CA2115926A1 - Hepatitis c assay utilizing recombinant antigens to ns1 - Google Patents

Abstract

The present invention provides unique recombinant antigens representing distinct antigenic regions of the NS1 region of the HCV
genome which can be used as reagents for the detection of antibodies and antigen in body fluids from individuals exposed to hepatitis C virus (HCV). The present invention also provides an assay for detecting the presence of an antibody to an HCV antigen in a sample by contacting the sample with the recombinant antigens.
Preferred assay formats include a screening assay, a confirmatory assay, a competition or neutralization assay and an immunodot assay.

Description

i WO 93/04088 . . n ~ PCl`/US92/07188 .~ L 1 c~ cJ ;~ ~) HEPATI~IS C ASSAY UTILZING RECOMBINANT ANTIGENS TO NS1 This is a cont~nualion-~n-part application of U.S. Ser~al No. 07/572,822, filed Augusl 24, 1990 and U.S. Ser~al No. 07,614,069, filed November 7, 1990, 5 which enpy common ownership and are incorporated herein by reference. This awlicalion also is related to co-filed pa1ent applicationS entitled ~HEPATITIS CAæAY UTIUZING RECOMBINANT ANTIGENS FROM NS5 REGION~(U. S. Serial No.
748,565) and ~HEPATITIS C ASSAY UTILIZING RECOMBINANT ANTIGENS TO C-100 REGION~(U. S. Serial No. 748,566) which enpy common ownership and are 10 incorporated herein by reference.
This inventbn relates generaUy to an assay for identilying the presence in a sample of an antibody which is immunologically reactive with a hepatitis C virusantigen and specifically to an assay for ~electing a complex of an an~ibody and recombinant anti~ens representing distinct regions of the HCV genome. Recombinant 15 antigens derived from the molecular cloning and expression in a heterologous expressbn system of the synthstic DNA sequences representing dist~nct antigenic regiions of the HCV g~nome can be used as reagents for the detection of an1ibodies and antben in body fluWs from individuals exposed to hepa1itls C virus (HCV).

2 0 ~4CKGK)UNDOFlHE INVENT~J
Acu1e viral hepatitis is clinically diagnosed by a well-defined set of patient symptoms, including jaundice, hepatic 1endemess, and an incr~ase in the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase.
Additional serologic immunoassays are generally performed to diagnose the specific 25 type of viral causative agent. Historically, patients presenting clinical hepatitis symptoms and not otherwise infected by hepatitis A, hepati~is B, Epstein-Barr orcytomegalovirus were clinically diagnosed as having non-A non-B hepatitis (NANBH) by default. The disease may result in chronic liver damage.
Each of the well-known, immunologica!ly characterized hepatitis-inducing 30 viruses, hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis D virus (HDV) belongs to a separate family of viruses and has a distinctive viral organization, protein structure, and mode of replication.
Attempts to identify the NANBH virus by virtue of genomic similarityl to one of the known hepatitis viruses have failed, suggesting that NANBH has a distinctorganization and structure. ~Fowler, ~L~L.. J- Med. Virol., 12:205-213 (1983) and Weiner, ~L~L.~I Med. Virol., 21:239-247 (1987)].
Progress in developing assays to detect anlibodies specific for NANBH has . ~:

WO 93~041N8 PCI~/US92/07188 be~n panicularly hamp~r~d by difficultles in corr~clly Identtfying antlg~ns associated wi1h NANBH. See, for example, Wands, J., ~L~.. U.S. Patent 4,870,076,Wands, et al.. Proc. Nat~l. Acad. Sci., 83:6608-6612 (1986), Ohori, ~L, J. Med.
Virol., 12:161-178 (1983), Bradley, ~, Proc. Nat'l. Acad. Sci., 84:6277-6281, (1987), Akalsuka, T., ~LaL.J- Med.~ , 20:43-56 (1986), Seto, B-, Qt ~, U.S. Patent Application Number 07/234,641 (available from U.S. Department of Commerce National Technical Information Service, Springfield, Virginia, No.
89138168), Takahashi, K., et al., European Patent Application No. 0 293 274, published November 30, 1988, and Seelig, R., ~LaL. in PCT Application 1 0 PCT/EP88/00123.
Recently, another hepatitis-inducing virus has been unequivocally identified as hepatilis C virus (HCV) by Houghton, M., ~Lal.. European Patent Application publication number 0 318 216, May ~, 1989. Related papers describing this virus include Kuo, G., ~.LaL, ~i~n~, 244:359-361 (1989) and Choo, Q., ~
1 5 ~icience, 244:362-364 (1989). Houghton, M., ~ reported isolating cDNA
sequences from HCV which encode an1igens which react immunologically with antibodies present in patients infected with NANBH, thus establishing that HCV is one ot the viral a~ents causing NANBH. The cDNA sequenc~s associatcd wi~h HCV
were isolated from a cDNA library prepared from the RNA obtained from pooled 2 0 se-um from a chimpanzee wilh chronic HCV infection. The cDNA library contained cDNA sequences of approximate mean size of about 200 base pairs. The cDNA
library was screened for encoded epitopes expressed in clones that could bind toantibodies in sera from patients who had previously experienced NANBH.
In the European Patent Application, Houghton, M., i~LaL also described the 2 5 preparation of several superoxide dismutase fusion polypeptides ~SOD) and lhe use of these SOD fusion polypeptides to develop an ~CV screening assay. The most complex SOû fusion polypeptide described in the European Patent Application, designated c100-3, was described as containing 154 amino acids of human SOD at lhe aminoterminus, 5 amino acid residues derived from the expression of a

3 0 synthetic DNA adapter containing a restriction site, EcoRI, 363 amino acids derived from 1he expression of a cloned HCV cDNA fragment, and 5 carboxyl terminal aminoacids derived from an MS2 cloning vector nucleotide sequence. The DNA sequence encoding this polypeptide was transformed into yeast cells using a plasmid. The transformed cells were cultured and expressed a 54,000 molecular weight 3~ polypeptide which was purified to about 80% purity by differential extraction.
Olher SOD fusion polypeptides designated SOD-NANBs 1 1 and SOD-NANB81 we(e expressed in recombinant bacteria. The .~!i fusion polypeptides ' WO93/04088 .~ ) PCr/US92/07188 were puritied by differential extraction and by chromatography using anion and catbn exchange columns. The purification proc~ures were able to produce SOD-NANBs 1 1 as about 80% pure and SO~-NAN38, as about 50% pure.
The recombinant SOD fusion polypeptides described by Houghton, M., ~, 5 were coated on microtiter wells or polystyrene beads and used to assay serum samples. Briefly, coated microtiter wells were incubated with a sample in a diluent. Atter incubation, the microtiter wells were washed and then developed using either a radioactively labelled sheep anti-human anti~ody or a mouse antihuman IgG-HRP (horseradish peroxidase) conjugate. These assays were used to 1 0 detect both post acute phase and chronic phase HCV infection.
Due to the preparative methods, assay specificity required adding yeast or i extracts to the samples in order to prevent undesired immunological reactions with any yeast or ~,~ antibQdies present in samples.
Ortho Diagnostic Systems Inc. have developed a immunoenzyme assay to 1~ detect antibodies to HCV antigens. The Ortho assay procedure is a three-stage test for serum/plasma carried out in a microwell coated with the recombinant yeasVhepalitis C virus SOD fusion polypeptide c100-3.
In the first stage, a test specimen is diluted directly in the test well and inaJbated for a specffled length of time. If antibodies to HCV antigens are present in 2 0 the speamen, antigen-antibody complexes will be f~rmed on the microwell surface.
If no antibodies are present, complexes will not be formed and the unbound serumor plasma proteins will be removed in a washing s1ep.
In the second stage, anti-human IgG murine monoclonal antibody horseradish peroxidase conjugate is added to the microwell. The conjugale binds specifically to 25 the antibody portion of the antigen-antibody complexes. If antigen-antibody oomplexes are not present, the unbound conjugate will also be removed by a washing step.
In the third stage, an enzyme detection system composed of o-pheny!enediamine 2HCI (OPD) and hydrogen peroxide is added to the test well. If 3 0 bound conjugate is present, the OPD will be oxidized, resultin~ in a colored end product. After formation of the colored end product, dilute sulfuric acid is added to the microwell to stop the color-forming detection reactîon.
The intensity of lhe colored end product is measured with a microwell reader. The assay may be used to screen patient serum and plasma.
3 ~ It is established that HCV may be transmitted by contaminated blood andblood products. în transfused patients, as many as 10% will suffer from post-transfusion hepatitis. Of these, approximately 90% are the result of infections WO 93/04088 ,~ ~ ~ J j ~ ~ PCl~/US92/07188 ~

diagnosed as HCV. The prevention of 1ransmission of HCV by bbod and bbod products requires reliable, sensitive and specific d~agnosis and prognostic tools to identify HCV carriers as well as oontaminated blood and bbod products. Thus, there exists a need for an HCV assay which uses reliable and efficient reagents and methods to accura1ely detecl the presence of HCV antibod~es ~n samples.

SU ULUMY OF THE lNv~moN
The present invention provides an improved assay for detecting the presen¢e of an anIibody to an HCV antben in a sample by contacting the sample with at least 1 0 one recombinant protein representing a distinct antigenic region of the HCV genome.
Recombinant an1igens which are derived from the molecular cloning and expression of synthetic DNA sequences in helerologous hosts are provided. Briefly, synthetic DNA sequences which encode the desired proteins representing distinct antigenic regions of the HCV genome are optimked for expression in ~QIi by 1 5 specific codon selection. Specifically, recombinant proteins representing tive distinct antigenic regions of NS1 of the HCV genome are described. The proteins are express~d as chimeric tusbns with .QQli CMP-K~O synthetase (CKS) gene. The flrst protein, expressed by plasm~d pHCV-77 (ident~f~ed as SEQ. ID. NO. 1) represents am~no acids 365-579 of the HCV sequence of NS1 and, based on analogy 2 0 to the genomic or~anization of other fbviv~ruses, has been named HCV CKS-NS1S1.
Note that the tenn pHCV-77 will also refer to the fusion protein itself and thatpHCV-77' will be the designation for a polypeptWe represent~ng the NS1 region -~ from about am~no ac~ds 365-579 of the HCV sequence prepared using other recombinant or synthetic methodologies. Other recombinant methodologies would indude the preparation of pHCV-77~, u~ilking different expression systems. The methodology for the preparation of synthetic peptides of HCV is described in U.S.
Serial No. 456,162, filed December 22, 1989, and U.S. Serial No. 610,180, tiled November 7, 1990, which enjoy common ownership and are incorporated herein by reference. The next protein is expressed by plasmid pHCV-65, identified as SEQ.
3 0 ID. NO. 2, and represents ar,1ino acids 565-731 of the NS1 region of the HCV
genome, pHCV-65 has been named HCV CKS-NS1S2 and is expressed by the plasmi pHCV-65. The fusion protein itself will also be referred to as pHCV-6S and pHCV-65~ shall be the designation for a polypeptide from the NS-1 region representingfrom about amino acids 565-731 of the HCV sequence prepared using other 3 5 recombinant or synthetic methodologies. The next recombinant antigen represents amino acids 717-847 ot the NS1 region of the HCV sequence, and is expressed by the plasmid pHCV-78 (identified by SEQ. ID. NO. 3). The fusion protein will be WO 93/040~ PCI`/US92/07188 referred lo as pHCV-78 and pHCV-78' shall be the designation for a polypeptide from the NS1 regbn representing from about amino acids 717-847 of the HCV
sequence prepared using other reoombinant or synthetb methodobgies. It has been designated cbne HCV CKS-NS1S3 based on the strategy used in its construction.
Figure 44 illustrates the position of pHCV-77, pHCV-65 and pHCV-78 in the NS1 re~ion of the HCV genome. The recombinant antigen produced by pHCV-80is identified as SEQ. ID.NO. 4 and ~s desbnated HCV CKS-NS1S1 -NS1 S2. The fusion protein is also designated by pHCV-80 and pHCV-80' refers to the polypeptide bcaled in 1he NS1 region of HCV, represen1ing amino acids 365-731 of 1he HCV
genome prepared using different recombinant methodologies. Figure 45 illustra1esthe posilion of pHCV-80 within the HCV genome. HCV CKS-Full Length NS1is 1he designation for the recombinant pro1ein pHCV-92 (SEQ.ID. NO. 5). It represents amino acids 365-847 of the HCV genome. The fusion proteins will be referred to as pHCV-92 and pHCV 92' shall be the designation tor the polypeptide from the NS1 1 5 region representing amino acWs 365-847 of the HCV sequence prepared using o1her recombinan1 or syn1hetic me1hodohgies. Figure 46 illustrates the position of pHCV-92 in the HCV genome. These antigens are used in the inventive immunoassays 10 de1ect 1he presencc ot HCV an1ibodies in samples.
One assay format acoording 10 the invention provides a screening assay for 2 0 iden1ifying 1he presence of an an1ibody 1ha1 is immunologically reactive wi1h an HCV
antigen. Briefly, a fluid sample is incubated with a solid support containing the oommonly bound recombinant proteins. Finally, the antibody-antigen complex is detected. In a modification of 1he screening assay 1he solid support additionally oon1ains recombinant polypeptide c100-3.
Ano1her assay format provides a confirmatory assay for unequivocally idenlifying lhe presence of an antibody that is immunologically reactive with an HCV
antigen. The confirmatory assay includes synthetic peptides or recombinant antigens representing the epitopes contained within the NS1 region of the HCV
genome, which are the same regions represented by the recombinant proteins 3 0 described in the screening assay. These are pHCV-77, pHCV-65, pHCV-78, pHCV-80 and pHCV-92. Recombinant proteins used in the confirmatory assay should have a heterologous source of antigen to that used in the primary screening assay (i.e.
should not be an E~Qli-derived recombinant antigen nor a recombinant antigen composed in part, of CKS sequences). Briefly, specimens repeatedly reactive in lhe 3 5 primary screening assay are relested in lhe confirmalory assay. Aliquols containing identical amounts of specimen are contacted with a synthetic peptide or recombinant antigen individually coated onto a solid support. Finally, the antibody-W0 93/04088 W i L J ~ ~ PCl~/US92/07188 anligen complex is detected. The polypeptides Ot recombinant proteins can be utilized as indicated or combined with other po~peptides and recombinant proteins a described herein and also described ~n U.S. Serial No. 456,162 entitled "Hepatitis C Assay", filed December 22, 1989, which enpys common ownership and is 5 incorporated herein by reference.
Another assay format provides a competition assay or neutralization assay directed to the confirmation ~hat positive results are not false by identifying the presenc~ of an antibody that is immunobgically reactive with an HCV anligen in aHuid sample where the sample is used to prepare first and second immunolo~ically10 equivalent aliquots. The first aliquot is contacted with solid support containing a bound polypeptide which contains at least one epitope of an HCV antigen under conditions suitable for complexing with the antibody to form a detectable antibody-polypeptide complex and the second aliguot is first contacted with the same solid support containing bound polypeptide. The preferred recombinant polypeptides 15 include pHCV-77, pHCV-65, pHCV-78, pHCV-80 and pHCV-92.
Another assay format provWes an immunodot assay for Wentifying the presence of an aniibody that is immunologically reactive with an HCV antigen by concurrently contacting a sample with recombinant polypeptides each containing distinct epitopes of an HCV antigen under conditions suitable for complexing the2 0 antibody with at least one of the polypeptWes and detecting the antibodypo~peptide complex by reacting the complex with cobrproducing reagents. The preferred recombinant polypeptides employed include those recombinant polypeptides derivedfrom pHCV-77,pHCV-66, pHCV-78, pHCV-80, as well as pHCV-92.
In all of the assays, the sample is preferably diluted before contacting the 2 5 polypeptide absorbed on a solid support. Samples may be ob~ained from different biological samples such as whole blood, serum, plasma, cerebral spinal fluid, and Iymphocyte or cell culture supernatants. .Solid support materials may include cellulose materials, such as paper and nnrocellulose, natural and synthetic polymeric materials, such as polyacrylamide, poiystyrene, and cotton, porous gels 3 0 such as silica gel, agarose, dextran and gelatin, and inorganic materials such as deactivated alumina, magnesium sulfate and glass. Suitable solid support materials may be used in assays in a variety of well known physical configurations, including microtiter wells, test tubes, beads, strips, membranes, and microparticles. A
preferred solid support for a non-immunodot assay is a polystyrene bead. A
3 5 preferred solid support for an immunodot assay is nitrocellubse.
Suitable methods and reagents for detecting an antibody-antigen complex in - an assay of the present invention are commercially available or known in the ,, WOg3/04088 ~ i') PCl'/US92/07188 relevant art. Represenlative methods may empby delection reagents such as enzymatic, radbisotopic, fluorescent, lumineæent, or chemiluminescent rea~ents.
These re~ents may be used to prepare hapten-labelled antihapten detectbn systems acoordin~ to known procedures, for example, a biotin-labelled antibiotin system may be used to detect an antibody-antben com~ex.
The present ~nvention also encompasses assay kits including polypept~s whbh oontain at bast one epitope of an HCV antigen bound to a solid support as well as needed sample preparatbn rea~ents, wash reagents, detection reagents and signal producing reagents.
1 0 Other aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the folbwing detailed description whichprovides illustrations o~ the invention in its presently preferred embodiments.
i strains containing plasmids useful for construcls of the invention have been deposited at the American Type Culture Collection, Rockville, Maryland on Au~ust 10, 1990, under the a~ssion Nbs. ATCC 68380(pHCV-23),ATCC68381 (pHCV-29), ATCC 68382(pHCV-31),ATCC 68383(pHCV-34) and on November ` 6,1990for~,~ strains conlaining ~lasmWs useful for constructs under the - a~sshn Nos. ATCC 684~ (pHCV-~),ATCC68459(pHCV-57), ATCC 6~60 (pHCV-103), ATCC 68461 (pHCV-102),ATCC 68462(pHCV-51),ATCC 68463 (pHCV-105),ATCC 68464(pHCV-107),ATCC 68465(pHCV-104),ATCC 6~66 (pHCV-45),ATCC 6~67(pHCV~8),ATCC 68468(pHCV-49), ATCC 6~69 (pHCV-58) and ATCC 68470(pHCV-101). E. coli strains ~ntaining plasmids uselul for constructs of lhe invention have been deposited at the A.T.C.C. on September 26,1991 under deposit num~rs ATCC 68690(pHCV-77),ATCC
68696(pHCV-65), ATCC 68689(pHCV-78),ATCG 68688~pHCV-80) and ATCC
68695 (pHCV-923.

