U.S. patent application number 09/930169 was filed with the patent office on 2003-01-02 for immunological enhancement agent comprising n-terminal peptide of p43 as an effective component.
Invention is credited to Kim, Sunghoon, Ko, Young-Gyu.
Application Number | 20030004309 09/930169 |
Document ID | / |
Family ID | 19710396 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030004309 |
Kind Code |
A1 |
Kim, Sunghoon ; et
al. |
January 2, 2003 |
Immunological enhancement agent comprising N-terminal peptide of
p43 as an effective component
Abstract
The present invention relates to an immunological enhancement
agent, more particularly, to an immunological enhancement agent
comprising peptide having an amino acid sequence represented by SEQ
ID NO:1 to SEQ ID NO:3 as an effective component. The peptides
according to the present invention comprising the N-terminal domain
of the p43 protein have excellent cytokine activity to improve an
immune response so that they can be used as an effective
immunological enhancement agent.
Inventors: |
Kim, Sunghoon; (Seoul,
KR) ; Ko, Young-Gyu; (Gyeonggi-do, KR) |
Correspondence
Address: |
Stephen A. Bent
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
19710396 |
Appl. No.: |
09/930169 |
Filed: |
August 16, 2001 |
Current U.S.
Class: |
530/350 ;
424/185.1 |
Current CPC
Class: |
A61K 2039/55522
20130101; C07K 14/52 20130101; A61K 38/00 20130101; A61P 37/04
20180101 |
Class at
Publication: |
530/350 ;
424/185.1 |
International
Class: |
A61K 039/00; C07K
014/435 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 5, 2001 |
KR |
2001-31310 |
Claims
What is claimed is:
1. An immunological enhancement agent comprising peptide
represented by SEQ ID NO:1 as an effective component.
2. An immunological enhancement agent comprising peptide
represented by SEQ ID NO:2 as an effective component.
3. An immunological enhancement agent comprising peptide
represented by SEQ ID NO:3 as an effective component.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an immunological
enhancement agent, more particularly, to an immunological
enhancement agent comprising specific peptides having immunological
activity as an effective component.
BACKGROUND OF THE INVENTION
[0002] Cells undergoing programmed cell-death (known as apoptosis)
are rapidly removed by monocyte-derived macrophages. This
phenomenon infers that apoptotic cells might secrete specific
factors and these factors cause chmotaxis to leucocyte and
monocyte. The endothelial monocyte-activating polypeptide II
(hereinafter, referred to as "EMAP II") is known as one of the
specific factors because it is released from the apoptotic cells
and causes chemotaxis.
[0003] The EMAP II is the C-terminal domain of the p43 protein--the
precursor of the EMAP II, consisting of 312 amino acids--and it is
cleaved to form by caspase-7, in which aspartic acid--the 146th
amino acid of p43 protein--is activated in apoptotic cells
(Quevillion, S. et al., J. Biol. Chem., 272:32573-32579, 1997;
