AU2004200398A1 - HCV-derived RNA polymerase gene - Google Patents

Description

AUSTRALIA

Patents Act 1990 CHUGAI SEIYAKU KABUSHIKI KAISHA, INTERNATIONAL REAGENTS CORPORATION, TETSUYA TOYODA COMPLETE SPECIFICATION STANDARD PATENT Invention Title: HCV-derived RNA polymerase gene The following statement is a full description of this invention including the best method of performing it known to us:- HCV-DERIVED RNA POLYMERASE GENE Technical Field The present invention relates to an RNA polymerase gene derived from hepatitis C virus (referred to as "HCV" herein), a method of screening using this gene or this RNA polymerase protein, and a substance able to be isolated by this screening method.

Prior Art Generally known viral hepatitis includes hepatitis A which is mainly orally transmitted, and hepatitis B transmitted by means of the blood. Moreover, apart from these hepatitis, there.is hepatitis called non-A, non-B hepatitis which is transmitted by means of blood transfusion. Since most of these infected with non-A, non-B hepatitis become chronic, and the incidence of development into cirrhosis and hepatoma is high, this is one disease for which the establishment of a certain means of treatment is urgently sought.

Though the causative agent of non-A, non-B hepatitis had been unclear for a long time, recently the causative virus was isolated by M. Houghton et al. (Japanese Patent Application Laid-Open (Kohyo) No. 2-500880), and was termed HCV is a single-stranded RNA virus belonging to the Flavivirdae, the length of its whole genomic RNA is about 9.4 kb. The genomic RNA is divided into 7 regions; core, El, E2/NS1, NS2, NS3, NS4, and NS5; and the genes related to virus growth, etc. are primarily included in downstream regions from NS3.

HCV RNA polymerase is related to the transcription and replication of genomic RNA, and plays an important role in the reproduction of HCV. The gene encoding this 2 polymerase is thought to be included in the above-mentioned NS5 region H. Yuan et al., Biochemical and Biophysical Research Communications 232, 231-235(1997), S. B.

Hwang et al., Virology 227, 439-446(1997), S. E. Behrens et al., The EMBO Journal 12-22(1996)).

The Problem to Be Solved by the Invention If the gene encoding HCV RNA polymerase can be isolated, it will become possible using this gene to easily screen for substances inhibiting RNA polymerase, and contribute greatly to the development of drugs for treating HCV. However, at present, although the nucleotide sequence of a portion of the NS5 region has been clarified (Japanese Patent Application Laid-Open (Kokai) No. 6-225770), the entire nucleotide sequence of the RNA polymerase gene has yet to be clarified.

The object of the present invention is to isolate the gene encoding the full length of HCV-derived RNA polymerase, to determine its nucleotide sequence, as well as to establish its expression system.

A further object of the present invention is to provide a screening method for a substance which inhibits the activity of this gene or this protein employing this gene or this RNA polymerase protein.

Means for Solving the Problem In order to solve the above problem, the present inventors, as result of deliberate and focused research have succeeded in isolating the gene encoding the full-length of HCV-derived RNA polymerase, thereby completing the present invention.

3 That is to say, the present invention relates to the following to A gene encoding the following protein or a protein consisting of the amino acid sequence represented in SEQ ID NO: 2; a protein consisting of an amino acid sequence derived from the amino acid sequence represented in SEQ ID NO: 2 by deletion, substitution or addition of one or several amino acid(s), and which has RNA polymerase activity.

A method of screening a substance which inhibits the activity of the gene of (1) above, or of the protein consisting of the amino acid sequence represented in SEQ ID NO: 2, wherein this method comprises the following steps: a step of contacting the gene of above or the protein consisting of the amino acid sequence represented in SEQ ID NO: 2, or a fragment of this protein, with a test sample; and, a step of selecting a substance which inhibits the activity of the gene of (1) above, or of the protein or the partial peptide fraction consisting of the amino acid sequence represented in SEQ ID NO: 2.

A substance able to be isolated by the method of above, wherein this substance inhibits the activity of the gene of above or of the protein consisting of the amino acid sequence represented in SEQ ID NO: 2.