BREFDESCRlPTlONOFTHEDRAWlNq~
FIGURE 1 illustrates lhe HCV genome.
3 0 FIGURE 2 illustrates the use of recombinant polypeptides to identify the presence of antibodies in a chimpanzee inoculated with HCV.
FIGURE 3 illustrates the sensitivity and specificity increase in using the screening assay using pHCV-34 and pHCV-31 antigens.
FIGURE 4 illustrates the construction of plasmid pHCV-34.
FIGURE 5 illustrates fusion protein pHCV-34.
FIGURE 6 illustrates the expression of pHCV-34 proteins in - FIGURE 7 illustrates the construction of plasmid pHCV-23.

"

WO93/04088 ~ PCr/US92/07188 ;~. 3 FIGURE 8 iilustrates the construction of plasmid pHCV-29.
FIGURE 9 il~ustrates ~he construction of plasmW pHCV-31.
FIGURE 10 illustrat~s the fusion protein pHCV-31.
FIGURE 11 illustrates the expression of pHCV-29 in ~j.
FIGURE 12 illustrates the expression of pHCV-23 in ,~.
FIGURE 13 illustrates the expression of pl~CV-31 in .~-FIGURE 14 illustrates the increased sensitivity usin~ the sueening assay utilizing the pHCV-34.
FIGURE 15 illustrates the increased specificity with the screening assay 10 utilizing pHCV-34 and pHCV-31.
FIGURE 16 illustrates the results in hemodialysis patien~s usin~ the screening and confirmatory assays.
FIGURE 17 illustrates earlier detection of HCV in a hemodialysis patient using the screening assay.
FIGURE 18 illustrates the results of the screening assay utilizing pH(::V-34 and ptlCV-31 on samples from individuals with acute NANBH.
FIGURE 19 illustrates the results of the confirmatory assay of the same population group as in Figure 18.
FIGURE 20 illustrates the results of the screening and confirmatory assays 2 0 on individuals infected with chronic NANBH.
FIGURE 21 illustrat~s preferred buffers, pH conditions, and spolting concentrations for the HCV immunodot assay.
FiGURE 22 illuslrates the results of the H{:V immunodot assay.
FIGURE 23 illustrates 1he fusion protein pHCV-4~.
FIGURE 24 illustrates the expression of pHCV-46 in ~.
FIGURE 25 illustrates the fusion protein pHCV-48.
FIGURE 26 illustrates the expression of pHCV-48 in ~Qli FIGURE 27 iliustrates the fusion protein pHCV-51.
FIGURE 28 illustrates lhe expression of pHCV-51 in E.co!i.
3 0 FiGURE 29 illustrates the fusion protein pHCV-50.
FIGURE 30 illustrales the expression of pHCV-50 in ~QIi-FIGURE 31 illustrates the fusion protein pHCV-49.
FIGURE 32 illustrates the expression of pHCV-49 in ~li FIGURE 33 illustrates an immunoblot of pHCV-23, pHCV-45, pHCV-48, pHCV-51, pHCV-50 and pHCV-49.
FIGURE 34 illustrates the fusion proteins pHCV-24, pHCV-57, pHCV-58.
FIGURE 35 illustrates the expression of pHCV-24, pHCV-57, and pHCV-58 W0 93/04088 ~ PCI /US92/07188 in ~
FIGURE 36 illustrates the fusion protein pHCV-105.
FIGURE 37 illustrates the expression of pHCV-105 in E,~
FIGURE 38 illustrates the fusion protein pHCV-103.
FIGURE 39 ~llustrates the fusion protein pHCV-101.
FIGURE 40 illustrates the fusion protein pHCV-102.
FIGURE 41 illustrates the expression of pHCV-102 in ~.
FIGURE 42 illus~rates the fusion protein pHCV-107.
FIGURE 43 illustrates the fusion protein pHCV-104.
1 0 FIGURE 44 illustrates the NS1 region of the HCV genome, and in particular, the locations of pHCV-77, pHCV-65 and pHCV-78.
FIGURE 45 illustrates the NS1 region of the HCV genome, and in particular, the location of pHCV-80.
FIGURE 46 illustrates the NS1 region of the HCV genome, and in particlar, 1 5 the location of pHCV-92.
FIGURE 47A ilustrates the expression of pHCV-77 in ., ~oli; and FIGURE
47B illustrates an immunblot of pHCV-77 in E. coli.
Fl¢URE 48A illus1rates the expression of pHCV-65 in E. ~oli and FIGURE
48B illustrates an immunoblot of pHCV-65 in ~
2.0 FIGURE 49A illustrates the expression of pHCV-80 in E. coli and FIGURE 49B illustrates an immunoblot of pHCV-80 in ~.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to an assay to detect an antibody to an HCV
2 5 antigen in a sample. Human serum or plasma is preferably diluted in a sample diluent and incubated with a polystyrene bead coated with a recombinant polypeptide 1hat represents a distinct antigenic region of the HCV g~nome. If antibodies arepresent in the sampie they will form a complex with tl e antigenic polypeptide and beoorne affixed to the polystyrene bead. After the complex has formed, unbound 3 0 materials and reagents are removed by washing the bead and the bead-antigeh-antibody complex is reacted with a solution containing horseradish peroxidase labeled goat antibodies directed against human antibodies. This pe~oxidase enzyme then binds to the antigen-antibody complex already fixed to the bead. In a finalreaction the horseradish peroxidase is contacted with o-phenylenediamine and hydrogen peroxide which results in a yellow-orange color. The intensity of the color is proportional to the amount of antibody which initially binds to the antigen fixed to the bead.

WO 93/04088 ~ 1 ~ J ~ ~ ~ PCr/US92/07188 The prefarred recombinant polypeptides having HCV antigenic epitopes were selected from portions of the HCV genome which encoded polypeptides which possessed amino acid sequences similar to other known immunologically reactive agents and which were identified as having some immunohgbal reactivity. (The immunological reactivity of a polypeptide was initially identified by reacting the cellular extract of E.ÇQIi clones which had been transformed with cDNA fragments of the HCV genome with HCV infected serum. Polypeptides expressed by clone containing the incorporated cDNA were immunologically reactive with serum known to contain antibody to HCV antigens.) An analysis of a given amino acid sequence, however, only provides rough guides to predicting immunological reactivity. There is no invariably predictable way to ensure immunological activity short of preparing a given amino acid sequence and testing the suspected sequence in an assay.
The use of recombinant polypeptides representing distinct antigenic regions 1 5 of the HCV genome to detect the presence of an anbbody to an HCV antigen isillustrated in Figure 2. The course of HCV Infection in the chimpanzee, Pan, wasfollowed with one assay using recombinant c100-3 polypeptide and with another improved assay, using the hNo recombinant antigens CKS-Core (pHCV-34) (SEQ.ID.N0 6 and 7) and pHCV-33c-BCD (pHCV-31) (SEa.lD.NO 8 and 9) - 2 0 expressed by the plasmids pHCV-34 and pHCV-31, respectively. The assay utilizing the recombinant pHCV-34 and pHCV-31 proteins detected plasma antibody three weeks prior to detection of anbbody by the assay using c100-3.
A summaly of the results of a study which followed the course of HCV
infection in Pan and six other chimpanzees using the two assays described above is 2 5 summarized in Figure 3. Both assays gave negative results before inoculation and both assays detected the presence of antibodies after the animal had been infected with HCV. However, in the comparison of the two assays, the improved screening assay using pHCV-34 and pHCV-31 detected seroconversion to HCV antigens at an earlier or equivalent bleed date in six of the seven chimpanzees. Data from these 3 0 chimpanzee studies clearly demonstr~ate that overall detection of HCV antibodies is greatly increased with the assay utilizing the pHCV-34 and pHCV-31 proteins.
This test is sufficiently sensitive to detect seroconversion during the acute phase of this disease, as defined as an elevation in ALT levels, in most animals. Equallyimportant is the high degree of specificity of the test as no pre-inoculation - ~ 35 specimens were reactive.
The polypeptides useful in the practice of this invention are produced using recombinant technologies. The DNA sequences which encode the desired polypeptides ~ .
. , .

' ~ WO g3/04088 .~, 1 L 3 J .~ ~ PCI/US92/07188 are preferably assembled from fragments of 1he total desired sequenee. ~ynthQtiCDNA fragments of Ihe HCV ~enome can be synthesked based on their eorresponding amino acid sequences. Once the amino aeid sequence is chosen, this is then reverse translated to determi!le the complementary DNA sequence using eodons optimized to 5 faeilitate expression in the ehosen system. The fragmenls are generally prepared using well known automated processes and apparatus. After the complete sequence has been prepared the desired sequenee is incorporated into an expression vectorwhieh is transformed into a host eell. The DNA sequenee is then expressed by thehost cell to give the desired polypeptide which is harvested from the host cell or 10 from the medium in which the host cell is cultured. When smaller peptides are to be made using recombinant technologies it may be advantageous to prepare a single DNA sequence which encodes several copies of the desired polypeptide in a connected chain. The long chain is then isolated and lhe chain is cleaved into the shorter, desired sequenoes.
The methodology of polymerase ehain reaction (PCR) may also be employed to devebp PCR amplified genes from any portion of the HCV genome, which in turn may then be ehned and expressed in a manner similar to the synthetie genes.
Vector systems whieh ean be used inelude plant, baeterial, yeast, inseet, and mammalian expression systems. It is preferred that the codons are optimized for 2 0 expression in the system used.
A preferred expression system utilizes a carrier gene for a fusion system where the reeombinant HCV proteins are expressed as a fusion protein of an .~1ienzyme, CKS (CTP:CMP-3-deoxy-m~nQ-oetulosonate cytidylyl transferase or CMP-KDO synthetase). The CKS method of protein synthesis is disclosed in U.S.
Patent Applieations Serial Nos. 167,067 and 276,263 filed March 11, 1988 and November 23, 1988, respectively, by Bolling (EPO 891029282) which enjoy common ownership and are incorporated herein by reference.
Other expression systems may be utilized including the lambda PL vector system whose features include a strong lambda pL promoter, a strong three-frame 3 0 translation terminator rrnBtl, and translation starting at an ATG codon.
In the present invention, the amino acid sequences encoding for the recombinant HCV antigens of interest were reverse translated using codons optimized to facilitate high level expression in ~ l- Individual oligonucleolides were synthesized by the method of oligonucleotide directed double-stranded breakrepair diselosed in U.S. Patent Application Serial No. 883,242, filed July 8, 1986 by Mandecki (EPO 87109357.1) which enjoys common ownership and is incorporated herein by reference. Alternatively, the individual oligonucleotides ' J
WO g3/040~8 PCI~/US92/07188 '~

may be synlhesked on the Applied Biosystem 380A DNA synthesizer using methods and rea~ents reoommended by the manufacturer. The DNA sequences of lhe individual oli~onucleotid~s were confirmed usin~ the Sanqer dideoxy chain termination method (Sanger et al., J. Mole. Biol., 162:729 (1982)). These indivWual gene fragments were Ihen annealed and ligated logether and cloned as EooRI-BamHI subfragmenls in the CKS fusion vector pJO200. After subsequent DNA sequence confirmation by the Sanger dideoxy chain termination method, the subfragments were di~ested with appropriate restriction enzymes, gel purified, ligated and c~oned again as an EcoRI-BamHI fragment in the CKS fusion vector pJ0200. The resulting clones were mapped to identify a hybrid gene consisting ofthe EcoRI-BamHI HCV fragment inserted at the 3' end of the CKS (CMP-KD0 synthetase) gene. The resultant fusion proteins, under control of the ~ promoter, consisl of 239 amino acids of the CKS p~olein fused lo the various re~ions of HCV.
The synthesis, cbning, and characterization of the recombinant polypeptides as well as the preferred formats for assays using these polypeptides are provided in the following examples. Examples 1 and 2 describe the synthesis and cloning of CKS-Core and CKS-33-BCD, respectively. Example 3 describes a screening assay.
Example 4 de~cribe~ a oonfirma~ory assay. Example 5 descrlbes a comp~lltion assay. Example 6 describes an immunodot assay. Example 7 describes the 2 0 synth~sis ar,~d cbnin~ of HCV CKS-NS5E, CKS-NSSF, CKS-NS5G, CKS-NS5H and CKSNS51. Example 8 describes the preparation of HCV CKS-C100 vectors.
Example 9 describes the preparation of HCV PCR derived expression vectors.
Example 10 desc.ribes the synthesis and characterization of pHCV-77 of NS1.
Example 1~ describes the synthesis and characterization of pHCV-65 of NS1.
Example 12 describes the synthesis and characterization of pHCV-78 of NS1.
Example 13 describes lhe synlhesis and characterization of pHCV-80 of NS1.
Example 14 describes the synthesis and characterization of pHCV-92 of NS1.

