U.S. patent application number 10/949975 was filed with the patent office on 2005-03-31 for novel modified nucleic acid sequences and methods for increasing mrna levels and protein expression in cell systems.
Invention is credited to Chen, Li How, Meade, Harry.
Application Number | 20050071890 10/949975 |
Document ID | / |
Family ID | 26742460 |
Filed Date | 2005-03-31 |
United States Patent
Application |
20050071890 |
Kind Code |
A1 |
Chen, Li How ; et
al. |
March 31, 2005 |
Novel modified nucleic acid sequences and methods for increasing
mRNA levels and protein expression in cell systems
Abstract
The invention provides modified recombinant nucleic acid
sequences (preferably DNA) and methods for increasing the mRNA
levels and protein expression of proteins which are known to be, or
are likely to be, difficult to express in cell culture systems,
mammalian cell culture systems, or in transgenic animals. The
preferred "difficult" protein candidates for expression using the
recombinant techniques of the invention are those proteins derived
from heterologous cells preferably those of lower organisms such as
parasites, bacteria, and virus, having DNA coding sequences
comprising high overall AT content or AT rich regions and/or mRNA
instability motifs and/or rare codons relative to the recombinant
expression system to be used.
Inventors: |
Chen, Li How; (Acton,
MA) ; Meade, Harry; (Newton, MA) |
Correspondence
Address: |
GTC BIOTHERAPEUTICS, INC.
175 CROSSING BOULEVARD, SUITE 410
FRAMINGHAM
MA
01702
US
|
Family ID: |
26742460 |
Appl. No.: |
10/949975 |
Filed: |
September 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10949975 |
Sep 24, 2004 |
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09175683 |
Oct 20, 1998 |
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60085649 |
May 15, 1998 |
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60062592 |
Oct 20, 1997 |
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Current U.S.
Class: |
800/7 ; 536/23.2;
800/14 |
Current CPC
Class: |
C07K 2319/02 20130101;
C12N 2830/008 20130101; A01K 2267/01 20130101; C12N 15/67 20130101;
C07K 14/4732 20130101; A01K 2227/10 20130101; A61K 39/00 20130101;
A61K 2039/51 20130101; A01K 2227/105 20130101; A01K 2217/05
20130101; C07K 2319/00 20130101; A61K 2039/53 20130101; C12N
15/8509 20130101; A61K 48/00 20130101; Y02A 50/30 20180101; C07K
14/445 20130101; A01K 67/0275 20130101 |
Class at
Publication: |
800/007 ;
536/023.2; 800/014 |
International
Class: |
A01K 067/027; C07H
021/04 |
Claims
1-26. (Cancelled)
27. A method for producing a parasite protein or fragment thereof
in milk of a non-human transgenic mammal, comprising: providing
said non-human transgenic mammal whose genome comprises a modified
nucleic acid sequence encoding said parasite protein or fragment
thereof operably linked to a promoter which directs expression in a
mammary gland, wherein said modified nucleic acid sequence has been
modified by replacing one or more AT-containing codons of the
nucleic acid sequence of said parasite protein or fragment thereof
as it naturally occurs in a parasite with a codon or codons
preferred by a mammalian cell for the purposes of expression and
encoding the same parasite protein or fragment thereof as derived
from said parasite; and allowing said non-human transgenic mammal
to express said parasite protein or fragment thereof in its milk,
to thereby produce said parasite protein or fragment thereof.
28. The method of claim 27, wherein all of the codons of the
naturally occurring nucleic acid sequence have been replaced with a
codon or codons preferred by a mammalian cell for the purposes of
expression and encoding the same parasite protein or fragment
thereof as found in the naturally occurring nucleic acid
sequence.
29. The method of claim 27, wherein said parasite protein or
fragment thereof is expressed in the milk of said non-human
transgenic mammal at a level of at least 0.5 mg/ml.
30. The method of claim 27, wherein said parasite protein or
fragment thereof is expressed in the milk of said non-human
transgenic mammal at a level which is between 1.0 mg/ml and 2.0
mg/ml.
31. The method of claim 27, wherein said parasite protein or
fragment thereof as expressed in said non-human transgenic mammal
can be detectably expressed in the milk of said transgenic
non-human mammal.
32. The method of claim 27, wherein all non-preferred codons are
replaced with a codon or codons preferred by a mammalian cell for
the purposes of expression.
33. The parasite protein or fragment thereof as produced by the
method of claim 27.
34. The method of claim 27 wherein said parasite protein or
fragment thereof is a protein, polypeptide or peptide derived from
the Plasmodium falciparum protein MSP-1.
35. The method of claim 27 wherein said mammalian cell for the
purposes of expression is a mammary epithelial cell.
36. The method of claim 27 wherein said promoter is selected from a
group of promoters consisting of: a) beta-casein; b) bovine
lactoglobulin; c) whey acid promoter; d) alpha-ovalbumin; and e)
caprine casein.
37. The method of claim 27 wherein said non-human transgenic mammal
is selected from a group of mammals consisting of: a) caprine; b)
bovine; c) porcine; d) rodent; and e) ovine.
38. The method of claim 27 wherein said modified nucleic acid
sequence is modified to provide for the expression of a modified
amino acid sequence such at least one glycosylation site on said
parasite protein or protein fragment thereof produced by said
non-human transgenic mammal is eliminated.
39. A method for producing a parasite protein or fragment thereof
in the milk of a non-human transgenic mammal, comprising: providing
said non-human transgenic mammal whose genome comprises a modified
nucleic acid sequence encoding said parasite protein or fragment
thereof operably linked to a promoter which directs expression in a
mammary gland, wherein said nucleic acid sequence of said parasite
protein or fragment thereof has been modified by replacing at least
a portion of an AUUUA mRNA instability motif in a coding sequence
of said parasite protein or fragment thereof as it naturally occurs
in a parasite with a codon or codons preferred by a mammalian cell
for the purposes of expression so as to remove said AUUUA mRNA
instability motif or prevent said AUUUA mRNA instability motif from
destabilizing mRNAs encoding said parasite protein or fragment
thereof while encoding an amino acid which is the same as the
replaced portion of said AUUUA mRNA instability motif; allowing
said non-human transgenic mammal to express said parasite protein
or fragment thereof in its milk, to thereby produce said parasite
protein or fragment thereof, and wherein the naturally occurring
nucleic acid sequence encoding said parasite protein or fragment
thereof contains at least one AUUUA instability motif; and,
40. The method of claim 39, wherein each of said AUUUA mRNA
instability motifs present in the naturally occurring nucleic acid
have been replaced by a codon or codons preferred by a mammalian
cell for the purposes of expression so as to remove said AUUUA mRNA
instability motif or prevent said AUUUA mRNA instability motif from
destabilizing mRNAs encoding said parasite protein or fragment
thereof.
41. The method of claim 39, wherein said modified nucleic acid
sequence further comprises at least one additional codon other than
a first codon replaced to lower AT content or a nucleic acid
sequence modification made to eliminate said AUUUA mRNA instability
motif which has been replaced with a codon or codons preferred by a
mammalian cell for the purposes of expression and encoding the same
parasite protein or fragment thereof as found in the naturally
occurring nucleic acid sequence.
42. The method of claim 39 wherein said parasite protein or
fragment thereof is a protein, polypeptide or peptide derived from
the Plasmodium falciparum protein MSP-1.
43. The method of claim 39 wherein said mammalian cell for the
purposes of expression is a mammary epithelial cell.
44. The method of claim 39 wherein said promoter is selected from a
group of promoters consisting of: a) beta-casein; b) bovine
lactoglobulin; c) whey acid promoter; d) alpha-ovalbumin; and e)
caprine casein.
45. The method of claim 39 wherein said non-human transgenic mammal
is selected from a group of mammals consisting of: a) caprine; b)
bovine; c) porcine; d) rodent; and e) ovine.
46. A method for producing a parasite protein or fragment thereof
in the milk of a non-human transgenic mammal, comprising: providing
a non-human transgenic mammal whose genome comprises a modified
nucleic acid sequence encoding said parasite protein or fragment
thereof operably linked to a promoter which directs expression in a
mammary gland, wherein said modified nucleic acid sequence has been
modified by: a) replacing at least a portion of an AUUUA mRNA
instability motif in the coding sequence of said parasite protein
or fragment thereof as it naturally occurs in a parasite with a
codon or codons preferred by a mammalian cell for the purposes of
expression so as to remove said AUUUA mRNA instability motif or
prevent said AUUUA mRNA instability motif from destabilizing mRNAs
encoding said parasite protein or fragment thereof while encoding
the same amino acid as the replaced portion of said AUUUA mRNA
instability motif; b) replacing one or more AT-containing codons of
said modified nucleic acid sequence as it naturally occurs in said
parasite with a codon or codons preferred by a mammalian cell for
the purposes of expression and encoding the same amino acid as the
replaced codon; c) allowing said non-human transgenic mammal to
express said parasite protein or fragment thereof in its milk, to
thereby produce said parasite protein or fragment thereof and
wherein the naturally occurring nucleic acid sequence encoding said
parasite protein or fragment thereof contains at least one AUUUA
instability motif; and,
47. The method of claim 46, wherein each of said AUUUA mRNA
instability motifs present in the naturally occurring nucleic acid
have been replaced by a codon or codons preferred by a mammalian
cell for the purposes of expression so as to remove said AUUUA mRNA
instability motif or prevent said AUUUA mRNA instability motif from
destabilizing mRNAs encoding said parasite protein or fragment
thereof.
48. The method of claim 46, wherein said modified nucleic acid
sequence further comprises at least one additional codon other than
a first codon replaced to lower AT content or a nucleic acid
sequence modification made to eliminate said AUUUA mRNA instability
motif which has been replaced with a codon or codons preferred by a
mammalian cell for the purposes of expression and encoding the same
parasite protein or fragment thereof as found in the naturally
occurring nucleic acid sequence.
