(b) together with the adjuvant of aluminum+CpG are used to immunize
the animal at hind leg muscle, tail end, and front leg armpit.
[0074] In a more specific embodiment, the immunization in step (c)
of the method of the invention comprises the following steps:
[0075] (A) the first immunization: 20 .mu.g of the expression
product of step (b) in 250 .mu.l buffer together with 250 .mu.l
Freund's complete adjuvant are used to immunize the animal at neck
and back, tail end, hind foot palm, hind leg inguen, and front leg
armpit; and 20 .mu.g of the expression product of step (b) in 500
.mu.l buffer together with the adjuvant of 25 .mu.l aluminum+10
.mu.g CpG are used to immunize the animal at hind leg muscle;
[0076] (B) the second immunization: 10 .mu.g of the expression
product of step (b) in 250 .mu.l buffer together with 250 .mu.l
Freund's complete adjuvant are used to immunize the animal at neck
and back, hind leg inguen, and front leg armpit; and 10 .mu.g of
the expression product of step (b) in 500 .mu.l buffer together
with the adjuvant of 25 .mu.l aluminum+10 .mu.g CpG are used to
immunize the animal at hind leg muscle;
[0077] (C) the third immunization: 10 .mu.g of the expression
product of step (b) in 500 .mu.l buffer together with the adjuvant
of 25 .mu.l aluminum+10 .mu.g CpG are used to immunize the animal
at hind leg muscle, tail end, and front leg armpit; and
[0078] (D) the fourth immunization: 10 .mu.g of the expression
product of step (b) in 500 .mu.l buffer together with the adjuvant
of 25 .mu.l aluminum+10 .mu.g CpG are used to immunize the animal
at hind leg muscle, tail end, and front leg armpit.
[0079] In one embodiment, the first immunization is performed on
the first day, the second immunization is performed on the fifth
day, the third immunization is performed on the eighth day, the
fourth immunization is performed on the eleventh day, and the
fusion is performed on the fourteenth day. The obtained antibodies
are mostly IgG subtype with matured affinity. Antibodies of IgG
subtype have the advantages of higher affinity, better stability
and easier for purification as compared to IgM.
[0080] The antibody obtained from lymphocyte of the immunized
animal in step (d) of the method of the invention can be obtained
through any methods known in the art. As used herein, the term
"lymphocyte" refers to a cell of lymphoid organs or a cell produced
in lymphoid organs, and the lymphoid organs include central
lymphoid organs (also referred to as primary lymphoid organ) and
peripheral lymphoid organ (also referred to as secondary lymphoid
organ). The former includes thymus, bursa of fabricius and
equivalent organs thereof (such as marrow of mammals), and the
latter includes spleen and lymph nodes etc.
[0081] In one embodiment, the antibody is obtained through at least
one process selected from: (1) fusing lymphocyte from the immunized
animal of step (c) with amyeloma cell, so that a hybridoma is
generated and then expressed to obtain the antibody; (2) isolating
antigen specific B cell from lymphocyte of the immunized animal of
step (c), and then using PCR to clone and express the gene of the
antibody so as to obtain the antibody; or (3) isolating mRNA from
lymphocyte of the immunized animal of step (c), and then obtaining
the antibody through phage display, or ribosome display, or yeast
display, or bacteria display, or Baculovirus display, or mammal
cell display, or mRNA display.
[0082] Detailed introductions for hybridoma technique can be seen
in Bazin, "Rat hybridomas and rat monoclonal antibodies", CRC
Press, 1990; Goding, "Monoclonal antibodies: principles and
practice", 3.sup.rd edition, Academic Press, 1996; Shepherd and
Dean "Monoclonal antibodies" Oxford University Press, 2000,
etc.
[0083] In step (d) of the method of the invention, the antibody can
also be obtained through phage display, or ribosome display, or
yeast display, or bacteria display, or Baculovirus display, or
mammal cell display, or mRNA display. These methods are all
conventional techniques in the art, the specific operations thereof
can be seen in corresponding textbooks or operation manuals, see,
e.g. Mondon P et al., Front. Biosci. 13:1117-1129, 2008. Using
phage display as an example, separate antibody genes are inserted
into the DNA of phage, so that the variable regions on the antibody
molecules that can bind the antigens are coupled to the capsid
protein of phage. After the phage infecting E. coli., single
stranded DNA is replicated in E. coli., and the phage is
reassembled and secreted into the culture medium, while the E.
coli. is not lysed. The phage is co-incubated with target antigens;
and after the bound phages are isolated, amplification and
purification are then conducted so that a great amount of clones
can be screened. The phage display technique can be seen in Liu, J.
et al., Chin. J. Cell Mol. Immunol., 2004: 20(6) 773-775;
CN03131796.0; WO 2009/109572, and WO 2009/085462.
[0084] The antibody produced in the method of the invention can be
monoclonal antibody, and can also be polyclonal antibody. In one
embodiment, the invention involves an antibody library screened and
prepared using the method of the invention against all the proteins
of interest of one specie. In another embodiment, the invention
involves an antibody library screened and prepared using the method
of the invention against all the epitopes of one protein of
interest.
[0085] As used herein, the terms "antibody", "monoclonal antibody",
"polyclonal antibody", "epitope" are common term in the art, the
meanings of which are in accordance with the general understanding
of a person skilled in the art and can also be referred to common
textbooks and manuals.
[0086] As used herein, the term "antibody library" refers to a
collection of antibodies comprising many different antibodies. An
antibody library can comprise antibodies against several different
proteins, and it can also comprise antibodies against different
epitopes of a same protein.
[0087] The "screen" used in step (e) of method of the invention
means using the peptide fragment of step (a) or the protein of
interest in its native conformation to screen the antibodies
obtained in step (d). In one embodiment, the antibodies produced in
step (d) is screened through affinity sorting in step (e) of the
method of the invention. The identification method for an antibody
can be seen in Griswold W R et al., Immunology letters, 1984:
229-232; Van Heyningen V et al., Journal of Immunological Methods,
62: 147-153, 1983; and Rath S et al., Journal of Immunological
Methods, 106: 245-249, 1988.
[0088] In one embodiment, the method of the invention can also
comprise a further step (f) of screening functional antibody and
detection antibody. For example, said detection antibody is
screened through Western blotting, IP, IF, IHC, flow cytometry,
ELISA, or any combination thereof; or said functional antibody is
screened through blocking or neutralizing assay
[0089] The detection antibody as used herein refers to an antibody
that can react with an antigen and can then be detected through
techniques in the art, such as Western blotting, IP, IF, IHC, flow
cytometry, or ELISA etc.
[0090] The functional antibody as used herein refers to an antibody
that can react with an antigen and can then affect (e.g. block or
neutralize) a biological function of the antigen.
[0091] As an example, antibodies against the protein of interest
are screened using the following method: the protein of interest or
fragment thereof is biotinylated, and then over-expressed in
eukaryotic cells like 293 cells; the over-expressed biotinylated
protein or fragment thereof is added into a plate coated with
streptavidin; then the antibodies obtained in step (e) of the
invention are added for ELISA assay, so as to obtain antibodies
with positive reaction.
[0092] One monoclonal antibody 4A1 of the invention is produced by
hybridoma strain 4A1, and said hybridoma strain 4A1 was deposited
in China Center for Type Culture Collection (CCTCC) on Jan. 28,
2011, with the deposition No. CCTCC C201107. Another monoclonal
antibody 1A6 of the invention is produced by hybridoma strain 1A6,
and said hybridoma strain 1A6 was deposited in China Center for
Type Culture Collection (CCTCC) on Jan. 28, 2011, with the
deposition No. CCTCC C201108. One monoclonal antibody 2F1 of the
invention is produced by hybridoma strain 2F1, and said hybridoma
strain 2F1 was deposited in China Center for Type Culture
Collection (CCTCC) on Jan. 28, 2011, with the deposition No. CCTCC
C201109.
[0093] In another aspect, the present invention provides an
antibody library produced according to the method of the invention.
In one embodiment, said antibody library comprises antibodies
against all the proteins of interest. In one embodiment, said
antibody library comprise antibodies against all the epitopes on
the surface of one protein of interest. In one embodiment, said
antibodies in the library are monoclonal antibodies.
[0094] In another aspect, the present invention also provides a
method for determining the epitope that the produced antibody is
directed to, comprising the step of using the peptide fragments
predicted and/or screened in above mentioned step (a) to construct
a detection antigen, which can be used to screen the produced
antibodies so as to determine the epitopes.
[0095] As used herein, the term "detection antigen" refers to a
fused protein constructed using the peptide fragments predicted
and/or screened in above steps. Said detection antigen can be used
for conducting screening against several proteins, wherein it
contains one epitope for each of the protein to be screened.
[0096] In one embodiment, the present invention utilizes a strategy
that can conducts a screening against N proteins, and the number of
epitopes and the number of proteins designed for each protein can
all be N (5.ltoreq.N.ltoreq.20), with the prerequisite that number
of epitopes selected for each protein is identical.
[0097] As an example, for 5 proteins, 5 antigen epitopes are
determined for each protein during the design of the immunization
antigens, which are represented by A, B, C, D, E respectively (see
table 1). For example, the epitopes for immunization antigen are
A1, B1, C1, D1, and E1 respectively. The number of epitopes and the
number of proteins here can both be N (5.ltoreq.N.ltoreq.20).
[0098] The detection antigen for 5 proteins uses the five
polypeptide epitopes in one column as one new protein sequence (see
table 1, e.g., detection antigen A comprises A1, A2, A3, A4, and
A5).
[0099] During the screening process, every fused positive cloning
well (obtained by screening of immunization antigens), is
respectively screened using 5 detection antigens, and through
typical ELISA screening, so as to determine which polypeptide
epitope each positive clone is directed to. Based on such results,
positive cells directed to each epitopes are preferably selected
for strain construction and subsequent identification, so as to
avoid the situation when the epitopes are unknown and the positive
clones are mostly directed to a certain most advantageous epitope
so that the obtained cell strains are homogeneous.
TABLE-US-00001 TABLE 1 The design method for immunization antigen
detec- detec- detec- detec- detec- tion tion tion tion tion antigen
antigen antigen antigen antigen A B C D E immunization antigen 1 A1
B1 C1 D1 E1 immunization antigen 2 A2 B2 C2 D2 E2 immunization
antigen 3 A3 B3 C3 D3 E3 immunization antigen 4 A4 B4 C4 D4 E4
immunization antigen 5 A5 B5 C5 D5 E5
[0100] In summary, the method of the invention uses bioinformatics
techniques to predict and/or select peptide fragments on the
surface of a protein of interest as potential epitopes. The method
of the invention can be used to effectively and rapidly obtain
antibodies against a protein of interest with low cost, and the
method of the invention can be used to obtain all the antibodies
against epitopes on the surface of the native conformation of a
protein of interest. Furthermore, the method of the invention can
also use combination screening to determine epitopes on the surface
of the native conformation of a protein of interest through,
identify the particular epitope that an antibody is directed to,
and further examine and screen the produced antibodies.
[0101] The several key factors for successful applications of an
antibody include the recognition site of the antibody (epitope),
affinity and specificity. As for monoclonal antibody, when the
epitopes are unknown, the obtained antibodies are generally focused
on a certain epitope with advantageous immunogenicity. The unity of
epitopes can result in failure when applying an antibody, which is
mainly related to the position of the epitope. In the case when the
epitopes that the antibodies recognize are known, then antibodies
of several epitopes are preferably obtained, so that the influences
of incorrect epitope position can be decreased, and the success
rate of cell strain applications can be greatly increased.
[0102] The advantage of the invention lies in the preparation of
large amount of antibodies. The method has the propertied of high
success rate (antibodies successfully used in Western applications
can be obtained for >90% of the proteins), and low-cost. The
success rates for proteins with high homology, receptor protein
domains, and proteins of mouse are all very high. The significance
of preparing antibodies against mouse proteins is in that, the
functional antibodies screened therefrom, can be used for antibody
treatment experiments in mouse models, which is important for
clinical researches. As for membrane receptor proteins, when using
functional domains to prepare antibodies, it will be more
advantageous in obtaining antibodies with blocking functions, and
benefits the developments of antibody medicaments. The antibody
library constructed using the method of the invention can be
further used for developing functional antibodies. Furthermore, the
high-throughput epitope screening strategy ensures that the
corresponding epitope recognition information is specifically clear
for every cell strain obtained, which is important both for
investigating antibody-protein interactions and for using several
epitopes to determine the expression information of a certain
protein.
[0103] The invention will be further illustrated using the
following specific examples. The following examples are only used
for describing the invention, with no intention of limiting the
present invention. Various modifications and alterations may be
made to the invention by one skilled in the art without departing
from the spirit and scope of the invention, and such modifications
and alterations are also encompassed in the protection scope as
defined in the appended claims.
EXAMPLES
Example 1
Modification of Vectors
A the Design for HBC Vector Modification
[0104] First a segment of designed multiple cloning site (MCS:
GGATCCTATCAGATCTATCGGGTACCGTATCGCGGCCGCTTCCAT ATGG AA TTC (SEQ ID
NO: 1)) was used to replace the c/e1 loop of the cDNA for Hepatitis
B virus nucleocapsid protein (HBc), i.e. the nucleotides encoding
the amino acids at positions 76-82 of HBc protein. The schematic
diagram and sequence of said MCS can be seen in FIG. 1. Then, the
nucleotide sequences encoding the two linkers L-N and L-C were
linked to the two ends of said MCS, and the nucleotides encoding
amino acids at position 76-82 of HBc protein were replaced, wherein
in the two ends of the two linkers L-N and L-C, the E represented
glutamic acid, G represented glycin, and S represented serine.
Subsequently, the nucleotide sequences encoding the sequence
elements 6.times.His tag (HHHHHH; SEQ ID NO:164), .beta.-gal
(MTMITDSL; SEQ ID NO:161), and linker (EFH) were added to in front
of the cDNA. The nucleotide sequence of the modified HBc is shown
in FIG. 2.
B Complete Gene Synthesis
[0105] DNA Works software (obtained from http colon slash slash
helixweb dot nih dot gov slaxh dnaworks/) was used to design HBc
nucleotide sequence through codon optimization, as shown below:
TABLE-US-00002 (SEQ ID NO: 2)
CCATGGGCAGCAGCCACCATCATCACCACCACATGACCATGATCACCGAT
AGCCTGGAGTTCCATATCGATCCGTACAAGGAATTTGGCGCGACCGTGGA
ACTGCTGAGCTTCCTGCCGAGCGACTTTTTTCCAAGCGTGCGTGACCTGC
TGGATACGGCGAGCGCACTGTATCGTGAAGCGCTGGAAAGCCCGGAACAT
TGCAGCCCGCATCATACCGCGCTGCGTCAGGCGATTCTGTGCTGGGGCGA
ACTGATGACCCTGGCGACCTGGGTGGGCGGCAATGAAGAAGGTGGTGGCG
GTAGCGGCGGTGGCGGATCCTATCAGATCTATCGGGTACCGTATCGCGGC
CGCTTCCATATGGAATTCGGTGGCGGCGGCAGCGGCGGTGGTGGCAGCGA
AGAAGACCTGGTTGTGAGCTATGTGAACACCAATATGGGCCTGAAGTTTC
GTCAGCTGCTGTGGTTTCATATTAGCTGCCTGACCTTTGGCCGCGAAACC
GTGATTGAATACCTGGTGAGCTTTGGCGTGTGGATTCGTACCCCACCGGC
GTATCGTCCGCCGAATGCGCCAATTCTGAGCACCCTGCCGGAAACGACCG
TTTAAGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAG
[0106] The optimized HBC nucleotide sequence was synthesized using
the following primers as shown in table 2, and primers as shown
were synthesized by SBSGENE co. ltd. (Shanghai, China).