REAGENTS AND ENZrMES
Media such as Luria-Bertani (LB) and Superbroth ll (Dri Form) were obtained from Gibco Laboratories Life Technologies, Inc., Madison Wisconsin.
Restriction enzymes, Klenow fragment of DNA polymerase 1, T4 DNA ligase, T4 polynucleotide kinase, nucleic acid molecular weight standards, M13 sequencing system, X-gal (5-bromo-4-chloro-3-indonyl-~-D-galactoside), IPTG
3 5 (isopropyl-~-D-thiogalactoside), glycerol, Dithiothreitol, 4-chloro-1-naphthol were purchased from Boehringer Mannheim Biochemicals, Indianapolis, Indiana; or New England Biolabs, Inc., Beverly, Massachusetts; or Bethesda Research ~;W093/04088 ~ 1 ~ J Y i~ ~ PCr/US92/07188 Laboratorbs Life Technobgies, Inc., Gaithetsburg, Maryland. Prestained protein molecular weight slandards, acrylamWe (crystallized, electrophoretic grade ~9996); N-N'-Methylene-bis-acrylamide (BIS); N,N,N',N',-~etramethylethylenediamine (TEMED) and sodium dodecylsulfate (SDS) were 5 purchased from BioRad Laboratories, Richmond, California. Lysozyme andampicillin were obtained from Sigma Chemical Co., St. Louis, Missouri.
Horseradish peroxidase (HRPO) labeled secondary antibodies were obtained from Kirke~aard 8 Perry Laboratories, Inc., Gaithersburg, Maryland. Seaplaque~
agarose (low melting agarose) was purchased from FMC Bioproducts, Rockland, 1 0 Maine.
TSOE10 contained 50mM Tris, pH 8.0, lOmM EDTA; 1X TG contained 100mM
Tris, pH 7.5 and 10% glycerol; 2X SDSIPAGE loading buffer consisted of 15%
glycerol, 5% SDS, lOOmM Tris base, 1 M B-mercaptoethanol and 0.8%
Bromophenol blue dye; TBS container 50 mM Tris, pH 8.0, and 150 mM sodium 15 chlorWe; Blocking solution consisted of 5% Carnation nonfat dry milk in TBS.

HOSTCQ I CULTUR,S. '~NAS~URCESANDVECTORS
.~QU JM103 cells, pUC8, pUC18, pUC19 and M13 clonin~ v~ctors were purchased from Pharmacia LKB Biotechnology, Inc., Piscataway, New Jersey;
20 Competent EpicureanrM coli stains XL1-Blue and JM109 were purchased from Stratagene Cbning Systems, LaJolla, California. RR1 cells were obtained from Coli Genetic Stock Center, Yale University, New Haven, Connecticut; and ~ i CAG456 cells from Dr. Carol Gross, University of Wisconsin, Madison, Wisconsin. Vector pRK248.clts was obtained from Dr. Donald R. Helinski, University of California, 25 San Diego, Califomia.

GENEF~I~llETtlODS
All restriction enzyme digestion were performed according to suppliers' instructions. At least 5 units of enzyme were used per microgram of DNA, and 3 0 sufficient ihcubation was allowed to complete digestion of DNA. Standard procedures were used for minicell Iysate DNA preparation, phenol-chloroform extraction, ethanol precipitation of DNA, restriction analysis of DNA on agarose, and low melting agarose gel purification of DNA fragments ~Maniatis et al., Molecular Clonina. A Laboratory Manual ~New York: Cold Spring Harbor, 1982]). Plasmid 3 5 isolations from ~ j strains used the alkali Iysis procedure and cesium chloride-ethidium bromide density gradient method (Maniatis et al., supra). Standard buffers were used for T4 DNA ligase and T4 polynucleotide kinase (Maniatis et al., WO 93/04088 ~ i Pcl/US92/07188'i supra) .

EXAMPLE 1. CKS CORE
A. Construction of the Plasmid pJ0200 The cloning vector pJO200 allows the fusion of recombinant proteins to the CKS protein. The plasmid consists of the plasmid pBR322 with a modified lac promoter fused to a KdsB gene fragment (encoding the first Z39 of the entire 248amino acids of the E~Ç~ CMP-KDO synthetase of CKS protein), and a synthetic linker fused to the end of the KdsB gene fragment. The cloning vector pJO200 is a 1 0 modification of vector pTB210. The synt~etic linker includes: mu~tiple restriction sites for insertion of genes; translational stop signals, and the trpA rho-independent transcriptional terminator. The CKS method of protein synthesis as well as CKS vectors including pTB210 are disclosed in U.S. Patent Application Serial Nos. 167,067 and 276,263, filed March 11, 1988 and November 23, 1 5 1988, respectively, by Bolling (EPO 891029282) which enjoy common ownership, and are herein incorporated by reference.

B. Preparation of HCV CKS-Core ~xpression Vector Six individual nucleotides representing amino aclds 1-150 of the HCV
O genome were ligated together and cloned as a 4B6 base pair EcoRI-BamHI fragment into the CKS fusion vector pJO200 as presented in Figure 4. The complete l)NA
sequence of this plasmid, designated pHCV-34, and the entire amino acW sequence of the pHCV-34 recombinant antigen produced is presented in SEQ.ID.NO 6 and 7. The resultant fusion protein HCV CKS-Core, consists of 239 arnino acids of CKS, seven 25 amino acids contributed by linker DNA sequences, and the first 15û amino acids of HCV as illustrated in Figure 5.
The pHCV-34 plasrnid and the CKS plasmid pTB210 were transfonned into ~.coli K-12 strain xL-I (recAI, endAI, gyrA96, thi-1, hsdRI7, supE44, relAI, lac/F', proAB, laclqZDM15, TN10) cells made competent by the calcium chl~ride 3 0 method. In these constructions the expression of the CKS fusion proteins was under the control of the iac promoter and was induced by the addition of IPTG. These plasmids replicated as independent elements, were nonmobilizable and were maintained at approximately 10-30 copies per cell.

35 C. Characterization of Recombinant HCV-Core In order to establish that clone pHCV-34 expressed the unique HCV-CKS
Core protein, the pHCV-34/XL-1 culture was grown overnight at 37C in growth `~WO 93/04088 ~ 1 1 J ~ ~ ~ PCI`/US92/07188 media consisting of yeast extract, trytone, phosphate salts, glucose, and ampicillin.
When the culture reached an OD600 of 1.0, IPTG was added to a final concentratbnof 1mM to induce oxpressbn. Samples (1.5 ml) were removed at 1 hour intervals, and cells were pelleted and resuspended to an OD600 of 1.0 in 2X SDS/PAGE loading 5 buffer. Aliquots (15ul) of the prepared samples were separa~ed on duplicate 12.5% SDS/PAGE gels.
One gel was fixed in a solution of 50% methanol and 10% acetic acid for 20 minutes at room temperature, and then stained with 0.25% Coomassie blue dye in asolulion of 50h methanol and 10h acetic acid for 30 minutes. Destaining was 1 0 carried out using a solution of 10% melhanol and 7% acetic acid for 3-4 hours, or until a clear baclcground was obtained.
~ Figure 6 presents the expression of pHCV-34 proteins in ~QIL Molecular weight standards were run in Lane M. Lane 1 contains the plasmid pJ0200-the CKS
vector without the HCV sequence. The arrows on the left indicate the mobilities o~
1 5 lhe molecular weighl markers from lop to bottom: 110,000; 84,000; 47,000;
33,000; 24,000; and 16,000 daltons. The arrows on lhe righl indicate lhe mobilities of lhe recombinanl HCV proteins. ane 2 contains lhe ,~Qli Iysate oontaining pHCV-34 expressing CKS-Core (amino acids 1 to 150) prior lo induction; and Lane 3 after 3 hours of induction. The results show 1hat the 2 0 reoombinant prolein pHCV-34 has an apparent mobility correspondin~ lo a molecular size of 48,000 daltons. This compares acceptably with the predicted molecular mass of 43,750 daltons.
Proteins from lhe second 12.5% SDS/PAGE gel were eleclrophoretically lransferred to nitrocellulose for immunoblotting. The nitrocellulose sheet ~ .
25 conlaining lhe lransferred proteins was incubated with Blocking Solution for one hour and incubated overnight at 4C with HCV patients' sera diluted in TBS
containing E.coli K-12 strain XL-I Iysate. The nitrocellulose sheet was washed three limes in TBS, then incubated with HRPO-labeled goat anti-human IgG, diluted in TBS containing 10% fetal calf sera. The nitrocellulose was washed three times3 0 with TBS and the color was developed in TBS containing 2 mglml 4-chloro-1-napthol, 0.02% hydrogen peroxide and 17% methanol. Clone HCV-34 demonstrated a strong immunoreactive band at 48,000 daltons with the HCV patients' sera. Thus, the major protein in the Coomassie stained protein gel was immunoreactive.
Normal human serum did not react with any component of pHCV-34.

EXAMPLE 2. HCV CKS-33C-BCD
AA Preearation of HCV CKS-33c-BCD Ex~ression Vector ,, WO 93/0408& f~ 1 ~ PCI /US92/07188 The construction of this recombinant clone expressing the HCV CKS-33-BCD
antigen was carried out in three steps described below. First, a clone expressing the HCV CKS-BCD antigen was constructed, designated pHCV-23. Second, a clone expressing the HCV CKS-33 antigen was constructed, designated pHCV-29. Lastly, the HCV BCD region was excised from pHCV-~3 and inserted into pHCV-29 to construct a clone expressing the HCV CKS-33-BCD antigen, designated pHCV-31 (SEQ.ID.NO. 8 and 9).
To construct the plasmid pHCV-23, thirteen individual oligonucleotides representing amino acids 1676-1931 of the HCV genome were ligated together and cloned as three separate EcoRI-BamHI subfragments into the CKS fusion vector pJ0200. After subsequent DNA sequence confirmation, the three subfragments, designated B, C, and D respectively, were digested with the appropriate restriction enzymes, gel purified, ligated together, a~d cloned as a 781 base pair EcoRI-BamHI
fragment in the CKS fusion vector pJO200, as illustrated in Figure 7. The resulting plasmid, designated pHCV-23, expresses the HCV CKS-BCD antigen under control of the lac promoter. The HCV CKS-BCD antigen consists of 239 amino acidsof CKS, seven amino acids contributed by linker DNA sequences, 2~6 amino acids from the HCV NS4 region (amino acids 1676-1931, and 10 additional amino acids contributed by linker DNA sequences.
To construct the plasmid pHCV-29 twelve individual oligonucleotides representing amino acids 1192-1457 of the HCV genome were ligated together and cbned as two separate EcoRI-BamHI subfragments in the CKS fusion vector pJ0200. After subsequent DNA sequence cs)nfirrnation, the two subfragments were digested with the appropriate restriction enzymes, gel purified, ligated together and cloned again as an 816 base pair EcoRI-BamHI fra~ment in the CKS fusion vector -pJO200, as illustrated in Figure 8. The resulting plasmid, designated pHCV-29, expresses the CKS-33 antigen under control of the iac promoter. The HCV CKS-33 antigen consists of 239 arnino acids of CKS, eight amino acids contributed by linker DNA sequences, and 266 amino acids from the HCV NS3 region (amino acids 1192-1457~.
To construct the plasmid pHCV-31, the 781 base pair EcoRI-BamHI
fragment from pHCV-23 representing the HCV-BCD region was linker-adapted to produce a Cla1-BamH1 fragment which was then gel purified and ligated into pHCV-29 at the Cla1-BamH1 sites as illustrated in Figure 9. The resulting plasmid, 3~ designated pHCV-31, expresses the pHCV-31 antigen under control of the lac promoter. The complete DNA sequence of pHCV-31 and the entire amino acid sequence of the HCV CKS-33-BCD recombinant antigen produced is presented in WO g3/04088 ~ 2 ~ PCI`/US92/07188 SEQ.ID.NO. 8 and 9. The HCV CKS-33-BCD antigen consists of 239 amino acids of CKS, ei~ht amino acids contributed by linker DNA sequences, 266 amino acids of the HCV NS3 region (amino acids 1192-1457), 2 amino acids contributed by linker DNA sequences, 256 amino acids of the HCV NS4 region ~amino acids 1676-1931), and 10 additional amino acids contributed by linker DNA sequences. Figure 12 presents a schematic representation of the pHCV-31 antigen.
The pHCV-31 plasmid was transformed into E~coli K-12 strain XL-I in a manner similar to the pHCV-34 and CKS-pTB210 plasmids of Example 1.