The descriptions contained in the specification of Japanese Patent Application No. 10-177817, which forms the basis of the right of priority of the present application, are incorporated herein in their entirety.

Disclosure of the Invention Below, the present invention will be explained in detail.

The gene of the present invention encodes a protein consisting of the amino acid sequence represented in SEQ ID NO: 2; or, a protein consisting of an amino acid sequence derived from the amino acid sequence represented in SEQ ID NO:2 by deletion, substitution, or addition of one or several amino acid(s) and having RNA polymerase activity.

The deletion, etc. of one or several amino acid can be performed by techniques in common use at the time of filing this application, such as, for example, site-specific mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982).

The gene of the present invention is able to be obtained from the blood of non-A, non-B hepatitis patients as described in the examples, or from the strain of E. coli into which a vector (pCALN/HCV RBZ) comprising the gene of the present invention was introduced, has been deposited at the National Institute of Bioscience and Human- Technology, Agency of Industrial Science and Technology Higashi, Tsukubashi, Ibaraki-ken, Japan) (Accession No. FERM BP-6763) on Oct. 31, 1997).

Further, the present invention relates to a screening method for a substance which inhibits the activity of this gene or this protein, employing the gene of the present invention or an RNA polymerase protein consisting of the amino acid sequence represented in SEQ ID NO: 2; and, to a substance able to be isolated by this screening method employing this gene or this RNA polymerase protein.

The RNA polymerase encoded by the gene of the present invention is an enzyme involved in the transcription and replication of HCV genomic RNA. Therefore, a substance which inhibits this enzyme is thought to be able to prevent the reproduction of HCV, and is promising as a drug for treating non-A, non-B hepatitis. By using the gene of the present invention it will be possible to produce HCV-derived RNA polymerase easily and in great quantities, and as a result of this, the screening of inhibitory substances for the RNA polymerase will become simpler.

The protein of the present invention that can be used for screening can be either a recombinant type, a wild type, or a partial peptide. Further it can be a purified peptide or a partial peptide thereof.

One embodiment of this method of screening comprises the steps of (a) contacting the gene of the present invention or the protein consisting of the amino acid sequence represented by SEQ ID NO: 2, or a fragment of this protein, with a test sample; and, selected a substance which inhibits the activity of the gene of the present invention or the protein consisting of the amino acid sequence represented by SEQ ID NO: 2. There is no particular limitation on what can be used as a test material in this screening method but for example, a cell extract, a cell culture supernatant, a protein, a peptide, or synthetic low molecular weight compound can be used.

Examples Isolation of HCV RNA from the Blood of a Hepatitis Patient and PCR Amplification of the Isolated RNA RNA was extracted from the blood of a non-A, non-B hepatitis patient by guanidine thiocyanate and phenol/chloroform method and RT-PCR was performed according to the method described in Japanese Patent Application Laid-Open (Kokai) No. 6-225770. Using this cDNA as a template, PCR (Science 230:1350(1985)) was performed using the primers described in Japanese Patent Application Laid-Open (Kokai) No. 6-225770, and four types of amplification fragment (C6-62 region, C6-66 region, C6-79 region, C6-82 region) were obtained. These amplification fragments were cloned using cloning vector pBM, and the nucleotide sequences of the amplification fragments were determined by Sanger's dideoxy- termination procedure (Science, 214, 1205(1981)). It should be noted that cloning vector pBM is a vector that was constructed such that mutations do not occur easily, taking into consideration the nature of the HCV gene to incorporate mutations easily during replication and cloning.

(Japanese Patent Application Laid-Open (Kokai) No. 6-225770).

The position of each amplification fragment was determined by a comparison of the homology of the amplification fragments within the clones obtained by the above method, and the previously reported non-A, non-B hepatitis virus gene, each of these amplification fragments were joined together by PCR, and the desired DNA fragment (amplified DNA) was cloned into a vector. pCALN/HCV RBZ was prepared using this cloning vector.

Determining the Nucleotide Sequence of the Fragment Encoding The fragment encoding NS5B was amplified by PCR with pCALN/HCV RBZ as a template using the following primers.