49. The method of claim 46, wherein all of the codons of the
naturally occurring nucleic acid sequence have been replaced with a
codon or codons preferred by a mammalian cell for the purposes of
expression and encoding the same parasite protein or fragment
thereof as found in the naturally occurring nucleic acid
sequence.
50. The method of claim 46, wherein said parasite protein or
fragment thereof is expressed in the milk of said non-human
transgenic mammal at a level of at least 0.5 mg/ml.
51. The method of claim 46, wherein said parasite protein or
fragment thereof is expressed in the milk of said non-human
transgenic mammal at a level which is between 1.0 mg/ml and 2.0
mg/ml.
52. The method of claim 46, wherein said parasite protein or
fragment thereof as expressed in said non-human transgenic mammal
can be detectably expressed in the milk of said transgenic
non-human mammal.
53. The method of claim 46, wherein all non-preferred codons are
replaced with a codon or codons preferred by a mammalian cell for
the purposes of expression.
54. The parasite protein or fragment thereof as produced by the
method of claim 46.
55. The method of claim 46 wherein said parasite protein or
fragment thereof is a protein, polypeptide or peptide derived from
the Plasmodium falciparum protein MSP-1.
56. The method of claim 46 wherein said mammalian cell for the
purposes of expression is a mammary epithelial cell.
57. The method of claim 46 wherein said promoter is selected from a
group of promoters consisting of: a) beta-casein; b) bovine
lactoglobulin; c) whey acid promoter; d) alpha-ovalbumin; and e)
caprine casein.
58. The method of claim 46 wherein said non-human transgenic mammal
is selected from a group of mammals consisting of: a) caprine; b)
bovine; c) porcine; d) rodent; and e) ovine.
59. The method of claim 46 wherein said modified nucleic acid
sequence is modified to provide for the expression of a modified
amino acid sequence such at least one glycosylation site on said
parasite protein or protein fragment thereof produced by said
non-human transgenic mammal is eliminated.
60. A transgenic non-human mammal whose germline comprises a
modified nucleic acid sequence encoding a parasite protein or
fragment thereof operably linked to a promoter which directs
expression in a mammary gland, wherein said modified nucleic acid
sequence has been modified by replacing at least a portion of an
AUUUA mRNA instability motif in a coding sequence as it naturally
occurs in a parasite with a codon or codons preferred by a
mammalian cell for the purposes of expression so as to remove said
AUUUA mRNA instability motif or prevent said AUUUA mRNA instability
motif from destabilizing mRNAs encoding said parasite protein or
fragment thereof while encoding the same amino acid as the replaced
portion of said AUUUA mRNA instability motif and by replacing one
or more AT-containing codons of the nucleic acid sequence of said
parasite protein or fragment thereof as it naturally occurs in the
parasite with a codon or codons preferred by a mammalian cell for
the purposes of expression and encoding the same amino acid as the
replaced codon, wherein said non-human transgenic mammal expresses
said parasite protein or fragment thereof in its milk and wherein
the naturally occurring nucleic acid sequence encoding said
parasite protein or fragment thereof contains at least one AUUUA
instability motif; and,
61. The mammal of claim 60, wherein each of said AUUUA mRNA
instability motifs present in the naturally occurring nucleic acid
have been replaced by a codon or codons preferred by a mammalian
cell for the purposes of expression so as to remove said AUUUA mRNA
instability motif or prevent said AUUUA mRNA instability motif from
destabilizing mRNAs encoding said parasite protein or fragment
thereof.
62. The mammal of claim 60, wherein said modified nucleic acid
sequence further comprises at least one additional codon other than
a first codon replaced to lower AT content or a nucleic acid
sequence modification made to eliminate said AUUUA mRNA instability
motif which has been replaced with a codon or codons preferred by a
mammalian cell for the purposes of expression and encoding the same
parasite protein or fragment thereof as found in the naturally
occurring nucleic acid sequence.
63. The mammal of claim 60, wherein all of the codons of the
naturally occurring nucleic acid sequence have been replaced with a
codon or codons preferred by a mammalian cell for the purposes of
expression and encoding the same parasite protein or fragment
thereof as found in the naturally occurring nucleic acid
sequence.
64. The mammal of claim 60, wherein said parasite protein or
fragment thereof is expressed in the milk of said non-human
transgenic mammal at a level of at least 0.5 mg/ml.
65. The mammal of claim 60, wherein said parasite protein or
fragment thereof is expressed in the milk of said non-human
transgenic mammal at a level which is between 1.0 mg/ml and 2.0
mg/ml.
66. The mammal of claim 60, wherein said parasite protein or
fragment thereof as expressed in said non-human transgenic mammal
can be detectably expressed in the milk of said transgenic
non-human mammal.
67. The mammal of claim 60, wherein all non-preferred codons are
replaced with a codon or codons preferred by a mammalian cell for
the purposes of expression.
68. The parasite protein or fragment thereof as produced by the
mammal of claim 60.
69. The mammal of claim 60 wherein said parasite protein or
fragment thereof is a protein, polypeptide or peptide derived from
the Plasmodium falciparum protein MSP-1.
70. The mammal of claim 60 wherein said mammalian cell for the
purposes of expression is a mammary epithelial cell.
71. The mammal of claim 60 wherein said promoter is selected from a
group of promoters consisting of: a) beta-casein; b) bovine
lactoglobulin; c) whey acid promoter; d) alpha-ovalbumin; and e)
caprine casein.
72. The mammal of claim 60 wherein said non-human transgenic mammal
is selected from a group of mammals consisting of: a) caprine; b)
bovine; c) porcine; d) rodent; and e) ovine.
73. The mammal of claim 60 wherein said modified nucleic acid
sequence is modified to provide for the expression of a modified
amino acid sequence such at least one glycosylation site on said
parasite protein or protein fragment thereof produced by said
non-human transgenic mammal is eliminated.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to heterologous gene expression. More
particularly, the invention relates to the expression of microbial
or parasitic organism genes in higher eukaryote cell systems.
[0003] 2. Summary of the Related Art
[0004] Recombinant production of certain heterologous gene products
is often difficult in in vitro cell culture systems or in vivo
recombinant production systems. For example, many researchers have
found it difficult to express proteins derived from bacteria,
parasites and virus in cell culture systems different from the cell
from which the protein was originally derived, and particularly in
mammalian cell culture systems. One example of a therapeutically
important protein which has been difficult to produce by mammalian
cells is the malaria merozoite surface protein (MSP-1).
[0005] Malaria is a serious heath problem in tropical countries.
Resistance to existing drugs is fast developing and a vaccine is
urgently needed. Of the number of antigens that get expressed
during the life cycle of P. falciparum, MSP-1 is the most
extensively studied and promises to be the most successful
candidate for vaccination. Individuals exposed to P. falciparum
develop antibodies against MSP-1, and studies have shown that there
is a correlation between a naturally acquired immune response to
MSP-1 and reduced malaria morbidity. In a number of studies,
immunization with purified native MSP-1 or recombinant fragments of
the protein has induced at least partial protection from the
parasite (Diggs et al, (1993) Parasitol. Today, 9:300-302). Thus
MSP-1 is an important target for the development of a vaccine
against P. falciparum.
[0006] MSP-1 is a 190-220 kDA glycoprotein. The C-terminal region
has been the focus of recombinant production for use as a vaccine.
However, a major problem in developing MSP-1 as a vaccine is the
difficulty in obtaining recombinant proteins in bacterial or yeast
expression systems that are equivalent in immunological potency to
the affinity purified native protein (Chang et al., (1992) J.
Immunol. 148:548-555.) and in large enough quantities to make
vaccine production feasible.
[0007] Improved procedures for enhancing expression of sufficient
quantities of proteins derived from parasite, bacterial and viral
organisms which have previously been difficult to produce
recombinantly would be advantageous. In particular, a recombinant
system capable of expressing MSP-1 in sufficient quantities would
be particularly advantageous.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides improved recombinant DNA
compositions and procedures for increasing the mRNA levels and
protein expression of proteins derived from heterologous cells,
preferably those of lower organisms such as bacteria, virus, and
parasite, which have previously been difficult to express in cell
culture systems, mammalian cell culture systems, or in transgenic
mammals. The preferred protein candidates for expression in an
expression system in accordance with the invention are those
proteins having DNA coding sequences comprising high overall AT
content or AT rich regions, and/or mRNA instability motifs and/or
rare codons relative to the recombinant expression systems.
[0009] In a first aspect, the invention features a modified known
nucleic acid, preferably a gene from a bacterium, virus or
parasite, capable of being expressed in a system, wherein the
modification comprises a reduced AT content, relative to the
unmodified sequence, and optionally further comprises elimination
of at least one or all mRNA instability motifs present in the
natural gene. In certain preferred embodiments the modification
further comprises replacement of one or more codons of the natural
gene with preferred codons of the cell system.
[0010] In a second aspect, the invention provides a process for
preparing a modified nucleic acid of the invention comprising the
steps of lowering the overall AT content of the natural gene
encoding the protein, and/or eliminating at least one or all mRNA
instability motifs and/or replacing one or more codons with a
preferred codon of the cell system of choice, all by replacing one
or more codons in the natural gene with codons recognizable to, and
preferably with codons preferred by the cell system of choice and
which code for the same amino acids as the replaced codon. This
aspect of the invention further includes modified nucleic acids
prepared according to the process of the invention.
[0011] In a third aspect, the invention also provides vectors
comprising nucleic acids of the invention and promoters active in
the cell line or organism of choice, and host cells transformed
with nucleic acids of the invention.
[0012] In a fourth aspect, he invention provides transgenic
expression vectors for the production of transgenic lactating
animals comprising nucleic acids of the invention as well as
transgenic non-human lactating animals whose germlines comprise a
nucleic acid of the invention.