1 0 B. Characterization of Recombinant HCV CKS-33-BCD
Characterization of pHCV CKS-33-BCD was carried out in a manner similar to pHCV CKS-Core of Example 1. pHCV-23, pHCV SDS/PAGE gels were run for E.coli Iysates containing the plasmidspHCV-29 (Figure 11), pHCV-23 (Figure 12), and pHCV-31 (Figure 13) expressing the recombinant fusion proteins CKS-1 5 33c, CKS-BCD, and CKS-33-BCD, respectively. For all three figures, molecular weight standards were run in Lane M, with the arrows on the left indicating mobilities of the molecular weight markers the from top to bottom: 110,000;
84,000; 47,000; 33,000; 24,000; and 16,000 daltons. In Figure 11, Lane 1 contained the ~Q~ Iysate containing pHCV-29 expressing HCV CKS-33c (arnino acids 1192 to 1457) prior to induction and lane 2 after 4 hours induction. Theseresults show that the recombinant pHCV-29 fusion protein has an apparent mobility corresponding to a molecular size of 60,000 daltons. This compares acceptably to the predicted molecular mass of 54,911.
In Figure 12, Lane 1 contained the E.coli Iysate containing pJO200-- the 2 5 CKS vector without the HCV sequence. Lane ~, contained pHCV-20 expressing the HCV C:KS-B (amino acids 1676 to 1790). Lane 3, contained the fusion protein pHCV-23 (amino acids 1676-1931). These results show that the recombinant pl~CV-23 fusion protein has an apparent mobility corresponding to a molecular size of ~5,000 daltons. This compares acceptably to the predicted molecular mass of 3 0 ~5,070 daltons.
In Figure 13, Lane 1 contained the E.coli Iysate containing pJO200 the CKS
vector without the HCV sequences. Lane 2 contained pHCV-31 expressing the CKS-33c-BCD fusion protein (amino acids 1192 to 1447 and 1676 to 1931) prior to induction and lane 3 after 2 hours induction. These results show that the 3~ recombinant pHCV-31 (CKS-33c-BCD) fusion protein has an apparent mobility corresponding to a molecular size of 90,000 daltons. This compares acceptably tothe predicted molecular mass of 82,995 daltons.

WO 93/04088 ~ PCI`/US92/07188 `

An immunoblot was also run on one of the SDS/PAGE gels derived from the pHCV-31~ 1 culture. Human serum from an HCV exposed irldividual reacted slrongly with lhe major pHCY-31 band at 90,000 daltons. Normal human serum did not react wi1h any component of the pHCV-31 (CKS-33-BCD) preparations.

E)(AMPLE 3. SCREENING ASSAY
The use of recombinant polypeptides which contain epitopes within cl00-3 as well as epitopes from other antigenic regions from the HCV genome, provide immunological assays which have increased sensitivity and may be more specific 10 than HCV immunological assays using epilopes within c100-3 alone.
In the presently preferred screenin~ assay, the procedure uses two ~
expressed recombinant proteins, CKS-Core (pHCV-34) and GKS-33-BCD (pHCV-31), representing three distinct regions of the HCV genome. These recombinant polypeptides were prepared following procedures described above. In the screening 1 5 assay, both recombinant antigens are coated onto the same polystyrene bead. In a modification of the screening assay the polystyrene bead may also be coated with the SOD-fusion polypeptide c100-3.
The polystyrene beads are first washed with distilled waler and propanol and then incubated with a solution containing recombinant pHCV-31 dillJted to 0.5 to2 0 2.0 ug/ml and pHCV-34 diluted to 0.1 to 0.~ ug/ml in 0.1 M NaH2P04-H20 with 0.4M NaC1 and 0.0022% Triton X-100, pH 6.~. The beads are incubated in the antigen solution for 2 hours (plus or minus 10 minutes) at 38-42C, washed in PBS and soaked in 0.1% (w/v) Triton X-100 in PBS ~r 60 minutes at 38-42C.
The beads are then washed two times in phosphat~ buffered saline (PBS), overcoated 25 with a solution of 5.0% (w/v) bovine serum albumin (BSA) in PBS for 60 rr~nules at 38-42C and washed one time in PBS. Finally, the beads are overcoated with 5%(w/v) sucrose in PBS, and dried under nitrogen or air.
The polystyrene beads coated with pHCV-31 and pHCV-34 are used in an antibody capture format. Ten microiiters of sample are added to the wells of the3 0 reaction tray along with 400 ul of a sample diluent and lhe recombinant coated bead.
The sample diluent consists of 10% (vtv) bovine serum and 20% (v/v) goat serum in 20 mM Tris phosphate buffer containing 0.1~% (v/v) Triton X^100, 1%(w/v) BSA, 1% .s~Qli Iysate and 500 ug/ml or less CKS Iysate. When lhe recombinant yeast c~00-3 polypeptide is used, antibodies to yeast antigens which may be 3 5 present in a sample are reacted with yeast extracts which are added to the sample diiuent (typically about 200 ug/ml). The addition of yeast extracts to the sample diluent is used to prevent false positive results. The final material is sterile '.~WO g3/04088 PCI/US92/07188 filtered and filled in plas~ic bonles, and preserved with 0.1% sodium azide.
After one hour of incubation at 40C, the beads are washed and 200 ul of conjugate is added to the wells of the reaction tray.
The preferred oonjugate is ~oat anti-human IgG horseradish peroxidase conjugate. Concentrated conjugate is titered to determine a working concentration.
A twenty-fold concentrate of the working conjugate solution is then prepared by diluting the concentrate in diluent. The 20X concentrate is sterile filtered andstored in plastic bottles.
The conjugate diluent includes 10% (v/v) bovine serum. 10% (v/v) goat serum and 0.15% Triton-X100 in 20 mM Tris buffer. pH 7.5 with 0.01%
gentamicin sulfate, 0.01% thimerosal and red dye. The conjugate is sterile filtered and filled in plastic bottles.
Anti-HCV positive control is prepared from plasma units positive for antibodies to HCV. The pool of units used includes plasma with antibodies reactive to 1 5 pHCV-31 and pHCV-34. The units are recalcified and heat inactivated at 59-61C
hr 12 hours with constant stirring. The pool is aliquoted and stored at -20C or at 2-8C. For each lot of positive control, the stock solution is diluSed with negative control containing 0.1% sodium azide as a preservative. The final material is s!erile filtered and filled in plastic bottles.
2 0 Anti-HCV negative control is prepared from recalcified human plasma, negative for antibodies to pHCV-31 and pHCV-34 proteins of HCV. The plasma is also negative for antibodies to human immunodeficiency virus (HIV) and negative for hepatitis B surface antigen (HBsAg). The units are pooled, and 0.1% sodium æide is added as a presen/ative. The final material is sterile filtered and ~illed in 2~ plastic bottles.
After one hour of incubation with the conjugate at 40C, the beads are washed, exposed to the OPD substrate for thirty minutes at room temperature and the reaction terminated by the addition of 1 N H2SO4. The absorbance is read at 492 nm.
3 0 In order to maintain acceptable specificity, the cutoff for the assay should be at least ~-7 standard deviations above the absorbance value of the normal population mean. In addition, it has generally been observed that acceptable specificity is obtained when the population mean runs at a sample to cutoff (S/CO) value of 0.2~ or less. Consistent with these criteria, a "preclinical" cutoff for the 3 ~ screening assay was selected which clearly separated most of the presumed "true negalive" from "true positiveN specimens. The cutoff value was calculated as thesum of 1he positive control mean absorbance value multiplied by 0.25 and the WO g3/04088 ~ PCI`/US~2/07188'~

negalive control mean absorbance value. The cutoff may be expressed algebraically as:
Cutoff value~0~25 PCx + NCx.
Testing may be performed by two methods which differ primarily in the 5 degree of automalion and the mechanism for reading the resulting color development in the assay. One method is referred to as the manual or OuantumlM method because C~uantum or Quantumatic is used to read absorbance at 492 nm. It is also called the manual method because sample pipening, washing and reagent additions are generally done manually by the technician, using appropriately calibrated pipettes, 1 0 dispensers and wash instruments. The second method is referred to as the PPC method and utilizes the automated Abbott Commander~ system. This system employs a pipetting device referred to as the Sample Management Center (SMC) anda wash/dispenselread device referred to~as the Parallel Processing Center (PPC) disclosed in E.P.O. Publication No. g1114072.~. The optical reader used in the PPC
15 has dual wavelength capabilities that can measure differential absorbencies (peak band and side band) from the sample wells. These readings are converted into results by the processo~s Control Center.

Screeninn Assav Performance 2 0 1. Serum/Plasma From Inoculated Chimeanzees As previously described, Table I summarizes the results of a study which followQd the course of HCV infectbn in seven chimpanzees using a screening assaywhich utilized 1he c1û0-3 polypeptide, and the screening assay which utilized pHCV-31 and pHCV-34. Both assays gave negative results b~fore inoculation and 2 5 both assays detected the presence of antibodies after the animal had been infected with HCV. However, in the comparison of the two assays, the assay utiiizing pHCV-31 and pHCV-34 detected seroconversion to HCV antigens at an earlier or equivalent bleed date in six of the seven chimpanzees. Data from these chimpanzee studies clearly demonstrate that overall detection of HCV antibodies is greatly increased 3 0 with the assay utilizing the pHCV-31 and pHCV-34 proleins. This test is sufficienlly sensi1ive to detecl seroconversion during the acute phase of this disease, as defined as an elevation in ALT levels, in most animals. Equally important is the high degree of specificity of the test as no pre-inoculation specimens were reactive.
2. Non-A. Non-B Panel 11 (H. Alter. NIH!
A panel of highly pedigreed human sera from Dr. H. Alter, NIH, Bethesda, W0 93/04088 ~ PCr/US92/07188 MD., containing infectious HCV sera, negative sera and o1her disease controls were tested. A total of 44 specimens were present in lhe panel.
Six of seven sera which were ~proven ~nfectious~ in chimpanzees were positive in both the screening assay using c100-3 as well as in the screening assay 5 utilking the recombinant proteins pHCV-31 and pHCV-34. These six reactive specim0ns were obtained from individuals with chronic hepatilis. All six of the reactive specimens were confirmed positive using synthetic peptide sp67. One specimen obtained during the acute phase of NANB post-transfusion hepatitis was non-reactive in both screening assays.
In the group labeled "probable infectious" were three samples taken from the same post transfusion hepatitis patient. The first two acute phase samples were negathe in both assays, but the third sample was reactive in bolh assay. The disease control samples and pedigree~_negative controls were uniformly negative.All sixteen specimens detected as positive by both screening assays were confirmed by the spll7 confirmatory assay (Figure 14). In addition, specimens 10and 29 were newly detected in the screening assay utilking the recom~inant pHCV-31 and pHCV-34 antigens and were reactive by the sp7~ confirmatory assay.
Spocimen 39 was initially reactive in the screening test utilizing pHCV-34 and pHCV-31, but upon retesting was negative and oould not be confirmed by the 2 0 oonfirmatory assays.
In summary, both screening tests identified 6 of 6 chronic NANBH carriers and 1 of 4 acute NANBH samples. Paired specimens trom an implicated donor were non-reactive in the screening ~est utilizing c100-3 but were reac1ive in lhe screening test with pHCV-31 and pHCY-34. Thus, the screening test utilizing the 2 5 recombinant antigens pHCV-31 and pHCV-34 appears to be more sensitive than the scrcening assay u1ilizing c100-3. None of the disease control specimens or pedigreed negative control specimens were reactive in either screening assay.

3 CBER Referencs Panel 3 0 A reference panel for antibody to Hepatitis C was received from the Center for Biologics Evaluation and Research (CBE~). This 10 member panel consists of eight reactive samples diluted in normal human sera negative for antibody to HCVand two sera that contain no detectable antibody to HCV. This panel was run on the Ortho first generation HCV EIA assay, the screening assay utilizing c100-3 and lhe 3 5 screening assay utilizing pHCV-31 and pHCV-34. The assay results are presented in Figure 15.
The screening assay utilizing pHCV-31 and pHCV-34 detected all six of the WO 93/04088 ~ 6 P~/US92/071~

HCV positive or borderline sample dilulions. The two non-reactive sample dilutions (709 and 710) appear 1O be diluted well iDeyond endpoint of anti~ody detectabili1y for both screening assays. A marked inaease was observed in the sample to cutoffvalues for three of the members on the screening assay utilking pHCV-31 and pHCV-34 compared to the screenin~ assay utilking c100-3 or the Ortho first generation test. All repeatably reactive specimens were confirmed.

EXAMPIF4.CON IRMATORYASSAY
The confirmatory assay provides a means for unequivocally iden1ifying the presence of an antii~ody that is immunologically reactive with an HCV antigen. The confirmatory assay includes synthetic peptides or recombinant antigens r~presenting major epitopes contained within the three distinct regions of the HCV
genome, which are the same regions represented by the two recombinant antigens described in the screening assay. Recombinant proteins used in the confirmatory assay should have a heterologous source of antigen to that used in the primary screening assay (i.e. should not be ~n ~ derived recombinant antigen nor a recombinant antigen composed in pan, of CKS sequences). Specimens repeatedly reactive in the primary screening assay are retested in the confirmatory assay.
Aliquots containing Wentical amounts of specimen are contacted with a synthetic 2 0 peptide or recombinanl antigen individually coated onto a polystyrene bead.
Seroreactivity for epitopes within the c100-3 region of the HCV genome are confirmed by use of the synthetic peptides sp67 and sp65. The synthetic peptide sp117 can also be used to confirm seroreactivity with the c100-3 region.
Seroreactivity for HCV epitopes within lhe putative core region of HCV are 2 5 confirmed by the use of the synthetic peptide sp75. In order to confirm seroreactivity for HCV epitopes within the 33c region of HCV, a recombinant antigen expressed as a chimeric protein with superoxide dismulase ~SOD) in yeastis used. Finally, the antibody-antigen complex is detected.
The assay protocols were similar to those described in Example 3 above. The 3 0 peptides are each individually coated on~o polystyrene beads and used in an antibody capture format similar to that described for the screening assay. Ten rnicroliters of specimen are added to the wells of a reaction tray along with 400 ui of a specimen diluent and a peptide coated bead. After one hour of incubation at 40C, the beads are washed and 200 ul of conjugate (identical to that described in Example 3) is3 5 added to the wells of the reaction tray. After one hour of incubation at 40C, the beads are washed, exposed to the OPD substrate for 30 minutes at room temperature and the reaction terminated by the addition of 1 N H2SO4. The absorbance is read at W093/04U88 23 PCI/US92/0718~

492 nm. The cutoff value for lhe peptide assay is 4 times the mean of the negative control absorbance value.