PCR primers: NS5B1 5'-ATC CCT CGA GAT GTC CTA CAC ATG GAC AGG-3 (SEQ ID NO: 3) NS5B2 5'-TAT GGA TCC AAG CTT CAC CGG TTG GGG AGC AGG T-3' (SEQ ID NO: 4) The reaction solution was prepared by adding to a 0.5 ml tube, 10 pl 10X PCR buffer II (500 mM KC1, 100 mM Tris-HCl pH8.3, 15 mM MgCl 2 16 Rl of 1.25 mM dNTP, 5 tl each of the 2 types of primers (20 gM) (NS5B1, NS5B2), 0.5 gl of 1 units/Il AmpliTaq DNA Polymerase (PERKIN ELMER) and adjusting to 100 pl with sterilized water. Thermal conditions were set such that, after initial heating at 95°C for minutes, 25 cycles were conducted with conditions of 95 0 C for 1 minute for denaturation, 55°C for 1 minute for annealing, and 72°C for 3 minutes for extension, and thereafter was finally maintained at 72°C for 10 minutes. A portion of the post-reaction solution was subjected to agarose gel electrophoresis, and the specifically amplified DNA fragment was confirmed.

After purifying this DNA fragment according to the Gene Clean (Biolol) method, the fragment was digested with Xhol and BamHI. The digested reaction solution was subjected to agarose gel electrophoresis, the desired DNA fragment was extracted from the gel, and the concentration of DNA in the extract was measured. The cloning vector pET-15b (Novagen) was similarly digested with Xhol and BamH, then purified. The digested reaction solution was subjected to agarose gel electrophoresis, the desired DNA fragment was extracted from the gel and the concentration of DNA in the extract was measured.

In respect the above-mentioned two types of DNA fragment extracted from the gel, a ligation reaction was performed according to the DNA ligation Kit ver.2 (Takara) method, and E. coli was transformed using a portion of the reaction solution. The transformed strain thereby obtained was cultured overnight on an LB-Amp plate (1% bactotryptone, 0.5% yeast extract, 1% NaC1, 1.4% agar, ampicillin 100 pg/ml).

Thereafter, the colonies appearing on the plate were each cultured (37°C, 16 hours) with a tube containing 2 ml LB-Amp medium bactotryptone, 0.5% yeast extract, 1% NaCI, ampicillin 100 gg/ml). The cultured fluid was centrifuged to collect the microorganism and plasmid DNA was extracted by mini-preparation method. The plasmid was digested with Xhol and BamHI, and then the digested product was subjected to agarose gel electrophoresis, and a plasmid clone into which the desired DNA fragment had been introduced, was obtained. This plasmid clonel was designated HCV pol.

The plasmid clone pET-15b HCV pol obtained above was digested with Xbal and BamHI, this product solution of the enzyme reaction was subjected to agarose gel electrophoresis and the desired DNA fragment was extracted. Similarly, baculovirus transfer vector pVL1392 (Pharmigen) was digested with Xbal and BamHI, this product solution of the enzyme reaction was subjected to agarose gel electrophoresis, and the desired DNA fragment was extracted.

By performing a ligation reaction with respect to the above-mentioned two types of DNA fragment extracted from agarose-gel, NS5B gene tagged with 6x His at the Nterminus was introduced into pVL1392. E. coli was transformed using this ligation reaction solution. The obtained transformed strain was cultured overnight on LB-Amp plate bactotryptone, 0.5% yeast extract, 1% NaCl, 1.4% agar, ampicillin 100 tg/ml), thereafter each colony appearing on the plate was cultured (37°C, 16 hours) with a tube containing 2 ml LB-Amp medium bactotryptone, 0.5% yeast extract, 1% NaC1, ampicillin 100 ptg/ml). After the cultured fluid was centrifuged, the plasmid DNA was miniprepped and DNA solution was prepared. After digested with XbaI and BamHI, the product solution of the enzyme reaction was subjected, to agarose gel electrophoresis, and a clone into which the desired DNA fragment had been introduced was obtained. This clone was designated pVL1392 His HCV pol. Using pVL1392 His HCV pol, a recombinant baculovirus (BacHisHCVpol) was produced with BaculoGold (Pharmigen). Production of the baculovirus was performed in accordance with the descriptions in "Baculovirus Expression Vector System: Procedures and Methods Manual" (Pharmigen).