[0013] In a fifth aspect, he invention provides a transgenic
expression vector for production of a transgenic lactating animal
species comprising a nucleic acid of the invention, a promoter
operatively coupled to the nucleic acid which directs mammary gland
expression of the protein encoded by the nucleic acid into the milk
of the transgenic animal.
[0014] In a sixth aspect, the invention provides a DNA vaccine
comprising a modified nucleic acid according to the invention. A
preferred embodiment of this aspect of the invention comprises a
fragment of a modified MSP-1 gene according to the invention.
DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 depicts the cDNA sequence of MSP-1.sub.42 modified in
accordance with the invention [SEQ ID NO 1] in which 306 nucleotide
positions have been replaced to lower AT content and eliminate mRNA
instability motifs while maintaining the same protein amino acid
sequence of MSP-1.sub.42. The large letters indicate nucleotide
substitutions.
[0016] FIG. 2 depicts the nucleotide sequence coding sequence of
the "wild type" or native MSP-1.sub.42 [SEQ ID NO 2].
[0017] FIG. 3a is a codon usage table for wild type MSP-1.sub.42
(designated "MSP wt" in the table) and the new modified
MSP-1.sub.42 gene (designated "edited MSP" in the table) and
several milk protein genes (casein genes derived from goats and
mouse). The numbers in each column indicate the actual number of
times a specific codon appears in each of the listed genes. The new
MSP-1.sub.42 synthetic gene was derived from the mammary specific
codon usage by first choosing GC rich codons for a given amino acid
combined with selecting the amino acids used most frequently in the
milk proteins.
[0018] FIG. 3b is a codon usage table comparing the number of times
each codon appears in both the wild type MSP-1.sub.42 (designated
"MSP wt" in the table) and the new modified MSP-1.sub.42 gene
(designated "edited MSP" in the table) as is also shown in the
table in FIG. 3a. The table in FIG. 3b, also compares the frequency
in which each codon appears in the wild type MSP-1.sub.42 and the
new modified MSP-1.sub.42 gene, to the frequency of appearance of
each codon in both E. coli genes and human genes. Thus, if the
expression system were E. coli cells, this table may be used to
determine what codons are recognized by, or preferred by E.
coli.
[0019] FIG. 4a-c depict MSP-1.sub.42 constructs GTC 479, GTC 564,
and GTC 627, respectively as are described in the examples.
[0020] FIG. 5 panel A is a Northern analysis wherein construct
GTC627 comprises the new MSP-1.sub.42 gene modified in accordance
with the invention, GTC479 is the construct comprising the native
MSP-1.sub.42 gene, and construct GTC469 is a negative control
DNA
[0021] FIG. 5 panel B is a Western analysis wherein the eluted
fractions after affinity purifications. Numbers are collected
fractions. The results show that fractions from GTC679 the modified
MSP-1.sub.42 synthetic gene construct reacted with polyclonal
antibodies to MSP-1 and the negative control GTC479 did not.
[0022] FIG. 6 depicts the nucleic acid sequences of OT1 [SEQ ID NO
3], OT2 [SEQ ID NO 4], MSP-8 [SEQ ID NO 5] MSP-2 [SEQ ID NO 6] and
MSP1 [SEQ ID NO 7] described in the Examples.
[0023] FIG. 7 is a schematic representation of plasmid BC574.
[0024] FIG. 8 is a schematic representation of BC620.
[0025] FIG. 9 is a schematic representation of BC670.
[0026] FIG. 10 is a representation of a Western blot of MSP in
transgenic milk.
[0027] FIG. 11 is a schematic representation of the nucleotide
sequence of MSP42-2 [SEQ ID NO 8].
[0028] FIG. 12 is a schematic representation of the BC-718.
[0029] FIG. 13 is a representation of a Western blot of BC-718
expression in transgenic milk.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. The issued US patents, allowed applications, published
foreign applications, and references cited herein are hereby
incorporated by reference. Any conflicts between these references
and the present disclosure shall be resolved in favor of the
present disclosure.
[0031] The invention provides modified recombinant nucleic acid
sequences (preferably DNA) and methods for increasing the mRNA
levels and protein expression of proteins which are known to be, or
are likely to be, difficult to express in cell culture systems,
mammalian cell culture systems, or in transgenic animals. The
preferred "difficult" protein candidates for expression using the
recombinant techniques of the invention are those proteins derived
from heterologous cells preferably those of lower organisms such as
parasites, bacteria, and virus, having DNA coding sequences
comprising high overall AT content or AT rich regions and/or mRNA
instability motifs and/or rare codons relative to the recombinant
expression system to be used.
[0032] In a first aspect, the invention features a modified known
nucleic acid, preferably a gene from a bacterium virus or parasite,
capable of being expressed in a cell system, wherein the
modification comprises a reduced AT content, relative to the
unmodified sequence, and optionally further comprises elimination
of at least one or all mRNA instability motifs present in the
natural gene. A "cell system" includes cell culture systems, tissue
culture systems, organ culture systems and tissues of living
animals. In certain preferred embodiments the modification further
comprises replacement of one or more codons of the natural gene
with preferred codons of the cell system. Each of these features
are achieved by replacing one or more codons of the natural gene
with codons recognizable to, and preferably preferred by the cell
system that encode the same amino acid as the codon which was
replaced in the natural gene. In accordance with the invention,
such "silent" nucleotide and codon substitutions should be
sufficient to achieve the goal lowering AT content and/or of
eliminating mRNA instability motifs, and/or reducing the number of
rare codons, white maintaining, and preferably improving the
ability of the cell system to produce mRNA and express the desired
protein.
[0033] Also included in the invention are those sequences which are
specifically homologous to the modified nucleic acids of the
invention under suitable stringent conditions, specifically
excluding the known nucleic acids from which the modified nucleic
acids are derived. A sequence is "specifically homologous" to
another sequence if it is sufficiently homologous to specifically
hybridize to the exact complement of the sequence. A sequence
"specifically hybridizes" to another sequence if it hybridizes to
form Watson-Crick or Hoogsteen base pairs either in the body, or
under conditions which approximate physiological conditions with
respect to ionic strength, e.g., 140 mM NaCl, 5 mM MgCl.sub.2.
Preferably, such specific hybridization is maintained under
stringent conditions, e.g., 0.2.times.SSC at 68.degree. C.
[0034] In preferred embodiments, the nucleic acid of the invention
is capable of expressing the protein in mammalian cell culture, or
in a transgenic animal at a level which is at least 25%, and
preferably 50% and even more preferably at least 100% or more of
that expressed by the natural gene in an in vitro cell culture
system or in a transgenic animal under identical conditions (i.e.
the same cell type, same culture conditions, same expression
vector).
[0035] As used herein, the term "expression" is meant mRNA
transcription resulting in protein expression. Expression may be
measured by a number of techniques known in the art including using
an antibody specific for the protein of interest. By "natural gene"
or "native gene" is meant the gene sequence, or fragments thereof
(including naturally occurring allelic variations), which encode
the wild type form of the protein and from which the modified
nucleic acid is derived. A "preferred codon" means a codon which is
used more prevalently by the cell system of choice. Not all codon
changes described herein are changes to a preferred codon, so long
as the codon replacement is a codon which is at least recognized by
the cell system. The term "reduced AT content" as used herein means
having a lower overall percentage of nucleotides having A (adenine)
or T (thymine) bases relative to the natural gene due to
replacement of the A or T containing nucleotide positions or A
and/or T containing codons with nucleotides or codons recognized by
the cell system of choice and which do not change the amino acid
sequence of the target protein. "Heterologous" is used herein to
denote genetic material originating from a different species than
that into which it has been introduced, or a protein produced from
such genetic material.
[0036] Particularly preferred cell systems of the invention include
mammalian cell culture systems such as COS cells and CHO cells, as
well as transgenic animals, particularly the mammary tissue of
transgenic animals. However, the invention also contemplates
bacteria, yeast, E. coli, and viral expression systems such as
baculovirus and even plant systems.
[0037] In a second aspect, the invention provides a process for
preparing a modified nucleic acid of the invention comprising the
steps of lowering the overall AT content of the natural gene
encoding the protein, and/or eliminating at least one or all mRNA
instability motifs and/or replacing one or more codons with a
preferred codon of the cell system of choice, all by replacing one
or more codons in the natural gene with codons recognizable to, and
peferably with codons preferred by the cell system of choice and
which code for the same amino acids as the replaced codon. Standard
reference works describing the general principals of recombinant
DNA technology include Watson, J. D. et al, Molecular Biology of
the Gene, Volumes I and II the Benjamin/Cummings Publishing
Company, Inc. publisher, Menlo Park, Calif. (1987) Darnell, J. E.
et al., Molecular Cell Biology, Scientific American Books, Inc.,
Publisher, New York, N.Y. (1986); Old, R. W., et al., Principles of
Gene Manipulation: An Introduction to Genetic Engineering, 2d
edition, University of California Press, publisher, Berkeley Calif.
(1981); Maniatis, T., et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed. Cold Spring Harbor Laboratory, publisher, Cold
Spring Harbor, N.Y. (1989) and Current Protocols in Molecular
Biology, Ausubel et al., Wiley Press, New York, N.Y. (1992). This
aspect of the invention further includes modified nucleic acids
prepared according to the process of the invention.
[0038] Without being limited to any theory, previous research has
indicated that a conserved AU sequence (AUUUA) from the 3'
untranslated region of GM-CSF mRNA mediates selective mRNA
degradation (Shaw, G. and Kamen, R. Cell 46:659-667). The focus in
the past has been on the presence of these instability motifs in
the untranslated region of a gene. The instant invention is the
first to recognize an advantage to eliminating the instability
sequences in the coding region of a gene.
[0039] In a third aspect, the invention also provides vectors
comprising nucleic acids of the invention and promoters active in
the cell line or organism of choice, and host cells transformed
with nucleic acids of the invention. Preferred vectors include an
origin of replication and are thus replicatable in one or more cell
type. Certain preferred vectors are expression vectors, and further
comprise at least a promoter and passive terminator, thereby
allowing transcription of the recombinant expression element in a
bacterial, fungal, plant, insect or mammalian cell.