Behrensdorf, M. A. et al., FEBS Lett., 466:143-147, 2000). The
structure and maturation of the EMAP II is similar to that of an
IL-1.beta., a cytokine involved in a proinflammatory response, and
14.5 kDa of the IL-1.beta. is cleaved to form from 33 kDa of
inactive pre-IL-1.beta. by ICE (caspase-1). An EMAP II is the
mediator of proinflammatory response that induces the expression of
tissue factor, tumor necrosis factor (hereinafter, referred to as
"TNF") and interleukin-8 (hereinafter, referred to as "IL-8") in
mononuclear phagocyte and polymorphonuclear leucocytes. Also, in a
tissue expressing a high level of EMAP II mRNA, macrophages are
accumulated. This means that an EMAP II is a chemotaxis material
leading macrophage to dead cells. It is known that the EMAP II acts
as a cytokine, and the 15 amino acids of the N-terminal domain of
an EMAP II play a vital role in the reaction (Quevillon, S. et al.,
J. Biol. Chem., 272:32573-32579, 1997; Kao, J. et al., J. Biol.
Chem., 269:9774-7982, 1994; Kao, J. et al., J. Biol. Chem.,
267:20239-20247, 1992; Kao, J. et al., J. Biol. Chem.,
269:25106-25119, 1994; Knies, U. E. et al., PNAS USA,
95:12322-12327, 1998). In the U.S. Pat. No. 5,641,867, it is
described that the N-terminal domain of an EMAP II that comprises
arginine-isoleucine-glycine-arginine-isoleucine-threonine is an
important residue in cytokine function of the EMAP II. Recently, it
was reported that the EMAP II repressed the growth of primary and
metastatic tumor in proliferating endothelial cells that are not
causing particular side-effect in normal cell (Schwarz, M. A. et
al., J. Exp. Med.,190:341-353, 1999).
[0004] On the other hand, the p43 protein is expressed extensively.
The expression level of p43 protein varies temporally and
spatially, especially in a developing mouse. For example, it was
shown that the expression of a p43 in the lung of mouse from 8 days
to 16 days after its birth was increased dramatically. In addition,
the p43 is highly expressed in the microglial cells in the lesions
of autoimmune disease such as encephalomyelitis, neuritis and
uveitis. The high expression level of the p43 in specific
developmental stage and tissues suggests that the p43 could have
unexpected functions in angiogenesis, inflammation, and apoptosis
(fas, M. P. R., and Marray, J. C., Int. J. Biochem. Cell. Biol.,
28:837-841, 1996; Schwarz, M. J. et al., Glia, 20:365-372, 1997;
Schuesner, H. J. et al., Glia, 20:365-372, 1997; Berger, A. C. et
al., J. Immunother., 23:519-527, 2000).
[0005] As mentioned previously, the EMAP II--C-terminal domain of
p43--has been studied extensively for its cytokine activities;
however, p43 (i.e. pro-EMAP II) and its N-terminal domain have not
been understood. Therefore, the present inventors have studied to
disclose that a p43 can act as a more effective cytokine and as an
immunological enhancement agent than the EMAP II. We did this by
comparing cytokine activity of a p43 with that of an EMAP II,
C-terminal domain of p43 in the PCT application
[0006] The present inventors compared the secretion pattern of a
p43 with that of an EMAP II in normal cells and apoptotic cells,
and reported that the cytokine acting in physiological condition
was the p43 and not the EMAP II. Since the EMAP II is secreted
during the late stage of apoptosis in which cells are completely
destroyed in normal cells, it is not active in the early stage of
apoptosis; whereas, the p43 is constitutively secreted from various
cells irrespectively of apoptosis (Ko YG et al., A cofactor of tRNA
synthetase, p43, is secreted to up-regulate proinflammatory genes,
J. Biol. Chem. 2001, Apl 5, 276).
[0007] While studying the p43, the present inventors achieved the
present invention by discovering that the peptides, including
N-terminal domain of p43, showed an excellent cytokine activity
with the result of estimating cytokine activities of
deletion-mutants of the p43 protein.
SUMMARY OF THE INVENTION
[0008] It is an object of the present invention to provide an
immunological enhancement agent that comprises the N-terminal
domain of the p43 showing an excellent immunological activity as an
effective component.
[0009] In order to accomplish the object above, the present
invention provides an immunological enhancement agent that
comprises a peptide having an amino acid sequence represented by
SEQ ID NO:1 to SEQ ID NO:3 as an effective component.
[0010] According to the present invention, the p43(1-147), the
p43(1-108) and the p43(91-256), which comprise the N-terminal
domain of the p43 protein, can act as a cytokine to increase the
amount of the TNF and the IL-8. As a result, they can improve an
immune response and be used as an effective immunological
enhancement agent.