TABLE-US-00003 TABLE 2 The primer sequences for complete gene
synthesis Name of primers Sequences of the primers (5'-3') H_01
CATGCCATGGGCAGCAGCCACCATCATCACCACCACATGA CC (SEQ ID NO: 3) H_02
CTCCAGGCTATCGGTGATCATGGTCATGTGGTGGTGATGA (SEQ ID NO: 4) H_03
TGATCACCGATAGCCTGGAGTTCCATATCGATCCGTACAA GG (SEQ ID NO: 5) H_04
CCACGGTCGCGCCAAATTCCTTGTACGGATCGATATGGAA (SEQ ID NO: 6) H_05
TGGCGCGACCGTGGAACTGCTGAGCTTCCTGCCGAGCGAC (SEQ ID NO: 7) H_06
CAGGTCACGCACGCTTGGAAAAAAGTCGCTCGGCAGGAAG (SEQ ID NO: 8) H_07
CAAGCGTGCGTGACCTGCTGGATACGGCGAGCGCACTGTA (SEQ ID NO: 9) H_08
TCCGGGCTTTCCAGCGCTTCACGATACAGTGCGCTCGCCG (SEQ ID NO: 10) H_09
CGCTGGAAAGCCCGGAACATTGCAGCCCGCATCATACCGC (SEQ ID NO: 11) H_10
CAGCACAGAATCGCCTGACGCAGCGCGGTATGATGCGGGC (SEQ ID NO: 12) H_11
GTCAGGCGATTCTGTGCTGGGGCGAACTGATGACCCTGGC (SEQ ID NO: 13) H_12
TTCATTGCCGCCCACCCAGGTCGCCAGGGTCATCAGTTCG (SEQ ID NO: 14) H_13
GGTGGGCGGCAATGAAGAAGGTGGTGGCGGTAGCGGCGGT (SEQ ID NO: 15) H_14
ACCCGATAGATCTGATAGGATCCGCCACCGCCGCTACCGC (SEQ ID NO: 16) H_15
GGATCCTATCAGATCTATCGGGTACCGTATCGCGGCCGCT (SEQ ID NO: 17) H_16
CCGCCGCCACCGAATTCCATATGGAAGCGGCCGCGATACG (SEQ ID NO: 18) H_17
ATTCGGTGGCGGCGGCAGCGGCGGTGGTGGCAGCGAAGAA (SEQ ID NO: 19) H_18
TCACATAGCTCACAACCAGGTCTTCTTCGCTGCCACCACC (SEQ ID NO: 20) H_19
CCTGGTTGTGAGCTATGTGAACACCAATATGGGCCTGAAG (SEQ ID NO: 21) H_20
AACCACAGCAGCTGACGAAACTTCAGGCCCATATTGGTGT (SEQ ID NO: 22) H_21
TCGTCAGCTGCTGTGGTTTCATATTAGCTGCCTGACCTTT (SEQ ID NO: 23) H_22
AATCACGGTTTCGCGGCCAAAGGTCAGGCAGCTAATATGA (SEQ ID NO: 24) H_23
GCCGCGAAACCGTGATTGAATACCTGGTGAGCTTTGGCGT (SEQ ID NO: 25) H_24
CGCCGGTGGGGTACGAATCCACACGCCAAAGCTCACCAGG (SEQ ID NO: 26) H_25
CGTACCCCACCGGCGTATCGTCCGCCGAATGCGCCAATTC (SEQ ID NO: 27) H_26
GTCGTTTCCGGCAGGGTGCTCAGAATTGGCGCATTCGGCG (SEQ ID NO: 28) H_27
ACCCTGCCGGAAACGACCGTTTAAGAGCTCCGTCGACAAG (SEQ ID NO: 29) H_28
CCGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCTT AAA (SEQ ID NO: 30)
[0107] 5 .mu.l of each of the synthesized primers H_01 to H_28 were
taken (with the concentration of 25 .mu.M/L) and mixed. Such mixed
primers were used as template, and the first round of PCR
amplification was conducted under the catalysis of PFU enzyme
(ShenNengBoCai co. ltd., Shanghai, China). The PCR conditions were
as: 94.degree. C., 2 min; 30 cycles (94.degree. C., 30 s;
60.degree. C., 30 s; 72.degree. C., 1 min); 72.degree. C., 10 min;
10.degree. C., 10 min. 2 .mu.l of the PCR product of the first
round was then taken as template, and primers H_01 and H_28 (with
the concentration of 25 .mu.M/L) were used for conducting the
amplification, with the amplification conditions as set forth
above. 1.5% agarose gel electrophoresis was performed, and the
target fragments (having the fragment of the above mentioned
optimized HBC nucleotide sequence) were recovered.
[0108] The above obtained HBc DNA was digested by NcoI/XhoI (NEB,
MA) for 4 h. The digested products were recovered (DNA recovery kit
obtained from TIANGEN Biotech (Beijing) co., ltd), ligated onto
digested pET28a vector (Novagen, Merck, Germany) by T4 ligase (NEB,
MA), and then transformed into TOP10 competent cells. After the
clones grown the next day were verified by colony PCR and
BamHI/EcoRI (NEB, MA) digestion, 3 of the positive clones were
picked for sequencing, and the sequencing primers were T7 promoter
primers (Biosune, co. ltd. Shanghai, China). The new vector that
was sequenced and successfully identified was named as H vector,
the structure of which is shown in FIG. 3.
Example 2
The Design, Expression and Purification of Desmin Immunization
Antigen
[0109] The Desmin protein has an identity of 97% with corresponding
mouse protein, and the full length amino acid sequence of Desmin
is:
TABLE-US-00004 (SEQ ID NO: 31) 1 MSQAYSSSQR VSSYRRTFGG APGFPLGSPL
SSPVFPRAGF GSKGSSSSVT SRVYQVSRTS 61 GGAGGLGSLR ASRLGTTRTP
SSYGAGELLD FSLADAVNQE FLTTRTNEKV ELQELNDRFA 121 NYIEKVRFLE
QQNAALAAEV NRLKGREPTR VAELYEEELR ELRRQVEVLT NQRARVDVER 181
DNLLDDLQRL KAKLQEEIQL KEEAENNLAA FRADVDAATL ARIDLERRIE SLNEEIAFLK
241 KVHEEEIREL QAQLQEQQVQ VEMDMSKPDL TAALRDIRAQ YETIAAKNIS
EAEEWYKSKV 301 SDLTQAANKN NDALRQAKQE MMEYRHQIQS YTCEIDALKG
TNDSLMRQMR ELEDRFASEA 361 SGYQDNIARL EEEIRHLKDE MARHLREYQD
LLNVKMALDV EIATYRKLLE GEESRINLPI 421 QTYSALNFRE TSPEQRGSEV
HTKKTVMIKT IETRDGEVVS EATQQQHEVL
[0110] Trans-membrane helixes, lysis sites, and signal peptides in
the protein sequence were predicted based on several artificial
neural networks and hidden Markov analysis, and the results showed
that the protein do not have trans-membrane domain or signal
peptide, and the distribution of hydrophilic and hydrophobic amino
acids was relatively even.
[0111] Peptides of 11-12 amino acids were generated according to
the design method for tandem polypeptide of immunization antigens
mentioned in the Section of Detailed Description of the Invention.
For every short peptide, the isoelectric point, accessibility,
immunogenicity, hydrophilicity, and specie specificity according to
BLAST methods were calculated. When the isoelectric point was
larger than 3.5, the weighted average value of the above parameters
were calculated for every short peptide
(0.2*immunogenicity+0.1*accessibility+0.2*hydrophilicity+0.5*spec-
ie specificity). These short peptides were sorted according to the
weighted average values, and the short peptides having the highest
score and with an inter-peptide overlap <3 are selected. 5
linear surface signature peptides with the length of 11-12 amino
acids were selected (see table 3), and these peptides were linked
using linker GGGGS (SEQ ID NO:158), to form tandem polypeptide of
immunization antigens.
[0112] According to the searching for said protein, it was
discovered that it do not contain trans-membrane structures or
signal-peptide. Analysis according to the above principle was
performed for the full length protein, and 5 surface signature
sequences were determined. The sequence of the finally determined
tandem polypeptide of immunization antigens of Desmin H vector was:
RETSPEQRGSEV-GGGGS-KVSDLTQAANK-GGGGS-RLKGREPTRVAE-GGGGS-VEVLTNQRARVD-GGGG-
S-EESRINLPIQTY(SEQ ID NO: 32).
TABLE-US-00005 TABLE 3 The selection of tandem polypeptide of
immunization antigens Starting site Ending site (based on (based on
Genbank: Genbank: AAF15400.1 amino AAF15400.1 amino Peptide
sequences length/aa acid numbering) acid numbering) RETSPEQRGSEV
(SEQ ID NO: 33) 12 429 440 KVSDLTQAANK (SEQ ID NO: 34) 11 299 309
RLKGREPTRVAE (SEQ ID NO: 35) 12 142 153 VEVLTNQRARVD (SEQ ID NO:
36) 12 166 177 EESRINLPIQTY (SEQ ID NO: 37) 12 412 423
B Complete Gene Synthesis Method for Tandem Polypeptide of
Immunization Antigens
[0113] After codon optimization, it was determined that the
nucleotide sequence (encoding the tandem polypeptide of
immunization antigens of SEQ ID NO:32) that needs to be completely
synthesized is:
TABLE-US-00006 (SEQ ID NO: 38)
GACACggatccCGTGAAACCAGCCCGGAACAGCGTGGCAGCGAAGTGGGT
GGTGGAGGTTCTAAAGTGAGCGATCTGACCCAGGCGGCGAATAAAGGGGG
AGGCGGCAGCCGTCTGAAAGGCCGTGAACCGACCCGTGTGGCGGAAGGTG
GGGGTGGAAGCGTGGAAGTGCTGACCAATCAGCGTGCGCGTGTGGATGGC
GGTGGCGGCTCGGAAGAAAGCCGTATTAATCTGCCGATTCAGACCTATGg aatttcgTGTC
[0114] According to complete gene synthesis method, all together 10
primer sequences were designed, see table 4. The complete gene
synthesis method can be seen in Example 1. Briefly, 5 .mu.l of the
synthesized primers 1_01 to 1_10 were taken (with the concentration
of 25 .mu.M) and mixed. Such mixed primers were used as template,
and the first round of PCR amplification was conducted under the
catalysis of PFU enzyme (ShenNengBoCai co. ltd., Shanghai, China).
The PCR conditions were as: 94.degree. C., 2 min; 30 cycles
(94.degree. C., 30 s; 60.degree. C., 30 s; 72.degree. C., 1 min);
72.degree. C., 10 min; 10.degree. C., 10 min. 2 .mu.l of the PCR
product of the first round was then taken as template, and primers
1_01 and 1_10 (with the concentration of 25 .mu.M) were used for
conducting the amplification, with the amplification conditions as
set forth above. 1.5% agarose gel electrophoresis was performed,
and the target fragments (i.e. the nucleotide sequence to be
completely synthesized) were recovered.
TABLE-US-00007 TABLE 4 The nucleotide sequences for the complete
gene synthesis of immunization antigens Nucleotides Primer number
of names Primer sequences(5'-3') each primer 1_01
GACACggatccCGTGAAACCAGCCCGGAACAGCGTGG 37 (SEQ ID NO: 39) 1_02
AGAACCTCCACCACCCACTTCGCTGCCACGCTGTTCCGGG 40 (SEQ ID NO: 40) 1_03
TGGGTGGTGGAGGTTCTAAAGTGAGCGATCTGACCCAGGC 40 (SEQ ID NO: 41) 1_04
CTGCCGCCTCCCCCTTTATTCGCCGCCTGGGTCAGATCGC 40 (SEQ ID NO: 42) 1_05
GGGGGAGGCGGCAGCCGTCTGAAAGGCCGTGAACCGACCC 40 (SEQ ID NO: 43) 1_06
CTTCCACCCCCACCTTCCGCCACACGGGTCGGTTCACGGC 40 (SEQ ID NO: 44) 1_07
GAAGGTGGGGGTGGAAGCGTGGAAGTGCTGACCAATCAGC 40 (SEQ ID NO: 45) 1_08
CACCGCCATCCACACGCGCACGCTGATTGGTCAGCACTTC 40 (SEQ ID NO: 46) 1_09
CGTGTGGATGGCGGTGGCGGCTCGGAAGAAAGCCGTATTA 40 (SEQ ID NO: 47) 1_10
GACACgaattcATAGGTCTGAATCGGCAGATTAATACGGC 51 TTTCTTCCGAG (SEQ ID NO:
48)
Example 3
Expression and Purification of the Tandem Polypeptide of
Immunization Antigens
[0115] The sequence of SEQ ID NO: 38 which was completely
synthesized in Example 2 was digested using BamHI/EcoRI, and then
was ligated through T4 ligase at room temperature for 2 h with the
H vector digested with BamHI/EcoRI and recovered in Example 1. The
ligated product was incubated on ice (4.degree. C.) for 0.5 h with
Rosetta competent cells (Novagen, Merck, Germany) prepared by
CaCl.sub.2 method, and was heat-activated at 42.degree. C. for 90
s. The heat-activated bacteria liquid was supplemented with 200
.mu.l-500 .mu.l LB liquid medium with no antibiotics. The
composition of the medium was: 10 g/L Tryptone (Oxoid, England), 5
g/L Yeast Extract (Oxoid, England), 10 g/L NaCl (Sinopharm Group,
Shanghai, China). The bacteria liquid was then slowly shaked on a
37.degree. C. shaker at 100 rpm for 45 min, and finally was plated
on LB plates with corresponding antibiotics. The bacteria plates
were cultured in 7.degree. C. incubator overnight.
[0116] The next day, the transformed colonies were picked into
auto-induction system culture medium, and the composition of the
medium was: 10 g/L Tryptone (Oxoid, England), 5 g/L yeast extract
(Oxoid, England), 3.3 g/L (NH.sub.4).sub.2SO.sub.4 (Sinopharm
Group, Shanghai, China), 6.8 g/L KH.sub.2PO.sub.4 (Sinopharm Group,
Shanghai, China), 7.1 g/L Na.sub.2HPO.sub.4 (Sinopharm Group,
Shanghai, China), 0.5 g/L glucose (Sinopharm Group, Shanghai,
China), 2.0 g/L a-Lactose (Sinopharm Group, Shanghai, China), 0.15
g/L MgSO.sub.4 (Sinopharm Group, Shanghai, China). The culture was
expressed at 37.degree. C., 250 rpm overnight. The next day, the
bacteria liquid was centrifuged (6,000 g, 5 min), the supernatant
was discarded, and the pellet was resuspended using lysis buffer
(50 mM Tris, 500 mM NaCl, 4M urea, 1 mM PMSF, pH7.4) and was lysed
overnight at room temperature.
[0117] 25 ml (5 times of the column volume) of the lysis buffer was
used for pre-equilibration of Ni.sup.2+-NTA column (Qiagen,
Germany), and the column was placed in still for future uses. After
centrifugation (15,000 g, 10 min) of the cell lysis liquid, the
supernatant was applied on the column and incubated at room
temperature for 1 h. After the incubation, the liquid to be lysed
naturally flew out from the column, 30 ml washing buffer (4M urea,
50 mM Tris, 500 mM NaCl, 30 mM imidazole, pH 7.4) was used for
washing, and finally 5 ml elution buffer (4M urea, 50 mM Tris, 500
mM NaCl, 500 mM imidazole, pH 7.4) was used for eluting and
collecting the protein. The eluted protein was dialysed into buffer
(phosphate buffer of pH5.8), and the composition of the buffer was:
25.39 g/L NaH.sub.2PO.sub.4--H.sub.2O (Sinopharm Group, Shanghai,
China), 5.73 g/L Na.sub.2HPO.sub.4-12H.sub.2O (Sinopharm Group,
Shanghai, China). The buffer was changed twice during the process,
and the dialysis was all together performed for 24 h.
[0118] 1.5 mg polypeptide was obtained after the dialysis, the
concentration was 0.3 mg/ml, and the purity was >95%.
Example 4
The Design, Expression and Purification of Detection Antigens
A the Design of Detection Antigens
[0119] The information for the polypeptide of immunization antigens
of the following 5 proteins including Desmin was collected, and the
sequences are in Table 5.
TABLE-US-00008 TABLE 5 The design of immunization antigens for
proteins Protein name The sequence of the polypeptide of
immunization antigens Desmin
RETSPEQRGSEV-GGGGS-KVSDLTQAANK-GGGGS-RLKGREPTRVAE-GG
GGS-VEVLTNQRARVD-GGGGS-EESRINLPIQTY (SEQ ID NO: 49) vimentin
LRPSTSRSLYAS-GGGGS-INETSQHHDDLE-GGGGS-AINTEFKNTRT-GGG
GS-EQLKGQGKSRLG-GGGGS-QREEAENTLQSF (SEQ ID NO: 50) CD3g
KKKWNLGSNAKD-GGGGS-DGVRQSRASDK-GGGGS-CKGSQNKSKPL-G
GGGS-VKVYDYQEDGSV-GGGGS-EAKNITWFKDGK (SEQ ID NO: 51) CD3e
DKNIGGDEDDK-GGGGS-EEMGGITQTPYK-GGGGS-CYPRGSKPEDA-GGG
GS-DHLSLKEFSELE-GGGGS-GQRDLYSGLNQR (SEQ ID NO: 52) CD79a
VQEGNESYQQSC-GGGGS-EDAHFQCPHNSS-GGGGS-RKRWQNEKLGLD-
GGGGS-EDISRGLQGTY-GGGGS-GPGEDPNGTLII (SEQ ID NO: 53)
[0120] The sequences of corresponding detection antigens were
determined according to the design strategy of detection antigens.
The main strategy was to respectively name 5 surface signature
peptide sequences of each protein as ABCDE according to the 5
sequences of the polypeptide of immunization antigens of 5
proteins, and then combined the A epitopes of each protein to get
detection antigen A. Similarly detection antigens B-E were
obtained. The specific information for the detection antigens was
shown in Table 6.
TABLE-US-00009 TABLE 6 The amino acids sequences for detection
antigens Detection
RETSPEQRGSEV-LRPSTSRSLYAS-KKKWNLGSNAKD-DKNIGGDEDDK- antigen A
VQEGNESYQQSC (SEQ ID NO: 54) Detection
KVSDLTQAANKN-INETSQHHDDLE-DGVRQSRASDK-EEMGGITQTPYK- antigen B
EDAHFQCPHNSS (SEQ ID NO: 55) Detection
RLKGREPTRVAE-AINTEFKNTRT-CKGSQNKSKPL-CYPRGSKPEDA- antigen C
RKRWQNEKLGLD (SEQ ID NO: 56) Detection
VEVLTNQRARVD-EQLKGQGKSRLG-VKVYDYQEDGSV-DHLSLKEFSELE- antigen D
EDISRGLQGTY (SEQ ID NO: 57) Detection
EESRINLPIQTY-QREEAENTLQSF-EAKNITWFKDGK-GQRDLYSGLNQR- antigen E
GPGEDPNGTLII (SEQ ID NO: 58)
B Complete Gene Synthesis of the Detection Antigens
[0121] DNA Works software was used for codon optimization of the
sequences of the designed detection antigens in Table 6, and the
primers for synthesizing the optimized nucleotide sequences of the
detection antigens were synthesized by SBSGENE co. ltd. (Shanghai,
China). The nucleotide sequences of the optimized 5 detection
antigens were:
[0122] Detection Antigen A:
TABLE-US-00010 (SEQ ID NO: 59)
CGTGAAACCAGCCCGGAACAGCGTGGCAGCGAAGTGCTGCGTCCGAGCA
CCAGCCGTAGCCTGTATGCGAGCAAGAAAAAATGGAATCTGGGCAGCAAT
GCGAAAGATGACAAAAACATTGGCGGCGACGAGGATGATAAGGTGCAGGA
AGGCAACGAAAGCTATCAGCAAAGCTGC
[0123] Detection Antigen B:
TABLE-US-00011 (SEQ ID NO: 60)
AAAGTGTCAGACCTGACCCAGGCGGCGAATAAGAACATTAACGAAACCA
GCCAGCATCACGATGATCTGGAAGATGGCGTGCGTCAGAGCCGTGCGAGC
GATAAAGAAGAAATGGGCGGCATTACCCAGACCCCGTATAAAGAGGATGC
ACATTTTCAGTGCCCGCATAATAGCAGC
[0124] Detection Antigen C:
TABLE-US-00012 (SEQ ID NO: 61)
CGTCTGAAAGGCCGTGAACCGACCCGTGTGGCGGAAGCGATTAACACCG
AATTTAAGAATACCCGTACCTGCAAAGGCAGCCAGAATAAATCGAAACCG
CTGTGCTATCCGCGTGGCAGCAAACCGGAAGATGCGCGTAAACGGTGGCA
GAATGAAAAACTGGGCCTGGAT
[0125] Detection Antigen D:
TABLE-US-00013 (SEQ ID NO: 62)
GTGGAAGTGCTGACCAATCAGCGTGCGCGTGTGGATGAACAGCTGAAAG
GCCAGGGCAAAAGCCGTCTGGGTGTGAAAGTGTATGATTATCAGGAAGAT
GGCAGCGTGGATCATCTGAGCCTGAAAGAATTTAGCGAACTGGAAGAAGA
CATCAGCCGTGGGCTGCAGGGCACCTAT
[0126] Detection Antigen E:
TABLE-US-00014 (SEQ ID NO: 63)
GAAGAAAGTCGTATTAATCTGCCGATTCAGACCTATCAGCGTGAGGAAG
CGGAAAATACCCTGCAGTCGTTTGAAGCGAAAAACATTACCTGGTTCAAA
GATGGCAAAGGCCAGCGGGATCTGTATAGCGGCCTGAACCAACGTGGTCC
GGGCGAAGATCCGAATGGCACCCTGATTATT
[0127] According to the nucleotide sequences of the detection
antigens and the BamH I and EcoR I digestions sites introduced into
the synthesized sequences, 8 primers were respectively designed,
and the sequences thereof were in Tables 7-11. The complete gene
synthesis method was the same to Example 1.