1. Panels containino Seecimens "At Risk" for HCV Infec1ion.
A group of 233 specimens representing 23 hemodialysis patients all with clinically diagnosed NANBH were supplied by Gary Gitnick, M.D. at the University of California, Los Angeles Center for the Health Sciences. These samples which weretested in by the screening assay utilizing c100-3 were subsequently tested in the screening assay which uses pHCV-31 and pHCV-34. A total of 7/23 patients 1 0 (30.44%) were reactive in the c100-3 screening assay, with a total of 36 repeat reactive specimens. Ten of 23 patients (43.48%) were reactive by the screening assay utilizing pHCV-31 and pHCV-34, with a total of 70 repea~able reactives among the available specimens (Figure~16). Two specimens were unavailable for testing. All of the 36 repeatedly reactive specimens detected in the c100-3 1 5 screening assay were confirmed by synthetic peptide confirmatory assays. A total of 34 of these 36 were repeatedly reactive on HCV EIA utilizing pHCV-34 and pHCV-31; two specimens were not available for testing. Of the 36 specimens additionally detected by the screening assay utilizing pHCV-34 and pHCV-31, 9 were confirmed by the core peptide confirmatory assay (sp75) and 27 were confirmed by the SOD-33c confirmatory assay.
In summary these data indicate that detection of anti-HCV by the screening assay utilizing pHCV-31 and pHCV-34 may occur al an equivalent bleed date or as many as 9 months earlier, when compared to the c100-3 screening assay. Figure 17 depicts earlier detection by the screening assay utilizing pHCV-34 and pHCV-31 2 5 in a hemodialysis patien1. --5. Acute/Chronic ~lon-A. Noo-B ~ is A population of specimens was identified from individuals diagnosed as having acute or chronic NANBH. Specimens from individuals with acute oases of 3 0 NANBH were received from Gary Gitnick, M.D. at the Universily of California, Los Angeles Center for Health Sciences. The diagnosis of acute hepatitis was based on the presence of a cytolytic syndrome (ALT levels greater than 2X the upper normal limit) on at least 2 serum samples for a duration of less than 6 months with or without other biological abnormalities and clinical symptoms. All specimens were35 also negative for IgM antibodies to Hepatitis A Virus (HAV) and were negative for Hepatitis B surface Ag when tested with commercially available tests. Specimens from cases of chronic NANBH were obtained from two clinical sites. Individuals WO 93/04088 PCI'/US92/07188 were diagnosed as having chronic NANBH based on the following criteria:
persistently elevated ALT levels, liver biopsy results, and/or lhe absence of detectable HBsAg. Specimens w~lh biopsy results were further categorked as either chronic active NANBH, chronic persistent NANBH, or chronic NANBH with 5 cirrhosis.
These specimens were test~d by both the c100-3 screaning assay and the screening assay utilizing pHCV-34 and pHCV-31. The laner testing was performed in replicates of two by both the Quantum and PPC methods.
~ommunity Acguired NANBH (Acute~
The c100-3 screening assay detected 2 of 10 specimens (20.00%) as repeatedly reactive, both of which were confirmed. The screening assay utilizingpHCV-34 and pHCV-31 detected both of these specimens plus and additional 2 specimens (Figure 18). These 2 specimens were confirmed by sp75 (see Figure 19).
15 Acute Post-Transfusion NANBH
The c100-3 assay detected 4 of 32 specimens (12.50%) as repeatedly reactive, all of which was confirmed. The screening assay utilizing pHCV-34 and pHCV-31 detected 3 out of these 4 specimens (75/O) as reactive. The one sample that was missed had an SJCO of 0.95 by the latter screening test. This sample was 2 0 confirmed by the sp67 peptide (Figure 18). In addition, the screening assay utilking pHCV-34 and pHCV-31 detected 11 specimens not reactive in the c100-3 screening assay. Of the 9 specimens available for confirrnation, 8 were oonfirmed by sp75 and 1 could not be confirmed but had an S/CO of 0.90 in the sp65 confirmatory test. (see Figure 19).
2 ~ Chronic NANBH
A summary of the results on these populations is shown in Figure 20.
Overail, 155 of 164 (94.5%) chronic NANBH samples were detected by the screening test utilizing pHCV-31 and pHCV-34 using either Quantum or PPC. The 155 reactive samples were all confirmed in alternate assays using synthetic .
3 0 peptides based on sequences from either the clO0, 33c or core regions of the HCV
genome. In contrast, only 138 of 164 (84.1%) specimens were positive by the c100-3 assay. All but one of the 138 c100-3 samples were detected as positive bythe screening assay utilizing pHCV-31 and pHCV-34. The one discordant specimen was nol confirmed by either synthetic or neutralization assays. Conversely, there 3~ were 17 confirmed specimens which were positive only by the screening assay utilizing pHCV-34 and pHCV-31.
The results indicate that the screening assay utilizing pHCV-34 and pHCV-WO93/04088 ~ ~ 1J~ PCr/US92/07188 31 is more sensitive than the current test in detecting HCV positive individualswithin chronically infected NANBH populations.

EXAMPLE 5. CompQtition ASSAY
The recombinant polypeptides containing antigenic HCV epitopes are useful for competition assays. To perfonn a neutralization assay, a recombinant polypeptide representing epitopes within the c100-3 region such as CKS-BCD
(pHCV-23) is solubilized and mixed with a sample diluent to a final concentration of 0.5-50 ug/ml. Ten microlilers of specimen or diluted specimen is added to a 1 0 reaction well followed by 400 ul of the sample diluent containing the recombinant polypeptide and if desired, the mixture may be preincubated for about fifteen minutes to two hours. A bead coated with c100-3 antigen is then added to the reaction well and incubated for one hour at 40C. After washing, 200 ulofa peroxidase labeled goat anti-human IgG in conjugate diluent is added and incubated 1 5 for one hour at 40C. After washing, OPD substrate is added and incubated at room temperature for thirty mirlutes. The reaction is terminated by the addition of 1 N
sulfuric acid and the absol~ance read at 492 nm.
Samples containing antibodies to the c100-3 antigen generate a reduced signal caused by the competitive binding of the peptides to these antibodies in 2 0 solution. The percentage of competitive binding may be calculated by comparing the absorbance value of the sample in the presence of a recombinant polypeptide to the absorbance value of the sample assayed in the absence of a recombinant polypeptide at lhe same dilution.

2 5 E)(AMPLE 6, IMMUNODOT AS~Y
The immunodot assay system uses a panel of purified recombinant polypeptides placed in an array on a nitrocellulose solid support. The prepared solid support is contacted with a sample and captures specific antibodies to HCVantigens. The captured an~ibodies are detected by a conjugate-specific reaction.3 0 Preferably, the conjugate specific reaction is quantified using a reflectance optics assemb3y within an instrument which has been described in U.S. Patent Applications Serial No. 07/227,408 filed August 2, 1988. The related U.S. Patent ApplicationsSerial Nos. 07l227,272, 071227,586 and 07/227,590 further describe specific methods and apparatus useful lo perform an imrnunodot assay. The assay has also been described in U.S. Application Serial No. 07/532,489 filed June 6, 1990.
Briefly, a nitrocellulose-base test cartridge is treated with multiple antigenicpolypeptides. Each polypeptide is contained within a specific reaction zone on the W0 93/04088 ~ ; PCI/US92/07188 .~ ' '',~

lest cartridge. After all the antigenic polypeptides have been placed on the nitrocellulose, excess bindin~ siles on the nitrocellubse are blocked. The test cartridge is then contacted with a sample such that each antigenic polypeptide in each reaction zone will react if lhe sample contains the appropriate antibody. After 5 reaction, the test cartridge is washed and any antigen-antibody reactions are identified using suitable well known reagents.
As described in the patent applications listed above, the entire process is amenaUe to automation. The specifications of these applications related to the method and apparatus for performing an immunodot assay are incorporated by 1 0 reference herein.
In a preferred immunodot assay, the recombinant polypeptides pHCV-23, pHCV-29, pHCV-34, and cl00-3 were diluted in the preferred buffers, pH
conditions, and sponing concentrations as summarized in Figure 21 and applied to a preassembled nitrocellulose ~est cartridge. After drying the cartridge overnight at 1 5 room temperature 37C, the non-specific binding capacity of the nitro-cellulose phase was bhcked. The blocking solution contained 1% porcine gelatin, 1% casein enzymatic hydrolysate, 5% Tween-20, 0.1% sodium azide, 0.5 M sodium chloride and 20 mM Tris, pH 7.5.
Forly normal donors were assayed by following the method described above.
2 0 The mean reflectance density value then was determined for each of the recombinant proteins. A cutoff value was calculated as the negative mean plus six standard deviations. Test cartridges were incubated with samples A00642 and 423 (see Figure 22). Sample A00642 was from a convalescent non-A, non-B hepatitis patient, diluted in negative human plasma from 1:100 to 1:12800. The other 2 ~ sample, 423, was from a paid plasma donor which ~ested positive in an assay using a recombinant c100-3 polypeptide, diluted in negative human plasma from 1:40 to 1:2560. After sample incubation, sequential incubations wi~h a biotin-conjugatedgoa~ anti-human immunoglobulin-specific antibody, an alkaline phosphatase-conjugated rabbit anti-biotin specific antibody, and 5-bromo-4-chloro-3-indolyl 3 0 phosphate produced a colored product at the site of the reaction. Sample to cutoff values (S/CO) were determined for all HCV recombinant proteins. Those S,'CO
values greater than or equal to 1.0 were considered reactive. The limiting dilution was defined as the lowest dilution at which the SICO was greater than or equal to 1Ø As seen in Figure 22, each sample tested positive for all HCV recombinant 3 ~ proteins. The data demonstrate that reactivity for sample A00642 was greatest with pHCV-29, and decreased for the remaining antigens pHCV-23, c100-3, and pHCV-34. Sample 423 most strongly reacted with the recombinant proteins WO 93/04088 ~ PCI'/US9Z/07188 expressing pHCV-29 and pHCV-34, and to a lesser extent with pHCV-23 and c100-3.

E XAMPLE 7. HCV CK~NS5 E)(PRESSION VECTORS
5 A. Preparation of HCV CKS-NSSE
Eight individual oligonucleobdes representing amino acids 1932-2191 of the HCV genome were ligated together and cloned as a 793 base pair EcoRI-BamHI
fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated pHCV-45 (SEQ.ID.NO 8), expresses the HCV CKS-NS5E antigen under control of the 1 0 lac promoter. The HCV CKS-NS5E antigen consists of 239 amino acids of CKS, nine amino acids contributed by linker DNA sequences, and 260 amino acids from the HCV NS41NS5 region (amino acids 1932-2191). Figure 23 presents a schematic representation of the recombinant antigen-expressed by pHCV~5. SEQ.ID.NO. 10 and 11 presents the DNA and amino acid sequence of the HCV CKS-NS5E recombinant 1 5 antigen produced by pHCV-45. Figure 24 presents the expression of pHCV-45 proteins in ~Q!l. Lane 1 contained the .~ Iysate containing pHCV45 expressing the HCV CKS-NS5E antigen (amino acids 1932-2191) prior to induction and lanes 2 and 3 after 2 and 4 hours post induction, respectively. These results show that the pHCV-45 fusion protein has an apparent mobility 2 0 conesponding to a molecular size of 55,000 daltons. This compares acceptably to the predicted molecular mass of 57,597 daltons.

B. Preparation of HCV CKS-NS5F
Eleven individual oligonucleotides representing amino acids 2188-2481 of 2 6 the HCV genome were ligated t~gether and cloned as a 89~ base pair EcoRI-BamHI
fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated pHCV-48, expresses the HCV CKS-NS5F antigen under control of the lac promoter.
The HCV CKS-NS5F antigen consists of 239 amino acids of CKS, eight amino acids contributed by linker DNA sequences, and 294 amino acids from the HCV NS5 region(amino acids 2188-2481). Figure 25 presents a schematic representation of the recombinant antigen expressed by pHCV-48. SEQ.ID.NO. 12 and 13 presents the DNA and amino acid sequence of the HCV CKS-NS5F recombinant antigen produced by pHCV-48. Figure 26 presents the expression of pHCV-48 proteins in ~Q!I- Lane 1 contained the E.coli Iysate containing pHCV-48 expressing the HCV CKS-NS5F
antigen (amino acids 2188-2481) prior to induction and lanes 2 and 3 after 2 an

4 hours post induction, respectively. These results show that the pHCV-48 fusionprotein has an apparent mobility corresponding to a molecular size of 65,000 WO 93/04088 w i ~ PCI`/US92/07188 ~, daltons. This compares acceptably to the predicted molecular mass of 58,985 daltons.

C. Preparation of HCVCKS-NS5G
Seven individual oligonucleotides representing amino acids 2480-2729 of the HCV genome were ligated togethe~ and cloned as a 769 base pair EcoRI-BamHI
fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated pHCV-51(SEQ.lD.NO.10), expresses the HCV CKS-NS5G antigen under control of the lac promoter. The HCVCKS-NS5G antigen consists of 239 amino acids of CKS, eight amino acids contributed by linker DNA sequences, and 250 amino acids from ~e HCV NS5 region (amino acids 2480-2729). Figure 27 presents a schematic representation of ~e recombinant antigen expressed by pHCV-51. SEQ.NO.ID NO.14 and 15 presents the DNA and amino acid sequence of the HCV CKS-NS5G recombinant antigen produced by pHCV-51. Figure 28 presents the expression of pHCV-51 1 5 proteins in ~ i- Lane 1 contained the E.coli Iysate containing pHCV-51 expre~ing the HCV CKS-NS5G antigen (amino acids 2480-2729) prior to induction and lanes 2 and 3 after 2 and 4 hoùrs post induction, respectively. These results show that the pHCV-51 fushn protein has an apparent mobility corresponding to a molecular ske of 55,000 daltons. This compares acceptably to 2 0 the predicted molecular mass of 54,720 daltons.

D. preparation of HCV CKS-NS6H
Si% individual oligonucleotides representing amino acids 2728-2867 of the HCV genome were ligated together and cloned as a 439 base pair EcoRI-BamHI
fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated pHCV-50 (SEQ.NO.11~.11) expresses the HCV CKS-NS5H antigen under control of the lac promoter. The HCV CKS-NS5H antigen consists of 239 amino acids of CKS, eight amino acids contributed by linker DNA sequences, and 140 amino acids from the HCV NS5 region (amino acids 2728-2867). Figure 29 presents a schematic 3 0 representation of the recombinant antigen expressed by pHCV-50. SEQ.ID.NO. 16 and 17 presents the DNA and amino acid sequence of the HCV CKS-NS5H recombinant antigen produced by pHCV-50. Figure 30 presents the expression of pHCV-50 proteins in E.coli. Lane 1 contained the E.coli Iysate containing pHCV-50 expressing the HCV CKS-NS5H antigen (amino acids 2728-2867) prior to induction and lanes 2 and 3 after 2 and 4 hours post induction, respectively. These results show that the pHCV-50 fusion protein has an apparent mobility corresponding to a molecular size of 45,000 daltons. This compares acceptably to W0 93/04088 ~ PC~r/US92/07188 the predicted molecular mass of 42,783 daltons.
E. Preparation of HCV CKS-NS51 Six individual oligonucleotides representing amino acids 2866-3011 of the HCV genome were ligated together and cloned as a 460 base pair EcoRI-BamHI

5 fragment into the CKS fusion vector pJ0200. The resulting plasmid, designated pHCV~9 (SEQ.NO.ID.NO. 12), expresses the HCV CKS-NS51 antigen under control of the lac promoter. The HCV CKS-NS51 antigen consists of 239 amino acids of CKS, eight amino acids contributed by linker DNA sequences, and 146 amino acids from the HCV NS5 region (amino acids 2866-3011). Figure 31 presents a schematic 10 representation of the recombinant antigen expressed by pHCV49. SEQ.ID.NO. 18 and 19 presents the DNA and amino acid sequence of the HCV CKS-NS61 recombinant antigen produced by pHCV-49. Figure 32 presents the expression of pHCV49 proteins in E.coli. Lane 1 contained thq E.coli Iysate containing pHCV-49 expressing HCV CKS-NS51 antigen (amino acids 2866-3011) prior to induction 15 and lanes 2 and 3 after 2 and 4 hours post induction, respectively. These results show that the pHCV-49 fusion protein has an apparent mobility corresponding to amolecular size of 42,000 daltons. This compares acceptably to the predicted molecular mass of 43,497 daltons.