Expression and Purification ofRNA Polymerase sf21 AE cells were infected with BacHisHCVpol, with moi=1 or 2, and three days after infection (72 hours later), cells were collected, and extracted with a buffer comprising 10 mM Tris/HCI pH7.9, 0.5M NaCl, 1.5 mM MgCl 2 7 riM 2-ME, 0.1% Triton X-100, 25% glycerol, 1 mM PMSF, and 10 ptg/ml leupeptin. The whole cell extract was first passed through a Ni-NTA column (QIAGEN, 60 mM imidazole), then purified with FPLC (MonoQcolumn, Pharmacia). RNA polymerase was eluted from MonoQ column with a buffer containing 0.1-1.0 M NaCl gradient.

Confirmation of RNA Polymerase Activity Preceding measurement of HCV RNA polymerase activity, in order to determine the optimal concentrations for magnesium acetate and KCl reactions, an RNA specific to HCV was produced as an RNA polymerase reaction template.

HCV cDNA downstream of the PvuII site (9240) of HCV was cloned into Bluescript KSII this vector was designated HCV RNA-9610. The prepared cloning vector Bluescript KSII HCV RNA-9610 was digested with Dral, PstI, Nhel, etc. and after purification, was supplied to an in vitro transcription reaction using T7 RNA polymerase. Due to the differences in the restriction enzymes that were used; Dral, PstI and Nhel; the synthesized RNA differed in length (377 nts in the case of Dral, 340 nts in the case of PstI, and 305 nts in the case of Nhel). These RNA were designated HCV RNA-9610, HCV RNA-9576, HCV RNA-9541, respectively. It should be noted that HCV RNA-9610 has two extra UMPs at its 3' terminus.

With the above-mentioned HCV RNA as a template, the optimal concentration of magnesium acetate in the HCV RNA polymerase activity measurement system was determined. HCV RNA polymerase (MonoQ column fraction) 5 jtl was added to 50 l1 of buffer (20 mM HEPES/KOH pH7.6, 50 mM KC1, 1 mM DTT, 25 utg/ml actinomycin D, 0.5 mM ATP,CTP,GTP, 50 tM UTP, 5 pCi[a-32P] UTP (15 TBq/mmol, Amersham), 10 pmole HCV RNA-9541, 400 U/ml RNase inhibitor (TOYOBO)), and in this solution, the concentration of magnesium acetate was set at eleven levels within a range of 0-10 mM and incubated at 29°C for 1.5 hours. Products were synthesized at each magnesium acetate concentration and subjected to electrophoresis with 4% PAGE/6M urea. The PSL of the product was measured with BAS. As a result, optimal magnesium acetate concentration was 3-4 mM.

Similarly, the optimal concentration of KCI in the HCV RNA polymerase activity measurement system was determined. Here polyA was used as a template instead of HCV RNA. 10 pl of HCV RNA polymerase fraction was added to 50 il of buffer (20 mM HEPES/KOH pH7.6, 5 mM magnesium acetate, 1 mM DTT, 25 tg/ml actinomycin D, 10 tM UTP, 2.5 tCi[a-32P] UTP (15 TBq/mmol, Amersham), 10 ug/ml poly A(Pharmacia), 100 jM UpU (Sigma), 400 U/ml RNase inhibitor (Takara)), and the KCl concentration was set at 10 levels within a range of 20-200 mM, and the amount of UMP uptake at each concentration was measured. This measurement itself was performed by firstly, by precipitation of the [a- 32 P] UMP that was taken up within TCA, collection on a glass filter (GF/C, Whatman), and measuring with a liquid scintillation counter (Aloka). With polyA as a template, the optimal salt concentration of KCl in the polymerase activity observation system, in the presence of UpU primer, was 100 mM.

Further, the KCl optimal concentration of the HCV RNA polymerase activity measurement system in the case where HCV RNA-9541 is used as a template, was determined. In the reaction system, the amount of HCV RNA polymerase was set at jl, the magnesium acetate concentration was set at 3.5 mM, 10 pmole of HCV RNA- 9541 was used as a template but otherwise the reaction system was identical to the experimental system for determining the optimal concentration of magnesium acetate.