[0128] 5 .mu.l of the synthesized primers 2_01 to 2_10 for
detection antigen A were taken (with the concentration of 25 .mu.M)
and mixed. Such mixed primers were used as template, and the first
round of PCR amplification was conducted under the catalysis of PFU
enzyme (ShenNengBoCai co. ltd., Shanghai, China). The PCR
conditions were as: 94.degree. C., 2 min; 30 cycles (94.degree. C.,
30 s; 60.degree. C., 30 s; 72.degree. C., 1 min); 72.degree. C., 10
min; 10.degree. C., 10 min 2 .mu.l of the PCR product of the first
round was then taken as template, and primers 2_01 and 2_10 (with
the concentration of 25 .mu.M) were used for conducting the
amplification, with the amplification conditions as set forth
above. 1.5% agarose gel electrophoresis was performed, and the
target fragments (i.e. the nucleotide sequence to be completely
synthesized) were recovered.
[0129] The complete gene synthesis methods for detection antigens
B-E were completely the same to that of detection antigen A, only
with the exception that the sequences of the primers were
different.
[0130] After being double-digested with the two enzymes BamH I and
EcoR I, the completely synthesized nucleotide sequences of the
detection antigens were ligated into pET32a vectors (Novagen,
Merck, Germany) digested by the same enzymes.
TABLE-US-00015 TABLE 7 The primers for the complete gene synthesis
of the detection antigen A Nucleotides Primer number of names
Primer sequences (5'-3) each primer 2_01
GACACggatccCGTGAAACCAGCCCGGAACAGCGTGGCA 39 (SEQ ID NO: 64) 2_02
CTGGTGCTCGGACGCAGCACTTCGCTGCCACGCTGTTCCG 40 (SEQ ID NO: 65) 2_03
GCGTCCGAGCACCAGCCGTAGCCTGTATGCGAGCAAGAAA 40 (SEQ ID NO: 66) 2_04
ATTGCTGCCCAGATTCCATTTTTTCTTGCTCGCATACAGG 40 (SEQ ID NO: 67) 2_05
TGGAATCTGGGCAGCAATGCGAAAGATGACAAAAACATTG 40 (SEQ ID NO: 68) 2_06
TATCATCCTCGTCGCCGCCAATGTTTTTGTCATCTTTCGC 40 (SEQ ID NO: 69) 2_07
CGGCGACGAGGATGATAAGGTGCAGGAAGGCAACGAAAGC 40 (SEQ ID NO: 70) 2_08
GACACgaattcGCAGCTTTGCTGATAGCTTTCGTTGCCTTCCTG 44 (SEQ ID NO: 71)
TABLE-US-00016 TABLE 8 The primers for the complete gene synthesis
of the detection antigen B Nucleotides Primer number of names
Primer sequences (5'-3') each primer 3_01
GACACggatccAAAGTGTCAGACCTGACCCAGGCGGCGAATAA 39 (SEQ ID NO: 72) 3_02
TGCTGGCTGGTTTCGTTAATGTTCTTATTCGCCGCCTGGG 40 (SEQ ID NO: 73) 3_03
AACGAAACCAGCCAGCATCACGATGATCTGGAAGATGGCG 40 (SEQ ID NO: 74) 3_04
CGCTCGCACGGCTCTGACGCACGCCATCTTCCAGATCATC 40 (SEQ ID NO: 75) 3_05
GAGCCGTGCGAGCGATAAAGAAGAAATGGGCGGCATTACC 40 (SEQ ID NO: 76) 3_06
CATCCTCTTTATACGGGGTCTGGGTAATGCCGCCCATTTC 40 (SEQ ID NO: 77) 3_07
AGACCCCGTATAAAGAGGATGCACATTTTCAGTGCCCGCA 40 (SEQ ID NO: 78) 3_08
GACACgaattcGCTGCTATTATGCGGGCACTGAAAATG 38 (SEQ ID NO: 79)
TABLE-US-00017 TABLE 9 The primers for the complete gene synthesis
of the detection antigen C Nucleotides Primer number of names
Primer sequences (5'-3') each primer 4_01
GACACggatccCGTCTGAAAGGCCGTGAACCGAC 34 (SEQ ID NO: 80) 4_02
TGTTAATCGCTTCCGCCACACGGGTCGGTTCACGGCCTTT 40 (SEQ ID NO: 81) 4_03
TGGCGGAAGCGATTAACACCGAATTTAAGAATACCCGTAC 40 (SEQ ID NO: 82) 4_04
TTCTGGCTGCCTTTGCAGGTACGGGTATTCTTAAATTCGG 40 (SEQ ID NO: 83) 4_05
TGCAAAGGCAGCCAGAATAAATCGAAACCGCTGTGCTATC 40 (SEQ ID NO: 84) 4_06
TCTTCCGGTTTGCTGCCACGCGGATAGCACAGCGGTTTCG 40 (SEQ ID NO: 85) 4_07
GGCAGCAAACCGGAAGATGCGCGTAAACGGTGGCAGAATG 40 (SEQ ID NO: 86) 4_08
GACACgaattcATCCAGGCCCAGTTTTTCATTCTGCCACCGTTTACG 47 (SEQ ID NO:
87)
TABLE-US-00018 TABLE 10 The primers for the complete gene synthesis
of the detection antigen D Nucleotides Primer number of names
Primer sequences (5'-3') each primer 5_01
GACACggatccGTGGAAGTGCTGACCAATCAGCGTGCGCGTG 42 (SEQ ID NO: 88) 5_02
GCCCTGGCCTTTCAGCTGTTCATCCACACGCGCACGCTGA 40 (SEQ ID NO: 89) 5_03
GCTGAAAGGCCAGGGCAAAAGCCGTCTGGGTGTGAAAGTG 40 (SEQ ID NO: 90) 5_04
TGCCATCTTCCTGATAATCATACACTTTCACACCCAGACG 40 (SEQ ID NO: 91) 5_05
ATGATTATCAGGAAGATGGCAGCGTGGATCATCTGAGCCT 40 (SEQ ID NO: 92) 5_06
TTCCAGTTCGCTAAATTCTTTCAGGCTCAGATGATCCACG 40 (SEQ ID NO: 93) 5_07
GAAAGAATTTAGCGAACTGGAAGAAGACATCAGCCGTGGG 40 (SEQ ID NO: 94) 5_08
GACACgaattcATAGGTGCCCTGCAGCCCACGGCTGATGTCTT 43 (SEQ ID NO: 95)
TABLE-US-00019 TABLE 11 The primers for the complete gene synthesis
of the detection antigen E Nucleotides Primer number of names
Primer sequences (5'-3') each primer 6_01
GACACggatccGAAGAAAGTCGTATTAATCTGCCGATTCAGACC 44 (SEQ ID NO: 96)
6_02 CCGCTTCCTCACGCTGATAGGTCTGAATCGGCAGATTAAT 40 (SEQ ID NO: 97)
6_03 CAGCGTGAGGAAGCGGAAAATACCCTGCAGTCGTTTGAAG 40 (SEQ ID NO: 98)
6_04 TTGAACCAGGTAATGTTTTTCGCTTCAAACGACTGCAGGG 40 (SEQ ID NO: 99)
6_05 CGAAAAACATTACCTGGTTCAAAGATGGCAAAGGCCAGCG 40 (SEQ ID NO: 100)
6_06 TTGGTTCAGGCCGCTATACAGATCCCGCTGGCCTTTGCCA 40 (SEQ ID NO: 101)
6_07 ATAGCGGCCTGAACCAACGTGGTCCGGGCGAAGATCCGAA 40 (SEQ ID NO: 102)
6_08 GACACgaattcAATAATCAGGGTGCCATTCGGATCTTCGCCCG 43 (SEQ ID NO:
103)
G the Expression and Purification of the Detection Antigens
[0131] The above DNA sequences of the 5 corresponding detection
antigens that were prepared by complete gene synthesis were
integrated into PET32a vectors (Novagen) through BamHI/EcoRI
restriction sites, and then 5 plasmids were obtained.
[0132] The composition of auto-induction system culture medium was:
10 g/L Tryptone (Oxoid, England), 5 g/L yeast extract (Oxoid,
England), 3.3 g/L(NH.sub.4).sub.2SO.sub.4(Sinopharm Group,
Shanghai, China), 6.8 g/L KH.sub.2PO.sub.4 (Sinopharm Group,
Shanghai, China), 7.1 g/L Na.sub.2HPO.sub.4 (Sinopharm Group,
Shanghai, China), 0.5 g/L glucose (Sinopharm Group, Shanghai,
China), 2.0 g/L a-Lactose (Sinopharm Group, Shanghai, China), 0.15
g/L MgSO.sub.4 (Sinopharm Group, Shanghai, China).
[0133] Said plasmids were respectively transformed into Rosseta
strains. Transformed colonies were picked into auto-induction
system culture medium (see example 3), and were expressed at
37.degree. C. 250 rpm overnight. The next day, the bacteria liquid
was centrifuged (6,000 g, 5 min), and the supernatant was
discarded. The pellet was resuspended using lysis buffer (50 mM
Tris, 500 mM NaCl, 4M urea, protease inhibitor, pH 7.4) and was
lysed overnight.
[0134] 25 ml (5 times of the column volume) of the lysis buffer was
used for pre-equilibration of Ni.sup.2+-NTA column, and the column
was placed in still for future uses. After centrifugation (15,000
g, 10 min) of the cell lysis liquid, the supernatant was applied on
the column and incubated at room temperature for 1 h. After the
incubation, the liquid to be lysed naturally flew out from the
column, 30 ml washing buffer (4M urea, 50 mM Tris, 500 mM NaCl, 30
mM imidazole, pH 7.4) was used for washing, and finally 5 ml
elution buffer (4M urea, 50 mM Tris, 500 mM NaCl, 500 mM imidazole,
pH 7.4) was used for eluting and collecting the protein.
[0135] The purified amount of detection antigen A was 0.8 mg, with
the purity of 80%; the purified amount of detection antigen B was
0.65 mg, with the purity of 90%; the purified amount of detection
antigen C was 1.2 mg, with the purity of 85%; the purified amount
of detection antigen D was 1.5 mg, with the purity of 75%; the
purified amount of detection antigen E was 0.9 mg, with the purity
of 95%.
Example 5
Preparation of Monoclonal Antibodies
A Immunization Method
[0136] Oligonucleotide adjuvant: 50 .mu.l aluminum adjuvant (Thermo
Fisher, USA)+1 .mu.g DNA adjuvant, and the sequence thereof was
tccatgacgttcctgacgtT (SEQ ID NO: 104), wherein the bases in lower
case were sites that need thio-modifications, the oligonucleotide
was synthesized by SBSGENE co. ltd. (Shanghai, China).
[0137] As for protein antigens in the experiments, the dosage for
each mouse and each immunization can be any dosage between 2-200
.mu.g, and the time interval for each immunization can be 2-14
days. The immunization method and dosages for the synthesized
protein Desmin (SEQ ID NO:32) were as follows.
[0138] Immunization method: 3 Balb/c mice, 8-10 weeks old, body
weight 18-20 g. 20 .mu.g antigen was completely emulsified with
Freund's complete adjuvant (Sigma), and the immunization sites were
hind foot palm, tail end, and front leg armpit as well as inguen,
wherein about 50 .mu.L was applied for each site. 20 .mu.g antigen
was completely mixed with the oligonucleotide adjuvant, and was
then used to immunize the mice at hind leg muscle, wherein 50-100
.mu.L was applied for each site.
[0139] On the eighth day, 10 .mu.g antigen was taken and completely
emulsified with Freund's complete adjuvant (Sigma), which was used
to immunize the mice at front leg armpit and inguen, wherein 50-100
.mu.L was applied for each site. 20 .mu.g antigen was completely
mixed with the oligonucleotide adjuvant, and was then used to
immunize the mice at hind leg muscle, wherein 50-100 .mu.L was
applied for each site.
[0140] On the twelfth day, 10 .mu.g antigen was completely mixed
with the oligonucleotide adjuvant, and was then used to immunize
the mice at hind leg muscle.
[0141] On the fourteenth day, blood was taken from eyepit of the
mice, and ELISA method was used to examine the serum titer, wherein
the serum titers of the mice were all higher than 1:32000.
B Cell Fusion and Screening
[0142] The preparation of relevant media: the basic medium used was
1640 medium (Thermo, USA), and the serum concentration in the
complete medium was 15%, wherein the serum was purchased from
Biowest, Spain, and HAT and HT stock solutions were purchased from
Sigma, Germany
[0143] On the fifteenth day, the mice were sacrificed by cervical
dislocation. The lymph-node cells of 2 mice were taken for the
fusion with SP20 cells (ATCC, USA), the cells were suspended (final
cell density was about 10.sup.6/ml), and then were plated on 4 384
plates, with 80 .mu.L for each well. The cell plates were cultured
at 37.degree. C., 5% CO.sub.2 for 6 days, and then were completely
changed to HT complete medium. 8 days after the fusion, 10 .mu.L
cell supernatant was taken, and diluted 5 times, for ELISA
assay.
[0144] The immunogen (H carrier protein carrying the polypeptide
sequence of SEQ ID NO:32) was diluted to 1 .mu.g/ml using 0.01M
Na.sub.2CO.sub.3/NaHCO.sub.3 buffer. 100 .mu.L was added into each
well, and coating was performed at 4 degree overnight. The plates
were swung to clean the solution therein, and were washed by PBST
for 3 times, with 250 .mu.L/well. 5% milk was used for blocking at
37.degree. C. for 1 h, and the plates were swung to clean the
solution therein, and were washed by PBST for 3 times, with 250
.mu.L/well. The cell fusion plate supernatant was taken, 20 .mu.L
for each well, 80 .mu.L of 5% milk was supplemented, and the plates
were then incubated at 37.degree. C. for 1 h. The plates were swung
to clean the solution therein, and were washed by PBST for 3 times,
with 250 .mu.L/well. 100 .mu.L of HRP labeled caprice-anti-mouse
antibody (Abmart, 1:8000) was added into each well, and the plates
were then incubated at 37.degree. C. for 1 h. The plates were swung
to clean the solution therein, and were washed by PBST for 3 times,
with 250 .mu.L/well. Horse radish peroxidase substrate TMB (Sigma)
solution was added, and the plates were then incubated at
37.degree. C. for 15 min 50 .mu.L of 2M H.sub.2SO.sub.4 solution
was added into each well to terminate the reaction, and the
absorption at 450 nm was read.
[0145] The detection antigens A-E were respectively diluted to 1
.mu.g/ml using 0.01M Na.sub.2CO.sub.3/NaHCO.sub.3 buffer, and 100
.mu.L was added into each well, and coating was performed at 4
degree overnight. The plates were swung to clean the solution
therein, and were washed by PBST for 3 times, with 250 .mu.L/well.
5% milk was used for blocking at 37.degree. C. for 1 h, and the
plates were swung to clean the solution therein, and were washed by
PBST for 3 times, with 250 .mu.L/well.
[0146] 50 .mu.L of supernatant was taken from wells that were
preliminarily identified as positive, 200 .mu.L of 5% milk was
supplemented, and 50 .mu.L, was respectively taken and added into
the ELISA plates for detection antigens 1-5. The plates were
incubated at 37.degree. C. for 1 h, swung to clean the solution
therein, and then washed by PBST for 3 times, with 250 .mu.L/well.
100 .mu.L of HRP labeled caprice-anti-mouse antibody (Abmart,
1:8000) was added into each well, and the plates were then
incubated at 37.degree. C. for 1 h. The plates were swung to clean
the solution therein, and were washed by PBST for 3 times, with 250
.mu.L/well. Horse radish peroxidase substrate TMB (Sigma) solution
was added, and the plates were then incubated at 37.degree. C. for
15 min 50 .mu.L of 2M H.sub.2SO.sub.4 solution was added into each
well to terminate the reaction, and the absorption at 450 nm was
read.
[0147] The data for the epitopes screening were shown in table 12.
The wells that were positive to different detection antigens were
just the antibodies against different antigen epitopes, and the
screening results can be seen in table 13. 5 polypeptide fragments
of SEQ ID NO:32 were named as epitopes A, B, C, D, E according to
the sequence from N-terminus to C-terminus.