2 0 F. Immunoblot of HCV CKS-NS5 Antigens Induced E.coli Iysates containing pHCV-23, pHCV~5, pHCV-48, pHCV-51, pHCV-50, or pHCV-49 were individually run on preparative SDSIPAGE gels to separate the various HCV CKS-NS5 or HCV CKS-BCD recombinant antigens assay from the majority of other E.coli proteins. Gel slices containing the separated 2 5 individual HCV CKS-NS5 or HCV CKS-BCD recombinant antigens were then electropheretically transferred to nitrocellulose, and the nitrocellulose sheet cut into strips. Figure 40 presents the results of a Western Blot analysis of various serum or plasma samples using these nitrocellulose strips. The arrows on the right indicate the position of each HCY CKS-BCD or HGV CKS-NS5 recombinant antigen, 30 from top to bottom pHCV-23 (HCV CKS-BCD), pHCV45(HCV CKS-NS5E),pHCV-48~HCV CKS-NS5F),pHCV-51 ~HCV CKS-NS5G),pHCV-50(HCV CKS-NS5H), pHCV-49tHCV CKS-NS51), and pJO200 ~CKS). Panel A contained five normal human plasma, panel B contained five normal human sera, panel C contained twentyhuman sera positive in the Abbott HCV EIA test, panel D contained two mouse sera3 5 directed against CKS, and panel E contained two normal mouse sera. Both the HCV
CKS-NS5E antigen expressed by pHCV45 and the HCV CKS-NS5F antigen expressed by pHCV-48 were immunoreactive when screened with human serum WO 93/04088 ~ i ~ 3 PCI`/US92/07188 samples containing HCV antibodies.

EXAMPLE 8. HCV CKS-C100 A. Preparation of HCV CKS-C100 Vectors Eighteen individual oligonucleotides representing amino acids 1569-1931 of the HCV genome were ligated together and cloned as four separate EcoRI-BamHI
subfragments into the CKS fusion vector pJ0200. After subsequent DNA sequences confirmation, the four subfragments were digested with the appropriate restriction enzymes, gel purified, ligated toge!her, and cloned as an 1102 base pair EcoRI-1 0 BamHI fragment in the CKS fusion vector pJ0200. The resulting plasmid, designated pHCV-24, expresses the HCV CKS-C100 antigen under control of the lac promoter. The HCV CKS-c100 antigen consists of 239 amino acids of CKS, eight amino acids contributed by linker DNA seguences, 363 amino acids from the HCV
NS4 region (amino acids 1569-1931) and 10 additional amino acids contributed 1 5 by linker DNA sequences. The HCV CKS-c100 antigen was expressed at very low levels by pHCV-24.
- Poor expression levels of this HCV CKS-c100 recombinant antigen were overcome by constructing two additional clones containing deletions in the extreme amino terrninal portion of the HCV c100 region. The first of these clones, des`ignated pHCV-57 (SEQ.ID.NO. 20 and 21), contains a 23 amino acid deletion (HCV amino acids 1575-1597) and was constructed by deleting a 69 base pair Ddel restric~on fragment. The second of these clones, designated pHCV-58 (SEQ.ID.NO.
22 and 23), contains a 21 amino acid deletion (HCV amino acids 1600-1620) and was constructed by deleting a 63 base pair NlalV-Haelll restriction fragment.
2 5 Figure 34 presents a schematic representation of the recombinant antigensexpressed by pHCV-24, pHCV-57, and pHCV-58. SEQ.ID. NO. 13 presents the DNA
and amino acid sequence of the HCV-C100D1 recombinant antigen produced by pHCV-57. SEQ.ID.NO.14 presents the DNA and amino acid sequence of the HCV-C100D2 recombinant antigen produced by pHCV-58. Figure 35 presents the expression of pHCV-24, pHCV-57, and pHCV-58 proteins in E.coli. Lane 1 contained the E.coli Iysate containing pHCV-24 expressing the HCV CKS-c100 antigen (amino acids 1569-1931) prior to induction and lanes 2 and 3 after 2 and4 hours post induction, respectively. Lane 4 contained the E.coli Iysate containing ~ ~ pHCV-57 expressing the HCV-CKS-C100D1 antigen (amino acids 1669-1574 and 5~ 35 1598-1931) prior to induction and~lanes 5 and 6 after 2 and 4 hours induction, respectively. Lane 7 contained the E.coli Iysate containing pHCV-58 expressing the HCV CKS-C100D2 antigen (amino acids 1569-1599 and 1621-1931) prior to :, `3 WO 93/04088 PCI lUS92/07188 induction, and lanes 8 and 9 after 2 and 4 hours induction, respectively. These results show that both the pHCV-57 and pHCV-58 fusion proteins express at significan~y higher levels than the pHCV-24 fusion protein and that both the pHCV-57 and pHCV-58 fusion proteins have an apparent mobility corresponding to 5 a molecular size of 65,000 daltons. This compares acceptably to the predicted molecular mass of 64,450 daltons for pHCV-~7 and 64,458 daltons for pHCV-58.

EXAMPLE 9. HCV PCR DERIVED EXPRESSION VECTORS
A. Preparation of HCV DNA Fraçlments RNA was extracted from the serum of various chimpanzees or humans infected with HCV by first subjecting the samples to digestion with Proteinase K and SDS for 1 hour at 37 centigrade followed by numerous phenol:chloroform extractions. The RNA was then concentrated by several ethanol precipitations andresuspended in water. RNA samples were then reverse transcribed according to 1~ supplier's instructions using a specific primer. A second primer was then added and PCR amplification was performed according to supplier's instructions. An aliquot of this PCR feaction was then subjected to an aWitional round of PCR using nested primers located internal to the first set of primers. In general, these primers also contained restriction endonuclease recognition sequences to be used for 2 0 subsequent cloning. An aliquot of this second round nested PCR reaction was then subjected to agarose gel electrophoresis and Southern blot analysis to confirm the specificity of the PCR reaction. The remainder of the PCR reaction was then digested with the appropriate restriction enzymes, the HCV DNA fragment of interest gel purified, and ligated to an appropriate cloning vector. This ligation was 2 5 then transformed into E.coii and single coionies were isolated and plasmid DNA
prepared for DNA sequences analysis. The DNA sequences was then evaluated to confirm that the specific HCV coding region of interest was intact. HCV DNA
fragments obtained in this manner were then cloned into appropriate vectors for expression analysis.
3 ~ B. PreDaration ot HCV CKS-NS3 Using the methods detailed above, a 474 base pair DNA fragment from the putative NS3 region of HCV was generated by PCR. This fragment represents HCV
amino acids #1473-1629 and was cloned into the CKS expression vector pJ0201 by blunt-end ligation. The resulting clone, designated pHCV-105, expresses the 3 ~ HCV CKS-NS3 antigen under control of the lac promoter. The HCV CKS-NS3 antigen consists of 239 amino acids of CKS, 12 amino acids contributed by linker DNA
sequences, 1~7 amino acids from the HCV NS3 region ~amino acids 1473-1629), W093/04088 ~ i i PCr/US92/07188 and 9 additional amino acids contributed by linker DNA sequences. Figure 36 prosents a schematic representation o~ the pHCV-105 anbgen. SEQ.ID.NO. 24 and 25 presents the DNA and amino acW sequence of the HCV CKS-NS3 recombinant antigen produced by pHCV-105. Figure 37 presents the expression of pHCV-105 5 proteins in E.~:QIj. Lane 1 contained me ~Q~ Iysate containing pHCV-105 expressing the HCV CKS-NS3 antigen (amino acids 1472-1629) prior to induction and lanes 2 and 3 after 2 and ~ hours induction, respectively. These results show that the pHCV-105 fusion protein has an apparent mobility corresponding to a molecular mass of 43,000 daltons. This compares acceptably to the predicted 1 0 molecular mass of 46,454 daltons.
C. Preparation of HCV CKS-5'ENV
- Using the methods detailed above, a 489 base pair DNA fragment from the putative envebpe region of HCV was gen~rated by PCR. This fragment represents the HCV amino acids 114-276 and was cloned into the CKS expression vector 1 5 pJ0202 using EcoRI-BamHI restriction sites. The resulting clone, designated pHCV-103 (SEQ.ID.NO. 26 and 27), expresses the HCV CKS-5'ENV antigen under control of the lac promoter. The HCV CKS-5'ENV antigen consists of 239 amino acids of CKS, 7 amino acids contributed by linker DNA sequences, 163 amino acidsfrom the HCV envelope region (amino acids 114-276), and 16 additional arnino 2 0 æids contributed by îinker DNA sequences. Figure 38 presents a schematic representation of the pHCV-103 antigen. SEQ.ID.NO. 26 and 27 presents the DNA
and amino acid sequence of the HCV CKS-5'ENV recombinant antigen produced by pWCV-103. Figure 37 presents me expression of pHCV-103 proteins in E.coli.
Lane 1 contained the E.coli Iysate containing pHCV-103 expressing the HCV CKS-25 5'ENV antigen (amino acids 114-276) prior to induction and lanes ~ and 6 after 2 and 4 hours induction, respectively. These results show that the pHCV-103 fusionprotein has an apparent mobility corresponding to a molecular mass of 47,000 daltons. This compares acceptably to the predicted molecular mass of 46,091 daltons.
3 0 D. Preparation of HCV CKS-3'ENV
Using the methods detailed above, a 621 base pair DNA fragment form the putative envelope region of HCV was generated by PCR. This fragment represents HCV amino acids 263469 and was cloned into the CKS expression vector pJ0202 using EcoRI restriction sites. The resulting clone, designated pHCV-101 36 (SEQ.ID.NO. 17), expresses the HCV CKS-3'ENV antigen under control of the lacpromoter. The HCV CKS-3'ENV antigen consists of 239 amino acids of CKS, 7 amino acids contributed by linker DNA sequences, 207 amino acids from the HCV

WO93/04088 ~ i ~ j PCr/US92/07188 envelope region (amino acids 263-469), and 15 additional amino acids contributedby linker DNA sequences. Figure 39 presents a schematic representation of the pHCV-101 antigen. SEQ.ID.NO. 28 and 29 presents the DNA and amino acW sequence of the HCV CKS-3'ENV recombinant antigen produced by pHCV-101. Figure 37 5 presents the expression of pHCV-101 prnteins in ~ i Lane 7 contained the ~
Iysate containing pHCV-101 expressing the HCV CKS-3'ENV antigen ~amino acids 263-469) prior to induction and lanes 8 and 9 after 2 and 4 hours induction, respectively. These resulting show that the pHCV-101 fusion protein has an apparent mobility corresponding to a molecular mass of 47,000 daltons. This 1 0 compares acceptably to the predicted molecular mass of 51,181 daltons.
E. Preparation of HCV CKS-NS2 Using the methods detailed above, a 636 base pair DNA fragment from the putative NS2 region of HCV was generated by PCR. This fragment represents the HCV amino acids 994-1205 and was cloned into the CKS expression vector pJ0201 1 5 using EcoRI restriction sites. The resulting clone, designated pHCV-102, expresses the HCV CKS-NS2 antigen under control of the lac promoter. The HCV CKS-NS2 antigen consists of 239 amino acids of CKS, 7 amino acids contributed by linker DNA sequences, 212 amino acids from the HCV NS2 region (amino acids 994-1205), and 16 additional amino acids contributed by linker DNA sequences. Figure40 presents a schematic representation of the pHCV-102 antigen. SEQ.Il).NO. 30 and 31 presents the DNA and amino acid sequence of the HCV CKS-Næ recombinant antigen produced by pHCV-102. Figure 41 presents the expression of pHCV^102 proteins in E.coli. Lane 1 contained the ~ Iysate containing pHCV-102 expressing the HCV CKS-NS2 antigen (amino acids g94-1205) prior to induction 2 5 and lanes 2 and 3 after 2 and 4 hours induction, respectively. These results show that the pHCV-102 fusion protein has an apparent mobili~ corresponding to a molecular mass of ~3,000 daltons. This compares acceptably to the predicted molecular mass of 51,213 daltons.
F. PreDaration of HCV CKS-NS1 3 0 Using the methods detailed above, a 654 base pair DNA fragment from the putative NS1 region of HCV was generated by PCR. This fragment represents HCV
amino acids 617-834 and was cloned into the CKS expression vector pJ0200 using EcoRI-BamHI restriction sites. The resulting clone, designated pHCV-107, expresses the HCV CKS-NS1 antigen under control of the lac promoter. The HCV
CKS-NS1 antigen consists of 239 amino acids of CKS, 10 amino acids contributed by iinker DNA sequences, and 218 amino acids from the HCV NS1 region (amino acids 617-834). Figure 42 presents a schematic representation of the pHCV-107 WO g3/04088 2 ~ ~ 3 ~ ~ ~i PCI`/USg2/07188 ~ ~`

antigen. SEQ.ID.NO. 32 and 33 presents the DNA and amino acid sequence of the HCV
CK~NS1 recombinant anbgen produced by pHCV-107.
G. Preparation of HCV CKS-ENV
Using the methods detailed above, a 1068 base pair DNA fragment from the 5 putative envebpe region of HCV was generated by PCR. This fragment represents HCV amino acids #114469 and was cloned into the CKS expression vector pJ0202 using EcoRI restriction sites. The resulting clone, designated pHCV-104, expresses the HCV CKS-ENV antigen under control of the lac promoter. The HCV CKS-ENV
anbgen consists of 239 amino acids of CKS, 7 amino acids contributed by linker 1 0 DNA sequences, 356 amino acids from the HCV envelope region (amino acids 114-469), and 15 additional amino acids contributed by linker DNA sequences. Figure 43 presents a schematic representation of the pHCV-104 antigen. SEQ.ID.NO. 34 and 35 presents the DNA and amino acid sequence of the HCV CKS-ENV recombinant antigen produced by pHCV-104.
EXAMPLE 10. HCY (~KS-NS1S1 A. Construction of the HCV CKS-NS1 S1 Expression Vector Eight individual oligonucleotides representing amino acids 365-579 of the HCV genome were ligated together and cloned as a 645 base pair EcoRI/BamHI
2 0 fragment into the CKS fusion vector pJO200. The amino acid sequence of this antigen is designated as pHCV-77 (SEQ. ID. NO. 1). The resultant fusion protein HCV CKS-NS1S1 consists of 239 amino acids of CKS, seven amino acids contributed by linked DNA sequences, and ~15 amino acids from the NS1 region of the HCV
gerlome.
25 B. Production and Characterization of the Recombinant Antiaen HCV-NS1S1 pHCV-77 was transformed into E.coli K-12 strain XL-1 (recA1, endA1, gyrA96, thi-1, hsdR17, SupE44, relA1, lac/f1, p10AB, lacl1ADM15, TN10) cells. Expression analysis and characterization of the recombinant protein was done using polyacrylamide gel electrophoresis as described in Example 1. The apparent3 0 molecular weight of the pHCV-77 antigen was the same as the expected molecular weight of 50,228 as visualized on a coumassie stained gel. The immunoreactivity as determined by Western blot analysis using human sera indicated that this recombinant antigen was indeed immunoreactive. FIGURE 47A presents the expression of pHCV-77 in E. coli. FIGURE 47B presents an immunoblot of the 3 ~ pHvV-77 antigen expressed in E. coli. Lane 1 contained the E. çoli Iysate containing pHCV-77 expressing the HCV CKS-NS1S1 antigen prior to induction and Lanes 2 and 3 are 2 and 4 hours post-induction, respectfully.