BRIEF DESCRIPTION OF DRAWINGS
[0011] FIG. 1 shows the relative location and the size of the
deletion-mutants of a p43 prepared in the present invention
compared to a normal p43.
[0012] FIG. 2 shows the result of the SDS-PAGE analysis of the
deletion-mutants of the p43 prepared in the present invention and
the p43 protein after purification.
[0013] FIG. 3 illustrates the amount of the TNF produced after
incubation of human monocyte THP-1 cells to which the purified p43
protein and its deletion-mutants are added respectively.
[0014] FIG. 4 illustrates the amount of the IL-8 produced after
incubation of human monocyte THP-1 cells to which the purified p43
protein and its deletion-mutants are added respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention will be described in detail.
[0016] According to the present invention, the peptides that have
an amino acid sequence--represented by SEQ ID NO:1 to SEQ ID NO:3
as an effective component of an immunological enhancement
agent--are the N-terminal domain of the p43 protein. More
particularly, these peptides consist of peptide having amino acids
from 1 to 147 of the p43 protein, peptide from 1 to 108 of the p43
protein, and peptide from 91 to 256 of the p43 protein.
[0017] In order to obtain the peptides that comprise the N-terminal
domain of the p43, first, the deletion-mutants of the p43 protein
were constructed. Then they were purified and added to cultivated
mammalian cells. Next, the degree of inducing the production of
cytokine was measured. Finally, the peptides showing a high level
of cytokine induction in cultivated mammalian cells could be
isolated from various deletion-mutants of the p43 protein.
[0018] To indicate the p43 protein or its deletion-mutants, for the
sake of convenience, the first and the last number of amino acid
sequence of the p43 protein was used in parentheses; for example,
the peptide having amino acids from 91 to 256 of the p43 was
represented by "p43(91-256)".
[0019] In one embodiment of the present invention, recombinant
vectors were respectively prepared according to known technique
(Park, S. G. et al., J. Biol. Chem., 274:166673-16676, 1999). The
recombinant vectors include the following genes respectively
encoding: the p43(1-312), which is the p43; the p43(148-312), which
is the C-terminal domain of the p43 or the EMAPII; the p43(1-147),
the p43(1-108), and the p43(91-256) which include N-terminal part
of the p43. The vectors for cloning the above genes are not limited
to specific vectors, but pET28a is preferable. The p43 protein and
its deletion mutants can be expressed by transforming host cell
with the prepared vectors including gene of the p43 or its deletion
mutants. The host cell for transformation is not limited
specifically, but E.coli is preferable.
[0020] In another embodiment of the present invention, the cytokine
activity of the p43 and its deletion-mutants protein was
investigated. The expressed proteins were added to a mammalian cell
cultured in serum-free media, and the amount of inducing production
of other cytokines was measured. The mammalian cell is not
specifically limited if only it is a growth factor dependent cell,
however, a human monocyte THP-1 cell is more preferable. Whether or
not the above proteins have immunological activity was investigated
by measuring the amount of cytokine produced that can be induced by
the p43 or its deletion-mutant proteins. More particularly, among
cytokines the amount of the TNF and the IL-8 produced was measured
in the present invention. In order to measure the amount of
produced cytokines, a well-known method in the art can be used,
particularly, in the present invention, ELISA (Enzyme-Linked Immuno
Sandwich Assay) was used. The p43(1-147), the p43(1-108) and the
p43(91-256), the peptides represented by SEQ ID NO:1 to SEQ ID NO:3
according to the present invention, showed cytokine activity and
induced the production of the TNF and the IL-8. According to the
results confirmed in the present invention, the produced amount of
the TNF and the IL-8 induced by the above peptides was higher by
2-3 times more than that by EMAP II. The produced amount of the TNF
and the IL-8 induced by the peptide represented by SEQ ID NO: 1 was
higher by 1.1 times more than by the p43 and higher by 2.5-3 times
more than by EMAP II.