[0148] Since each detection antigen only carried one protein
surface signature peptide related to the immunization antigen,
every detection antigen positive clone (OD larger than 0.5), was
directed to the first polypeptide of the immunization antigen (the
polypeptide of SEQ ID NO:32). For example, the well of detection
antigen A4 was positive for ELISA detection (see table 12,
OD=2.092), since the detection antigen A carried the sequence of
RETSPEQRGSEV-the first surface linear signature peptide of SEQ ID
NO:32 polypeptide (see tables 5 and 6), it can be determined from
such results that the recognition epitope of the antibody clone in
A4 well was the first surface linear signature
peptide--RETSPEQRGSEV. Such strategy was used to determine the
sequences recognized by different positive wells.
TABLE-US-00020 TABLE 12 The epitopes screening results for the
wells of fusion positive clone Detection antigens 1 2 3 4 5 6 7 8 9
10 11 12 Detection 0.058 0.077 0.132 2.092 0.089 0.066 0.085 0.069
0.073 0.084 2.148 0.117 A antigen A 0.08 0.071 0.074 0.08 0.072
0.089 0.106 0.089 1.942 0.093 0.107 0.088 B Detection 0.087 2.595
0.062 0.066 0.085 0.069 0.084 2.045 0.089 0.088 0.081 0.069 C
antigen B 0.074 2.181 0.061 0.072 0.089 1.942 0.093 0.093 0.107
0.065 0.063 0.063 D Detection 0.062 0.132 2.595 0.103 0.085 0.066
0.101 0.093 0.13 2.027 0.058 0.063 E antigen C 0.07 0.074 0.066
0.13 0.092 0.069 2.075 0.082 0.075 0.125 0.058 0.058 F Detection
0.068 0.068 0.136 0.076 0.06 1.994 0.075 0.139 1.923 0.067 0.053
0.070 G antigen D 0.085 0.062 1.575 0.059 0.066 0.083 0.067 0.064
0.076 0.072 0.064 0.080 H Detection 0.076 0.061 0.086 0.06 0.067
0.09 0.064 0.101 0.106 0.077 0.073 0.068 I antigen E 0.054 0.066
0.065 0.073 0.073 0.062 0.083 0.07 0.065 0.068 0.062 0.066 J
TABLE-US-00021 TABLE 13 The epitopes screening results and
corresponding clone number Number of cell Epitopes The sequences of
epitopes strains A RETSPEQRGSEV (SEQ ID NO: 105) 3 B KVSDLTQAAN
(SEQ ID NO: 106) 4 C RLKGREPTRVAE (SEQ ID NO: 107) 3 D VEVLTNQRARVD
(SEQ NO: 108) 3 E EESRINLPIQTY (SEQ ID NO: 109) 0
[0149] 2 positive wells of each epitopes were respectively picked
to conduct limiting dilution for sub-cloning. After 3 rounds of
sub-clonings, 10 strains of stable hybridoma cell strains were
obtained, and corresponding cell strain culture supernatants were
collected for the antibody verification in Example 6. These cell
strains were respectively directed to the 4 polypeptide epitopes
(SEQ ID NO: 105-108) of Desmin, wherein 7 cell strains were
identified as having an affinity KD lower than 10 nM.
Example 6
The Data for Antibody Verification
A the Experiment Procedure of Western Blotting
[0150] C2C12 cells (ATCC, USA) were lysed with RIPA buffer (50 mM
Tris pH7.4, 150 mM NaCl, 1% Triton-X-100, 1% sodium deoxycholate,
0.1% SDS lysis buffer) containing protease inhibitor (Roche),
quantified by BCA (ShenNengBoCai co. ltd.), and diluted by 5.times.
loading buffer. After 10 minutes denaturation at 100.degree. C.,
20-30 ng was loaded for each lane, and 10% SDS-PAGE gel
electrophoresis was performed. After PVDF membrane transfer, 5%
skimmed milk was used for blocking for 1 h. The supernatant of
monoclonal cell strain against Desmin (the cell culture supernatant
of the cell strain selected in Example 5) 1:5 was added, and then
incubation at room temperature was conducted for 1 hour.
1.times.PBST was used for washing 3.times.5 min. The secondary
antibody-HRP coupled anti-mouse IgG (Abmart, M210015) was added at
1:5000, and then incubation at room temperature was conducted for
30 min 1.times.PBST was used for washing 3.times.5 min. ECL Plus
(Amersham, USA) was used for the detection.
B the Experiment Procedure of Immuno-Fluorescence (IF)
[0151] 5.times.10.sup.3 BHK cells (ATCC, USA) were inoculated on a
cell slide (SUPER GRADE MICROSCOPE SLIDES) and cultured at
37.degree. C. overnight, after being fixed with chilled methanol
(-20.degree. C.) for 15 min, washed with 1.times.PBS for 2.times.3
min, and blocked with 1% BSA at room temperature for 1 h, they were
then incubated at room temperature for 1 hour with the addition of
1:1 supernatant of monoclonal antibody cell strain culture as
primary antibody, and washed with 1.times.PBST for 3.times.5 min;
they were incubated at room temperature for 1 hour with the
addition of secondary antibody anti-mouse Dylight 549 (Abcam) at
1:400 and DAPI at 1:4000, washed with 1.times.PBST for 3.times.5
min, and then examined with fluorescence microscope (Olympus) with
a picture being taken.
[0152] The antibodies against different epitopes were verified by
Western. The 7 cell strains obtained in Example 5 can specifically
recognize endogenous Desmin of C2C12 cell line, wherein the
antibody 4A1 produced by the cell strain named 4A1 (deposited in
China Center for Type Culture Collection (CCTCC) on Jan. 28, 2011,
with the deposition No. CCTCC C201107) had the best affinity and
specificity, and favorite results were obtained in both Western
blotting and immuno-fluorescence (see, FIGS. 4 and 5).
Example 7
Preparation of Domain-Specific Antibodies of Membrane Receptor
Protein GPR116
A the Selection of Protein Surface Signature Domain:
[0153] GPR116 protein is a membrane receptor protein, the specie
thereof is human, and the NCBI No. thereof is NP_001091988.1. The
full length protein is as following:
TABLE-US-00022 (SEQ ID NO: 110) 1 MKSPRRTTLC LMFIVIYSSK AALNWNYEST
IHPLSLHEHE PAGEEALRQK RAVATKSPTA 61 EEYTVNIEIS FENASFLDPI
KAYLNSLSFP IHGNNTDQIT DILSINVTTV CRPAGNEIWC 121 SCETGYGWPR
ERCLHNLICQ ERDVFLPGHH CSCLKELPPN GPFCLLQEDV TLNMRVRLNV 181
GFQEDLMNTS SALYRSYKTD LETAFRKGYG ILPGFKGVTV TGFKSGSVVV TYEVKTTPPS
241 LELIHKANEQ VVQSLNQTYK MDYNSFQAVT INESNFFVTP EIIFEGDTVS
LVCEKEVLSS 301 NVSWRYEEQQ LEIQNSSRFS IYTALFNNMT SVSKLTIHNI
TPGDAGEYVC KLILDIFEYE 361 CKKKIDVMPI QILANEEMKV MCDNNPVSLN
CCSQGNVNWS KVEWKQEGKI NIPGTPETDI 421 DSSCSRYTLK ADGTQCPSGS
SGTTVIYTCE FISAYGARGS ANIKVTFISV ANLTITPDPI 481 SVSEGQNFSI
KCISDVSNYD EVYWNTSAGI KIYQRFYTTR RYLDGAESVL TVKTSTREWN 541
GTYHCIFRYK NSYSIATKDV IVHPLPLKLN IMVDPLEATV SCSGSHHIKC CIEEDGDYKV
601 TFHTGSSSLP AAKEVNKKQV CYKHNFNASS VSWCSKTVDV CCHFTNAANN
SVWSPSMKLN 661 LVPGENITCQ DPVIGVGEPG KVIQKLCRFS NVPSSPESPI
GGTITYKCVG SQWEEKRNDC 721 ISAPINSLLQ MAKALIKSPS QDEMLPTYLK
DLSISIDKAE HEISSSPGSL GAIINILDLL 781 STVPTQVNSE MMTHVLSTVN
VILGKPVLNT WKVLQQQWTN QSSQLLHSVE RFSQALQSGD 841 SPPLSFSQTN
VQMSSMVIKS SHPETYQQRF VFPYFDLWGN VVIDKSYLEN LQSDSSIVTM 901
AFPTLQAILA QDIQENNFAE SLVMTTTVSH NTTMPFRISM TFKNNSPSGG ETKCVFWNFR
961 LANNTGGWDS SGCYVEEGDG DNVTCICDHL TSFSILMSPD SPDPSSLLGI
LLDIISYVGV 1021 GFSILSLAAC LVVEAVVWKS VTKNRTSYMR HTCIVNIAAS
LLVANTWFIV VAAIQDNRYI 1081 LCKTACVAAT FFIHFFYLSV FFWMLTLGLM
LFYRLVFILH ETSRSTQKAI AFCLGYGCPL 1141 AISVITLGAT QPREVYTRKN
VCWLNWEDTK ALLAFAIPAL IIVVVNITIT IVVITKILRP 1201 SIGDKPCKQE
KSSLFQISKS IGVLTPLLGL TWGFGLTTVF PGTNLVFHII FAILNVFQGL 1261
FILLFGCLWD LKVQEALLNK FSLSRWSSQH SKSTSLGSST PVFSMSSPIS RRFNNLFGKT
1321 GTYNVSTPEA TSSSLENSSS ASSLLN
[0154] Protein surface signature domain was predicted according to
http colon slash slash pfam dot sanger dot ac dot uk slash search
slash sequence, and the protein fragment of the amino acid residues
at positions 166-307 was used for constructing immunization antigen
and detection antigen, wherein the fragment of the amino acid
residues at positions 166-307 comprised two important domains.
B Amplification of the Target Fragment
[0155] The forward primer: CGCGGATCCCTTCAGGAAGATGTTACCCTGAA (SEQ ID
NO: 111) and the reverse primer: CGCGAATTCAACATCTA
TTTTCTTCTTGCACT(SEQ ID NO: 112) were designed according to the
amino acid sequences of the two ends of the above fragment and the
insertion of restriction sites.
[0156] The purchased GPR116 CDNA plasmid (Yeli co. ltd., China) was
use as template, and the amount of the template was 50 ng. PCR
conditions were: 94.degree. C., 2 min; 94.degree. C. 30
s-60.degree. C. 30 s-72.degree. C. 1 min, 30 cycles; 72.degree. C.,
10 min; 10.degree. C., 10 min.
[0157] The target fragments around 450 bp were recovered from gel.
After the BamHI/EcoRI restriction sites were integrated to the H
vector of Example 1, 5 plasmids were obtained. The target fragment
was respectively inserted into H vectors and PET32a vectors
digested by the same enzymes, and then were respectively used for
expressing immunization antigen and detection antigen.
[0158] The constructed H and PET32a expression vectors were
respectively transformed to Rosetta competent cells (Novagen,
Merck, Germany) The expression and purification methods for
immunization antigens and detection antigens can respectively be
seen in Example 3 and Example 4. The produced amount of
immunization antigen was 1.7 mg, with the purity of 80%; produced
amount of detection antigen was 2.3 mg, with the purity of 95%.
C Immunization Method
[0159] The immunization method and dosage for protein GPR116 were
as follows.
[0160] 3 Balb/c mice (Shanghai sippr bk laboratory animals co.
ltd., Shanghai) which were 8-10 weeks old were selected for the
immunization.
[0161] On the first day, 20 .mu.g (the dosage can be 2-200 ug) of
the above prepared immunization antigen was completely emulsified
with Freund's complete adjuvant (Sigma), and the immunization sites
were front leg armpit and inguen, 4 sites all together, wherein
about 50 .mu.L was applied for each site.
[0162] On the fourteenth day, 10 .mu.g antigen was taken and
completely emulsified with Freund's complete adjuvant (Sigma), and
then used to immunize the mice at front leg armpit and inguen.
[0163] On the 21st day, blood was taken from eyepit of the mice,
and ELISA method was used to examine the serum titer, wherein the
serum titers of the mice were all higher than 1:32000.
[0164] On the 28th day, the mouse with the highest titer was
selected, and 50 .mu.g antigen was used for strengthening at
abdominal cavity.
D Cell Fusion and Hybridoma Screening
[0165] On the 31st day, the mice were sacrificed by cervical
dislocation. Spleen cells of the mice were fused with SP20 cells.
The cells were suspended in HAT complete medium, and the plated for
4 384 well plates, with 80 .mu.L in each well. The cell plates were
cultured at 37.degree. C., 5% CO.sub.2 for 6 days, and then totally
changed to HT medium. 8 days after the fusion, 10 .mu.L cell
supernatant was taken, and diluted 5 times, which was used for
ELISA assay.
[0166] The operation of the primary screening ELISA was the same to
that of Example 5, with the difference that the coating antigen was
detection antigen which was expressed by PET32a and which was fused
with TRX. The detection antigen per se also contained the selected
domain sequence of aa 166-307, and the other components thereof
were totally different to H vector. Since the detection also
contained other parts of the H vector besides the sequence of the
domain parts, and these other parts also produced corresponding
antibodies. The detection antigen fused with TRX was used to
confirm that the obtained cell strain recognized said surface
signature domain rather than other parts expressed by the H vector.
The concentration for coating the plates was 1 .mu.g/ml, and the
solutions for coating the plated was pH9.6 bicarbonate buffer
(Na.sub.2CO.sub.3/NaHCO.sub.3).
[0167] 100 clones with highest OD values were selected for
sub-cloning and cell strain construction. An antibody library (68
strains) against the surface signature domains of GPR116 protein
(the specific domains selected above) was obtained. Cell strains
with various applications can be selected from the library. The
antibodies secreted by more than 50 cell strains were assayed as
having an affinity less than 10 nM, indicating that affinity of the
antibodies in the antibody library was in a relatively high
level.
E Verification Data
[0168] The basis verification method of Western was the same to
that of Example 6, and the lysate selected for the verification was
Y79 cell (ATCC, USA) lysate. FIG. 6 showed the Western results of a
monoclonal antibody 1A6 produced by one cell strain 1A6 of the
above 50 cell strains, and said cell strain was deposited in China
Center for Type Culture Collection (CCTCC) on Jan. 28, 2011, with
the deposition No. CCTCC C201108. 50 cell strains can be used to
specifically detect endogenous GPR116 protein. More than 50% of the
cell strains were successful in the verification applications of
co-immuno-precipitation and immuno-fluorescence.
Example 8
Preparation of Antibodies of Mouse Protein-Aof1
A Design and Expression of the Immunization Antigen
[0169] Aof1 protein (a protein containing flavin amine oxidase
domain) is a self-protein of mouse. As for self-protein,
traditional recombinant protein expression method can hardly be
used to prepare antibodies with high affinity. Using the method of
the invention, an antibody library with high affinity was
successfully prepared.
[0170] The full length amino acids of Aof1 protein is:
TABLE-US-00023 (SEQ ID NO: 113) 1 MAASRGRSKK RSNLELSPDN LPLRSSGRQA
KKKAVEIPDE DEDGSSEKKY RKCEKAGCTA 61 AYPVCFASAS ERCAKNGYTS
RWYHLSCGEH FCNECFDHYY RSHKDGYDKY SAWKRVWTSN 121 GKTEPSPKAF
MADQQLPYWV QCTKPECGKW RQLTKEIQLT PHMARTYRCG MKPNTITKPD 181
TPDHCSFPED LRVLEVSNHW WYPMLIQPPL LKDSVAAPLL SAYYPDCVGM SPSCTSTHRA
241 TVTAATTTTG SASPGEMEPS KAAPSSLVLG MNRYFQPFYQ PNECGKALCV
RPDVMELDEL 301 YEFPEYSRDP TMYLALRNLI LALWYTNCKE ALTPQKCIPH
IIVRGLVRIR CVQEVERILY 361 FMTRKGLINT GVLTVAAGQH LLPKHYHNKS
VLVVGAGPAG LAAARQLHNF GMKVTVLEAK 421 DRIGGRVWDD KSFKGVVVGR
GPQIVNGCIN NPVALMCEQL GISMRKLGER CDLIQEGGRI 481 TDPTVDKRMD
FHFNALLDVV SEWRKDKTLL QDVPLGEKIE EIYRAFVKES GIQFSELEGQ 541
VLQFHLSNLE YACGSSLHQV SARSWDHNEF FAQFAGDHTL LTPGYSTIIE KLAEGLDIRL
601 KSPVQSIDYT GDEVQVTTTD GMGHSAQKVL VTVPLAILQR GAIQFNPPLS
EKKMKAINSL 661 GAGIIEKIAL QFPYRFWDSK VQGADFFGHV PPSASQRGLF
AVFYDMDSQQ SVLMSVITGE 721 AVASLRTMDD KQVLQQCMGI LRELFKEQEI
PEPTKYFVTR WSTEPWIQMA YSFVKTFGSG 781 EAYDIIAEEI QGTVFFAGEA
TNRHFPQTVT GAYLSGVREA SKIAAF
[0171] According to searching in the protein, it was discovered
that the protein does not contain trans-membrane domain or signal
peptide. Based on the above mentioned principle, the full length of
the protein was analyzed, and 5 surface signature peptide sequences
were determined.
[0172] The method for constructing the tandem polypeptide of
immunization antigens was the same to that of Example 2. 7 short
peptides were selected, and the sequences can be seen in Table
14.