[0040] In a fourth aspect, he invention provides transgenic
expression vectors for the production of transgenic lactating
animals comprising nucleic acids of the invention as well as
transgenic non-human lactating animals whose germlines comprise a
nucleic acid of the invention. Such transgenic expression vectors
comprise a promoter capable of being expressed as part of the
genome of the host transgenic animal. General principals for
producing transgenic animals are known in the art. See for example
Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual,
Cold Spring Harbor Laboratory, (1986); Simons et al, Bio/Technology
6:179-183, (1988); Wall et al., Biol. Reprod. 32:645-651, (1985);
Buhler et al., Bio/Technology, 8:140-143 (1990); Ebert et al.,
Bio/Technology 9:835-838 (1991); Krimenfort et al., Bio/Technology
9:844-847 (1991); Wall et al., J. Cell. Biochem. 49:113-120 (1992).
Techniques for introducing foreign DNA sequences into mammals and
their germ cells were originally developed in the mouse. See e.g.,
Gordon et al., Proc. Natl. Acad. Sci. USA 77:7380-7384, (1980);
Gordon and Ruddle, Science 214: 1244-1246 (1981); Palmiter and
Brinster, Cell 41: 343-345, 1985; Brinster et al., Proc Natl. Acad.
Sci., USA 82:4438-4442 (1985) and Hogan et al. (ibid.). These
techniques were subsequently adapted for use with larger animals
including cows and goats. Up until very recently, the most widely
used procedure for the generation of transgenic mice or livestock,
several hundred linear molecules of the DNA of interest in the form
of a transgenic expression construct are injected into one of the
pro-nuclei of a fertilized egg. Injection of DNA into the cytoplasm
of a zygote is also widely used. Most recently cloning of an entire
transgenic cell line capable of injection into an unfertilized egg
has been achieved (KHS Campbell et al., Nature 380 64-66,
(1996)).
[0041] In a fifth aspect; he invention provides a transgenic
expression vector for production of a transgenic lactating animal
species comprising a nucleic acid of the invention, a promoter
operatively coupled to the nucleic acid which directs mammary gland
expression of the protein encoded by the nucleic acid into the milk
of the transgenic animal. The mammary gland expression system has
the advantages of high expression levels, low cost, correct
processing and accessibility. Known proteins, such as bovine and
human alpha-lactalbumin have been produced in lactating transgenic
animals by several researchers. (Wright et al, Bio/Technology
9:830-834 (1991); Vilotte et al, Eur. J. Biochem., 186:43-48
(1989); Hochi et al., Mol Reprod. And Devel. 33:160-164 (1992);
Soulier et al., FEBS Letters 297(1,2):13-18 (1992)) and the system
has been shown to produce high levels of protein.
[0042] Preferred promoters are active in the mammary tissue.
Particularly useful are promoters that are specifically active in
genes encoding milk specific proteins such as genes found in
mammary tissue, i.e. are more active in mammary tissue than in
other tissues under physiological conditions where milk is
synthesized. Most preferred are promoters that are both specific to
and efficient in mammary tissue. Among such promoters, the casein,
lactalbumin and lactalglobulin promoters are preferred, including,
but not limited to the alpha, beta and gamma casein promoters and
the alpha lactalbumin and beta-lactalglobulin promoters. Preferred
among the promoters are those from rodent, goats and cows. Other
promoters include those that regulate a whey acidic protein (WAP)
gene.
[0043] In a preferred embodiment of the invention, a modified
nucleic acid encoding MSP-1 or fragments thereof capable of
expression in a cell culture system, mammalian cell culture system
or in the milk of a transgenic animal is provided. Nucleic acid
sequences encoding the natural MSP-1 gene are modified in
accordance with the invention. First the overall AT content is
reduced by replacing codons of the natural gene with codons
recognizable to, and preferably with codons preferred by the cell
system of choice, that encode the same amino acid but are
sufficient to lower the AT content of the modified nucleic acid as
compared to the native MSP-1 gene or gene fragment. Second, mRNA
instability motifs (AUUUA, Shaw and Kamen, supra) in the native
gene or gene fragment are eliminated from the coding sequence of
the gene by replacing codons of the natural gene with codons
recognizable to, and preferably prefrred by the cell system of
choice that encode the same amino acid but are sufficient to
eliminate the mRNA instability motif. Optionally, any other codon
of the native gene may be replaced with a preferred codon of the
expression system of choice as described.
[0044] In a sixth aspect, the invention provides a DNA vaccine
comprising a modified nucleic acid according to the invention. In
certain preferred embodiments, the DNA vaccine comprises a vector
according to the invention, The DNA vaccine according to the
invention may be in the form of a "naked" or purified modified
nucleic acid according to the invention, which may or may not be
operatively associated with a promoter. A nucleic acid is
operatively associated with a promoter if it is associated with the
promoter in a manner which allows the nucleic acid sequence to be
expressed. Such DNA vaccines may be delivered without
encapsulation, or they may be delivered as part of a liposome, or
as part of a viral genome. Generally, such vaccines are delivered
in an amount sufficient to allow expression of the nucleic acid and
elicit an antibody response in an animal, including a human, which
receives the DNA vaccine. Subsequent deliveries, at least one week
after the first delivery, may be used to enhance the antibody
response. Preferred delivery routes include introduction via
mucosal membranes, as well as parenteral administration.
[0045] A preferred embodiment of this aspect of the invention
comprises a fragment of a modified MSP-1 gene according to the
invention. Such fragment preferably includes from about 5% to about
100% of the overall gene sequence and comprises one or more
modification according to the invention.
[0046] Examples of codon usage from E coli and human are shown in
FIG. 3b. FIG. 3b shows the frequency of codon usage for the MSP-1
native gene as well as the modified MSP-1 gene of the invention and
also compares the frequency of codon usage to that of E. coli and
human genes. Codon usage frequency tables are readily available and
known to those skilled in the art for a number of other expression
systems such as yeast, baculovirus and the mammalian, systems.
[0047] The following examples illustrate certain preferred modes of
making and practicing the present invention, but are not meant to
limit the scope of the invention since alternative methods may be
utilized to obtain similar results.
EXAMPLES
[0048] Creation of Novel Modified MSP-1.sub.42 Gene
[0049] In one embodiment, a novel modified nucleic acid encoding
the C-terminal fragment of MSP-1 is provided. The novel, modified
nucleic acid of the invention encoding a 42 kD C-terminal part of
MSP-1 (MSP-1.sub.42) capable of expression in mammalian cells of
the invention is shown in FIG. 1. The natural MSP-1.sub.42 gene
(FIG. 2) was not capable of being expressed in mammalian cell
culture or in transgenic mice Analysis of the natural MSP-1.sub.42
gene suggested several characteristics that distinguish it from
mammalian genes. First, it has a very high overall AT content of
76%. Second, the mRNA instability motif, AUUUA, occurred 10 times
in this 1100 bp DNA segment (FIG. 2). To address these differences
a new MSP-1.sub.42 gene was designed. Silent nucleotide
substitution was introduced into the native MSP-1.sub.42 gene at
306 positions to reduce the overall AT content to 49.7%. Each of
the 10 AUUUA mRNA instability motifs in the natural gene were
eliminated by changes in codon usage as well. To change the codon
usage, a mammary tissue specific codon usage table, FIG. 3a, was
created by using several mouse and goat mammary specific proteins.
The table was used to guide the choice of codon usage for the
modified MSP-1.sub.42 gene as described above. For example as shown
in the Table in FIG. 3a, in the natural gene, 65%, (25/38) of the
Leu was encoded by TTA, a rare codon in the mammary gland. In the
modified MSP-1.sub.42 gene, 100% of the Leu was encoded by CTG, a
preferred codon for Leu in the mammary gland.
[0050] An expression vector was created using the modified
MSP-1.sub.42 gene by fusing the first 26 amino acids of goat
beta-casein to the N-terminal of the modified MSP-1.sub.42 gene and
a SalI-Xho I fragment which carries the fusion gene was subcloned
into the XhoI site of the expression vector pCDNA3. A His6 tag was
fused to the 3' end of the MSP-1.sub.42 gene to allow the gene
product to be affinity purified. This resulted in plasmid GTC627
(FIG. 4c).
[0051] To compare the natural MSP-1.sub.42 gene construct to the
modified MSP-1.sub.42 nucleic acid of the invention, an expression
vector was also created for the natural MSP-1.sub.42 gene and the
gene was added to mammalian cell culture and injected into mice to
form transgenic mice as follows:
[0052] Construction of the Native MSP-1.sub.42 Expression
Vector
[0053] To secrete the truncated the merozoite surface protein-I
(MSP-1) of Plasmodium falciparum, the wild type gene encoding the
42 KD C-terminal part of MSP-1 (MSP-1.sub.42) was fused to either
the DNA sequence that encodes the first 15 or the first 25 amino
acids of the goat beta-casein. This is achieved by first PCR
amplify the MSP-1 plasmid (received from Dr. David Kaslow, NIH)
with primers MSP1 and MSP2 (FIG. 6), then cloned the PCR product
into the TA vector (Invitrogen). The Bg1II-XhoI fragments of the
PCR product was ligated with oligos OT1 and OT2 (FIG. 6) into the
expression vector pCDNA3. This yielded plasmid GTC564 (FIG. 4b),
which encodes the 15 amino acid beta-casein signal peptide and the
first 11 amino acids of the mature goat beta-casein followed by the
native MSP-1.sub.42 gene. Oligos MSP-8 and MSP-2 (FIG. 6) were used
to amplify MSP-1 plasmid by PCR, the product was then cloned into
TA vector. The XhoI fragment was exercised and cloned into the XhoI
site of the expression vector pCDNA3 to yield plasmid GTC479 (FIG.
