JPH044880A - Dna coding hepatitis non-a and non-b antigen and polypeptide - Google Patents

Abstract

NEW MATERIAL:A DNA containing a base sequence of the formula etc. USE:Diagnostics for hepatitis non-A and non-B. PREPARATION:For example, a precipitant such as polyethylene glycol is added to human high-GPT (glutamic-pyruvic transaminase) plasma collected to concen trate virus followed by extracting RNA by guanidine-thiocyanate technique, and with this RNA as template, cDNA is synthesized using reverse transcriptase and put to restriction enzyme treatment and then bound to a vector to prepare cDNA library, which is then screened through plaque hybridization using a probe to select a plaque containing DNA coding hepatitis non-A and non-B antigen of the formula. From this plaque, the DNA is taken out and put to restriction enzyme treatment, thus obtaining the objective DNA containing a base sequence of the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to DNA and polypeptides, and more particularly to DNA chains and polypeptides encoding non-A, non-B hepatitis antigens.

<Prior art and problems to be solved by the invention> It was known that viral hepatitis includes infectious hepatitis (hepatitis A) and serum hepatitis (hepatitis B) before the causative virus was discovered. The causative viruses of hepatitis A and hepatitis B have been discovered, and diagnosis can be made by immunological methods. With the establishment of screening methods for hepatitis B, it was expected that post-transfusion hepatitis could be prevented; however, in reality, although post-transfusion hepatitis has been reduced, it has not been eradicated, meaning that post-transfusion hepatitis is neither hepatitis A nor hepatitis B. It has become clear that viral hepatitis exists, namely non-A, non-B hepatitis (also referred to as hepatitis C). Non-A, non-B hepatitis accounts for the majority of post-transfusion hepatitis, and an increase in hepatocytosis associated with infection with non-A, non-B hepatitis viruses has also been noted. After the existence of non-A, non-B hepatitis became clear, many researchers conducted research into the causative virus of non-A, non-B hepatitis, but the causative virus was not identified for a long time. Therefore, the diagnosis of non-A, non-B hepatitis is made by testing for hepatitis A and hepatitis B, as well as testing for Epstein-Barr virus, cytomegalovirus, etc. If these tests are negative, non-A, non-B hepatitis is diagnosed. has been diagnosed as. As described above, the diagnosis of non-A, non-B hepatitis has the problem of requiring time and effort to obtain results.

Recently, complementary DNA was obtained based on RNA collected from chimpanzee serum infected with non-A, non-B hepatitis (
cDNA) was cloned and its nucleotide sequence was presented. A diagnostic method for non-A, non-B hepatitis and a vaccine for non-A, non-B hepatitis using polypeptides (antigens) prepared thereby have been proposed (see European Patent Publication No. 8216, A1-31). However, it is said that non-A, non-B hepatitis viruses differ depending on the region, etc., and the diagnostic method for non-A, non-B hepatitis using the antigen described in the above publication,
There are doubts as to whether it is possible to diagnose all types of non-A, non-B hepatitis, and it has been pointed out that vaccines may not be sufficiently effective.

Means for Solving the Problems> The present invention was made in view of the above problems, and as a result of intensive research by the present inventors on antigenic substances that specifically react with non-A, non-B hepatitis antibodies, human We discovered that a DNA fragment encoding a non-A, non-B hepatitis antigen was present in cDNA prepared from RNA collected from GTP-high plasma of patients, and determined the base sequence and amino acid sequence of the DNA. Completed the invention. That is, the DN of the present invention
The A chain contains the base sequence shown in FIG. 1, the base sequence shown in FIG. 2, the base sequence shown in FIG. 3, or the base sequence shown in FIG. Furthermore, the polypeptide of the present invention is shown in FIG.
It contains the amino acid sequence shown in FIG. 4, FIG. 5, or FIG. 6, which is encoded by the base sequence shown in FIG. 2 or 3, respectively.

The present invention will be explained in detail below.

In the present invention, isolation of a DNA strand containing a base sequence encoding a non-A, non-B hepatitis antigen and determination of the base sequence can be performed, for example, by the following method.