TABLE-US-00024 TABLE 14 The information for surface signature
peptide Starting site Ending site (based on (based on SEQ ID SEQ ID
Specific Numbering Peptide sequences length/aa NO: 113) NO: 13)
Score 1 KKYRKCEKAG 10 48 57 90.2 (SEQ ID NO: 114) 2 AASRGRSKKR 10 2
11 89.8 (SEQ ID NO: 115) 3 RSSGRQAKKK 10 24 33 88.6 (SEQ ID NO:
116) 4 VRGLVRIRCV 10 343 352 89.9 (SEQ ID NO: 117) 5 KYSAWKRVWT 10
109 118 90.8 (SEQ ID NO: 118) 6 RILYFMTRKG 10 357 366 90.7 (SEQ ID
NO: 119) 7 MARTYRCGMK (SEQ ID NO: 120) 10 163 172 91.1
Specific Score Calculation:
[0173] 1, The protein was cut into segments of 10 aa (gradually
cutting, 1-10, 2-11, 3-12 . . . )
[0174] 2, Blast was conducted for all the 10 aa peptide segments
against the specie of the protein
[0175] 3, The formula for calculating the specific score of each
target peptide was: i) the most homogenous 20 sequences of each
target polypeptide were selected; ii) the number of identical amino
acids in each of the 20 sequences when compared to the target
polypeptide was listed; iii) the average of these numbers of
identical amino acids was calculated; iv) the specific score of the
target polypeptide was: 100--the average
[0176] 4, The polypeptide fragments with highest specific score
were selected, and surface signature peptides were selected based
on the specific score in combination with the factors of
antigenicity, hydrophilicity, trans-membrane structure, signal
peptide etc.
[0177] Finally, it was determined that the sequence of the tandem
polypeptide displayed by H carrier of the Aof1 protein was:
KKYRKCEKAG-GGGGS-AASRGRS KKR-GGGG S-RS
SGRQAKKK-GGGGS-VRGLVRIRCV-GGGGS-KYSAWKRVWT-GGGGS-RIL
YFMTRKG-GGGGS-MARTYRCGMK(SEQ ID NO: 121)
[0178] After codon optimization, it was determined that the
complete gene sequence to be synthesized was:
TABLE-US-00025 (SEQ ID NO: 122)
GACACggatccAAGAAATACCGTAAATGCGAGAAAGCAGGAGGTGGCGGC
GGAAGCGCGGCTTCCCGTGGCCGTTCAAAAAAACGTGGCGGTGGAGGGTC
CCGGAGCAGCGGCCGTCAGGCGAAAAAGAAGGGTGGTGGGGGATCTGTGC
GTGGCCTGGTGCGTATTCGTTGCGTTGGGGGGGGTGGATCAAAATACTCT
GCGTGGAAACGTGTGTGGACCGGCGGAGGCGGCAGTCGTATCCTGTATTT
CATGACCCGTAAAGGAGGAGGGGGAGGCTCGATGGCGCGTACCTATCGTT
GTGGGATGAAAgaattcGTGTC
[0179] Based on the above, the primers used for the complete gene
synthesis were determined and can be seen in the following table.
The complete gene synthesis method was the same to that of Example
4.
[0180] 5 .mu.l of the synthesized primers 164_01 to 164_14 were
taken (with the concentration of 25 .mu.M) and mixed. Such mixed
primers were used as template, and the first round of PCR
amplification was conducted under the catalysis of PFU enzyme
(ShenNengBoCai co. ltd., Shanghai, China). The PCR conditions were
as: 94.degree., 2 min; 30 cycles (94.degree. C., 30 s; 60.degree.
C., 30 s; 72.degree. C., 1 min); 72.degree. C., 10 min; 10.degree.
C., 10 min. 2 .mu.l of the PCR product of the first round was then
taken as template, and primers 164_01 and 164_10 (with the
concentration of 25 .mu.M) were used for conducting the
amplification, with the amplification conditions as set forth
above. 1.5% agarose gel electrophoresis was performed, and the
target fragments (i.e. the nucleotide sequence to be completely
synthesized) were recovered.
TABLE-US-00026 TABLE 15 The list of the primers for the complete
gene synthesis of Aof1 The number Primer of bases in names Primer
sequences (5'-3') each primer 164_01
GACACggatccAAGAAATACCGTAAATGCGAGAAAGCAGGA 41 (SEQ ID NO: 123)
164_02 GCCGCGCTTCCGCCGCCACCTCCTGCTTTCTCGCATTTAC 40 (SEQ ID NO: 124)
164_03 GCGGAAGCGCGGCTTCCCGTGGCCGTTCAAAAAAACGTGG 40 (SEQ ID NO: 125)
164_04 CTGCTCCGGGACCCTCCACCGCCACGTTTTTTTGAACGGC 40 (SEQ ID NO: 126)
164_05 AGGGTCCCGGAGCAGCGGCCGTCAGGCGAAAAAGAAGGGT 40 (SEQ ID NO: 127)
164_06 GCCACGCACAGATCCCCCACCACCCTTCTTTTTCGCCTGA 40 (SEQ ID NO: 128)
164_07 GGGATCTGTGCGTGGCCTGGTGCGTATTCGTTGCGTTGGG 40 (SEQ ID NO: 129)
164_08 CAGAGTATTTTGATCCACCCCCCCCAACGCAACGAATACG 40 (SEQ ID NO: 130)
164_09 GGGGTGGATCAAAATACTCTGCGTGGAAACGTGTGTGGAC 40 (SEQ ID NO: 131)
164_10 GATACGACTGCCGCCTCCGCCGGTCCACACACGTTTCCAC 40 (SEQ ID NO: 132)
164_11 GAGGCGGCAGTCGTATCCTGTATTTCATGACCCGTAAAGG 40 (SEQ ID NO: 133)
164_12 ATCGAGCCTCCCCCTCCTCCTTTACGGGTCATGAAATACA 40 (SEQ ID NO: 134)
164_13 GAGGGGGAGGCTCGATGGCGCGTACCTATCGTTGTGGGAT 40 (SEQ ID NO: 135)
164_14 GACACgaattcTTTCATCCCACAACGATAGGTACG 35 (SEQ ID NO: 136)
B Expression of the Tandem Polypeptide of Immunization Antigens
[0181] The expression method was the same to that of Example 3. All
together 1.5 mg of recombinantly expressed tandem polypeptide of
immunization antigens was obtained, with the purity of the protein
as 85%.
C Synthesis of the Detection Antigens
[0182] The construction strategy of the detection antigens was the
same to that of Example 4, based on 7 immunogen including the Aof1
protein (see table 16), 7 detection antigens were constructed (see
table 17). The expression and purification methods were the same to
that of Example 4.
[0183] The purified amount of detection antigen 1 was 0.75 mg, with
the purity of 85%; the purified amount of detection antigen 2 was
0.9 mg, with the purity of 70%; the purified amount of detection
antigen 3 was 1.3 mg, with the purity of 75%; the purified amount
of detection antigen 4 was 0.95 mg, with the purity of 95%; the
purified amount of detection antigen 5 was 1.9 mg, with the purity
of 65%; the purified amount of detection antigen 6 was 1.1 mg, with
the purity of 80%; the purified amount of detection antigen 7 was
0.9 mg, with the purity of 85%.
TABLE-US-00027 TABLE 16 Tandem polypeptide sequences of 7
immunization antigens The Protein Name sequence of the tandem
polypeptide of immunization antigens Aof1
GKLLGQGAFGggggsPDSPETSKEVggggsGGSVKDQLKAggggsRKYTRQILEGgg
ggsVKLGDFGASKggggsMDEQEALNSIggggsLTHHFAQLMY (SEQ ID NO: 137)
Histone AQTQGTRRKVggggsSVASAVKLNKggggsRDGIDDESYEggggsPDFKLHISPSggg
deacetylase 1 gsGGRKNSSNFKggggsKGVKEEVKLAggggsKPVMSKVMEM (SEQ ID
NO: 138) OsSPX1
AADGGEEEAggggsQDRVARAAGREggggsMKFGKSLSSQggggsKDLKKRLKLIg
gggsEERQAKRARVggggsGDSSPEEQQEggggsKIPVIEQAAK (SEQ ID NO: 139) Vivax
QKMEVQGNLFggggsKKKHANDLQHggggsETYDPEGKFLggggsPKRDDDNAK
GggggsEKKHSSETPQggggsEKRNYTNLKKggggsERNEPYNIVD (SEQ ID NO: 140)
Knowlesi DGVYSKKKHAggggsRAKKNNVEKIggggsKNFMEEKDKQggggsPKPDDAKAK
GggggsDAQIKKQENKggggsKKKITNHSNRggggsEKRNYTNLKK (SEQ ID NO: 141)
Chabaudi KTNKTFKIKKggggsNKKKHENDLRggggsETYDPKGEFLggggsPGFLYNEQDKgg
ggsEKYKPLIEQVggggsKTPENINAVKggggsEKRNYTNLKK (SEQ ID NO: 142) Ber
NKKKHENDLKggggsETYDPKGEFLggggsDTKNQGLKVDggggsGEMGLDFDRL SEQ ID NO:
143)
TABLE-US-00028 TABLE 17 7 detection antigens Detection antigen
Sequence Detection
KKYRKCEKAGAQTQGTRRKVAADGGEEEAQKMEVQGNLFDGVYSKKKHA antigen 1
KTNKTFKIKKNKKKHENDLK (SEQ ID NO: 144) Detection
AASRGRSKKRSVASAVKLNKQDRVARAAGREKKKHANDLQHRAKKNNVEK antigen 2
INKKKHENDLRETYDPKGEFL (SEQ ID NO: 145) Detection
RSSGRQAKKKRDGIDDESYEMKFGKSLSSQETYDPEGKFLKNFMEEKDKQET antigen 3
YDPKGEFLDTKNQGLKVD (SEQ ID NO: 146) Detection
VRGLVRIRCVPDFKLHISPSKDLKKRLKLIPKRDDDNAKGPKPDDAKAKGPGF antigen 4
LYNEQDKGEMGLDFDR (SEQ ID NO: 147) Detection
KYSAWKRVWTGGRKNSSNFKEERQAKRARVEKKHSSETPQDAQIKKQENKE antigen 5
KYKPLIEQVEKYKPLIEQV (SEQ ID NO: 148) Detection
RILYFMTRKGKGVKEEVKLAGDSSPEEQQEEKRNYTNLKKKKKITNHSNRKT antigen 6
PENINAVKEKRNYTNLKK (SEQ ID NO: 149) Detection
MARTYRCGMKKPVMSKVMEMKIPVIEQAAKERNEPYNIVDEKRNYTNLKKE antigen 7
KRNYTNLICKDRNEPYNIVD (SEQ ID NO150)
C Immunization Method
[0184] The immunization method and dosage for protein Aof1 were as
follows.
[0185] On the first day, 10 .mu.g antigen (H carrier protein
carrying the polypeptide sequence of SEQ ID NO:121) was completely
mixed with the oligonucleotide adjuvant (detailed method can be
seen in the explanation of the oligonucleotide adjuvant in Example
5), and was then used to immunize the mice at hind leg muscle, and
two sites all together, wherein 50-100 .mu.L was applied for each
site.
[0186] On the eighth day, 20 .mu.g antigen was taken and mixed with
the oligonucleotide adjuvant, and was then used to immunize the
mice at tail end and hind leg muscle, wherein 50-100 .mu.L was
applied for each site.
[0187] On the twelfth day, 10 .mu.g antigen was completely mixed
with the oligonucleotide adjuvant, and was then used to immunize
the mice at hind leg muscle.
[0188] On the fourteenth day, blood was taken from eyepit of the
mice, and ELISA method was used to examine the serum titer, wherein
the serum titers of the mice were all higher than 1:32000.
D Cell Fusion and Epitopes Screening
[0189] On the fifteenth day, lymph-node cells of 2 mice were taken
for conducting the fusion. The methods for cell fusion, positive
clone screening, and epitopes screening, were the same to that of
Example 5.
[0190] The wells that were positive to different detection antigens
were just the antibodies against different antigen epitopes, and
the screening results can be seen in table 18. Antibodies against
all together 6 epitopes were obtained, and the numbers of positive
clones were different.
TABLE-US-00029 TABLE 18 The data for the recognition epitopes of
the monoclonal antibodies of Aof1 protein Clone Epitopes The
sequence of epitopes number A GKLLGQGAFG (SEQ ID NO: 151) 5 B
PDSPETSKEV (SEQ ID NO: 152) 8 C GGSVKDQLKA (SEQ ID NO: 153) 10 D
RKYTRQILEG (SEQ ID NO: 154) 0 E VKLGDFGASK (SEQ ID NO: 155) 38 F
MDEQEALNSI (SEQ ID NO: 156) 15 G LTHHFAQLMY (SEQ ID NO: 157) 6
[0191] 2-5 positive wells of each epitopes were respectively picked
to conduct limiting dilution for sub-cloning. After 3 rounds of
sub-clonings, 30 strains of stable hybridoma cell strains were
obtained, which were directed to 6 epitopes.
E the Data for Antibody Verification
[0192] The basic method of Western verification was the same to
that of Example 6, and the cell line used was Hela cervical cancer
cell line (ATCC, USA). FIG. 7 showed the Western results of a
monoclonal antibody 2F1 produced by one cell strain 2F1 of the
above 30 cell strains, and said cell strain was deposited in China
Center for Type Culture Collection (CCTCC) on Jan. 28, 2011, with
the deposition No. CCTCC C201109. 5 epitopes, 15 cell strains were
successful in Western applications.