. ~

W0 93/04088 ~ 2 ~ PCI/US92/0~188 EXAMPLE 11. HCV CK~NSlS2 A. Construction of the HCV CKS-NS1S2 Expression Vector Six individual oligonucleotides representing amino acids 565-731 of the 5 HCV genome was ligated together and cloned as a 501 base pair EcoRI/BamHI
fragment into the CKS fusion vector p~O200. The complete amino acid sequence of this antigen is designated as pHCV-65 (SEQ. ID. NO. 2). The resultant fusion protein HCV CKS-NSlS2 oonsists of 239 amino acids of CKS, eight amino acids contributed by linker DNA sequences, and 167 amino acids from the NS1 region of 1 0 the HCV genome.
B. Production and Characterization of the Recombinant Antioen HCV-NS1S2 pHCV-65 was transformed into E.coli K-12 strain XL-1 (recA1, endA1, gyrA96, thi-1, hsdR17, SupE44, relA1, lac/f1, p10AB, laclqAMD15, TN10) cells.
Expression analysis and characterization of the recombinant protein was done using 1~ polyacrylamide gel electrophoresis as described in Example 1. The apparent molecular weight of the pHCV-66 antigen was the same as the expected molecular weight ot 46,223 as visualized on a coumassie stained gel. The immunoreactivity as determined by Western blot analyis using human sera indicated that this recombinant antigen was indeed immunoreactive. FIGURE 48A presents the 2 0 expression of pHCV-65 in E. coli. FIGURE 48B presents an immunobiot of the pHCV-65 antigen expressed in E. coli. Lane 1 contained the E. coli Iysate containing pHCV-65 expressing the HCV CKS-NS1S2 antigen prior to induction and Lanes 2 and 3 are 2 and 4 hours post-induc~ion, respectively.

EXAMPLE 12. CKS-NS1S3 A. Construction of the HCV CKS-NS1 S3 Expression Vector Six individual oligonucleotides representing amino acids 717-847 of the HCV genome were ligated together and cloned as a 393 base pair EcoRltBamHI
fragment into the CKS fusion vector pJO200. The complete amino acid sequence of 3 0 this antigen is designated as pHCV-78 (SEQ. ID. NO. 3). The resultant fusion protein HCV CKS-NS1S3 consists of 239 amino acids of CKS, eight amino acids contributed by linker DNA sequences, and 131 amino acids from the NS1 region of the HCV genome.
B. Production and C~haracterization of the Recombiant Antiaen HCV-NS1S3 pHCV-78 was transformed into E.coli K-12 strain XL-1 (recA1, endA1, gyrA96, thi-1, hsdR17, SupE44, relA1, lactf1, p10AB, laclqADM15, TN10) cells.
Expression analysis and characterization of the recombinant protein was done using WO 93/04088 ~ i ~ PCl'/US92/07188 polyacryiamide gel electrophoresis as described in Example 1. Analysis of the coumassie stained gel indicated very low levels of expression of the protein with an expected molecular weight of 42,1141. Western blot analysis also failed to show any immunoreactivity and we are continuing to identify human sera that is specific to this region of NS1.

EXAMPLE 13. CKS-NS1S1-NS1S2 A. Construction of the HCV CKS-NS1S1-NSlS2 Expression Vector The construction of pHCV-80 (NS1S1-NS1S2) involved using the SACI/BamHI insert from pHCV-65 and ligating that into the Sacl/BamHI vector backbone of pHCV-77. The resultant HCV gene represents amino acids 365-731 of the HCV genome. This resulted in a 1101 base pair EcoRI/BamHI fragment of HCV
cloned into the CKS fusion vector pJO20~ The complete amino acid sequence of this antigen is designated as pHCV-80 (SEQ. ID. NO. 4). The resultant fusion protein 1 5 HCV CKS NS1S1-NS1S2 consists of 239 amino acids of CKS, seven amino acids contributed by linker DNA sequences, and 367 amino acids from the NS1 region of ~e HCV genorne.
B. Production and Characterization of the Recombinant Antiaen HCV-NS1S1:~1S1S2 pHCV-80 was transformed into E.coli K-12 strain XL-1 (recA1, endA1, gyrA96, thi-1, hsdR17, SupE44, relA1, lac/fl, p10AB, laclqADM15, TN10) cells.
Expression analysis and characterization of the recombinant protein was done using polyac~ylamide gel electrophoresis as described in Example 1. The apparent molecular weight of the pHCV-80 antigen was the same as the expected molecular weight of 68,454 as visualized on a coumassie stained gel. The immunoreactivity as 2 5 determined by Western blot analysis using human sera indicated that this recombinant antigen was very immunoreactive. FIGURE 49A presents the expression of pHCV-80 in E. coli. FIGURE 49B presents an immunoblot of pHCV-80 antigen expressed in E. coli. Lane 1 contained the E. coli Iysate containing pHCV-80 expressing the HCV CKS-NS1S1-NS1S2 antigen prior to induction and Lanes 2 and 3 are 2 and 4 hours post-induction, respectively.

E)(AMPLE 14. HCV CKS-FULL LENGTH NS1 A. Construction of the HCV CKS-full lenath NS1 Expression Vector The construction of pHCV-92 (SEQ. ID. NO. 5) full length NS1) involved using the Xhol/BamHI insert from pHCV-78 (SEQ. ID. NO. 3) and ligating that intothe XhoVBamHI vector backbone of pHCV-80 (SEQ. ID. NO. 4) The resultant HCV
gene represents amino acids 36~-847 of the HCV genorne. This resulted in a 1449 ! WO 93/04088 ~ i i J ~ ~ 6 Pcr/usg2!o7188 base pair EcoRI/BamHI fragment of HCV cloned into CKS fusion vector pJO200. The complete amino acid sequence of this antigen is designated as pHCV-92 (SEQ. ID. NO.
5). The resultant fusion protein HCV CKS-full length NS1 consists of 239 amino acids of CKS, seven amino acids contributed by linker DNA sequences, and 483 5 amino acids from the NS1 region of the HCV genome.
B. Production and Characterization of the Recombinant Antigen pHCV-92 pHCV-92 was transformed into E.coli K-12 strain XL-1 ~recA1, endA1, gyrA96, thi-1, hsdR17, SupE44, relA1, lac/f1, p10AB, laclqADM15, TN10) cells.
Expression analysis and characterization of the recombinant protein was done using 10 polyacrylameide gel electrophoresis as described in Example 1. The expressionlevels as seen by counassie stained gel were virtually undectable and the Western blot indicated no immunoreactivity. We are still in the process of identifying sera that will recognize this region of HCV N~1.

The present invention thus provides unique recombinant antigens representing dis~inct antigenic regions of the HCV genome which can be used as reagents for ~e detection and/or confirmation of antibodies and antigens in testsamples from individuals exposed to HCV. The NS1 protein is considered to be a non-structural membrane glycoprotein and to be able to elicit a protective immune 2 ~ response of the host against iethal viral infection.
The recombinant antigens, either alone or in combination, can be used in the assay formats provided herein and exemplified in the Examples. It also is contemplated that these recombinant antigens can be used to develop specific inhibitors of viral replication and used for therapeutic purposes, such as for 2 5 vaccines. Other applications and modifications of the use of these antigens and the specific embodiments of this inventions as set forth herein, will be apparent tothose skilied in the art. Accordingly, the invention is intended to be limited only in accordance with the appended claims.
, .

PCrNS 9 2 / 0 7 1 8 8 BO/US 23DEC~992 3~

SEQUENCE LISTING
~1) GENERAL INFORMATION:
(i) APPLICANT: DEV~RE, S.
DESAI, S.
DAILEY, S.
(ii) TITLE OF INVENTION: HCV SYNTHETIC PEPTIDE FROM NSl REGION
(iii) NUMBER OF SEQUENCES: 35 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: ABBOTT LABORATORIES
(B) STREET: ONE ABBOTT PARK ROAD
(C) CITY: ABBOTT PARK
(D) STATE: ILLINOIS
(E) COUNTRY: U.S.
~F-) ZIP: 60065-3500 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
tD) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
- (C~ CLASSIFICATION:
~viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: POREMBSKI, PRISCILLA E.
(B) REGISTR~TION NU~BER: 33,207 (C) REF~RENCE/DOCKET NUMBER: d834PC.02 (ix) TELECOMMUNICATION INFORMATION: -(A) TELEPHONE: 708-937-6365 (8~ TELEFAX: 708-937-9556 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: ~63 amino acids (B) TYPE: amino acid (C) STRP~JDEDNESS: single (D) TOPOLOGY: linear (ii) NOLECULE TYPE: pep~ide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu '}~ ~ r 92G PCTIlJS 92/0718 8 3g Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His VA1 Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Vial Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Aisp Asn Leu Ala Gln Arg Gln Val Gly ~et Thr Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Vial Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val ('-ly Asp isn Phe L~u Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile 18~ 1~5 190 Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu G:lu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu sp Pro Ser Thr Asn Ser Thr Met Val Gly Asn Trp Ala Lys Val Leu al Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr His Val Thr Gly Gly Ser Ala Gly His Thr Val Ser Gly Phe Val Ser Leu Leu Ala Pro Gly Ala Lys Gln Asn Val Gln Leu I le Asn Thr Asn Gly Ser Trp 2gO 295 300 PCTlllS 9 2/ 07 1 8 8 B0/ljS 23 DEC lm Hi~ Leu Asn Ser Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn Thr Gly ~05 310 315 320 Trp Leu Ala Gly Leu Phe Tyr His His Lys Phe Asn Ser Ser Gly Cy5 Pro Glu Arg Leu Ala Ser Cys Arg Pro Leu Thr Asp Phe A~p Gln Gly Trp Gly Gln Ile Ser Tyr Ala Asn Gly Ser Gly Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys Ser Val Cys Gly Pro Val Tyr.Cys Phe~Thr Pro Ser Pro Val Val Val Gly Ihr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Glu Asn 4.05 410 415 Asp Thr Asp Val Phe ~al Leu Asn Asn Thr Arg Pro Pro Leu Gly Asn 420 ~25 430 Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys Val Cys g35 440 g45 Gly Ala Pro Pro Cys Val Ile Gly Gly Ala Gly A~n Asn Thr Leu 450 ~55 460 (2) INFORMATION FOR SEQ ID No~2:
ti) SEQU~NCE CHARACTERISTICS.
(A) LENGIH: 414 amino acids (B) TYPE: amino acid (C) STRANDXDNESS: single (D~ TOPOLOGY: linear (ii) ~GLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala g5 Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu `~ PCTIUS 92/07l8a ~ O / 1~ S 2 3 DEC 1992 Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala lu Val ~'al Glu Lys Cys Ala Phe S~r A~p Asp Thr Val Ile Val Asn al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val 115 120 1~5 Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Al~ Val Lys Val 130 135 lgO
Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr I le ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly A~p sn Phe Leu Arg His Leu Gly Ile Iyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met lgS 200 205 Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Yal Ala Gln Glu Val Pro Gly Thr Gly Val A p Thr Pro Glu Asp Leu 225 230 23~ 24Q
sp Pro Ser Thr Asn Ser Met Gly Ala Pro Pro Cys Val Ile Gly Gly la Gly Asn Asn Thr Leu His Cy~ Pro Thr Asp CyY Phe Arg Lys His 260 2~5 270 Pro Asp Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro 275 2~0 285 Arg Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Thr Pro Cys Thr Ile Asn Thr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu 305 310 315 . 320 is Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp ,eu Glu Asp Arg Asp Arg Ser Glu Leu S,er Pro Leu Leu Leu Thr Thr - hLi~f~2~ PCTI~JS 92/071 8 8 K0/US 23DEClg~