(a) Isolate RNA encoding a non-A, non-B hepatitis antigen; (b) synthesize single-stranded cDNA from the RNA; and (C) synthesize double-stranded cDNA from the RNA; , integrate it into a phage or plasmid to create a cDNA library.
Screen a DNA library by plaque hybridization, colony hybridization, etc., and clone a phage or plasmid containing the desired DNA: (e) Amplify the cloned phage or plasmid and collect the DNA. After that, the cDNA obtained by cutting with a restriction enzyme is subcloned, and then the base sequence is determined.

The above steps will be explained in more detail.

(Preparation of RNA) RNA encoding a non-A, non-B hepatitis antigen can be prepared, for example, by adding a precipitant such as polyethylene glycol to human GTP-high plasma to concentrate the virus, and then using the AGTC method using guanidine thiocyanate [ Chon+czynsk
iP,-et at, (1987) An
al, Biochea+.

Vol. 182.156].

The RNA thus obtained can be used as is in the next step.

(Synthesis of cDNA) Synthesis of cDNA can be performed using, for example, Gubler-Hoff'
man's method [Gene, Vol, 25.283 (1
983)], Okayama-Barb's method [Mo1. Ce1
1. BIol, Vol. 2.181 (1982) 1
This can be done using the following methods. More specifically, for example, using the above RNA as a template, a single-stranded cDNA complementary to the RNA is synthesized by reverse transcriptase using a random primer of about 6 bases. Then, ribonuclease H
RNA is degraded and double-stranded cDNA is synthesized using DNA polypolyase I. This synthesis method can be used as a cDNA synthesis kit sold by Amajam, Boehringer, etc.

(Preparation of cDNA library) The above double-stranded cDNA is ligated to both ends with synthetic linkers or an appropriate region (e.g., polyC) is added using terminal transferase, and the double-stranded cDNA is transferred to a plasmid vector or λ
A cDNA library can be created by ligating to a phage vector.

For example, double-stranded cDNA is methylated with EcoRI methylase to convert EcoR present in the cDNA.
After protecting the 1 cleavage site, the EcoRI Linker was ligated with DNA ligase derived from T4 phage, and then cut with the restriction enzyme EcoRl to obtain a double-stranded cDNA with EcoRI sticky ends, which was then inserted into the EcoR1 site of the phage vector λgtll. A cDNA library is prepared by performing in vitro socaging.

In addition, commercially available cDNAs of λgill and λgtlO
A library kit (manufactured by Stratagene, Amajam, Promega Biotic) can also be used.

(Cloning of cDNA) As a method for cloning a target cDNA from a cDNA library, for example, Brachts 1 hybridization [5science, Vol. 19[i, 18
0 (1977)], colony hybridization [Ge, Vol. 10.83 (1980) 1, etc. can be used.

As the probe, a complementary DNA fragment synthesized from the cDNA base sequence obtained in a preliminary experiment and labeled with a radioactive isotope (for example, 32P) is used.

The cloned phage or plasmid is cultured and amplified using an appropriate host, and then the DNA is extracted.

(Subcloning) Subcloning of the obtained DNA is performed as follows. A cDNA fragment and a vector DNA fragment are prepared by cleaving the cDNA-containing DNA and vector DNA with restriction enzymes. Next, the mixture of both is treated with T4 DNA ligase, and then transformed into Escherichia coli etc. to obtain a transformant into which the cDNA has been integrated. Vector DNAs used include pUc18, pUcL9, pUc118, and the like. In addition, restriction enzymes include EcoRI, I
lndm, Pstl, Bara! It etc. are mentioned.

(Determination of the base sequence of cDNA) The base sequence of cDNA was determined by Maxa-Gllber.
Method in EnzyIIlolo
gy, Vol, 85.499゜Acad, Pres.
(1980)], dideoxy method [Sange
r, P.

(1981) 5science, 214.1205-
This can be carried out by a conventional method such as 12101.

In this way, the base sequence of the cDNA encoding the non-A, non-B hepatitis antigen can be determined, and the DNA strand of the present invention
It had the base sequence shown in Fig. 2 and Fig. 3. Next, the amino acid sequences of the polypeptides translated by each DNA strand are shown in FIGS. 4, 5, and 6, respectively, and the polypeptides of the present invention have such amino acid sequences. In addition, in this specification and drawings, the meanings of the symbols of amino acid residues in the polypeptide are as follows.