[0021] The immonological enhancement agent according to the present
invention that comprises the N-terminal part of the p43 as an
effective component is not limited to a specific form, if only it
is a feasible form for being administered to human and animals.
Also, it can be a form supported with carrier.
[0022] As the carrier, at least one of a solid, a liquefied
diluent, a filling agent, and other assistant can be used with the
ratio of 0.1.about.99.5%. The immunological enhancement agent can
be administered orally or non-orally. The non-oral administration
can be tissue partial-, hypodermal-, intramuscular-, arterial-,
intravenous-, and rectal administration. The preferable
administration form can be prepared in conventional techniques.
[0023] Oral administration can be done by using a solid or liquid
form; for example, bulk powders, powders, granules, tablets,
capsules, syrups, suspensions, etc. If necessary, the unit
administration form for oral administration can be microcapsulated.
The extension of the activity period and the sustained release of a
formulation can be obtained by coating the formulation or inserting
its active components to polymer or wax.
[0024] The administration amount of the immunological enhancement
agent can be determined preferably by taking into consideration for
the age of patient, the body weight, a method of administration,
any disease and its state, and so on.
[0025] The present invention will be described in more detail by
the following examples. However, it should be understood that these
examples are provided only for the illustration of the present
invention but not intended to limit the scope of the invention in
any manner.
EXAMPLE 1
[0026] Preparation of Recombinant Vector Including p43(1-147) Gene,
Protein Expression, and its Purification
[0027] The recombinant vector, including the p43(1-147) gene, was
prepared by known method in the art (Park, S. G. et al., J. Biol.
Chem., 274:16673-16676, 1999). Plasmid pM388 provided from Dr.
Kiyotaka Shiba in Japan Cancer Institute was restricted with NdeI
and XhoI to obtain the p43 gene, and it was used as a template. The
PCR was performed using the forward and reverse primer represented
by SEQ ID NO:4 and SEQ ID NO:5 respectively, under the condition as
follows; 1 min at 95.degree. C., 1 min at 58.degree. C., and 1 min
at 72.degree. C. The resulting PCR product was restricted with
EcoRI and Sall and inserted into pET28a (Novagen, Madison, USA) to
prepare the recombinant vector.
[0028] E.coli BL21(DE3) was transformed with the above recombinant
vector including the p43(1-147) gene. The transformed BL21 was
cultured in 100 ml of LB medium (Luria Broth; Ig NaCl, 1 g
Bacto-tryptone, 0.5 g yeast extracts) and the p43(1-147) gene was
expressed as a His-tag fusion protein form. The expressed
p43(1-147) protein was purified by using nickel affinity
chromatography and mono Q or S ion-exchange chromatography
according to known method in the art (Park, S. G. et al., J. Biol.
Chem., 274:16673-16676, 1999). To remove lipopolysaccharide, which
induces inflammatory response, the purified protein was dialyzed
with the pyrogen-free buffer solution (10 mM potassium phosphate
(pH 6.0), 100 mM NaCl) overnight. After dialysis, the protein was
loaded to a polymyxin resin (Bio-Rad) that was equilibrated with
the same buffer, incubated for 20 minutes and eluted. The
concentration of the remaining lipopolysaccharide was measured
using the Limulus Amebocyte lysate QCL-1000 kit (Bio Whittacker).
As a result, the concentration of the lipopolysaccharide was below
20 pg/ml--not able to induce inflammatory response. The purified
protein was then subjected to SDS-PAGE. As shown in FIG. 2, it was
confirmed that the p43(1-147) protein having the molecular weight
of 21 kDa was isolated purely.
EXAMPLE 2
[0029] Preparation of Recombinant Vector Including p43(1-108) Gene,
Protein Expression, and its Purification
[0030] The recombinant vector, including the p43(1-108) gene, was
prepared according to the same method described in the Example 1.