Sequence CWU 1
1
164154DNAArtificialmultiple cloning site 1ggatcctatc agatctatcg
ggtaccgtat cgcggccgct tccatatgga attc 542639DNAArtificialHBc
nucleotide sequence after codon optimization 2ccatgggcag cagccaccat
catcaccacc acatgaccat gatcaccgat agcctggagt 60tccatatcga tccgtacaag
gaatttggcg cgaccgtgga actgctgagc ttcctgccga 120gcgacttttt
tccaagcgtg cgtgacctgc tggatacggc gagcgcactg tatcgtgaag
180cgctggaaag cccggaacat tgcagcccgc atcataccgc gctgcgtcag
gcgattctgt 240gctggggcga actgatgacc ctggcgacct gggtgggcgg
caatgaagaa ggtggtggcg 300gtagcggcgg tggcggatcc tatcagatct
atcgggtacc gtatcgcggc cgcttccata 360tggaattcgg tggcggcggc
agcggcggtg gtggcagcga agaagacctg gttgtgagct 420atgtgaacac
caatatgggc ctgaagtttc gtcagctgct gtggtttcat attagctgcc
480tgacctttgg ccgcgaaacc gtgattgaat acctggtgag ctttggcgtg
tggattcgta 540ccccaccggc gtatcgtccg ccgaatgcgc caattctgag
caccctgccg gaaacgaccg 600tttaagagct ccgtcgacaa gcttgcggcc gcactcgag
639342DNAArtificialPrimer 3catgccatgg gcagcagcca ccatcatcac
caccacatga cc 42440DNAArtificialPrimer 4ctccaggcta tcggtgatca
tggtcatgtg gtggtgatga 40542DNAArtificialPrimer 5tgatcaccga
tagcctggag ttccatatcg atccgtacaa gg 42640DNAArtificialPrimer
6ccacggtcgc gccaaattcc ttgtacggat cgatatggaa
40740DNAArtificialPrimer 7tggcgcgacc gtggaactgc tgagcttcct
gccgagcgac 40840DNAArtificialprimer 8caggtcacgc acgcttggaa
aaaagtcgct cggcaggaag 40940DNAArtificialprimer 9caagcgtgcg
tgacctgctg gatacggcga gcgcactgta 401040DNAArtificialprimer
10tccgggcttt ccagcgcttc acgatacagt gcgctcgccg
401140DNAArtificialprimer 11cgctggaaag cccggaacat tgcagcccgc
atcataccgc 401240DNAArtificialprimer 12cagcacagaa tcgcctgacg
cagcgcggta tgatgcgggc 401340DNAArtificialprimer 13gtcaggcgat
tctgtgctgg ggcgaactga tgaccctggc 401440DNAArtificialprimer
14ttcattgccg cccacccagg tcgccagggt catcagttcg
401540DNAArtificialprimer 15ggtgggcggc aatgaagaag gtggtggcgg
tagcggcggt 401640DNAArtificialprimer 16acccgataga tctgatagga
tccgccaccg ccgctaccgc 401740DNAArtificialprimer 17ggatcctatc
agatctatcg ggtaccgtat cgcggccgct 401840DNAArtificialprimer
18ccgccgccac cgaattccat atggaagcgg ccgcgatacg
401940DNAArtificialprimer 19attcggtggc ggcggcagcg gcggtggtgg
cagcgaagaa 402040DNAArtificialprimer 20tcacatagct cacaaccagg
tcttcttcgc tgccaccacc 402140DNAArtificialprimer 21cctggttgtg
agctatgtga acaccaatat gggcctgaag 402240DNAArtificialprimer
22aaccacagca gctgacgaaa cttcaggccc atattggtgt
402340DNAArtificialprimer 23tcgtcagctg ctgtggtttc atattagctg
cctgaccttt 402440DNAArtificialprimer 24aatcacggtt tcgcggccaa
aggtcaggca gctaatatga 402540DNAArtificialprimer 25gccgcgaaac
cgtgattgaa tacctggtga gctttggcgt 402640DNAArtificialprimer
26cgccggtggg gtacgaatcc acacgccaaa gctcaccagg
402740DNAArtificialprimer 27cgtaccccac cggcgtatcg tccgccgaat
gcgccaattc 402840DNAArtificialprimer 28gtcgtttccg gcagggtgct
cagaattggc gcattcggcg 402940DNAArtificialprimer 29accctgccgg
aaacgaccgt ttaagagctc cgtcgacaag 403042DNAArtificialprimer
30ccgctcgagt gcggccgcaa gcttgtcgac ggagctctta aa 4231470PRTHomo
sapiens 31Met Ser Gln Ala Tyr Ser Ser Ser Gln Arg Val Ser Ser Tyr
Arg Arg 1 5 10 15 Thr Phe Gly Gly Ala Pro Gly Phe Pro Leu Gly Ser
Pro Leu Ser Ser 20 25 30 Pro Val Phe Pro Arg Ala Gly Phe Gly Ser
Lys Gly Ser Ser Ser Ser 35 40 45 Val Thr Ser Arg Val Tyr Gln Val
Ser Arg Thr Ser Gly Gly Ala Gly 50 55 60 Gly Leu Gly Ser Leu Arg
Ala Ser Arg Leu Gly Thr Thr Arg Thr Pro 65 70 75 80 Ser Ser Tyr Gly
Ala Gly Glu Leu Leu Asp Phe Ser Leu Ala Asp Ala 85 90 95 Val Asn
Gln Glu Phe Leu Thr Thr Arg Thr Asn Glu Lys Val Glu Leu 100 105 110
Gln Glu Leu Asn Asp Arg Phe Ala Asn Tyr Ile Glu Lys Val Arg Phe 115
120 125 Leu Glu Gln Gln Asn Ala Ala Leu Ala Ala Glu Val Asn Arg Leu
Lys 130 135 140 Gly Arg Glu Pro Thr Arg Val Ala Glu Leu Tyr Glu Glu
Glu Leu Arg 145 150 155 160 Glu Leu Arg Arg Gln Val Glu Val Leu Thr
Asn Gln Arg Ala Arg Val 165 170 175 Asp Val Glu Arg Asp Asn Leu Leu
Asp Asp Leu Gln Arg Leu Lys Ala 180 185 190 Lys Leu Gln Glu Glu Ile
Gln Leu Lys Glu Glu Ala Glu Asn Asn Leu 195 200 205 Ala Ala Phe Arg
Ala Asp Val Asp Ala Ala Thr Leu Ala Arg Ile Asp 210 215 220 Leu Glu
Arg Arg Ile Glu Ser Leu Asn Glu Glu Ile Ala Phe Leu Lys 225 230 235
240 Lys Val His Glu Glu Glu Ile Arg Glu Leu Gln Ala Gln Leu Gln Glu
245 250 255 Gln Gln Val Gln Val Glu Met Asp Met Ser Lys Pro Asp Leu
Thr Ala 260 265 270 Ala Leu Arg Asp Ile Arg Ala Gln Tyr Glu Thr Ile
Ala Ala Lys Asn 275 280 285 Ile Ser Glu Ala Glu Glu Trp Tyr Lys Ser
Lys Val Ser Asp Leu Thr 290 295 300 Gln Ala Ala Asn Lys Asn Asn Asp
Ala Leu Arg Gln Ala Lys Gln Glu 305 310 315 320 Met Met Glu Tyr Arg
His Gln Ile Gln Ser Tyr Thr Cys Glu Ile Asp 325 330 335 Ala Leu Lys
Gly Thr Asn Asp Ser Leu Met Arg Gln Met Arg Glu Leu 340 345 350 Glu
Asp Arg Phe Ala Ser Glu Ala Ser Gly Tyr Gln Asp Asn Ile Ala 355 360
365 Arg Leu Glu Glu Glu Ile Arg His Leu Lys Asp Glu Met Ala Arg His
370 375 380 Leu Arg Glu Tyr Gln Asp Leu Leu Asn Val Lys Met Ala Leu
Asp Val 385 390 395 400 Glu Ile Ala Thr Tyr Arg Lys Leu Leu Glu Gly
Glu Glu Ser Arg Ile 405 410 415 Asn Leu Pro Ile Gln Thr Tyr Ser Ala
Leu Asn Phe Arg Glu Thr Ser 420 425 430 Pro Glu Gln Arg Gly Ser Glu
Val His Thr Lys Lys Thr Val Met Ile 435 440 445 Lys Thr Ile Glu Thr
Arg Asp Gly Glu Val Val Ser Glu Ala Thr Gln 450 455 460 Gln Gln His
Glu Val Leu 465 470 3279PRTArtificialtandem antigen 32Arg Glu Thr
Ser Pro Glu Gln Arg Gly Ser Glu Val Gly Gly Gly Gly 1 5 10 15 Ser
Lys Val Ser Asp Leu Thr Gln Ala Ala Asn Lys Gly Gly Gly Gly 20 25
30 Ser Arg Leu Lys Gly Arg Glu Pro Thr Arg Val Ala Glu Gly Gly Gly
35 40 45 Gly Ser Val Glu Val Leu Thr Asn Gln Arg Ala Arg Val Asp
Gly Gly 50 55 60 Gly Gly Ser Glu Glu Ser Arg Ile Asn Leu Pro Ile
Gln Thr Tyr 65 70 75 3312PRTArtificialantigen 33Arg Glu Thr Ser Pro
Glu Gln Arg Gly Ser Glu Val 1 5 10 3411PRTArtificialantigen 34Lys
Val Ser Asp Leu Thr Gln Ala Ala Asn Lys 1 5 10
3512PRTArtificialantigen 35Arg Leu Lys Gly Arg Glu Pro Thr Arg Val
Ala Glu 1 5 10 3612PRTArtificialantigen 36Val Glu Val Leu Thr Asn
Gln Arg Ala Arg Val Asp 1 5 10 3712PRTArtificialantigen 37Glu Glu
Ser Arg Ile Asn Leu Pro Ile Gln Thr Tyr 1 5 10
38261DNAArtificialnucleic acid encoding tandem antigen 38gacacggatc
ccgtgaaacc agcccggaac agcgtggcag cgaagtgggt ggtggaggtt 60ctaaagtgag
cgatctgacc caggcggcga ataaaggggg aggcggcagc cgtctgaaag
120gccgtgaacc gacccgtgtg gcggaaggtg ggggtggaag cgtggaagtg
ctgaccaatc 180agcgtgcgcg tgtggatggc ggtggcggct cggaagaaag
ccgtattaat ctgccgattc 240agacctatgg aatttcgtgt c
2613937DNAArtificialprimer 39gacacggatc ccgtgaaacc agcccggaac
agcgtgg 374040DNAArtificialprimer 40agaacctcca ccacccactt
cgctgccacg ctgttccggg 404140DNAArtificialprimer 41tgggtggtgg
aggttctaaa gtgagcgatc tgacccaggc 404240DNAArtificialprimer
42ctgccgcctc cccctttatt cgccgcctgg gtcagatcgc
404340DNAArtificialprimer 43gggggaggcg gcagccgtct gaaaggccgt
gaaccgaccc 404440DNAArtificialprimer 44cttccacccc caccttccgc
cacacgggtc ggttcacggc 404540DNAArtificialprimer 45gaaggtgggg
gtggaagcgt ggaagtgctg accaatcagc 404640DNAArtificialprimer
46caccgccatc cacacgcgca cgctgattgg tcagcacttc
404740DNAArtificialprimer 47cgtgtggatg gcggtggcgg ctcggaagaa
agccgtatta 404851DNAArtificialprimer 48gacacgaatt cataggtctg
aatcggcaga ttaatacggc tttcttccga g 514979PRTArtificialantigen 49Arg
Glu Thr Ser Pro Glu Gln Arg Gly Ser Glu Val Gly Gly Gly Gly 1 5 10
15 Ser Lys Val Ser Asp Leu Thr Gln Ala Ala Asn Lys Gly Gly Gly Gly
20 25 30 Ser Arg Leu Lys Gly Arg Glu Pro Thr Arg Val Ala Glu Gly
Gly Gly 35 40 45 Gly Ser Val Glu Val Leu Thr Asn Gln Arg Ala Arg
Val Asp Gly Gly 50 55 60 Gly Gly Ser Glu Glu Ser Arg Ile Asn Leu
Pro Ile Gln Thr Tyr 65 70 75 5079PRTArtificialantigen 50Leu Arg Pro
Ser Thr Ser Arg Ser Leu Tyr Ala Ser Gly Gly Gly Gly 1 5 10 15 Ser
Ile Asn Glu Thr Ser Gln His His Asp Asp Leu Glu Gly Gly Gly 20 25
30 Gly Ser Ala Ile Asn Thr Glu Phe Lys Asn Thr Arg Thr Gly Gly Gly
35 40 45 Gly Ser Glu Gln Leu Lys Gly Gln Gly Lys Ser Arg Leu Gly
Gly Gly 50 55 60 Gly Gly Ser Gln Arg Glu Glu Ala Glu Asn Thr Leu
Gln Ser Phe 65 70 75 5178PRTArtificialantigen 51Lys Lys Lys Trp Asn
Leu Gly Ser Asn Ala Lys Asp Gly Gly Gly Gly 1 5 10 15 Ser Asp Gly
Val Arg Gln Ser Arg Ala Ser Asp Lys Gly Gly Gly Gly 20 25 30 Ser
Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro Leu Gly Gly Gly Gly 35 40
45 Ser Val Lys Val Tyr Asp Tyr Gln Glu Asp Gly Ser Val Gly Gly Gly
50 55 60 Gly Ser Glu Ala Lys Asn Ile Thr Trp Phe Lys Asp Gly Lys 65
70 75 5278PRTArtificialantigen 52Asp Lys Asn Ile Gly Gly Asp Glu
Asp Asp Lys Gly Gly Gly Gly Ser 1 5 10 15 Glu Glu Met Gly Gly Ile
Thr Gln Thr Pro Tyr Lys Gly Gly Gly Gly 20 25 30 Ser Cys Tyr Pro
Arg Gly Ser Lys Pro Glu Asp Ala Gly Gly Gly Gly 35 40 45 Ser Asp
His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gly Gly Gly 50 55 60
Gly Ser Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg 65 70 75
5379PRTArtificialantigen 53Val Gln Glu Gly Asn Glu Ser Tyr Gln Gln
Ser Cys Gly Gly Gly Gly 1 5 10 15 Ser Glu Asp Ala His Phe Gln Cys
Pro His Asn Ser Ser Gly Gly Gly 20 25 30 Gly Ser Arg Lys Arg Trp
Gln Asn Glu Lys Leu Gly Leu Asp Gly Gly 35 40 45 Gly Gly Ser Glu
Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr Gly Gly 50 55 60 Gly Gly
Ser Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile 65 70 75
5459PRTArtificialantigen 54Arg Glu Thr Ser Pro Glu Gln Arg Gly Ser
Glu Val Leu Arg Pro Ser 1 5 10 15 Thr Ser Arg Ser Leu Tyr Ala Ser
Lys Lys Lys Trp Asn Leu Gly Ser 20 25 30 Asn Ala Lys Asp Asp Lys
Asn Ile Gly Gly Asp Glu Asp Asp Lys Val 35 40 45 Gln Glu Gly Asn
Glu Ser Tyr Gln Gln Ser Cys 50 55 5559PRTArtificialantigen 55Lys
Val Ser Asp Leu Thr Gln Ala Ala Asn Lys Asn Ile Asn Glu Thr 1 5 10
15 Ser Gln His His Asp Asp Leu Glu Asp Gly Val Arg Gln Ser Arg Ala
20 25 30 Ser Asp Lys Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr
Lys Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser 50 55
5657PRTArtificialantigen 56Arg Leu Lys Gly Arg Glu Pro Thr Arg Val
Ala Glu Ala Ile Asn Thr 1 5 10 15 Glu Phe Lys Asn Thr Arg Thr Cys
Lys Gly Ser Gln Asn Lys Ser Lys 20 25 30 Pro Leu Cys Tyr Pro Arg
Gly Ser Lys Pro Glu Asp Ala Arg Lys Arg 35 40 45 Trp Gln Asn Glu
Lys Leu Gly Leu Asp 50 55 5759PRTArtificialantigen 57Val Glu Val
Leu Thr Asn Gln Arg Ala Arg Val Asp Glu Gln Leu Lys 1 5 10 15 Gly
Gln Gly Lys Ser Arg Leu Gly Val Lys Val Tyr Asp Tyr Gln Glu 20 25
30 Asp Gly Ser Val Asp His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu
35 40 45 Glu Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr 50 55
5860PRTArtificialantigen 58Glu Glu Ser Arg Ile Asn Leu Pro Ile Gln
Thr Tyr Gln Arg Glu Glu 1 5 10 15 Ala Glu Asn Thr Leu Gln Ser Phe
Glu Ala Lys Asn Ile Thr Trp Phe 20 25 30 Lys Asp Gly Lys Gly Gln
Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg 35 40 45 Gly Pro Gly Glu
Asp Pro Asn Gly Thr Leu Ile Ile 50 55 60 59177DNAArtificialantigen
encoding sequence 59cgtgaaacca gcccggaaca gcgtggcagc gaagtgctgc
gtccgagcac cagccgtagc 60ctgtatgcga gcaagaaaaa atggaatctg ggcagcaatg
cgaaagatga caaaaacatt 120ggcggcgacg aggatgataa ggtgcaggaa
ggcaacgaaa gctatcagca aagctgc 17760177DNAArtificialantigen encoding
sequence 60aaagtgtcag acctgaccca ggcggcgaat aagaacatta acgaaaccag
ccagcatcac 60gatgatctgg aagatggcgt gcgtcagagc cgtgcgagcg ataaagaaga
aatgggcggc 120attacccaga ccccgtataa agaggatgca cattttcagt
gcccgcataa tagcagc 17761171DNAArtificialantigen encodin sequence
61cgtctgaaag gccgtgaacc gacccgtgtg gcggaagcga ttaacaccga atttaagaat
60acccgtacct gcaaaggcag ccagaataaa tcgaaaccgc tgtgctatcc gcgtggcagc
120aaaccggaag atgcgcgtaa acggtggcag aatgaaaaac tgggcctgga t
17162177DNAArtificialantigen encoding sequence 62gtggaagtgc
tgaccaatca gcgtgcgcgt gtggatgaac agctgaaagg ccagggcaaa 60agccgtctgg
gtgtgaaagt gtatgattat caggaagatg gcagcgtgga tcatctgagc
120ctgaaagaat ttagcgaact ggaagaagac atcagccgtg ggctgcaggg cacctat
17763180DNAArtificialantigen encoding sequence 63gaagaaagtc
gtattaatct gccgattcag acctatcagc gtgaggaagc ggaaaatacc 60ctgcagtcgt
ttgaagcgaa aaacattacc tggttcaaag atggcaaagg ccagcgggat
120ctgtatagcg gcctgaacca acgtggtccg ggcgaagatc cgaatggcac