N: asparagine D: aspartic acid A: alanine R: arginine ■ = isoleucine G nigericin Q: glutamine E, glutamic acid C nigistein S: serine Y: tyrosine W, tryptophan T: threonine ■: valine H: histidine F: phenylalanine P nibroline M :Methionine:Lysine L:Leucine Also, the base sequences shown in Figures 1, 2 and 3 and the amino acid sequences shown in Figures 4, 5 and 6, and the European patent When compared with the cDNA base sequence and amino acid sequence described in Publication No. Al-318216, the DNA chain and polypeptide of the present invention are found to be a part of the non-structural region 5 (NS5) of the non-A, non-B hepatitis antigen. It is assumed that this applies.

<Effects of the Invention> The DNA chain of the present invention encodes a non-A, non-B hepatitis antigen, and the polypeptide translated from the DNA is useful for the diagnosis of non-A, non-B hepatitis and the development of non-A, non-B hepatitis vaccines. It is useful for manufacturing, and has the advantage that it can be safely supplied in large quantities using genetic engineering techniques.

<Examples> Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited to these Examples.

Example 1 Concentration of Virus from GPT-High Plasma G of Human Plasma
PT (glutamate pyruvate transaminase)
Polyethylene glycol 4000 was added to 38.5 g of pooled plasma with a high value at a concentration of 4% to obtain 638 g of virus precipitate fraction.

2. RNA extraction AGTC method using guanidine thiocyanate [Ch
omczynskl P, et al. (1987
) Anal, Blochem, Vol. 16
RNA was extracted using 2.1581. 4M guanidine thiocyanate, 25mM sodium citrate, pH
After homogenizing 7,0,5% Sarcosyl in 0.1M 2-mercaptoethanol, extracting with 1/10 volume of 2M sodium acetate (pH 4), equal volume of phenol, 2/10 volume of chloroform + isoamyl alcohol, 2- RNA was precipitated with PropaTool.

After dissolving it again in 4M guanidine solution, it was precipitated with ethanol. The DNA thought to be human is RNA.
[Ma
xwell IIt. et at, (1977),
Nucl. Acids Res, Vol. 4.

241].

3. cDNA Synthesis cDNA was synthesized using cDNA Synthesis System Plus (manufactured by Amajam). The method of use is according to Amajam's protocol, and the brimer is randomly selected.
0 reverse transcriptase reaction using xanucleotides was performed at 42°C for 1 hour, and
, DNA polymerase I reaction at 12°C for 1 hour, 22
After 1 hour at 70°C, the reaction was carried out at 70°C for 10 minutes to inactivate the enzyme. Furthermore, after reacting with T4 DNA polymerase at 37°C for 10 minutes, phenol/chloroform extraction was performed twice and chloroform extraction was performed once. An equivalent amount of 4M ammonium acetate was added to the upper layer, and then 2 times the amount of ethanol was added, and dry ice ethanol was added. After standing for 10 minutes in
400 gg of ethanol was added, and after standing in dry ice-ethanol for 10 minutes, centrifugation was performed, and the precipitate was washed with 70% ethanol to recover cDNA.

The obtained cDNA was extracted with λg using a kit manufactured by Amajam.
inserted into tll. cD integrated into this vector
NA is amplified when bacteriophage infects host E. coli Y 1090 cells and multiplies. Thus, under certain conditions, the phenomenon of bacteriolysis makes it visible to the naked eye as a plaque.

At the same time, this cDNA is used as a fusion protein with β-galactosidase, which is pre-integrated into the phage DNA.
The amino acid sequence encoded by A is expressed. This-
Find clones that react with antibodies from among the amino acid sequences (peptides, proteins) of a group. This fusion protein is
Screening was performed using non-B hepatitis patient serum absorbed with Escherichia coli cells. That is, patient serum was absorbed with bacterial cells and then diluted 8 times as a primary antibody, and peroxidase-labeled anti-human IgG was used as a primary antibody against the antigen plotted on a nitrocellulose membrane from veterinary germs forming plaques.
After reacting with sheep IgG as a secondary antibody, positive plaques were picked up by color reaction. In this way, positive clone AM-1 was obtained.

5. Preparation of cDNA library inserted into λgtlO The cDNA was reacted with EcoRI Methylase 400 at 37°C for 1 hour, and the cDNA was recovered by chloroform extraction and ethanol precipitation. This cDNA and E
coRI 11nker (pGGAATTCC)2
ug was reacted at 16° C. for 2 hours using DNALIgatlon kH (manufactured by Takara Shuzo Co., Ltd.), and DNA was recovered by ethanol precipitation. This DNA was converted into EcoRI 50
Digested overnight at 37°C with U and Quick 5pin c
olumn Linkers 6 (manufactured by Boehringer)
cDNA and Hnker were separated. cDNA was recovered by ethanol precipitation.