The concentration of KCl was set at nine levels within a range of 50-200 mM. The result of this measurement was that the optimal concentration of KCl was 50 mM. Here, the cause of the difference in KCl optimal concentration was presumed to be due to the secondary structure of the template.

12 Inhibition of the Uptake of UMP of HCV RNA Polymerase by Rabbit Anti-HCV RNA Polymerase Antibodies (HCV RNA polymerase) within pET-15b HCV pol was expressed in E.

coli, and then purified with a Ni-NTA (QIAGEN) column. A rabbit was immunized using this protein as an antigen, and antibodies were (anti-HCVpol) produced.

First, 20 pl of HCV RNA polymerase purified with Ni-NTA and incubated at room temperature for 30 minutes under the conditions of a final concentration of 3.1, 6.3, 12.5, 25, 50 tg/ml of rabbit IgG anti-HCV RNA polymerase; or as a control, normal rabbit IgG. The composition of the buffer that was used was 20 mM HEPES/KOH pH7.6, 100 mM KC1.

After incubation, this HCV RNA polymerase solution was re-adjusted such that it was composed of 3 mM magnesium acetate, 1 mM DTT, 25 ug/ml actinomycin D, tM UTP (Pharmacia), 2.5 pCi [a- 32 P] UTP (15 TBq/mmol, Amersham), 10 ug/ml poly A(Pharmacia), 100 pM UpU (Sigma), 400 U/ml Prime RNase inhibitor Inc.), and incubated at 29°C for 1.5 hours. The [a-32P] UMP that was taken up was precipitated in TCA, collected on a glass filter (GF/C, Whatman), and measured with a liquid scintillation counter (Aloka). As a result, inhibition of the uptake of UMP of HCV RNA polymerase by the rabbit anti-HCV RNA polymerase anti-body was observed.

All publications, patents and patent publications cited herein are incorporated into this specification in their entirety.

Effect of the Invention The present invention provides a novel RNA polymerase gene derived from hepatitis C virus and a method of screening using this gene or this RNA polymerase 13 protein. Screening of an inhibitory substance of this RNA polymerase can be easily performed by using this gene.

Claims (6)

1. An isolated nucleic acid encoding a protein comprising the amino acid sequence represented in SEQ ID NO: 2.

2. An isolated nucleic acid encoding a protein comprising the amino acid sequence in which one amino acid is deleted from or added to the amino acid sequence represented in SEQ ID NO: 2, and having RNA polymerase activity.

3. An isolated protein comprising the amino acid sequence represented in SEQ ID NO: 2 and having RNA polymerase activity.

4. An isolated protein comprising the amino acid sequence in which one amino acid is deleted from or added to the amino acid sequence represented in SEQ ID NO: 2, and having RNA polymerase activity.

A method of identifying a substance which inhibits RNA polymerase activity of the protein of claim 3, wherein said method comprises the following steps: contacting the protein with test substance; measuring an activity of the protein; and determining if the substance inhibits the activity of the protein.