4a), which encoded 15 amino acid goat beta-casein signal peptide
fused to the wild-type MSP-1.sub.42 gene. A His6 tag was added to
the 3' end of MSP-1.sub.42 gene in GTC 564 and GTC 479.
[0054] Native MSP-1.sub.42 Gene is not Expressed in COS-7 Cells
[0055] Expression of the native MSP gene in cultured COS-7 cells
was assayed by transient transfection assays. GTC479 and GTC564
plasmids DNA were introduced into COS-7 cells by lipofectamine
(Gibco-BRL) according to manufacturer's protocols. Total cellular
RNA was isolated from the COS cells two days post-transfection. The
newly synthesized proteins were metabolically labeled for 10 hours
by adding .sup.35S methionine added to the culture media two
days-post transfection.
[0056] To determine the MSP mRNA expression in the COS cells, a
Northern blot was probed with a .sup.32P labeled DNA fragment from
GTC479. No MSP RNA was detected in GTC479 or GTC564 transfectants
(data not shown). Prolonged exposure revealed residual levels of
degraded MSP mRNA. The .sup.35S labeled culture supernatants and
the lysates were immunoprecipitated with a polyclonal antibody
raised against MSP. Immunoprecipitation experiments showed that no
expression from either the lysates or the supernatants of the
GTC479 or GTC564 transfected cells (data not shown). These results
showed that the native MSP-1 gene was not expressed in COS
cells.
[0057] Native MSP-1.sub.42 Gene is not Expressed in the Mammary
Gland of Transgenic Mice
[0058] The SalI-XhoI fragment of GTC479, which encoded the 15 amino
acids of goat beta-casein signal peptide, the first 11 amino acids
of goat beta-casein, and the native MSP-1.sub.42 gene, was cloned
into the XhoI site of the beta-casein expressed in vector BC350.
This yielded plasmid BC574 (FIG. 7). A SalI-NotI fragment of BC574
was injected into the mouse embryo to generate transgenic mice.
Fifteen lines of transgenic mice were established. Milk from the
female founder mice was collected and subjected to Western analysis
with polycolonal antibodies against MSP. None of the seven mice
analyzed were found to express MSP-1.sub.42 protein in their milk.
To further determine if the mRNA of MSP-1.sub.42 was expressed in
the mammary gland, total RNA was extracted from day 11 lactating
transgenic mice and analyzed by Northern blotting. No MSP-1.sub.42
mRNA was detected by any of the BC 574 lines analyzed. Therefore,
the MSP-1.sub.42 transgene was not expressed in the mammary gland
of transgenic mice. Taken together, these experiments suggest that
native parasitic MSP-1.sub.42 gene could not be expressed in
mammalian cells, and the block is as the level of mRNA
abundance.
[0059] Expression of MSP in the Mammalian Cells
[0060] Transient transfection experiments were performed to
evaluate the expression of the modified MSP-1.sub.42 gene of the
invention in COS cells. GTC627 and GTC479 DNA were introduced into
the COS-7 cells. Total RNA was isolated 48 hours post-transfection
for Northern analysis. The immobilized RNA was probed with .sup.32P
labeled SalI-XhoI fragment of GTC627. A dramatic difference was
observed between GTC479 and GTC627. While no MSP-1.sub.42 mRNA was
detected in the GTC479 transfected cells as shown previously,
abundant MSP-1.sub.42 mRNA was expressed by GTC627 (FIG. 5, Panel
A). GTC 469 was used as a negative control and comprises the insert
of GTC564 cloned into cloning vector PU19, a commercially available
cloning vector. A metabolic labeling experiment with .sup.35S
methionine followed by immunoprecipitation with polyclonal antibody
(provided by D. Kaslow NIAID, NIH) against MSP showed that
MSP-1.sub.42 protein was synthesized by the transfected COS cells
(FIG. 5, Panel B). Furthermore, MSP-1.sub.42 was detected in the
transfected COS supernatant, indicating the MSP-1.sub.42 protein
was also secreted. Additionally, using Ni-NTA column, MSP-1.sub.42
was affinity purified from the GTC627 transfected COS
supernatant.
[0061] These results demonstrated that the modification of the
parasitic MSP-1.sub.42 gene lead to the expression of MSP mRNA in
the COS cells. Consequently, the MSP-1.sub.42 product was
synthesized and secreted by mammalian cells.
[0062] Polyclonal antibodies used in this experiment may also be
prepared by means well known in the art (Antibodies: A Laboratory
Manual, Ed Harlow and David Lane, eds. Cold Spring Harbor
Laboratory, publishers (1988)). Production of MSP serum antibodies
is also described in Chang et al., Infection and Immunity (1996)
64:253-261 and Chang et al., (1992) Proc Natl. Acad. Sci. USA
86:6343-6347.
[0063] The results of this analysis indicate that the modified
MSP-1.sub.42 nucleic acid of the invention is expressed at a very
high level compared to that of the natural protein which was not
expressed at all. These results represent the first experimental
evidence that reducing the AT % in a gene leads to expression of
the MSP gene in heterologous systems and also the first evidence
that removal of AUUUA mRNA instability motifs from the MSP coding
region leads to the expression of MSP protein in COS cells.
[0064] Thus, the data presented here suggest that certain
heterologous proteins that may be difficult to express in cell
culture or transgenic systems because of high AT content and/or the
presence of instability motifs, and or the usage of rare codons
which are unrecognizable to the cell system of choice may be
reengineered to enable expression in any given system with the aid
of codon usage tables for that system. The present invention
represents the first time that a DNA sequence has been modified
with the goal of removing suspected sequences responsible for
degradation resulting in low RNA levels or no RNA at all. The
results shown in the FIG. 5, Panel A Northern (i.e. no RNA with
native gene and reasonable levels with a modified DNA sequence in
accordance with the invention), likely explains the increase in
protein production.
[0065] The following examples describe the expression of MSP1-42 as
a native non-fusion (and non-glycosylated) protein in the milk of
transgenic mice.
[0066] Construction of MSP Transgene
[0067] To fuse MSP1-42 to the 15 amino acid .beta.-casein signal
peptide, a pair of oligos, MSP203 and MSP204 (MS P203:
ggccgctcgacgccaccatgaaggtcc- tcataattgcc
tgtctggtggctctggccattgcagtcactccctccgtcat, MSP204:
cgatgacggagggagtgacggctg
caatggccagagccaccagacaggcattatgaggaccttcatggtggc- gtcgagc), which
encode the 15 amino acid--casein signal and the first 5 amino acid
of the MSP1-42 ending at the Cla I site, was ligated with a Cla
I-Xho I fragment of BC620 (Fit. 8) which encodes the rest of the
MSP1-42 gene, into the Xho I site of the expression vector pCDNA3.
A Xho I fragment of this plasmid (GTC669) was then cloned into the
Xho I site of milk specific expression vector BC350 Lo generate
B670 (FIG. 9)
[0068] Expression of MSP1-42 in the Milk of Transgenic Mice
[0069] A Sal I-Not I fragment was prepared from plasmid BC670 and
microinjected into the mouse embryo to generate transgenic mice.
Transgenic mice was identified by extracting mouse DNA from tail
biopsy followed by PCR analysis using oligos GTC17 and MSP101
(sequences of oligos: GTC17, GATTGACAAGTAATACGCTGTTTCCTC, Oligo MSP
101, GGATTCAATAGATACGG). Milk from the female founder transgenic
mice was collected at day 7 and day 9 of lactation, and subjected
to western analysis to determine the expression level of MSP-1-42
using an polyclonal anti-MSP antibody and monoclonal anti MSP
antibody 5.2 (Dr. David Kaslow. NIH). Results indicated that the
level of MSP-1-42 expression in the milk of transgenic mice was at
1-2 mg/ml (FIG. 10).
[0070] Construction of MSP1-42 Glycosylation Sites Minus
Mutants
[0071] Our analysis of the milk produced MSP revealed that the
transgenic MSP protein was N-glycosylated. To eliminate the
N-glycosylation sites in the MSP1-42 gene, Asn. (N) at positions
181 and 262 were substituted with Gln.(Q). The substitutions were
introduced by designing DNA oligos that anneal to the corresponding
region of MSP1 and carry the AAC to CAG mutations. These oligos
were then used as PCR primers to produce DNA fragments that encode
the N to Q substitutions.
[0072] To introduce N262-Q mutation, a pair of oligos, MSPGYLYCO-3
(CAGGGAATGCTGCAGATCAGC) AND MSP42-2 (AATTCTCGAGTTAGTG
GTGGTGGTGGTGGTGATCGCAGAAAATACCATG, FIG. 11), were used to PCR
amplify plasmid GTC627, which contains the synthetic MSP1-42 gene.
The PCR product was cloned into pCR2.1 vector (Invitrogen). This
generated plasmid GTC716.
[0073] To introduce N181-Q mutation, oligos MSPGLYCO-1
(CTCCTTGTTCAGG AACTTGTAGGG) and MSPGLCO-2 (GTCCTGCAGTACACATATGAG.
FIG. 4) were used to amplify plasmid GTC 627. The PCR product was
cloned into pCR2.1. This generated plasmid GTC700.
[0074] The MSP double glycosylation mutant was constructed by the
following three steps: first, a Xho I-Bsm I fragment of BC670 and
the Bsm I-Xho I fragment of GTC716 is ligated into the Xho I site
of vector pCR2.1. This resulted a plasmid that contain the MSP-1-42
gene with N262-Q mutation. EcoN I-Nde I fragment of this plasmid
was then replaced by the EcoN I-Nde I fragment from plasmid GTC716
to introduce the second mutation, N181-Q. A Xho I fragment of this
plasmid was finally cloned into BC350 to generate BC718 (FIG.
12).