This cDNA and 1 μg of λgttoarms (Stratagene) were added to Iigation bufTer (50
m M Tri 5-HCJ7SpH8,0,7
mM MgC1) 2.1mM DTT, 1mM
ATP) in T4 DNAIjgase (manufactured by Takara Shuzo Co., Ltd.)
After reacting with 1.4 U at 22° C. for 1 hour, the reaction was carried out at 4° C. overnight, and a total amount of 5 μg was subjected to 1 nvHro packaging.

in vjtro packaglng is Giga p
ack Gold (manufactured by Stratagene) was used.

5 μg of the Iiga-tion reaction solution prepared above was added to
In addition to the reeze-thaw extract, 15 μL of 5onic extract was added and mixed gently by pipetting. After reacting at 22°C for 2 hours, SM (0.1M Na cD
.

8.1mM Mg5o4 0.01% gelatin, 50
mM Tri 5-HCIII pH
7,5) 0°5νR and 20 μg of chloroform were added and stored at 4°C. Indicator bacteria is Escherichia coli C600hfl
+ and C600hrl- were used.

TB medium containing 10 m M M g SO and 0.2% maltose (Bacto-tryp per ρ of water)
ton (dissolved 10 g of SNaCg) at 37°C overnight with shaking, and cultured A6 with 10 m M M g S 04.
00 was adjusted to 2. In vHro package
The ng phage solution was diluted with SM, 100 μg of each phage dilution and 100 μg of the above indicator bacteria were mixed, and after reaction at 37°C for 15 minutes, 0.7% NZ was incubated at 45°C.
Y top agar (NZ amine per 1 p of water)
Type A 10 g, NaCΩ5gs
Yeast extract 5g, Mg5O+
17H202g, Bacto-agar 7g
(autoclave sterilized), add 2.5vf, 37
1.5%NZYbottoIipl kept at ℃
ate (NZ amlnel Ogs per 1 ρ of water
N a C1) 5 gSYeast extrac
t5 gSMg S 04 ・7H202gSBac
15 g of to-agar was added and spread on top (autoclave sterilized). After the jopagar was solidified, it was cultured overnight at 37°C, the number of plaques was examined, and the phage titer was determined.

Total titer is 2,3 x 106 pf'
u (plaquerorming unit).

6. Probe From the nucleotide sequence of the positive clone (AM-1) CDNA obtained in 4 above (see Figure 7), the complementary strand 511Iler (5°-GGT
GATGTTGCCGCCCATCTCCTGCCGC
CATAGAAGATTGGCCTCGATGAG-3
°, Fig. 8) using an automatic DNA synthesizer (＾ppHed Bl
381A (manufactured by OSYSTEMS) and used as a probe for plaque hybridization.

250ng of synthetic NA was added to [7P]ATP (P B 10218.5000Ci/mmol manufactured by AmaJam)
250μci, 50mM Tri 5-HCI) (
pH7゜6), 10mM Mg (,Q 2.5mM
DTT.

Polynucleotide kinase (Boehringer) 10U
The reaction was carried out at 37° C. for 30 minutes in a total of 20 μΩ, and the reaction was stopped by adding 1 μg of 0.5M EDTA (pH 8°0). This reaction solution is Nen5orb■-
20 (manufactured by NEN) column, free [γ
IJ ATP and labeled DNA were separated and purified.

7. Plaque hybridization The steps for preparing a filter and plaque hybridization are shown below.

(1)-Next Screening ∎ C600 (hrl+) was used as the master plate indicator bacteria. Diameter 15c
+n L-Bottom plate (B per 1g of water
acto-tryptonlOg, NaCρ5g
, Yeast extract 5 g.

Add 5 g of Bacto-agar and sterilize in an autoclave) with a phage solution prepared so that there are approximately 40,000 plaques per plate and C600 (hf'l+).
was incubated at 37°C for 15 minutes. And 45℃
0.7% L-101) agarose
2. 511 and L-bottom plat
It spread to e. After solidification, the plate was incubated at 37°C for 5 to 6 hours, and when the plaque diameter became 1 + am or less, it was stored at 4°C.