The recombinant vector pET28a (Novagen) containing the p43(1-312)
gene was restricted with AspI and SalI. The restricted recombinant
vector was treated with the klenow fragment to fill up the DNA ends
and re-ligated. And then, the recombinant vector, including the
p43(1-108) gene, was prepared by treating ligase and making the
vector self-ligation. BL21 was transformed with the recombinant
vector, including the p43(1-108) gene, according to the same method
described in the Example 1. The expressed p43(1-108) protein was
purified and analyzed by SDS-PAGE.
[0031] As shown in FIG. 2, it was confirmed that the p43(1-108)
protein having the molecular weight of 20 kDa was isolated
purely.
EXAMPLE 3
[0032] Preparation of Recombinant Vector Including p43(91-256)
Gene, Protein Expression, and its Purification
[0033] The recombinant vector, including the p43(91-256) gene, was
prepared according to the same method described in the Example 1,
except that the PCR was performed using the forward and reverse
primer represented by SEQ ID NO:6 and SEQ ID NO:7 respectively,
under the condition as follows; 30 sec at 95.degree. C., 30 sec at
50.degree. C., and 40 sec at 72%C. BL21 was transformed with the
recombinant vector, including the p43(91-256) gene, according to
the same method described in the Example 1. The expressed
p43(91-256) protein was purified and analyzed by SDS-PAGE.
[0034] As shown in FIG. 2, it was confirmed that the p43(91-256)
protein having the molecular weight of 29 kDa was isolated
purely.
EXAMPLE 4
Measurement of Cytokine Activity
[0035] To measure a cytokine activity of the deletion-mutant
proteins of the p43 that was purified in the Example 1-3, the
following experiment was performed.
[0036] The human monocyte THP-1 cells (supplied from the ATCC and
cultured selecting the sensitive cells to lipopolysaccharide) were
inoculated to the RPMI1640 medium containing 10% fetal bovine serum
(FBS) and 50 .mu.g/ml streptomycin and penicillin, and cultured in
5% CO.sub.2 at 37.degree. C. The cultured cells were washed twice
with serum-free RPMI1640, and then 2.times.10.sup.6 cells/ml were
inoculated into 24-well plate containing 0.5 ml of serum-free
RPMI1640 medium. The cells were cultured for 2 hours under the same
condition, and stimulated for 4 hours by adding 100 nM of the
proteins purified in the Example 1-3 respectively. The supernatants
were collected, and the concentration of the TNF and the IL-8 was
measured using the ELISA kit (PharMingen) according to the
manufacturer's instructions. The stimulation experiments were
repeated twice. The results of the experiments were shown in FIG. 3
and FIG. 4, respectively.
COMPARATIVE EXAMPLE 1
[0037] Preparation of Recombinant Vector Including p43(1-312) Gene,
Protein Expression, and Measurement of Cytokine Activity
[0038] The recombinant vector, including the p43(1-312) gene, was
prepared according to the same method described in the Example 1,
except that the PCR was performed using the forward and reverse
primer represented by SEQ ID NO:8 and SEQ ID NO:9 respectively, and
the pM338 as a template, and NdeI and XhoI were used as restriction
enzymes. BL21 was transformed with the recombinant vector,
including the p43(1-312) gene, according to the same method
described in the Example 1. The expressed p43(1-312) protein was
purified and analyzed by SDS-PAGE.
[0039] As shown in FIG. 2, it was confirmed that the p43(1-312)
protein having the molecular weight of 42 kDa was isolated
purely.
[0040] To measure a cytokine activity of the purified p43(1-312)
protein, the each produced amount of the TNF and the IL-8 was
measured according to the same method described in the Example 4.
The results were shown in FIG. 3 and FIG. 4, respectively.