[0075] Expression of Nonglycosylated MSP1 in Transgenic Animals
[0076] BC718 has the following characteristics: it carries the MSP
1-42 gene under the control of the .beta.-casein promoter so it can
be expressed in the mammary gland of the transgenic animal during
lactation. Further, it encodes a 15 amino acid .beta.-casein leader
sequence fused directly to MSP 1-42, so that the MSP1-42, without
any additional amino acid at its N-terminal, can be secreted into
the milk. Finally, because the N-Q substitutions, the MSP produced
in the milk of the transgenic animal by this construct will not be
N-glycosylated. Taken together, the transgenic MSP produced in the
milk by BC718 is the same as the parasitic MSP.
[0077] A SalI/XhoI fragment was prepared from plasmid BC718 and
microinjected into mouse embryos to generate transgenic mice.
Transgenic animals were identified as described previously. Milk
from female founders was collected and analyzed by Western blotting
with antibody 5.2. The results, shown in FIG. 13, indicate
expression of nonglycosylated MSP1 at a concentration of 0.5 to 1
mg/ml.
[0078] Equivalents
[0079] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents are considered to be within the scope of this
invention, and are covered by the following claims.
Sequence CWU 1
1
19 1 1065 DNA Artificial Sequence altered MSP sequence; preferably,
a bacterium, virus, or parasite 1 gcc gtc act ccc tcc gtc atc gat
aac atc ctg tcc aag atc gag aac 48 Ala Val Thr Pro Ser Val Ile Asp
Asn Ile Leu Ser Lys Ile Glu Asn 1 5 10 15 gag tac gag gtg ctg tac
ctg aag ccg ctg gca ggg gtc tac cgg agc 96 Glu Tyr Glu Val Leu Tyr
Leu Lys Pro Leu Ala Gly Val Tyr Arg Ser 20 25 30 ctg aag aag cag
ctg gag aac aac gtg atg acc ttc aac gtg aac gtg 144 Leu Lys Lys Gln
Leu Glu Asn Asn Val Met Thr Phe Asn Val Asn Val 35 40 45 aag gat
atc ctg aac agc cgg ttc aac aag cgg gag aac ttc aag aac 192 Lys Asp
Ile Leu Asn Ser Arg Phe Asn Lys Arg Glu Asn Phe Lys Asn 50 55 60
gtg ctg gag agc gat ctg atc ccc tac aag gat ctg acc agc agc aac 240
Val Leu Glu Ser Asp Leu Ile Pro Tyr Lys Asp Leu Thr Ser Ser Asn 65
70 75 80 tac gtg gtc aag gat ccc tac aag ttc ctg aac aag gag aag
aga gat 288 Tyr Val Val Lys Asp Pro Tyr Lys Phe Leu Asn Lys Glu Lys
Arg Asp 85 90 95 aag ttc ctg agc agt tac aac tac atc aag gat agc
att gat acc gat 336 Lys Phe Leu Ser Ser Tyr Asn Tyr Ile Lys Asp Ser
Ile Asp Thr Asp 100 105 110 atc aac ttc gcc aac gat gtc ctg gga tac
tac aag atc ctg tcc gag 384 Ile Asn Phe Ala Asn Asp Val Leu Gly Tyr
Tyr Lys Ile Leu Ser Glu 115 120 125 aag tac aag agc gat ctg gat tca
atc aag aag tac atc aac gat aag 432 Lys Tyr Lys Ser Asp Leu Asp Ser
Ile Lys Lys Tyr Ile Asn Asp Lys 130 135 140 cag gga gag aac gag aag
tac ctg ccc ttc ctg aac aac atc gag acc 480 Gln Gly Glu Asn Glu Lys
Tyr Leu Pro Phe Leu Asn Asn Ile Glu Thr 145 150 155 160 ctg tac aag
acc gtc aac gat aag att gat ctg ttc gtg atc cac ctg 528 Leu Tyr Lys
Thr Val Asn Asp Lys Ile Asp Leu Phe Val Ile His Leu 165 170 175 gag
gcc aag gtc ctg aac tac aca tat gag aag agc aac gtg gag gtc 576 Glu
Ala Lys Val Leu Asn Tyr Thr Tyr Glu Lys Ser Asn Val Glu Val 180 185
190 aag atc aag gag ctg aat tac ctg aag acc atc cag gat aag ctg gcc
624 Lys Ile Lys Glu Leu Asn Tyr Leu Lys Thr Ile Gln Asp Lys Leu Ala
195 200 205 gat ttc aag aag aac aac aac ttc gtc ggg atc gcc gat ctg
agc acc 672 Asp Phe Lys Lys Asn Asn Asn Phe Val Gly Ile Ala Asp Leu
Ser Thr 210 215 220 gat tac aac cac aac aac ctg ctg acc aag ttc ctg
agc acc ggt atg 720 Asp Tyr Asn His Asn Asn Leu Leu Thr Lys Phe Leu
Ser Thr Gly Met 225 230 235 240 gtc ttc gaa aac ctg gcc aag acc gtc
ctg agc aac ctg ctg gat ggg 768 Val Phe Glu Asn Leu Ala Lys Thr Val
Leu Ser Asn Leu Leu Asp Gly 245 250 255 aac ctg cag ggg atg ctg aac
atc agc cag cac cag tgt gtg aag aag 816 Asn Leu Gln Gly Met Leu Asn
Ile Ser Gln His Gln Cys Val Lys Lys 260 265 270 cag tgt ccc cag aac
agc ggg tgt ttc aga cac ctg gat gag aga gag 864 Gln Cys Pro Gln Asn
Ser Gly Cys Phe Arg His Leu Asp Glu Arg Glu 275 280 285 gag tgt aag
tgt ctg ctg aac tac aag cag gaa ggt gat aag tgt gtg 912 Glu Cys Lys
Cys Leu Leu Asn Tyr Lys Gln Glu Gly Asp Lys Cys Val 290 295 300 gaa
aac ccc aat cct act tgt aac gag aac aat ggt gga tgt gat gcc 960 Glu
Asn Pro Asn Pro Thr Cys Asn Glu Asn Asn Gly Gly Cys Asp Ala 305 310
315 320 gat gcc aag tgt acc gag gag gat tca ggg agc aac ggg aag aag
atc 1008 Asp Ala Lys Cys Thr Glu Glu Asp Ser Gly Ser Asn Gly Lys
Lys Ile 325 330 335 acc tgt gag tgt acc aag cct gat tct tat cca ctg
ttc gat ggt atc 1056 Thr Cys Glu Cys Thr Lys Pro Asp Ser Tyr Pro
Leu Phe Asp Gly Ile 340 345 350 ttc tgt agt 1065 Phe Cys Ser 355 2
1088 DNA Plasmodium falciparum CDS (1)...(1083) 2 gca gta act cct
tcc gta att gat aac ata ctt tct aaa att gaa aat 48 Ala Val Thr Pro
Ser Val Ile Asp Asn Ile Leu Ser Lys Ile Glu Asn 1 5 10 15 gaa tat
gag gtt tta tat tta aaa cct tta gca ggt gtt tat aga agt 96 Glu Tyr
Glu Val Leu Tyr Leu Lys Pro Leu Ala Gly Val Tyr Arg Ser 20 25 30
tta aaa aaa caa tta gaa aat aac gtt atg aca ttt aat gtt aat gtt 144
Leu Lys Lys Gln Leu Glu Asn Asn Val Met Thr Phe Asn Val Asn Val 35
40 45 aag gat att tta aat tca cga ttt aat aaa cgt gaa aat ttc aaa
aat 192 Lys Asp Ile Leu Asn Ser Arg Phe Asn Lys Arg Glu Asn Phe Lys
Asn 50 55 60 gtt tta gaa tca gat tta att cca tat aaa gat tta aca
tca agt aat 240 Val Leu Glu Ser Asp Leu Ile Pro Tyr Lys Asp Leu Thr
Ser Ser Asn 65 70 75 80 tat gtt gtc aaa gat cca tat aaa ttt ctt aat
aaa gaa aaa aga gat 288 Tyr Val Val Lys Asp Pro Tyr Lys Phe Leu Asn
Lys Glu Lys Arg Asp 85 90 95 aaa ttc tta agc agt tat aat tat att
aag gat tca ata gat acg gat 336 Lys Phe Leu Ser Ser Tyr Asn Tyr Ile
Lys Asp Ser Ile Asp Thr Asp 100 105 110 ata aat ttt gca aat gat gtt
ctt gga tat tat aaa ata tta tcc gaa 384 Ile Asn Phe Ala Asn Asp Val
Leu Gly Tyr Tyr Lys Ile Leu Ser Glu 115 120 125 aaa tat aaa tca gat
tta gat tca att aaa aaa tat atc aac gac aaa 432 Lys Tyr Lys Ser Asp
Leu Asp Ser Ile Lys Lys Tyr Ile Asn Asp Lys 130 135 140 caa ggt gaa
aat gag aaa tac ctt ccc ttt tta aac aat att gag acc 480 Gln Gly Glu
Asn Glu Lys Tyr Leu Pro Phe Leu Asn Asn Ile Glu Thr 145 150 155 160
tta tat aaa aca gtt aat gat aaa att gat tta ttt gta att cat tta 528