6. A method of identifying a substance which inhibits RNA polymerase activity of the protein of claim 4, wherein said method comprises the following steps: contacting the protein with test substance; measuring an activity of the protein; and determining if the substance inhibits the activity of the protein. Dated this third day of February 2004 Chugai Seiyaku Kabushiki Kaisha, International Reagents Corporation, Tetsuya Toyoda Patent Attorneys for the Applicant: F B RICE CO SEQUENCE LISTING <100> <160> <200> <210> <211> GENERAL INFORMATION: NUMBER OF SEQ ID NOS: 4 SEQUENCE CHARACTERISTICS: SEQ ID NO 1 LENGTH: 1773 <212> TYPE: DNA <213> ORGANISM: HCV <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1773) <400> SEQUENCE: 1 tca atg tcc tac aca tgg aca ggc gcc Ser Met Ser tyr Thr trp Thr Gly Ala atc acg cca tgc gcc gcg Ile Thr Pro Cys Ala Ala gag gaa agc Glu Glu Ser cac cac aac His His Asn cag aag aag Gin Lys Lys ttg ccc atc aac Leu Pro Ile Asn ttg agc aac tct Leu Ser Asn Ser ttg cgt Leu Arg atg gtc tat gct Met Val tyr Ala aca tcc cgc agc gca ggc cta cgg Thr Ser Arg Ser Ala Gly Leu Arg gtc acc ttt Val Thr Phe ctg caa gtc Leu Gln Val gac gac cac tac Asp Asp His tyr cgg gac Arg Asp gtg ctc aag Val Leu Lys atg aag gcg aag Met Lys Ala Lys tcc aca gtt aag Ser Thr Val Lys aaa ctc cta tcc Lys Leu Leu Ser gcc aga tcc Ala Arg Ser agc aag gcc Ser Lys Ala 115 gac act gag Asp Thr Glu 130 a s ata Ile ttt Phe gaa gaa gcc tgt Glu Glu Ala Cys ctg acg ccc cca Leu Thr Pro Pro cat tcg His Ser ggc tat ggg Gly tyr Gly aag gac gtc cgg Lys Asp Val Arg aac cta tcc Asn Leu Ser 110 ttg ctg gaa Leu Leu Glu aac cac atc Asn His Ile tcc gtg tgg aag Ser Val trp Lys aca cca att gac acc acc gtc atg Thr Pro Ile Asp Thr Thr Val Met aaa agt gag gtt Lys Ser Glu Val tgc gtc caa cca gag Cys Val Gin Pro Glu 150 gga ggc cgc Gly Gly Arg gct cgc ctt Ala Arg Leu ttc cca gac Phe Pro Asp ggg gtt cgt gta Gly Val Arg Val aag atg gcc Lys Met Ala ctt tat Leu tyr 175 gac gtg gtc Asp Val Val ttc cag tac Phe Gln tyr 195 ctt cct cag Leu Pro Gin gtg atg ggc tcc Val Met Gly Ser tca tac gga Ser tyr Gly 190 aat gcc tgg Asn Ala trp cct gga cag Pro Gly Gin gtc gag ttc ctg Val Glu Phe Leu aaa tca Lys Ser 210 aag aaa tgc cct Lys Lys Cys Pro ttt tca tat Phe Ser tyr acc cgc tgt ttt Thr Arg Cys Phe tcg aca gtc act Ser Thr Val Thr gac atc cgt Asp Ile Arg gag tca att Glu Ser Ile tgt tgt gac Cys Cys Asp ccc gaa gcc aga Pro Glu Ala Arg 250 cag gcc ata aag Gln Ala Ile Lys tcg ctc Ser Leu 255 ggg cag Gly Gln aca gag cgg ctt Thr Glu Arg Leu att ggg ggt ccc ctg acc aat tca aaa Ile Gly Gly Pro Leu Thr Asn Ser Lys aac tgt ggc Asn Cys Gly 275 cgc cgg tgc Arg Arg Cys gcg agt ggc gtg Ala Ser Gly Val 270 acg acc agc Thr Thr Ser gcc tgt cga Ala Cys Arg tgc ggt Cys Gly 290 gct gca Ala Ala aat acc ctt aca Asn Thr Leu Thr tac ttg aag gcc tyr Leu Lys Ala aag ctc cgg Lys Leu Arg acg atg ctc Thr Met Leu aac gga gac gac Asn Gly Asp Asp gtc atc tgt Val Ile Cys gcg gga acc Ala Gly Thr gat gag gcg Asp Glu Ala aac cta Asn Leu 335 cga gtc ttc Arg Val Phe ccg ccc cga Pro Pro Arg 355 gct atg act Ala Met Thr tct gcc ccc Ser Ala Pro ccc ggg gac Pro Gly Asp 350 tgt tcc tcc Cys Ser Ser