COMPARATIVE EXAMPLE 2
[0041] Preparation of Recombinant Vector Including p43(148-312)
Gene, Protein Expression, and Measurement of Cytokine Activity
[0042] The recombinant vector, including the p43(148-312) gene,
encoding EMAP II was prepared according to the same method
described in the Example 1, except that the PCR was performed using
the forward and reverse primer represented by SEQ ID NO:10 and SEQ
ID NO:11 respectively, and pM338 as a template. BL21 was
transformed with the recombinant vector, including the p43(148-312)
gene, according to the same method described in the Example 1. The
expressed p43(148-312) protein was purified and analyzed by
SDS-PAGE.
[0043] As shown in FIG. 2, it was confirmed that the p43(148-312)
protein having the molecular weight of 26 kDa was isolated purely.
To measure a cytokine activity of the purified p43(148-312)
protein, the each produced amount of the TNF and the IL-8 was
measured according to the same method described in the Example 4.
The results were shown in FIG. 3 and FIG. 4, respectively.
[0044] Observing that the produced amount of the TNF induced by the
p43(1-312)(p43 protein), the p43(148-312)(EMAP II) and the
deletion-mutants of the p43 protein, i.e., p43(1-147), p43(1-108),
and p43(91-256), as shown in FIG. 3, in case of p43(1-132), the
produced amount of TNF was 1,500 pg/ml, p43(148-312) was 500 pg/ml,
p43(1-147) was 1,650 pg/ml, p43(1-108) was 1,000 pg/ml, and
p43(91-256) was 2,200 pg/ml. From these results, it was confirmed
that the amount of the TNF produced by the p43(1-312)(p43 protein),
and proteins comprising the N-terminal part of the p43 protein,
i.e., p43(1-147), p43(1-108), and p43(91-256) was much more than
that by the p43(148-312)(EMAP II).
[0045] Observing that the produced amount of the IL-8 induced by
the above proteins, as shown in FIG. 4, in case of p43(1-132), the
produced amount of IL-8 was 1,495 pg/ml, p43(148-312) was 650
pg/ml, p43(1-147) was 1,650 pg/ml, p43(1-108) was 1,050 pg/ml, and
p43(91-256) was 1,950 pg/ml. From these results, it was confirmed
that the amount of the IL-8 produced by p43(1-312)(p43 protein) and
proteins comprising the N-terminal part of the p43 protein, i.e.,
p43(1-147), p43(1-108), and p43(91-256) was much more than that by
p43(148-312)(EMAP II).
[0046] In conclusion, it was confirmed that the p43(1-147), the
p43(1-108), and the p43(91-256)--which comprise the N-terminal part
of the p43 protein--showed by far higher cytokine activity than the
EMAP II. The result illustrates that the N-terminal part of the p43
plays a vital role in the cytokine activity. Therefore, the
peptides of the present invention comprising the N-terminal domain
of the p43 can be used as an immunological enhancement agent
showing an excellent cytokine activity.
Sequence CWU 1
1
11 1 147 PRT Unknown Organism Description of Unknown Organism
Mammalian protein sequence 1 Met Ala Asn Asn Asp Ala Val Leu Lys