Leu Tyr Lys Thr Val Asn Asp Lys Ile Asp Leu Phe Val Ile His Leu 165
170 175 gaa gca aaa gtt cta aat tat aca tat gag aaa tca aac gta gaa
gtt 576 Glu Ala Lys Val Leu Asn Tyr Thr Tyr Glu Lys Ser Asn Val Glu
Val 180 185 190 aaa ata aaa gaa ctt aat tac tta aaa aca att caa gac
aaa ttg gca 624 Lys Ile Lys Glu Leu Asn Tyr Leu Lys Thr Ile Gln Asp
Lys Leu Ala 195 200 205 gat ttt aaa aaa aat aac aat ttc gtt gga att
gct gat tta tca aca 672 Asp Phe Lys Lys Asn Asn Asn Phe Val Gly Ile
Ala Asp Leu Ser Thr 210 215 220 gat tat aac cat aat aac tta ttg aca
aag ttc ctt agt aca ggt atg 720 Asp Tyr Asn His Asn Asn Leu Leu Thr
Lys Phe Leu Ser Thr Gly Met 225 230 235 240 gtt ttt gaa aat ctt gct
aaa acc gtt tta tct aat tta ctt gat gga 768 Val Phe Glu Asn Leu Ala
Lys Thr Val Leu Ser Asn Leu Leu Asp Gly 245 250 255 aac ttg caa ggt
atg tta aac att tca caa cac caa tgc gta aaa aaa 816 Asn Leu Gln Gly
Met Leu Asn Ile Ser Gln His Gln Cys Val Lys Lys 260 265 270 caa tgt
cca caa aat tct gga tgt ttc aga cat tta gat gaa aga gaa 864 Gln Cys
Pro Gln Asn Ser Gly Cys Phe Arg His Leu Asp Glu Arg Glu 275 280 285
gaa tgt aaa tgt tta tta aat tac aaa caa gaa ggt gat aaa tgt gtt 912
Glu Cys Lys Cys Leu Leu Asn Tyr Lys Gln Glu Gly Asp Lys Cys Val 290
295 300 gaa aat cca aat cct act tgt aac gaa aat aat ggt gga tgt gat
gca 960 Glu Asn Pro Asn Pro Thr Cys Asn Glu Asn Asn Gly Gly Cys Asp
Ala 305 310 315 320 gat gcc aaa tgt acc gaa gaa gat tca ggt agc aac
gga aag aaa atc 1008 Asp Ala Lys Cys Thr Glu Glu Asp Ser Gly Ser
Asn Gly Lys Lys Ile 325 330 335 aca tgt gaa tgt act aaa cct gat tct
tat cca ctt ttc gat ggt att 1056 Thr Cys Glu Cys Thr Lys Pro Asp
Ser Tyr Pro Leu Phe Asp Gly Ile 340 345 350 ttc tgc agt cac cac cac
cac cac cac taact 1088 Phe Cys Ser His His His His His His 355 360
3 88 DNA Artificial Sequence synthetically generated
oligonucleotide 3 tcgacgagag ccatgaaggt cctcatcctt gcctgtctgg
tggctctggc cattgcaaga 60 gagcaggaag aactcaatgt agtcggta 88 4 88 DNA
Artificial Sequence synthetically generated oligonucleotide 4
gatctaccga ctacattgag ttcttcctgc tctcttgcaa tggccagagc caccagacag
60 gcaaggatga ggaccttcat ggctctcg 88 5 60 DNA Artificial Sequence
oligonucleotide for PCR 5 taactcgagc gaaccatgaa ggtcctcatc
cttgcctgtc tggtggctct ggccattgca 60 6 48 DNA Artificial Sequence
oligonucleotide for PCR 6 aattctcgag ttagtggtgg tggtggtggt
gactgcagaa ataccatc 48 7 31 DNA Artificial Sequence oligonucleotide
for PCR 7 aatagatctg cagtaactcc ttccgtaatt g 31 8 1142 DNA
Artificial Sequence altered MSP sequence; preferably, a bacterium,
virus, or parasite 8 atg aag gtc ctc ata att gcc tgt ctg gtg gct
ctg gcc att gca gcc 48 Met Lys Val Leu Ile Ile Ala Cys Leu Val Ala
Leu Ala Ile Ala Ala 1 5 10 15 gtc act ccc tcc gtc atc gat aac atc
ctg tcc aag atc gag aac gag 96 Val Thr Pro Ser Val Ile Asp Asn Ile
Leu Ser Lys Ile Glu Asn Glu 20 25 30 tac gag gtg ctg tac ctg aag
ccc ctg gca gga gtc tac agg agc ctg 144 Tyr Glu Val Leu Tyr Leu Lys
Pro Leu Ala Gly Val Tyr Arg Ser Leu 35 40 45 aag aag cag ctg gag
aac aac gtg atg acc ttc aac gtg aac gtg aag 192 Lys Lys Gln Leu Glu
Asn Asn Val Met Thr Phe Asn Val Asn Val Lys 50 55 60 gat atc ctg
aac agc agg ttc aac aag agg gag aac ttc aag aac gtg 240 Asp Ile Leu
Asn Ser Arg Phe Asn Lys Arg Glu Asn Phe Lys Asn Val 65 70 75 80 ctg
gag agc gat ctg atc ccc tac aag gat ctg acc agc agc aac tac 288 Leu
Glu Ser Asp Leu Ile Pro Tyr Lys Asp Leu Thr Ser Ser Asn Tyr 85 90
95 gtg gtc aaa gat ccc tac aag ttc ctg aac aag gag aag aga gat aag
336 Val Val Lys Asp Pro Tyr Lys Phe Leu Asn Lys Glu Lys Arg Asp Lys
100 105 110 ttc ctg agc agt tac aat tac atc aag gat agc att gac acc
gat atc 384 Phe Leu Ser Ser Tyr Asn Tyr Ile Lys Asp Ser Ile Asp Thr
Asp Ile 115 120 125 aac ttc gcc aac gat gtc ctg gga tac tac aag atc
ctg tcc gag aag 432 Asn Phe Ala Asn Asp Val Leu Gly Tyr Tyr Lys Ile
Leu Ser Glu Lys 130 135 140 tac aag agc gat ctg gat agc atc aag aag
tac atc aac gat aag cag 480 Tyr Lys Ser Asp Leu Asp Ser Ile Lys Lys
Tyr Ile Asn Asp Lys Gln 145 150 155 160 gga gag aac gag aag tac ctg
ccc ttc ctg aac aac atc gag acc ctg 528 Gly Glu Asn Glu Lys Tyr Leu
Pro Phe Leu Asn Asn Ile Glu Thr Leu 165 170 175 tac aag acc gtc aac
gat aag att gat ctg ttc gtg atc cac ctg gag 576 Tyr Lys Thr Val Asn
Asp Lys Ile Asp Leu Phe Val Ile His Leu Glu 180 185 190 gcc aag gtc
ctg cag tac aca tat gag aag agc aac gtg gag gtc aag 624 Ala Lys Val
Leu Gln Tyr Thr Tyr Glu Lys Ser Asn Val Glu Val Lys 195 200 205 atc
aag gag ctg aat tac ctg aag acc atc cag gat aag ctg gcc gat 672 Ile
Lys Glu Leu Asn Tyr Leu Lys Thr Ile Gln Asp Lys Leu Ala Asp 210 215
220 ttc aag aag aac aac aac ttc gtc gga atc gcc gat ctg agc acc gat
720 Phe Lys Lys Asn Asn Asn Phe Val Gly Ile Ala Asp Leu Ser Thr Asp
225 230 235 240 tac aac cac aac aac ctg ctg acc aag ttc ctg agc acc
gga atg gtc 768 Tyr Asn His Asn Asn Leu Leu Thr Lys Phe Leu Ser Thr
Gly Met Val 245 250 255 ttc gaa aac ctg gcc aag acc gtc ctg agc aac
ctg ctg gat gga aac 816 Phe Glu Asn Leu Ala Lys Thr Val Leu Ser Asn
Leu Leu Asp Gly Asn 260 265 270 ctg cag gga atg ctg cag atc agc cag
cac cag tgt gtg aag aag cag 864 Leu Gln Gly Met Leu Gln Ile Ser Gln
His Gln Cys Val Lys Lys Gln 275 280 285 tgt ccc cag aac agc gga tgc
ttc aga cac ctg gat gag agg gag gag 912 Cys Pro Gln Asn Ser Gly Cys
Phe Arg His Leu Asp Glu Arg Glu Glu 290 295 300 tgc aag tgc ctg ctg
aac tac aag cag gaa gga gat aag tgt gtg gaa 960 Cys Lys Cys Leu Leu
Asn Tyr Lys Gln Glu Gly Asp Lys Cys Val Glu 305 310 315 320 aac ccc
aat cct act tgt aac gag aac aat gga gga tgc gat gcc gat 1008 Asn
Pro Asn Pro Thr Cys Asn Glu Asn Asn Gly Gly Cys Asp Ala Asp 325 330
335 gcc aag tgt acc gag gag gat tca gga agc aac gga aag aag atc acc
1056 Ala Lys Cys Thr Glu Glu Asp Ser Gly Ser Asn Gly Lys Lys Ile
Thr 340 345 350 tgc gag tgt acc aag cct gat tct tat cca ctg ttc gat
ggt att ttc 1104 Cys Glu Cys Thr Lys Pro Asp Ser Tyr Pro Leu Phe
Asp Gly Ile Phe 355 360 365 tgc agt cac cac cac cac cac cac taa ctc
gag gat cc 1142 Cys Ser His His His His His His * Leu Glu Asp 370
375 9 355 PRT Artificial Sequence altered MSP sequence; preferably,
a bacterium, virus, or parasite 9 Ala Val Thr Pro Ser Val Ile Asp
Asn Ile Leu Ser Lys Ile Glu Asn 1 5 10 15 Glu Tyr Glu Val Leu Tyr