cctgattatt 1806439DNAArtificialprimer 64gacacggatc ccgtgaaacc
agcccggaac agcgtggca 396540DNAArtificialprimer 65ctggtgctcg
gacgcagcac ttcgctgcca cgctgttccg 406640DNAArtificialprimer
66gcgtccgagc accagccgta gcctgtatgc gagcaagaaa
406740DNAArtificialprimer 67attgctgccc agattccatt ttttcttgct
cgcatacagg 406840DNAArtificialprimer 68tggaatctgg gcagcaatgc
gaaagatgac aaaaacattg 406940DNAArtificialprimer 69tatcatcctc
gtcgccgcca atgtttttgt catctttcgc 407040DNAArtificialprimer
70cggcgacgag gatgataagg tgcaggaagg caacgaaagc
407144DNAArtificialprimer 71gacacgaatt cgcagctttg ctgatagctt
tcgttgcctt cctg 447243DNAArtificialprimer 72gacacggatc caaagtgtca
gacctgaccc aggcggcgaa taa
437340DNAArtificialprimer 73tgctggctgg tttcgttaat gttcttattc
gccgcctggg 407440DNAArtificialprimer 74aacgaaacca gccagcatca
cgatgatctg gaagatggcg 407540DNAArtificialprimer 75cgctcgcacg
gctctgacgc acgccatctt ccagatcatc 407640DNAArtificialprimer
76gagccgtgcg agcgataaag aagaaatggg cggcattacc
407740DNAArtificialprimer 77catcctcttt atacggggtc tgggtaatgc
cgcccatttc 407840DNAArtificialprimer 78agaccccgta taaagaggat
gcacattttc agtgcccgca 407938DNAArtificialprimer 79gacacgaatt
cgctgctatt atgcgggcac tgaaaatg 388034DNAArtificialprimer
80gacacggatc ccgtctgaaa ggccgtgaac cgac 348140DNAArtificialprimer
81tgttaatcgc ttccgccaca cgggtcggtt cacggccttt
408240DNAArtificialprimer 82tggcggaagc gattaacacc gaatttaaga
atacccgtac 408340DNAArtificialprimer 83ttctggctgc ctttgcaggt
acgggtattc ttaaattcgg 408440DNAArtificialprimer 84tgcaaaggca
gccagaataa atcgaaaccg ctgtgctatc 408540DNAArtificialprimer
85tcttccggtt tgctgccacg cggatagcac agcggtttcg
408640DNAArtificialprimer 86ggcagcaaac cggaagatgc gcgtaaacgg
tggcagaatg 408747DNAArtificialprimer 87gacacgaatt catccaggcc
cagtttttca ttctgccacc gtttacg 478842DNAArtificialprimer
88gacacggatc cgtggaagtg ctgaccaatc agcgtgcgcg tg
428940DNAArtificialprimer 89gccctggcct ttcagctgtt catccacacg
cgcacgctga 409040DNAArtificialprimer 90gctgaaaggc cagggcaaaa
gccgtctggg tgtgaaagtg 409140DNAArtificialprimer 91tgccatcttc
ctgataatca tacactttca cacccagacg 409240DNAArtificialprimer
92atgattatca ggaagatggc agcgtggatc atctgagcct
409340DNAArtificialprimer 93ttccagttcg ctaaattctt tcaggctcag
atgatccacg 409440DNAArtificialprimer 94gaaagaattt agcgaactgg
aagaagacat cagccgtggg 409543DNAArtificialprimer 95gacacgaatt
cataggtgcc ctgcagccca cggctgatgt ctt 439644DNAArtificialprimer
96gacacggatc cgaagaaagt cgtattaatc tgccgattca gacc
449740DNAArtificialprimer 97ccgcttcctc acgctgatag gtctgaatcg
gcagattaat 409840DNAArtificialprimer 98cagcgtgagg aagcggaaaa
taccctgcag tcgtttgaag 409940DNAArtificialprimer 99ttgaaccagg
taatgttttt cgcttcaaac gactgcaggg 4010040DNAArtificialprimer
100cgaaaaacat tacctggttc aaagatggca aaggccagcg
4010140DNAArtificialprimer 101ttggttcagg ccgctataca gatcccgctg
gcctttgcca 4010240DNAArtificialprimer 102atagcggcct gaaccaacgt
ggtccgggcg aagatccgaa 4010343DNAArtificialprimer 103gacacgaatt
caataatcag ggtgccattc ggatcttcgc ccg 4310420DNAArtificialDNA
adjuvant 104tccatgacgt tcctgacgtt 2010512PRTArtificialepitope
105Arg Glu Thr Ser Pro Glu Gln Arg Gly Ser Glu Val 1 5 10
10610PRTArtificialepitope 106Lys Val Ser Asp Leu Thr Gln Ala Ala
Asn 1 5 10 10712PRTArtificialepitope 107Arg Leu Lys Gly Arg Glu Pro
Thr Arg Val Ala Glu 1 5 10 10812PRTArtificialepitope 108Val Glu Val
Leu Thr Asn Gln Arg Ala Arg Val Asp 1 5 10
10912PRTArtificialepitope 109Glu Glu Ser Arg Ile Asn Leu Pro Ile
Gln Thr Tyr 1 5 10 1101346PRTHomo sapiens 110Met Lys Ser Pro Arg
Arg Thr Thr Leu Cys Leu Met Phe Ile Val Ile 1 5 10 15 Tyr Ser Ser
Lys Ala Ala Leu Asn Trp Asn Tyr Glu Ser Thr Ile His 20 25 30 Pro
Leu Ser Leu His Glu His Glu Pro Ala Gly Glu Glu Ala Leu Arg 35 40
45 Gln Lys Arg Ala Val Ala Thr Lys Ser Pro Thr Ala Glu Glu Tyr Thr
50 55 60 Val Asn Ile Glu Ile Ser Phe Glu Asn Ala Ser Phe Leu Asp
Pro Ile 65 70 75 80 Lys Ala Tyr Leu Asn Ser Leu Ser Phe Pro Ile His
Gly Asn Asn Thr 85 90 95 Asp Gln Ile Thr Asp Ile Leu Ser Ile Asn
Val Thr Thr Val Cys Arg 100 105 110 Pro Ala Gly Asn Glu Ile Trp Cys
Ser Cys Glu Thr Gly Tyr Gly Trp 115 120 125 Pro Arg Glu Arg Cys Leu
His Asn Leu Ile Cys Gln Glu Arg Asp Val 130 135 140 Phe Leu Pro Gly
His His Cys Ser Cys Leu Lys Glu Leu Pro Pro Asn 145 150 155 160 Gly
Pro Phe Cys Leu Leu Gln Glu Asp Val Thr Leu Asn Met Arg Val 165 170
175 Arg Leu Asn Val Gly Phe Gln Glu Asp Leu Met Asn Thr Ser Ser Ala
180 185 190 Leu Tyr Arg Ser Tyr Lys Thr Asp Leu Glu Thr Ala Phe Arg
Lys Gly 195 200 205 Tyr Gly Ile Leu Pro Gly Phe Lys Gly Val Thr Val
Thr Gly Phe Lys 210 215 220 Ser Gly Ser Val Val Val Thr Tyr Glu Val
Lys Thr Thr Pro Pro Ser 225 230 235 240 Leu Glu Leu Ile His Lys Ala
Asn Glu Gln Val Val Gln Ser Leu Asn 245 250 255 Gln Thr Tyr Lys Met
Asp Tyr Asn Ser Phe Gln Ala Val Thr Ile Asn 260 265 270 Glu Ser Asn
Phe Phe Val Thr Pro Glu Ile Ile Phe Glu Gly Asp Thr 275 280 285 Val
Ser Leu Val Cys Glu Lys Glu Val Leu Ser Ser Asn Val Ser Trp 290 295
300 Arg Tyr Glu Glu Gln Gln Leu Glu Ile Gln Asn Ser Ser Arg Phe Ser
305 310 315 320 Ile Tyr Thr Ala Leu Phe Asn Asn Met Thr Ser Val Ser
Lys Leu Thr 325 330 335 Ile His Asn Ile Thr Pro Gly Asp Ala Gly Glu
Tyr Val Cys Lys Leu 340 345 350 Ile Leu Asp Ile Phe Glu Tyr Glu Cys
Lys Lys Lys Ile Asp Val Met 355 360 365 Pro Ile Gln Ile Leu Ala Asn
Glu Glu Met Lys Val Met Cys Asp Asn 370 375 380 Asn Pro Val Ser Leu
Asn Cys Cys Ser Gln Gly Asn Val Asn Trp Ser 385 390 395 400 Lys Val
Glu Trp Lys Gln Glu Gly Lys Ile Asn Ile Pro Gly Thr Pro 405 410 415
Glu Thr Asp Ile Asp Ser Ser Cys Ser Arg Tyr Thr Leu Lys Ala Asp 420
425 430 Gly Thr Gln Cys Pro Ser Gly Ser Ser Gly Thr Thr Val Ile Tyr
Thr 435 440 445 Cys Glu Phe Ile Ser Ala Tyr Gly Ala Arg Gly Ser Ala
Asn Ile Lys 450 455 460 Val Thr Phe Ile Ser Val Ala Asn Leu Thr Ile
Thr Pro Asp Pro Ile 465 470 475 480 Ser Val Ser Glu Gly Gln Asn Phe
Ser Ile Lys Cys Ile Ser Asp Val 485 490 495 Ser Asn Tyr Asp Glu Val
Tyr Trp Asn Thr Ser Ala Gly Ile Lys Ile 500 505 510 Tyr Gln Arg Phe
Tyr Thr Thr Arg Arg Tyr Leu Asp Gly Ala Glu Ser 515 520 525 Val Leu
Thr Val Lys Thr Ser Thr Arg Glu Trp Asn Gly Thr Tyr His 530 535 540
Cys Ile Phe Arg Tyr Lys Asn Ser Tyr Ser Ile Ala Thr Lys Asp Val 545
550 555 560 Ile Val His Pro Leu Pro Leu Lys Leu Asn Ile Met Val Asp
Pro Leu 565 570 575 Glu Ala Thr Val Ser Cys Ser Gly Ser His His Ile
Lys Cys Cys Ile 580 585 590 Glu Glu Asp Gly Asp Tyr Lys Val Thr Phe
His Thr Gly Ser Ser Ser 595 600 605 Leu Pro Ala Ala Lys Glu Val Asn
Lys Lys Gln Val Cys Tyr Lys His 610 615 620 Asn Phe Asn Ala Ser Ser
Val Ser Trp Cys Ser Lys Thr Val Asp Val 625 630 635 640 Cys Cys His
Phe Thr Asn Ala Ala Asn Asn Ser Val Trp Ser Pro Ser 645 650 655 Met
Lys Leu Asn Leu Val Pro Gly Glu Asn Ile Thr Cys Gln Asp Pro 660 665
670 Val Ile Gly Val Gly Glu Pro Gly Lys Val Ile Gln Lys Leu Cys Arg
675 680 685 Phe Ser Asn Val Pro Ser Ser Pro Glu Ser Pro Ile Gly Gly
Thr Ile 690 695 700 Thr Tyr Lys Cys Val Gly Ser Gln Trp Glu Glu Lys
Arg Asn Asp Cys 705 710 715 720 Ile Ser Ala Pro Ile Asn Ser Leu Leu
Gln Met Ala Lys Ala Leu Ile 725 730 735 Lys Ser Pro Ser Gln Asp Glu
Met Leu Pro Thr Tyr Leu Lys Asp Leu 740 745 750 Ser Ile Ser Ile Asp
Lys Ala Glu His Glu Ile Ser Ser Ser Pro Gly 755 760 765 Ser Leu Gly
Ala Ile Ile Asn Ile Leu Asp Leu Leu Ser Thr Val Pro 770 775 780 Thr
Gln Val Asn Ser Glu Met Met Thr His Val Leu Ser Thr Val Asn 785 790
795 800 Val Ile Leu Gly Lys Pro Val Leu Asn Thr Trp Lys Val Leu Gln
Gln 805 810 815 Gln Trp Thr Asn Gln Ser Ser Gln Leu Leu His Ser Val
Glu Arg Phe 820 825 830 Ser Gln Ala Leu Gln Ser Gly Asp Ser Pro Pro
Leu Ser Phe Ser Gln 835 840 845 Thr Asn Val Gln Met Ser Ser Met Val
Ile Lys Ser Ser His Pro Glu 850 855 860 Thr Tyr Gln Gln Arg Phe Val
Phe Pro Tyr Phe Asp Leu Trp Gly Asn 865 870 875 880 Val Val Ile Asp
Lys Ser Tyr Leu Glu Asn Leu Gln Ser Asp Ser Ser 885 890 895 Ile Val
Thr Met Ala Phe Pro Thr Leu Gln Ala Ile Leu Ala Gln Asp 900 905 910
Ile Gln Glu Asn Asn Phe Ala Glu Ser Leu Val Met Thr Thr Thr Val 915
920 925 Ser His Asn Thr Thr Met Pro Phe Arg Ile Ser Met Thr Phe Lys
Asn 930 935 940 Asn Ser Pro Ser Gly Gly Glu Thr Lys Cys Val Phe Trp
Asn Phe Arg 945 950 955 960 Leu Ala Asn Asn Thr Gly Gly Trp Asp Ser
Ser Gly Cys Tyr Val Glu 965 970 975 Glu Gly Asp Gly Asp Asn Val Thr
Cys Ile Cys Asp His Leu Thr Ser 980 985 990 Phe Ser Ile Leu Met Ser
Pro Asp Ser Pro Asp Pro Ser Ser Leu Leu 995 1000 1005 Gly Ile Leu
Leu Asp Ile Ile Ser Tyr Val Gly Val Gly Phe Ser 1010 1015 1020 Ile
Leu Ser Leu Ala Ala Cys Leu Val Val Glu Ala Val Val Trp 1025 1030
1035 Lys Ser Val Thr Lys Asn Arg Thr Ser Tyr Met Arg His Thr Cys
1040 1045 1050 Ile Val Asn Ile Ala Ala Ser Leu Leu Val Ala Asn Thr
Trp Phe 1055 1060 1065 Ile Val Val Ala Ala Ile Gln Asp Asn Arg Tyr
Ile Leu Cys Lys 1070 1075 1080 Thr Ala Cys Val Ala Ala Thr Phe Phe
Ile His Phe Phe Tyr Leu 1085 1090 1095 Ser Val Phe Phe Trp Met Leu
Thr Leu Gly Leu Met Leu Phe Tyr 1100 1105 1110 Arg Leu Val Phe Ile
Leu His Glu Thr Ser Arg Ser Thr Gln Lys 1115 1120 1125 Ala Ile Ala
Phe Cys Leu Gly Tyr Gly Cys Pro Leu Ala Ile Ser 1130 1135 1140 Val
Ile Thr Leu Gly Ala Thr Gln Pro Arg Glu Val Tyr Thr Arg 1145 1150
1155 Lys Asn Val Cys Trp Leu Asn Trp Glu Asp Thr Lys Ala Leu Leu
1160 1165 1170 Ala Phe Ala Ile Pro Ala Leu Ile Ile Val Val Val Asn
Ile Thr 1175 1180 1185 Ile Thr Ile Val Val Ile Thr Lys Ile Leu Arg
Pro Ser Ile Gly 1190 1195 1200 Asp Lys Pro Cys Lys Gln Glu Lys Ser
Ser Leu Phe Gln Ile Ser 1205 1210 1215 Lys Ser Ile Gly Val Leu Thr
Pro Leu Leu Gly Leu Thr Trp Gly 1220 1225 1230 Phe Gly Leu Thr Thr
Val Phe Pro Gly Thr Asn Leu Val Phe His 1235 1240 1245 Ile Ile Phe
Ala Ile Leu Asn Val Phe Gln Gly Leu Phe Ile Leu 1250 1255 1260 Leu
Phe Gly Cys Leu Trp Asp Leu Lys Val Gln Glu Ala Leu Leu 1265 1270
1275 Asn Lys Phe Ser Leu Ser Arg Trp Ser Ser Gln His Ser Lys Ser
1280 1285 1290 Thr Ser Leu Gly Ser Ser Thr Pro Val Phe Ser Met Ser
Ser Pro 1295 1300 1305 Ile Ser Arg Arg Phe Asn Asn Leu Phe Gly Lys
Thr Gly Thr Tyr 1310 1315 1320 Asn Val Ser Thr Pro Glu Ala Thr Ser
Ser Ser Leu Glu Asn Ser 1325 1330 1335 Ser Ser Ala Ser Ser Leu Leu
Asn 1340 1345 11132DNAArtificialprimer 111cgcggatccc ttcaggaaga
tgttaccctg aa 3211232DNAArtificialprimer 112cgcgaattca acatctattt
tcttcttgca ct 32113826PRTMus musculus 113Met Ala Ala Ser Arg Gly
Arg Ser Lys Lys Arg Ser Asn Leu Glu Leu 1 5 10 15 Ser Pro Asp Asn
Leu Pro Leu Arg Ser Ser Gly Arg Gln Ala Lys Lys 20 25 30 Lys Ala
Val Glu Ile Pro Asp Glu Asp Glu Asp Gly Ser Ser Glu Lys 35 40 45
Lys Tyr Arg Lys Cys Glu Lys Ala Gly Cys Thr Ala Ala Tyr Pro Val 50
55 60 Cys Phe Ala Ser Ala Ser Glu Arg Cys Ala Lys Asn Gly Tyr Thr
Ser 65 70 75 80 Arg Trp Tyr His Leu Ser Cys Gly Glu His Phe Cys Asn
Glu Cys Phe 85 90 95 Asp His Tyr Tyr Arg Ser His Lys Asp Gly Tyr
Asp Lys Tyr Ser Ala 100 105 110 Trp Lys Arg Val Trp Thr Ser Asn Gly
Lys Thr Glu Pro Ser Pro Lys 115 120 125 Ala Phe Met Ala Asp Gln Gln
Leu Pro Tyr Trp Val Gln Cys Thr Lys 130 135 140 Pro Glu Cys Gly Lys
Trp Arg Gln Leu Thr Lys Glu Ile Gln Leu Thr 145 150 155 160 Pro His
Met Ala Arg Thr Tyr Arg Cys Gly Met Lys Pro Asn Thr Ile 165 170 175
Thr Lys Pro Asp Thr Pro Asp His Cys Ser Phe Pro Glu Asp Leu Arg 180
185 190 Val Leu Glu Val Ser Asn His Trp Trp Tyr Pro Met Leu Ile Gln
Pro 195 200 205 Pro Leu Leu Lys Asp Ser Val Ala Ala Pro Leu Leu Ser
Ala Tyr Tyr 210 215 220 Pro Asp Cys Val Gly Met Ser Pro Ser Cys Thr
Ser Thr His Arg Ala 225 230 235 240 Thr Val Thr Ala Ala Thr Thr Thr
Thr Gly Ser Ala Ser Pro Gly Glu 245 250 255 Met Glu Pro Ser Lys Ala
Ala Pro Ser Ser Leu Val Leu Gly Met Asn 260 265 270 Arg Tyr Phe Gln
Pro Phe Tyr Gln Pro Asn Glu Cys Gly Lys Ala Leu 275 280 285 Cys Val
Arg Pro Asp Val Met Glu Leu Asp Glu Leu Tyr Glu Phe Pro 290 295 300
Glu Tyr Ser Arg Asp Pro Thr Met Tyr Leu Ala Leu Arg Asn Leu Ile 305
310 315 320 Leu Ala Leu Trp Tyr Thr Asn Cys Lys Glu Ala Leu Thr Pro
Gln Lys 325 330 335 Cys Ile Pro His Ile Ile Val Arg Gly Leu Val Arg
Ile Arg Cys Val 340 345 350 Gln Glu Val Glu Arg Ile Leu Tyr Phe Met