■Filter The filter is colony #+Iaque 5cree
n (NEN) was used. -C6 cultured at night in TB medium containing 0.2% maltose, 10mM MgSO4
00 (hr++>) was diluted 20 times with TB medium, a filter was immersed therein and dried on the filter paper for 2 to 4 hours.

■ Replica and amplification of the phage to the filter Place the filter from ■ above on the master plate, and after 5 minutes, peel it off and contact it on a new L-agar plate.
Place the plated side up and incubate at 37°C for 6-12 hours.

■ Filters with amplified denatured plaques were placed on 3MM filter paper soaked with 0.5M NaOH and 1.5M NaC (l) for 5 minutes, and then washed with 0.5M Tris-HCI (pH 8,0).
, 5 on 3MM filter paper soaked with 1.5M NaCl).
2xSSPE (0,36M NaC11
s 20mM Na2 HPO4, 2mM EDT
Soak in A) for 2 minutes. After drying at room temperature, it was baked at 80° C. for 1 hour.

■Prehybridization The filter from ■ above was mixed with 5 x SSC (0,75
MNaCΩ, 75mM sodium citrate), 20mM
NaH2PO4(+)H7,0), 7% SDS
(sodium dodecyl sulfate), 10X Denha
rdt's (0, 2% Bovine serum a
lbumin, 0. 2%Polyvinylpyr
rolldone, 0, 226 Flcoll) 1
0%dextran 5ulf'ate, 100
ug/i! Incubated for 12 hours at 50"C in a plastic bag containing prehybridization solution containing YeasttRN^.

■Remove the prehybridization solution from the hybridization bag and
XSSC, 7%SDS, 10XDenh
ardtS110%dextran 5ulfate
s 100 tt g / 11Yeast tRNA
, 32P-labeled probe (2, Qxl 06 epm
/11f, 8, 6 X 106 cpi/pmol
) was added and heated at 50'C.
Incubated for 114 hours.

■ Wash filter with 3XSSC, 20mM NaH2PO4
(pH 7,5), 5% SDS, 10x Denhard
After washing 4 times with T's solution at 50°C for 1 hour, 1x SSC
, 1% SDS solution at 65°C for 2 hours.

(2) Exposure to X-ray film The washed filter was exposed to X-ray film at -80°C overnight, then developed, and the position of the signal was confirmed. 1.2x
106 pru were screened and 5 signals were obtained.

(2) The gel from the master plate corresponding to the position of the secondary screening signal was scraped off, suspended in SM, and re-spread on a plate with a diameter of 15 cm to the extent that approximately 800 plaques appeared, and screening was performed again. As a result, three types of single plaques were obtained.

8. Put 100 μg of phage amplification indicator bacteria into a dry heat sterilized test tube kept at 37°C, and add NZY tol) agar2 kept at 50°C.
．． After adding 5y1, the mixture was spread on a NZY bottom plate kept at 37°C using a light portex.

Once the top agar had solidified, the single plaque from 7 above was poked once with the thin end of an autoclaved toothpick, and then poked into the plate 4 to 5 times.

When the Co(7) plate was cultured at 37°C for 8 hours, bacterial Iysls caused by phage was observed. Using the thick end of a Pasteur pipette that had been sterilized by dry heat, remove the transparent part of the Iysls with phage, and remove the whole agar.
λdiluent (10mM Tri
5-HCI).

The phages were suspended in pH 8, 0, 2mM MgC1)z), portexed, and then extracted overnight at 4°C. Portex the extract and transfer 5 μg of it to E, coilCGOO.
After mixing with OHrl-indicator bacteria (100 ul) in a Corning disposable 15111 centrifuge tube, the mixture was left to stand in a 37° C. thermostat for 20 to 30 minutes. Here, 61! NZY m
edium was added and cultured with shaking at 37°C for 4 to 6 hours.

When 1ysis of bacteria is observed, stop culturing and transfer to a tabletop centrifuge.3. 000rpI11. Centrifugation was performed for 20 minutes, and the resulting supernatant was transferred to a short test tube that had been sterilized by dry heat. 100 μg of chloroform was added and stored at 4°C.