Leu Lys Pro Leu Ala Gly Val Tyr Arg Ser 20 25 30 Leu Lys Lys Gln
Leu Glu Asn Asn Val Met Thr Phe Asn Val Asn Val 35 40 45 Lys Asp
Ile Leu Asn Ser Arg Phe Asn Lys Arg Glu Asn Phe Lys Asn 50 55 60
Val Leu Glu Ser Asp Leu Ile Pro Tyr Lys Asp Leu Thr Ser Ser Asn 65
70 75 80 Tyr Val Val Lys Asp Pro Tyr Lys Phe Leu Asn Lys Glu Lys
Arg Asp 85 90 95 Lys Phe Leu Ser Ser Tyr Asn Tyr Ile Lys Asp Ser
Ile Asp Thr Asp 100 105 110 Ile Asn Phe Ala Asn Asp Val Leu Gly Tyr
Tyr Lys Ile Leu Ser Glu 115 120 125 Lys Tyr Lys Ser Asp Leu Asp Ser
Ile Lys Lys Tyr Ile Asn Asp Lys 130 135 140 Gln Gly Glu Asn Glu Lys
Tyr Leu Pro Phe Leu Asn Asn Ile Glu Thr 145 150 155 160 Leu Tyr Lys
Thr Val Asn Asp Lys Ile Asp Leu Phe Val Ile His Leu 165 170 175 Glu
Ala Lys Val Leu Asn Tyr Thr Tyr Glu Lys Ser Asn Val Glu Val 180 185
190 Lys Ile Lys Glu Leu Asn Tyr Leu Lys Thr Ile Gln Asp Lys Leu Ala
195 200 205 Asp Phe Lys Lys Asn Asn Asn Phe Val Gly Ile Ala Asp Leu
Ser Thr 210 215 220 Asp Tyr Asn His Asn Asn Leu Leu Thr Lys Phe Leu
Ser Thr Gly Met 225 230 235 240 Val Phe Glu Asn Leu Ala Lys Thr Val
Leu Ser Asn Leu Leu Asp Gly 245 250 255 Asn Leu Gln Gly Met Leu Asn
Ile Ser Gln His Gln Cys Val Lys Lys 260 265 270 Gln Cys Pro Gln Asn
Ser Gly Cys Phe Arg His Leu Asp Glu Arg Glu 275 280 285 Glu Cys Lys
Cys Leu Leu Asn Tyr Lys Gln Glu Gly Asp Lys Cys Val 290 295 300 Glu
Asn Pro Asn Pro Thr Cys Asn Glu Asn Asn Gly Gly Cys Asp Ala 305 310
315 320 Asp Ala Lys Cys Thr Glu Glu Asp Ser Gly Ser Asn Gly Lys Lys
Ile 325 330 335 Thr Cys Glu Cys Thr Lys Pro Asp Ser Tyr Pro Leu Phe
Asp Gly Ile 340 345 350 Phe Cys Ser 355 10 361 PRT Plasmodium
falciparum 10 Ala Val Thr Pro Ser Val Ile Asp Asn Ile Leu Ser Lys
Ile Glu Asn 1 5 10 15 Glu Tyr Glu Val Leu Tyr Leu Lys Pro Leu Ala
Gly Val Tyr Arg Ser 20 25 30 Leu Lys Lys Gln Leu Glu Asn Asn Val
Met Thr Phe Asn Val Asn Val 35 40 45 Lys Asp Ile Leu Asn Ser Arg
Phe Asn Lys Arg Glu Asn Phe Lys Asn 50 55 60 Val Leu Glu Ser Asp
Leu Ile Pro Tyr Lys Asp Leu Thr Ser Ser Asn 65 70 75 80 Tyr Val Val
Lys Asp Pro Tyr Lys Phe Leu Asn Lys Glu Lys Arg Asp 85 90 95 Lys
Phe Leu Ser Ser Tyr Asn Tyr Ile Lys Asp Ser Ile Asp Thr Asp 100 105
110 Ile Asn Phe Ala Asn Asp Val Leu Gly Tyr Tyr Lys Ile Leu Ser Glu
115 120 125 Lys Tyr Lys Ser Asp Leu Asp Ser Ile Lys Lys Tyr Ile Asn
Asp Lys 130 135 140 Gln Gly Glu Asn Glu Lys Tyr Leu Pro Phe Leu
Asn
Asn Ile Glu Thr 145 150 155 160 Leu Tyr Lys Thr Val Asn Asp Lys Ile
Asp Leu Phe Val Ile His Leu 165 170 175 Glu Ala Lys Val Leu Asn Tyr
Thr Tyr Glu Lys Ser Asn Val Glu Val 180 185 190 Lys Ile Lys Glu Leu
Asn Tyr Leu Lys Thr Ile Gln Asp Lys Leu Ala 195 200 205 Asp Phe Lys
Lys Asn Asn Asn Phe Val Gly Ile Ala Asp Leu Ser Thr 210 215 220 Asp
Tyr Asn His Asn Asn Leu Leu Thr Lys Phe Leu Ser Thr Gly Met 225 230
235 240 Val Phe Glu Asn Leu Ala Lys Thr Val Leu Ser Asn Leu Leu Asp
Gly 245 250 255 Asn Leu Gln Gly Met Leu Asn Ile Ser Gln His Gln Cys
Val Lys Lys 260 265 270 Gln Cys Pro Gln Asn Ser Gly Cys Phe Arg His
Leu Asp Glu Arg Glu 275 280 285 Glu Cys Lys Cys Leu Leu Asn Tyr Lys
Gln Glu Gly Asp Lys Cys Val 290 295 300 Glu Asn Pro Asn Pro Thr Cys
Asn Glu Asn Asn Gly Gly Cys Asp Ala 305 310 315 320 Asp Ala Lys Cys
Thr Glu Glu Asp Ser Gly Ser Asn Gly Lys Lys Ile 325 330 335 Thr Cys
Glu Cys Thr Lys Pro Asp Ser Tyr Pro Leu Phe Asp Gly Ile 340 345 350
Phe Cys Ser His His His His His His 355 360 11 379 PRT Artificial
Sequence altered MSP sequence; preferably, a bacterium, virus, or
parasite 11 Met Lys Val Leu Ile Ile Ala Cys Leu Val Ala Leu Ala Ile
Ala Ala 1 5 10 15 Val Thr Pro Ser Val Ile Asp Asn Ile Leu Ser Lys
Ile Glu Asn Glu 20 25 30 Tyr Glu Val Leu Tyr Leu Lys Pro Leu Ala
Gly Val Tyr Arg Ser Leu 35 40 45 Lys Lys Gln Leu Glu Asn Asn Val
Met Thr Phe Asn Val Asn Val Lys 50 55 60 Asp Ile Leu Asn Ser Arg
Phe Asn Lys Arg Glu Asn Phe Lys Asn Val 65 70 75 80 Leu Glu Ser Asp
Leu Ile Pro Tyr Lys Asp Leu Thr Ser Ser Asn Tyr 85 90 95 Val Val
Lys Asp Pro Tyr Lys Phe Leu Asn Lys Glu Lys Arg Asp Lys 100 105 110
Phe Leu Ser Ser Tyr Asn Tyr Ile Lys Asp Ser Ile Asp Thr Asp Ile 115
120 125 Asn Phe Ala Asn Asp Val Leu Gly Tyr Tyr Lys Ile Leu Ser Glu
Lys 130 135 140 Tyr Lys Ser Asp Leu Asp Ser Ile Lys Lys Tyr Ile Asn
Asp Lys Gln 145 150 155 160 Gly Glu Asn Glu Lys Tyr Leu Pro Phe Leu
Asn Asn Ile Glu Thr Leu 165 170 175 Tyr Lys Thr Val Asn Asp Lys Ile
Asp Leu Phe Val Ile His Leu Glu 180 185 190 Ala Lys Val Leu Gln Tyr
Thr Tyr Glu Lys Ser Asn Val Glu Val Lys 195 200 205 Ile Lys Glu Leu
Asn Tyr Leu Lys Thr Ile Gln Asp Lys Leu Ala Asp 210 215 220 Phe Lys
Lys Asn Asn Asn Phe Val Gly Ile Ala Asp Leu Ser Thr Asp 225 230 235
240 Tyr Asn His Asn Asn Leu Leu Thr Lys Phe Leu Ser Thr Gly Met Val
245 250 255 Phe Glu Asn Leu Ala Lys Thr Val Leu Ser Asn Leu Leu Asp
Gly Asn 260 265 270 Leu Gln Gly Met Leu Gln Ile Ser Gln His Gln Cys
Val Lys Lys Gln 275 280 285 Cys Pro Gln Asn Ser Gly Cys Phe Arg His
Leu Asp Glu Arg Glu Glu 290 295 300 Cys Lys Cys Leu Leu Asn Tyr Lys
Gln Glu Gly Asp Lys Cys Val Glu 305 310 315 320 Asn Pro Asn Pro Thr
Cys Asn Glu Asn Asn Gly Gly Cys Asp Ala Asp 325 330 335 Ala Lys Cys
Thr Glu Glu Asp Ser Gly Ser Asn Gly Lys Lys Ile Thr 340 345 350 Cys
Glu Cys Thr Lys Pro Asp Ser Tyr Pro Leu Phe Asp Gly Ile Phe 355 360
365 Cys Ser His His His His His His Leu Glu Asp 370 375 12 82 DNA
Artificial Sequence synthetically generated oligonucleotide 12
ggccgctcga cgccaccatg aaggtcctca taattgcctg tctggtggct ctggccattg
60 cagccgtcac tccctccgtc at 82 13 80 DNA Artificial Sequence
synthetically generated oligonucleotide 13 cgatgacgga gggagtgacg
gctgcaatgg ccagagccac cagacaggca attatgagga 60 ccttcatggt
ggcgtcgagc 80 14 27 DNA Artificial Sequence oligonucleotide for PCR
14 gattgacaag taatacgctg tttcctc 27 15 17 DNA Artificial Sequence
oligonucleotide for PCR 15 ggattcaata gatacgg 17 16 21 DNA
Artificial Sequence oligonucleotide for PCR 16 cagggaatgc
tgcagatcag c 21 17 49 DNA Artificial Sequence oligonucleotide for
PCR 17 aattctcgag ttagtggtgg tggtggtggt gatcgcagaa aataccatg 49 18
24 DNA Artificial Sequence oligonucleotide for PCR 18 ctccttgttc
aggaacttgt aggg 24 19 21 DNA Artificial Sequence oligonucleotide
for PCR 19 gtcctgcagt acacatatga g 21
* * * * *