Arg Leu Glu Gln Lys Gly Ala 1 5 10 15 Glu Ala Asp Gln Ile Ile Glu
Tyr Leu Lys Gln Gln Val Ser Leu Leu 20 25 30 Lys Glu Lys Ala Ile
Leu Gln Ala Thr Leu Arg Glu Glu Lys Lys Leu 35 40 45 Arg Val Glu
Asn Ala Lys Leu Lys Lys Glu Ile Glu Glu Leu Lys Gln 50 55 60 Glu
Leu Ile Gln Ala Glu Ile Gln Asn Gly Val Lys Gln Ile Ala Phe 65 70
75 80 Pro Ser Gly Thr Pro Leu His Ala Asn Ser Met Val Ser Glu Asn
Val 85 90 95 Ile Gln Ser Thr Ala Val Thr Thr Val Ser Ser Gly Thr
Lys Glu Gln 100 105 110 Ile Lys Gly Gly Thr Gly Asp Glu Lys Lys Ala
Lys Glu Lys Ile Glu 115 120 125 Lys Lys Gly Glu Lys Lys Glu Lys Lys
Gln Gln Ser Ile Ala Gly Ser 130 135 140 Ala Asp Ser 145 2 108 PRT
Unknown Organism Description of Unknown Organism Mammalian protein
sequence 2 Met Ala Asn Asn Asp Ala Val Leu Lys Arg Leu Glu Gln Lys
Gly Ala 1 5 10 15 Glu Ala Asp Gln Ile Ile Glu Tyr Leu Lys Gln Gln
Val Ser Leu Leu 20 25 30 Lys Glu Lys Ala Ile Leu Gln Ala Thr Leu
Arg Glu Glu Lys Lys Leu 35 40 45 Arg Val Glu Asn Ala Lys Leu Lys
Lys Glu Ile Glu Glu Leu Lys Gln 50 55 60 Glu Leu Ile Gln Ala Glu
Ile Gln Asn Gly Val Lys Gln Ile Ala Phe 65 70 75 80 Pro Ser Gly Thr
Pro Leu His Ala Asn Ser Met Val Ser Glu Asn Val 85 90 95 Ile Gln
Ser Thr Ala Val Thr Thr Val Ser Ser Gly 100 105 3 166 PRT Unknown
Organism Description of Unknown Organism Mammalian protein sequence
3 Met Val Ser Glu Asn Val Ile Gln Ser Thr Ala Val Thr Thr Val Ser 1
5 10 15 Ser Gly Thr Lys Glu Gln Ile Lys Gly Gly Thr Gly Asp Glu Lys
Lys 20 25 30 Ala Lys Glu Lys Ile Glu Lys Lys Gly Glu Lys Lys Glu
Lys Lys Gln 35 40 45 Gln Ser Ile Ala Gly Ser Ala Asp Ser Lys Pro
Ile Asp Val Ser Arg 50 55 60 Leu Asp Leu Arg Ile Gly Cys Ile Ile
Thr Ala Arg Lys His Pro Asp 65 70 75 80 Ala Asp Ser Leu Tyr Val Glu
Glu Val Asp Val Gly Glu Ile Ala Pro 85 90 95 Arg Thr Val Val Ser
Gly Leu Val Asn His Val Pro Leu Glu Gln Met 100 105 110 Gln Asn Arg
Met Val Ile Leu Leu Cys Asn Leu Lys Pro Ala Lys Met 115 120 125 Arg
Gly Val Leu Ser Gln Ala Met Val Met Cys Ala Ser Ser Pro Glu 130 135
140 Lys Ile Glu Ile Leu Ala Pro Pro Asn Gly Ser Val Pro Gly Asp Arg
145 150 155 160 Ile Thr Phe Asp Ala Phe 165 4 27 DNA Artificial
Sequence Description of Artificial Sequence PCR Primer 4 ccggaattca
tggcaaataa tgatgct 27 5 24 DNA Artificial Sequence Description of
Artificial Sequence PCR Primer 5 ctggtcgacg tcggcacttc cagc 24 6 32
DNA Artificial Sequence Description of Artificial Sequence PCR
Primer 6 cggaattcat ggtttctgaa aatgtgatac ag 32 7 30 DNA Artificial
Sequence Description of Artificial Sequence PCR Primer 7 ccggtcgact
cagaaagcat caaagtaatt 30 8 18 DNA Artificial Sequence Description
of Artificial Sequence PCR Primer 8 catatggcaa ataatgat 18 9 18 DNA
Artificial Sequence Description of Artificial Sequence PCR Primer 9
ctcgagggaa gcatttta 18 10 27 DNA Artificial Sequence Description of
Artificial Sequence PCR Primer 10 ccggaattct ctaagccaat agatgtt 27
11 27 DNA Artificial Sequence Description of Artificial Sequence
PCR Primer 11 ccggtcgact tatttgattc cactgtt 27
* * * * *