9. Extraction of phage DNA An equivalent amount of activated DE-52 suspension was added to the phage solution (Lysate) amplified in 8 above. 20 phage liquid
After mixing by inverting ~30 times, the mixture was allowed to stand at room temperature for 30 minutes, and centrifuged at 10.00 rpm for 10 minutes using a centrifuge.

Centrifugation was repeated twice.

1xl of 5M NaC,Q and '3.4yl of isopropanol were added to the supernatant, and the mixture was allowed to stand at -40°C for 1 hour. This was centrifuged at 10,000 rpm for 10 minutes.

After discarding the supernatant, the precipitate was washed twice with 70% cold ethanol. This precipitate was dried under reduced pressure to give a concentration of 0, 1 mg/11
proteinase K: 10%SDS:TE-5
pr mixed at a ratio of 0 uΩ: 1.20 ul: 311
It was dissolved in 200 μg of oteinase K solution and reacted for 10 minutes at room temperature. 200 μg of TE-saturated phenol:chloroform-1:1 was added and portexed, followed by centrifugation at 15,000 rpm for 2.5 minutes in a microcentrifuge. Transfer the aqueous phase to a new sample cup and add 20 μg
3M sodium acetate (pH 6.0) and 500 μg of ethanol were added, and the mixture was left standing in dry ice and ethanol for 15 minutes or more. This was heated in a microcentrifuge for 15,
000rpm.

After centrifugation for 5 minutes, the resulting phage DNA precipitate was dried under reduced pressure.

10. Subcloning cDNA into E. coli vector The phage DNA from 9 above was digested with EcoRl and 1%
Perform low melting point gel electrophoresis, cut out the cDNA band, perform phenol extraction twice, and D by ethanol precipitation.
NA was collected. The E. coli vector pUc18 digested with EcoRI and the recovered cDNA were transformed into DNA Llg.
I 1g by atlon klt (manufactured by Takara Shuzo Co., Ltd.)
An atton reaction was performed to transform Escherichia coli HBIOI:I competent cells (manufactured by Takara Shuzo Co., Ltd.), and 20 μg/'I
! The cells were spread onto an ampicillin-containing L-plate and incubated overnight at 37°C to obtain ampicillin-resistant bacteria into which the cDNA had been incorporated.

11. Determination of cDNA base sequence The cDNA base sequence was determined using the dldeoxy method [Sange
r, P (1981) 5science, 214.1
205-1210], 7-D E A Z
A Sequenclng Kit (manufactured by Takara Shuzo Co., Ltd.)
This was done using 3 clones (pKS-01SpKsO
The number of bases of O2, pKsOO3) is 526 bp and 28 bp, respectively.
1 bp, 125 bp, and each base sequence is the first
It was as shown in Figures 2 and 3. As a result of translating each sequence into amino acids and examining the relationship with stop codons, we found that the base sequence in Figure 1 corresponds to the amino acid sequence in Figure 4, the base sequence in Figure 2 corresponds to the amino acid sequence in Figure 5, and the base sequence in Figure 3 corresponds to the amino acid sequence in Figure 5. It was found that the nucleotide sequence shown in the figure encodes the amino acid sequence shown in FIG.

The nucleotide sequences shown in FIGS. 1, 2, and 3, the amino acid sequences shown in FIGS. 4, 5, and 6, and the cDNA described in European Patent Publication No. Al-318216 Comparing the nucleotide sequence and amino acid sequence of The homology of was as shown in the table below.

table

[Brief explanation of drawings]

Figures 1, 2 and 3 are diagrams showing the base sequence of the DNA encoding the non-A, non-B hepatitis antigen of the present invention, and Figures 4, 5, and 6 are Figure 1, respectively. Figures 2 and 3 show the amino acid sequence translated from the nucleotide sequences, and Figures 7 and 8 respectively show the nucleotide sequence of positive clone AM-1 and the nucleotide sequence of the synthetic probe that is its complementary strand. It is a diagram. Figure 1 CGCCCGGACTACAACC Figure 4 Figure 5 Figure 6 GGNITRVESEN Figure 2 CAGACTACAAC Figure 3 GAAC Figure 7 CCCGCAGAGATCCTGCGGAAG Figure 8

Claims (1)

[Claims] 1. A DNA strand containing the base sequence shown in FIG. 1, FIG. 2, FIG. 3, or FIG. 8. 2. A polypeptide comprising the amino acid sequence shown in FIG. 4, FIG. 5, or FIG. 6.