Thr Arg Lys Gly Leu Ile 355 360 365 Asn Thr Gly Val Leu Thr Val Ala
Ala Gly Gln His Leu Leu Pro Lys 370 375 380 His Tyr His Asn Lys Ser
Val Leu Val Val Gly Ala Gly Pro Ala Gly 385 390 395 400 Leu Ala Ala
Ala Arg Gln Leu His Asn Phe Gly Met Lys Val Thr Val 405 410 415 Leu
Glu Ala Lys Asp Arg Ile Gly Gly Arg Val Trp Asp Asp Lys Ser 420
425 430 Phe Lys Gly Val Val Val Gly Arg Gly Pro Gln Ile Val Asn Gly
Cys 435 440 445 Ile Asn Asn Pro Val Ala Leu Met Cys Glu Gln Leu Gly
Ile Ser Met 450 455 460 Arg Lys Leu Gly Glu Arg Cys Asp Leu Ile Gln
Glu Gly Gly Arg Ile 465 470 475 480 Thr Asp Pro Thr Val Asp Lys Arg
Met Asp Phe His Phe Asn Ala Leu 485 490 495 Leu Asp Val Val Ser Glu
Trp Arg Lys Asp Lys Thr Leu Leu Gln Asp 500 505 510 Val Pro Leu Gly
Glu Lys Ile Glu Glu Ile Tyr Arg Ala Phe Val Lys 515 520 525 Glu Ser
Gly Ile Gln Phe Ser Glu Leu Glu Gly Gln Val Leu Gln Phe 530 535 540
His Leu Ser Asn Leu Glu Tyr Ala Cys Gly Ser Ser Leu His Gln Val 545
550 555 560 Ser Ala Arg Ser Trp Asp His Asn Glu Phe Phe Ala Gln Phe
Ala Gly 565 570 575 Asp His Thr Leu Leu Thr Pro Gly Tyr Ser Thr Ile
Ile Glu Lys Leu 580 585 590 Ala Glu Gly Leu Asp Ile Arg Leu Lys Ser
Pro Val Gln Ser Ile Asp 595 600 605 Tyr Thr Gly Asp Glu Val Gln Val
Thr Thr Thr Asp Gly Met Gly His 610 615 620 Ser Ala Gln Lys Val Leu
Val Thr Val Pro Leu Ala Ile Leu Gln Arg 625 630 635 640 Gly Ala Ile
Gln Phe Asn Pro Pro Leu Ser Glu Lys Lys Met Lys Ala 645 650 655 Ile
Asn Ser Leu Gly Ala Gly Ile Ile Glu Lys Ile Ala Leu Gln Phe 660 665
670 Pro Tyr Arg Phe Trp Asp Ser Lys Val Gln Gly Ala Asp Phe Phe Gly
675 680 685 His Val Pro Pro Ser Ala Ser Gln Arg Gly Leu Phe Ala Val
Phe Tyr 690 695 700 Asp Met Asp Ser Gln Gln Ser Val Leu Met Ser Val
Ile Thr Gly Glu 705 710 715 720 Ala Val Ala Ser Leu Arg Thr Met Asp
Asp Lys Gln Val Leu Gln Gln 725 730 735 Cys Met Gly Ile Leu Arg Glu
Leu Phe Lys Glu Gln Glu Ile Pro Glu 740 745 750 Pro Thr Lys Tyr Phe
Val Thr Arg Trp Ser Thr Glu Pro Trp Ile Gln 755 760 765 Met Ala Tyr
Ser Phe Val Lys Thr Phe Gly Ser Gly Glu Ala Tyr Asp 770 775 780 Ile
Ile Ala Glu Glu Ile Gln Gly Thr Val Phe Phe Ala Gly Glu Ala 785 790
795 800 Thr Asn Arg His Phe Pro Gln Thr Val Thr Gly Ala Tyr Leu Ser
Gly 805 810 815 Val Arg Glu Ala Ser Lys Ile Ala Ala Phe 820 825
11410PRTArtificialsignature peptide 114Lys Lys Tyr Arg Lys Cys Glu
Lys Ala Gly 1 5 10 11510PRTArtificialsignature peptide 115Ala Ala
Ser Arg Gly Arg Ser Lys Lys Arg 1 5 10 11610PRTArtificialsignature
peptide 116Arg Ser Ser Gly Arg Gln Ala Lys Lys Lys 1 5 10
11710PRTArtificialsignature peptide 117Val Arg Gly Leu Val Arg Ile
Arg Cys Val 1 5 10 11810PRTArtificialsignature peptide 118Lys Tyr
Ser Ala Trp Lys Arg Val Trp Thr 1 5 10 11910PRTArtificialsignature
peptide 119Arg Ile Leu Tyr Phe Met Thr Arg Lys Gly 1 5 10
12010PRTArtificialsignature peptide 120Met Ala Arg Thr Tyr Arg Cys
Gly Met Lys 1 5 10 121100PRTArtificialtandem polypeptide 121Lys Lys
Tyr Arg Lys Cys Glu Lys Ala Gly Gly Gly Gly Gly Ser Ala 1 5 10 15
Ala Ser Arg Gly Arg Ser Lys Lys Arg Gly Gly Gly Gly Ser Arg Ser 20
25 30 Ser Gly Arg Gln Ala Lys Lys Lys Gly Gly Gly Gly Ser Val Arg
Gly 35 40 45 Leu Val Arg Ile Arg Cys Val Gly Gly Gly Gly Ser Lys
Tyr Ser Ala 50 55 60 Trp Lys Arg Val Trp Thr Gly Gly Gly Gly Ser
Arg Ile Leu Tyr Phe 65 70 75 80 Met Thr Arg Lys Gly Gly Gly Gly Gly
Ser Met Ala Arg Thr Tyr Arg 85 90 95 Cys Gly Met Lys 100
122322DNAArtificialencoding seqence 122gacacggatc caagaaatac
cgtaaatgcg agaaagcagg aggtggcggc ggaagcgcgg 60cttcccgtgg ccgttcaaaa
aaacgtggcg gtggagggtc ccggagcagc ggccgtcagg 120cgaaaaagaa
gggtggtggg ggatctgtgc gtggcctggt gcgtattcgt tgcgttgggg
180ggggtggatc aaaatactct gcgtggaaac gtgtgtggac cggcggaggc
ggcagtcgta 240tcctgtattt catgacccgt aaaggaggag ggggaggctc
gatggcgcgt acctatcgtt 300gtgggatgaa agaattcgtg tc
32212341DNAArtificialprimer 123gacacggatc caagaaatac cgtaaatgcg
agaaagcagg a 4112440DNAArtificialprimer 124gccgcgcttc cgccgccacc
tcctgctttc tcgcatttac 4012540DNAArtificialprimer 125gcggaagcgc
ggcttcccgt ggccgttcaa aaaaacgtgg 4012640DNAArtificialprimer
126ctgctccggg accctccacc gccacgtttt tttgaacggc
4012740DNAArtificialprimer 127agggtcccgg agcagcggcc gtcaggcgaa
aaagaagggt 4012840DNAArtificialprimer 128gccacgcaca gatcccccac
cacccttctt tttcgcctga 4012940DNAArtificialprimer 129gggatctgtg
cgtggcctgg tgcgtattcg ttgcgttggg 4013040DNAArtificialprimer
130cagagtattt tgatccaccc cccccaacgc aacgaatacg
4013140DNAArtificialprimer 131ggggtggatc aaaatactct gcgtggaaac
gtgtgtggac 4013240DNAArtificialprimer 132gatacgactg ccgcctccgc
cggtccacac acgtttccac 4013340DNAArtificialprimer 133gaggcggcag
tcgtatcctg tatttcatga cccgtaaagg 4013440DNAArtificialprimer
134atcgagcctc cccctcctcc tttacgggtc atgaaataca
4013540DNAArtificialprimer 135gagggggagg ctcgatggcg cgtacctatc
gttgtgggat 4013635DNAArtificialprimer 136gacacgaatt ctttcatccc
acaacgatag gtacg 35137100PRTArtificialantigen 137Gly Lys Leu Leu
Gly Gln Gly Ala Phe Gly Gly Gly Gly Gly Ser Pro 1 5 10 15 Asp Ser
Pro Glu Thr Ser Lys Glu Val Gly Gly Gly Gly Ser Gly Gly 20 25 30
Ser Val Lys Asp Gln Leu Lys Ala Gly Gly Gly Gly Ser Arg Lys Tyr 35
40 45 Thr Arg Gln Ile Leu Glu Gly Gly Gly Gly Gly Ser Val Lys Leu
Gly 50 55 60 Asp Phe Gly Ala Ser Lys Gly Gly Gly Gly Ser Met Asp
Glu Gln Glu 65 70 75 80 Ala Leu Asn Ser Ile Gly Gly Gly Gly Ser Leu
Thr His His Phe Ala 85 90 95 Gln Leu Met Tyr 100
138100PRTArtificialantigen 138Ala Gln Thr Gln Gly Thr Arg Arg Lys
Val Gly Gly Gly Gly Ser Ser 1 5 10 15 Val Ala Ser Ala Val Lys Leu
Asn Lys Gly Gly Gly Gly Ser Arg Asp 20 25 30 Gly Ile Asp Asp Glu
Ser Tyr Glu Gly Gly Gly Gly Ser Pro Asp Phe 35 40 45 Lys Leu His
Ile Ser Pro Ser Gly Gly Gly Gly Ser Gly Gly Arg Lys 50 55 60 Asn
Ser Ser Asn Phe Lys Gly Gly Gly Gly Ser Lys Gly Val Lys Glu 65 70
75 80 Glu Val Lys Leu Ala Gly Gly Gly Gly Ser Lys Pro Val Met Ser
Lys 85 90 95 Val Met Glu Met 100 139100PRTArtificialantigen 139Ala
Ala Asp Gly Gly Glu Glu Glu Ala Gly Gly Gly Gly Ser Gln Asp 1 5 10
15 Arg Val Ala Arg Ala Ala Gly Arg Glu Gly Gly Gly Gly Ser Met Lys
20 25 30 Phe Gly Lys Ser Leu Ser Ser Gln Gly Gly Gly Gly Ser Lys
Asp Leu 35 40 45 Lys Lys Arg Leu Lys Leu Ile Gly Gly Gly Gly Ser
Glu Glu Arg Gln 50 55 60 Ala Lys Arg Ala Arg Val Gly Gly Gly Gly
Ser Gly Asp Ser Ser Pro 65 70 75 80 Glu Glu Gln Gln Glu Gly Gly Gly
Gly Ser Lys Ile Pro Val Ile Glu 85 90 95 Gln Ala Ala Lys 100
140100PRTArtificialantigen 140Gln Lys Met Glu Val Gln Gly Asn Leu
Phe Gly Gly Gly Gly Ser Lys 1 5 10 15 Lys Lys His Ala Asn Asp Leu
Gln His Gly Gly Gly Gly Ser Glu Thr 20 25 30 Tyr Asp Pro Glu Gly
Lys Phe Leu Gly Gly Gly Gly Ser Pro Lys Arg 35 40 45 Asp Asp Asp
Asn Ala Lys Gly Gly Gly Gly Gly Ser Glu Lys Lys His 50 55 60 Ser
Ser Glu Thr Pro Gln Gly Gly Gly Gly Ser Glu Lys Arg Asn Tyr 65 70
75 80 Thr Asn Leu Lys Lys Gly Gly Gly Gly Ser Glu Arg Asn Glu Pro
Tyr 85 90 95 Asn Ile Val Asp 100 141100PRTArtificialantigen 141Asp
Gly Val Tyr Ser Lys Lys Lys His Ala Gly Gly Gly Gly Ser Arg 1 5 10
15 Ala Lys Lys Asn Asn Val Glu Lys Ile Gly Gly Gly Gly Ser Lys Asn
20 25 30 Phe Met Glu Glu Lys Asp Lys Gln Gly Gly Gly Gly Ser Pro
Lys Pro 35 40 45 Asp Asp Ala Lys Ala Lys Gly Gly Gly Gly Gly Ser
Asp Ala Gln Ile 50 55 60 Lys Lys Gln Glu Asn Lys Gly Gly Gly Gly
Ser Lys Lys Lys Ile Thr 65 70 75 80 Asn His Ser Asn Arg Gly Gly Gly
Gly Ser Glu Lys Arg Asn Tyr Thr 85 90 95 Asn Leu Lys Lys 100
142100PRTArtificialantigen 142Lys Thr Asn Lys Thr Phe Lys Ile Lys
Lys Gly Gly Gly Gly Ser Asn 1 5 10 15 Lys Lys Lys His Glu Asn Asp
Leu Arg Gly Gly Gly Gly Ser Glu Thr 20 25 30 Tyr Asp Pro Lys Gly
Glu Phe Leu Gly Gly Gly Gly Ser Pro Gly Phe 35 40 45 Leu Tyr Asn
Glu Gln Asp Lys Gly Gly Gly Gly Ser Glu Lys Tyr Lys 50 55 60 Pro
Leu Ile Glu Gln Val Gly Gly Gly Gly Ser Lys Thr Pro Glu Asn 65 70
75 80 Ile Asn Ala Val Lys Gly Gly Gly Gly Ser Glu Lys Arg Asn Tyr
Thr 85 90 95 Asn Leu Lys Lys 100 143100PRTArtificialantigen 143Asn
Lys Lys Lys His Glu Asn Asp Leu Lys Gly Gly Gly Gly Ser Glu 1 5 10
15 Thr Tyr Asp Pro Lys Gly Glu Phe Leu Gly Gly Gly Gly Ser Asp Thr
20 25 30 Lys Asn Gln Gly Leu Lys Val Asp Gly Gly Gly Gly Ser Gly
Glu Met 35 40 45 Gly Leu Asp Phe Asp Arg Leu Gly Gly Gly Gly Ser
Glu Lys Tyr Lys 50 55 60 Pro Leu Ile Glu Gln Val Gly Gly Gly Gly
Ser Glu Lys Arg Asn Tyr 65 70 75 80 Thr Asn Leu Lys Lys Gly Gly Gly
Gly Ser Asp Arg Asn Glu Pro Tyr 85 90 95 Asn Ile Val Asp 100
14469PRTArtificialantigen 144Lys Lys Tyr Arg Lys Cys Glu Lys Ala
Gly Ala Gln Thr Gln Gly Thr 1 5 10 15 Arg Arg Lys Val Ala Ala Asp
Gly Gly Glu Glu Glu Ala Gln Lys Met 20 25 30 Glu Val Gln Gly Asn
Leu Phe Asp Gly Val Tyr Ser Lys Lys Lys His 35 40 45 Ala Lys Thr
Asn Lys Thr Phe Lys Ile Lys Lys Asn Lys Lys Lys His 50 55 60 Glu
Asn Asp Leu Lys 65 14571PRTArtificialantigen 145Ala Ala Ser Arg Gly
Arg Ser Lys Lys Arg Ser Val Ala Ser Ala Val 1 5 10 15 Lys Leu Asn
Lys Gln Asp Arg Val Ala Arg Ala Ala Gly Arg Glu Lys 20 25 30 Lys
Lys His Ala Asn Asp Leu Gln His Arg Ala Lys Lys Asn Asn Val 35 40
45 Glu Lys Ile Asn Lys Lys Lys His Glu Asn Asp Leu Arg Glu Thr Tyr
50 55 60 Asp Pro Lys Gly Glu Phe Leu 65 70
14670PRTArtificialantigen 146Arg Ser Ser Gly Arg Gln Ala Lys Lys
Lys Arg Asp Gly Ile Asp Asp 1 5 10 15 Glu Ser Tyr Glu Met Lys Phe
Gly Lys Ser Leu Ser Ser Gln Glu Thr 20 25 30 Tyr Asp Pro Glu Gly
Lys Phe Leu Lys Asn Phe Met Glu Glu Lys Asp 35 40 45 Lys Gln Glu
Thr Tyr Asp Pro Lys Gly Glu Phe Leu Asp Thr Lys Asn 50 55 60 Gln
Gly Leu Lys Val Asp 65 70 14769PRTArtificialantigen 147Val Arg Gly
Leu Val Arg Ile Arg Cys Val Pro Asp Phe Lys Leu His 1 5 10 15 Ile
Ser Pro Ser Lys Asp Leu Lys Lys Arg Leu Lys Leu Ile Pro Lys 20 25
30 Arg Asp Asp Asp Asn Ala Lys Gly Pro Lys Pro Asp Asp Ala Lys Ala
35 40 45 Lys Gly Pro Gly Phe Leu Tyr Asn Glu Gln Asp Lys Gly Glu
Met Gly 50 55 60 Leu Asp Phe Asp Arg 65 14870PRTArtificialantigen
148Lys Tyr Ser Ala Trp Lys Arg Val Trp Thr Gly Gly Arg Lys Asn Ser
1 5 10 15 Ser Asn Phe Lys Glu Glu Arg Gln Ala Lys Arg Ala Arg Val
Glu Lys 20 25 30 Lys His Ser Ser Glu Thr Pro Gln Asp Ala Gln Ile
Lys Lys Gln Glu 35 40 45 Asn Lys Glu Lys Tyr Lys Pro Leu Ile Glu
Gln Val Glu Lys Tyr Lys 50 55 60 Pro Leu Ile Glu Gln Val 65 70
14970PRTArtificialantigen 149Arg Ile Leu Tyr Phe Met Thr Arg Lys
Gly Lys Gly Val Lys Glu Glu 1 5 10 15 Val Lys Leu Ala Gly Asp Ser
Ser Pro Glu Glu Gln Gln Glu Glu Lys 20 25 30 Arg Asn Tyr Thr Asn
Leu Lys Lys Lys Lys Lys Ile Thr Asn His Ser 35 40 45 Asn Arg Lys
Thr Pro Glu Asn Ile Asn Ala Val Lys Glu Lys Arg Asn 50 55 60 Tyr
Thr Asn Leu Lys Lys 65 70 15070PRTArtificialantigen 150Met Ala Arg
Thr Tyr Arg Cys Gly Met Lys Lys Pro Val Met Ser Lys 1 5 10 15 Val
Met Glu Met Lys Ile Pro Val Ile Glu Gln Ala Ala Lys Glu Arg 20 25
30 Asn Glu Pro Tyr Asn Ile Val Asp Glu Lys Arg Asn Tyr Thr Asn Leu
35 40 45 Lys Lys Glu Lys Arg Asn Tyr Thr Asn Leu Lys Lys Asp Arg
Asn Glu 50 55 60 Pro Tyr Asn Ile Val Asp 65 70
15110PRTArtificialepitope 151Gly Lys Leu Leu Gly Gln Gly Ala Phe
Gly 1 5 10 15210PRTArtificialepitope 152Pro Asp Ser Pro Glu Thr Ser
Lys Glu Val 1 5 10 15310PRTArtificialepitope 153Gly Gly Ser Val Lys
Asp Gln Leu Lys Ala 1 5 10 15410PRTArtificialepitope 154Arg Lys Tyr
Thr Arg Gln Ile Leu Glu Gly 1 5 10 15510PRTArtificialepitope 155Val
Lys Leu Gly Asp Phe Gly Ala Ser Lys 1 5 10
15610PRTArtificialepitope 156Met Asp Glu Gln Glu Ala Leu Asn Ser
Ile 1 5 10 15710PRTArtificialepitope 157Leu Thr His His Phe Ala Gln
Leu Met Tyr 1 5 10 1585PRTArtificiallinker 158Gly Gly Gly Gly Ser 1
5 15961DNAArtificialmultiple cloning site 159gtggcggatc ctatcagatc
tatcgggtac cgtatcgcgg ccgcttccat atggaattcg 60g
6116061DNAArtificialmultiple cloning site 160ccgaattcca tatggaagcg
gccgcgatac ggtacccgat agatctgata ggatccgcca 60c
611618PRTArtificialBeta-gal 161Met Thr Met Ile Thr Asp Ser Leu 1 5
16212PRTArtificiallinker 162Glu Glu Gly Gly Gly Gly Ser Gly Gly Gly
Gly
Ser 1 5 10 16312PRTArtificiallinker 163Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Glu 1 5 10 1646PRTArtificiallinker 164His His His
His His His 1 5
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