gaa tac gac Glu tyr Asp cta ata aca Leu Ile Thr aat gtg Asn Val 370 acc cgc Thr Arg tcg gtc gcg cac Ser Val Ala His tct ggc aaa Ser Gly Lys gta tac tac ctc Val tyr tyr Leu gac ccc tcc Asp Pro Ser act cca gtt Thr Pro Val 405 ccc ctt gca cgg Pro Leu Ala Arg gcg tgg gag aca Ala trp Glu Thr cac His aat tcc tgg cta Asn Ser trp Leu aac atc att atg Asn Ile Ile Met tat gcg tyr Ala 415 tcc atc Ser Ile ccc acc tta Pro Thr Leu ctt cta gcc Leu Leu Ala 435 ggg gcc tgt Gly Ala Cys tgg gca trp Ala agg atg att Arg Met Ile atg acc cat ttc Met Thr His Phe 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1773 gag caa ctt Glu Gin Leu aaa gcc ctg gat Lys Ala Leu Asp cag atc tac Gin Ile tyr atc att gaa Ile Ile Glu tac tcc att gag cca ctt gac cta tyr Ser Ile Glu Pro Leu Asp Leu 450 cga ctc Arg Leu cat ggt ctt His Gly Leu ttt tca ctc Phe Ser Leu agt tac tct cca Ser tyr Ser Pro gag atc aat agg Glu Ile Asn Arg tca tgc ctc Ser Cys Leu aaa ctt ggg gta Lys Leu Gly Val cca ccc Pro Pro 495 ctg ctg Leu Leu ttg cga gtc Leu Arg Val tcc cag ggg Ser Gin Gly 515 gca gta agg Ala Val Arg 530 aga cat cgg gcc Arg His Arg Ala agt gtc cgc gct Ser Val Arg Ala agg gct gcc act tgt ggt aag tac Arg Ala Ala Thr Cys Gly Lys tyr ttc aac tgg Phe Asn trp gcg tcc cag Ala Ser Gln acc aag ctc Thr Lys Leu ctc act cca atc Leu Thr Pro Ile ttg gac ttg tcc agc Leu Asp Leu Ser Ser tgg trp gtg gct ggt Val Ala Gly agc ggg gga gac Ser Gly Gly Asp 550 tat cac agc ctg tyr His Ser Leu tct cgt gcc cga ccc cgc Arg Ala Arg Pro Arg 570 tgg ttc atg ttg trp Phe Met Leu tgc cta Cys Leu 575 ctc cta ctt Leu Leu Leu ggg gta ggc Gly Val Gly atc tac ctg ctc ccc aac cga Ile tyr Leu Leu Pro Asn Arg 585 590 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211> LENGTH: 591 <212> TYPE: PRT <213> ORGANISM: <400> SEQUENCE: Ser Met Ser tyr 1 Glu Glu Ser Lys His His Asn Met Gin Lys Lys Val Arg Asp Val Leu Lys Leu Leu Ser Ala Arg Ser Lys 100 Ser Lys Ala Val 115 Asp Thr Glu Thr 130 Phe Cys Val Gin 145 Val Phe Pro Asp Asp Val Val Ser 180 Phe Gln tyr Ser 195 Lys Ser Lys Lys 210 Asp Ser Thr Val 225 Gin Cys Cys Asp Thr Glu Arg Leu 260 Asn Cys Gly tyr 275 Cys Gly Asn Thr 290 Ala Ala Lys Leu 305 Val Val Ile Cys Arg Val Phe Thr 340 Pro Pro Arg Pro 355 Asn Val Ser Val 370 Thr Arg Asp Pro 385 Arg His Thr Pro Pro Thr Leu trp 420 Leu Leu Ala Giri 435 Gly Ala Cys tyr 450 Thr Gly Ala Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser tyr Ser Pro Gly 465 470 475 480 Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro Pro 485 490 495 Leu Arg Val trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu Leu 500 505 510 Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys tyr Leu Phe Asn trp 515 520 525 Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Ser Gln 530 535 540 Leu Asp Leu Ser Ser trp Phe Val Ala Gly tyr Ser Gly Gly Asp Ile 545 550 555 560 tyr His Ser Leu Ser Arg Ala Arg Pro Arg trp Phe Met Leu Cys Leu 565 570 575 Leu Leu Leu Ser Val Gly Val Gly Ile tyr Leu Leu Pro Asn Arg 580 585 590 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 3 atccctcgag atgtcctaca catggacagg <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 4 tatggatcca agcttcaccg gttggggagc aggt 34