Thr Gln Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile His Leu Gly Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu 385 390 395 g00 Tyr Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val 405 gl0 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 378 ami~o acids (B) TYPE: amino ~cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met S~r Phe Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro ~et Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu A3p Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu Val Val Glu Lys Cy9 Ala Phe Ser Asp Asp Thr Val Ile Val Aqn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val Pro Ile ~is Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile Pro `2~ 0/US 23 DEC 199 Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu Hi~ Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala V~l Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu A~p 225 230 235 2~0 Pro Ser Thr Asn Ser Thr Met Glu Tyr Val V~l Leu Leu Phe Leu L~u Leu Ala Asp Ala Arg Val Cy9 Ser Cy9 Leu Trp Met Met Leu Leu Ile Ser Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Ile Leu Asn Ala Ala Ser Leu Ala Gly Thr His Gly Leu Val Ser Phe Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Lys Gly Lys Trp Val Pro Gly Al~ Val Tyr Thr Phe Tyr Gly Met Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Gln Arg Ala Tyr Al~ Leu A~p Thr G;u Val Ala Al~ Ser Cy~ Gly Gly Val 340 3~5 3S0 Val Leu Val Gly Leu Met Ala Leu Thr Leu Ser Pro Tyr Tyr Lys Arg Tyr Ile Ser Trp Cys Leu Trp Trp Leu Gln ~2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 622 amino acids (B) TYPE: amino a-cid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide - PCTllJS 9 2 / 0 7 1 8 0/~S 23 DEC 1992 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu ro Gly Lyq Pro Leu Val Asp Ile Asn Gly Lyq Pro Met Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln S~r Gly Thr Glu Arg Leu Ala ~ 75 80 lu Val Val Glu Lys Cys Ala Phe Ser A3p Asp Thr Val Ile Val A~n ~l Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 135 1~0 Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile ro ~rp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly A~p sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Ar~ Ala Gly Phe Ile 180 1~5 190 Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala 210 ~ 215 220 Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu qp Pro Ser Thr Asn Ser Thr Met Val Gly Asn Trp Ala Lys Val Leu al Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr His Val Thr ly Gly Ser Ala Gly His Thr Val Ser Gly Phe Val Ser Leu Leu Ala PCT/lJS 9z/O718.
- - ~O/lJS 23 OEC 1992 Pro Gly Ala Lys Gln Asn Val Gln Leu Ile Asn Thr Asn Gly Ser Trp His Leu A~n Ser Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn Thr Gly Trp LQU Ala Gly Leu Phe T~r His His Lys Phe A~n Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Pro Leu Thr Asp Phe Asp Gln Gly 3gO 3g5 350 Trp Gly Gln Ile Ser Tyr Ala Asn Gly Ser Gly Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys Ser Val C-ys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Glu Asn gO5 410 415 Asp Thr Asp Val Phe Val Leu Asn A~n Thr Arg Pro Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp Met A~n Ser Thr Gly Phe Thr Lys Vnl Cys Gly Ala Pro Pro Cys Val Ile Gly Pro Pro Cys ~al Ile Gly Gly Ala ~50 455 g60 Gly Asn A~n Thr L~u Hi~ Cys Pro Thr Asp Cys Phe Arg Ly~ His Pro 465 470 ~75 ~80 Asp Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr Ile Phe Ly~ Ile Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Thr Thr Thr 5g5 550 555 560 Gln Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser ` i- PCTIUS ~/0718 8 ~
3~b ~O/US 23 aEC 1992 Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Xaa (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ?38 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Ser Phe Val Val Ile Ile Pro Ala Arg ~yr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His V~l Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu A~p Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val cy9 Met Thr Arg Ala Asp Hi~ Gln Ser Gly Thr Glu Arg Leu Ala Glu V~l Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn Val Gln Gly A~p Glu Pro ~et Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 135 1~0 Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Th~ Val Gly A~p PCT/US 9 2 / 0 7 1 8 8 .
b ~0/liS 23 DEC ~992 Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Asg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser Thr Met Val Gly Asn Trp Ala Lys Val Leu Val Val Leu Leu Leu Phe Alà Gly Val Asp Ala Glu Thr His Val Thr Gly Gly Ser Ala Gly His Thr Val Ser Gly Phe Val Ser Leu Leu Ala 275 28~ 285 Pro Gly Al~ Lys Gln Asn Val Gln Leu Ile Asn Thr Asn Gly Ser Trp His Leu Asn Ser Thr Ala Leu Asn Cys Asn Asp Ser Leu Asn Thr Gly 305 310 315 . 320 Trp Leu Ala Gly Leu Phe Tyr His His Lys Phe As~ Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser ~ys Arg Pro Leu Thr Asp Phe Asp Gln Gly . 340 345 350 Trp Gly Gln Ile Ser Tyr Ala Asn Gly Ser Gly Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys Ser Val ~yg Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Glu Asn gO5 410 415 Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro Pro Leu Gly Asn 420 q25 g30 Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile Gly Pro Pro Cys Val Ile Gly Gly Ala ' I

Gly Asn Asn Thr Leu His Cys Pro Thr Asp Cys Phe Arg Lys Hi~ Pro sp Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg y.s Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Iyr Pro Cys Thr Ile Asn Tyr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg qr8 A-~p Leu 530 535 5~0 Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Thr Thr rhr ln Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser hr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu Trp Met Met Leu Leu Ile Ser Gln Ala Glu Ala Ala I~eu Glu Asn eu Val Ile Leu Asn Ala Ala Ser Leu Ala Gly 'rhr His Gly Leu Val er Phe Leu Val Phe Phe Cys Phe Ala Trp Iyr Leu Lyc Gly Lys Trp Val Pro Gly Ala Val l~rr Thr Phe ~r Gly Met Trp Pro Leu Leu Leu 675 ~80 685 Leu Lau Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Val A1A Ala Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr eu Ser Pro Tyr Tyr Lys Arg Tyr I le Ser Trp Cys Leu Trp Trp Leu ln Xaa - PCTIUS 9 2/ ~ 7 1 8 8 ~LL,~ /l,S- 23 o~c lm (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~481 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNESS: 9 ingle (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 130..1317 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:~:
GAATTAATTC CCATTAATGT GAGTTAGCTC ACTCATTAGG CACCCCAGGC m ACACTTT 60 AT~TICCGGC TCGTATTTTG TGTG~AATTG TGAGCGGATA ACAA~TGGGC ATCCAGTAAG 120 GAG~TTTAA ATG AGT TTT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG . 168 Met Ser Phe Va} Val Ile Ile Pro Ala Arg Tyr Ala Ser 1 ' 5 10 ACG CGT CT~ CCC GGT AAA CCA TTG G~T GAT ATT AAC GGC AAA CCC ATG 216 Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Lou Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile 30 -.35 g0 45 ATC GTG GCA ACC GAT CAT GA~ GAT GTT GCC CGC GCC GTT GAA GCC GCT 312 lle V~l Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu V~l Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu C~T CTG GCG GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG 408 Arg Leu Ala Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr CTG GCG GTG CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG . 552 .

,~:
,,, Y~ 0/liS- 23 DEC 1992 Leu Ala VA1 Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala 130 135 lgO

VA1 Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly A~p Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Al~

Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Va' Leu Trp Tyr Gly Glu Lys Ile Hi~ Val Ala V 1 Ala Gln Glu Val Pro Gly Thr Gly Yal Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser Met Ser Thr Asn Pro Lys Pro Gln Lyq Ly~ Asn Lys Arg A~n Thr A n Arg Arg Pro Gln A9P Val Lys TTC CCG GGT GGT GGT C~G ATC GTT GGT GGT GTT TAC CTG C~G CCG CGT 984 Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg 270 275 280 2a5 Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro GAA GGT CGT ACC TGG GCT CAG CCG GGT TAC CCG TGG CCG CTG TAC GGT 112 ?3 Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly A~n Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser ~ 1 L J .j ~ ti PCTIUS 9 2 / 0 7 1 8 8 Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala TAACCCATGG ATCCTCTAGA CTGC~GGCAT GCTAAGTAAG TAGATCTTGA GCGCGTTCGC 1377 CGTTACGATT TTCCTCAATT TTTCTTTTCA ACAATTGATC TCATTCAGGT GACATCTTTT 1~97 TCCCCATTAT GATTCT$CTC GCTTCCGGCG GCATCGGGAT GCCCGCGTTG CAGGCCATGC 1797 TGTCCAGGCA GGTAGATGAC G~CCATCAGG GACAGCTTCA AGGATCGCTC GCGGCTCTTA 1857 CCAGCCTAAC TTCGATCACT GGACCGCTGA TCGTCACGGC GATTTA~GCC GCCTCGGCGA 1917 GCAC~TGGAA CGGGTTGGCA TGGATTGTAG GCGCCGCCCT ATACCTTGTC TGCCTCCCCG 1977 TGCGCAA~CC AACCCTTGGC AGAACATATC CATCGCGTCC GCCATCTCCA GCAGCCGCAC 2157 GCGGCGCA~C TCGGGCAGCG TTGGGTCCTG GCCACGGGTG CCCATGATCG TGCTCCTGTC 2217 ~GTATTAACG AAGCGCTTCT TCCGCTTCCT CGCTCACl'GA CTCGCTGCGC TCGGTCGTTC 2517 2 ~ PCTIJS 9 2 / 0 7 1 8 8 ~0/luS 23DEC1992 AGGCCGCGTT GCTGGCG m TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC 2697 CCTTTCTCCC TTCGGGAAGC GTGGCGC m CTCAATGCTC ACGCTGTAGG TATCTCAGTT 2877 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTA m GGTATCTGCG 3117 CCACCGCTGG TAGCGGTGGT TTTTTTG m GCAAGCAGCA GATTACGCGC AGAAAAAAAG 3237 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT 3gl7 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCG AGA m ATCA GCAATAAACC 3597 . .
AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC m ATCCGCC TCCATCCAGT 3657 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAA~GCGG 3837 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GA~GTAAGTT GGCCGCAGTG TTATCACTCA 3897 8~us 23DEC1992 C~CGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT 4437 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 396 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: prOteiQ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu ~0 55 60 Va~ Cy Met Thr Arg Ala Asp Hi3 Gln Ser Gly Thr Glu Arg Leu Ala lu Val Val Glu Lys Cy9 Ala Phe Ser A~p Asp Thr Val Ilç Val Asn al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val 115 120 ' 125 Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr-Ala Leu Tyr Phe Ser Arg Ala Thr Ile ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile - PCTllJS 92/0718 8 Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly T~r Gly Val Asp Thr Pro Glu Asp Leu sp Pro Ser Thr Asn Ser Met Ser Thr Asn Pro Lys Pro Gl~ Lys Lys sn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro 290 295 30~
Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Axg 305 . 310 315 320 hr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly A~n Glu Gly ys Gly TTP Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro m r Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly ~ys Val 355 360 36~
Ile Asp Thr Leu Thr Cy~ Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala AIg Ala (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5600 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE IYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 130..2472 PcTNs 9 2 / 0 7 1 8 8 O/US 23 D~ 1992 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GAATTAATTC CCATTAATGT GAGTTAGCTC ACTCATTAGG CACCCCAGGC TTTACAC m 60 Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val ~eu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile V~l Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala GGC G~T GAA GTA TGT ATG AC~ CGC GCC GAT CAT CAG TCA GGA ACA GAA . 360 Gly Gly Glu V~l Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu 65 ' 70 75 CGT CTG GCG GAA GTT GTC GAA AAA TGC GCA TT~C AGC GAC GAC ACG GTG 408 Arg Lou Ala Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val 80 a5 90 Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Lou Ala Gln Arg Gln Val Gly Met Ala m r CTG GCG GTG CCA ATC CAC AAT GCG GAA GAA GCG m AAC CCG AAT GCG 552 Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg GCC ACC ATT CCT TGG GAT CGT GAT CGT m GCA GAA GGC CTT GAA ACC 648 Ala Thr Ile Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala .

.. . . . . .... ... .. .. . .. .. . .. . .. . ..

P~IUS 9 2 / 0 7 1 8 ~US 23D~Cl992 Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile Hi~ Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val A~p Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser Met Ala Val Asp Phe Ile Pro 240 245 _ 250 Val Glu Asn Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr A~p Asn TCT TCT CCG CCG GTT GTT CCG CAG TCT TTC CAG GTT GCT CAC C~G CAT 984 Ser Ser Pro Pro Val Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala GCT CAG GGT TAC AAA GTT CTG GrT CTG AAC CCG TCT GTT GCT GCT ACT 1080 Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr ~ Leu Gly Phe Gly Ala Tyr Met Ser Lys Al~ His Gly Ile Aqp Pro A~n A~T CGT ACT GGT GTA CGT ACT ATC ACT ACT GGT TCT CCG ATC ACT TAC 1176 Ile Ary Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr TCT A.T TAC GGT AAA TTC CTG GCT GAC GGT GGT TGC TCT GGT GGT GCT 1224 Ser Thr Tyr Gly Lys Phs Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Sex Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala 385 390 ~95 CGT CTG GTT GTT C~G GCT ACT GCT ACT CCG CCG G5T TCT GTT ACT GTT 1368 Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val` Thr Val 2~ ~O/~s 23 nEC ~992 Pro Hi~ Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile CCG TTC TAs- GGT AAA GCT ATC CCG CTC GAG GTT ATC AAA GGT GGT CGT 14 6 4 Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Arg ~is Leu I le Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala AAG CTT GTT GCT CTG GGT ATC AAC GCT GTT GCT TAC TAC CGT GGT Cl~ 15 6 0Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala Tyr l~r Arg Gly Leu 465 470 ~ 475 GAC GTT TCT GTT ATC CCG ACT TCT GGT GAC GTT GTT GTT GTG GCC ACT i 6 0 8 Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val VA1 Val Val Ala Thr GAC GCT CTG ATG ACT GGT TAC ACT GGT GAC TTC GAC TCT GTT ATC GAT 16 5 6A~p Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp TGC AAC ACT TGC AAT TCG TCG ACC GGT TGC Gl~ GTT ATC GTT GGT CGT 17 0 4Cys Asn Thr Cys Asn Ser Ser Thr Gly Cys Val Val Ile Val Gly Arg GTT GlT C:TG TCT GGT AAA CCG BCC Aq~ ATC CCG GAC CGT GAA Gl~ CTG 17 52 ~al Val Leu Sex Gly Lys Pro Ala Ile Ile Pro Asp Ar~ Glu Val L~u , 530 535 540 TAC CGT GAG TTC GAC GAA ATG GAA GAA 5Y~C TCT CAG CAC CTG CCG TAC 18 0 0Tyr Arg Blu Phe Asp Glu Met Glu Glu Cy~ Ser Gln His Leu Pro Tyr ATC GAA C~G GGT ATG ATG CTG BCT GAA CAG TTC AAA CAG AAA GCT CT& 1848 Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu 560 56~ 570 Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala Glu Val Ile Ala Pro Ala GTT CAG ACC AAC TGG CAG AAA CTC GAG ACC TTC TGG GCT AAA CAC ATG 19 4 4Val Gln Thr Asn Trp Gln Lys Leu Glu Thr Phe Trp Ala Lys His Met TGG AAC TTC ATC TCT GGT ATC CAG TAC CTG GCT GGT CTG TCT ACC CTG l 9 9 2 Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu PCTIUS 92/0718~
12b 1~0/l~S 23 DEC 1992 Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala Val m r Ser Pro Leu Thr Thr Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu Gly Lys Val Leu Ile Asp Ile Leu Ala Gly Tyr Gly Ala Gly V~l Ala Gly GCT CTG GTT GCT TTC AAA ATC ATG TCT GGT GAA GTT CCG TCT ACC GAA 2 2 80Ala Leu V~l Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp L u Val Asn Leu Leu Pro Ala Ile L~u Ser Pro Gly Ala Leu 'Jal ~TT G~T GTT GTT TGC GCT GCT ATC CTG CGT CGT CAC GTT GGC CCG GGT 2 3 7 6 Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly 735 740 7~5 GAA G~T GCT GTT CAG TGG ATG AAC CGT CTG ATC GCT TTC GCT TCT CGT 2424 Glu Gly Ala V~l Gln Trp Met A~n Ary Leu Ile Ala Phe Ala Ser Arg 750 755 76~ 765 Gly A~n ~is Val S~r Pro Trp A~p Pro Leu Asp Cys Arg Hi~ Ala Lys TAAGTAGATC lTGAtCBCGT TCGCGCTGAA ATGCGCTAAT TTCACTTCAC GACA~`TTCAG 2532 CCAATTTT~;;G GAGGAGTGTC GTACCGTTAC GAT m CCTC AAT m TCTT TTCAACAATT 2 5 9 2 GATCTCATTC AGGTGACATC T m ATATTG GCGCTCATTA TGAAAGCAGT AGCTTTTATG 2652 AGGGTAATCT GAATGGAACA GCTGCGTGCC GAATTAAGCC A m ACTGGG CGAAAAACTC 2712 CAGGGAAACC CAATGCCGTT AATGGCAAGA AGCTTAGCCC GCCTAAT~AG CGGGCTTTTT 2832 ` - , PCTIUS 92/0718~.
i 1 v 8 ~b K0/ IJ S 2 3 DEC l992 ss CGGCGA m A TGCCGCCTCG GCGAGCACAT GGAACGGGTT GGCATGGATT GTAGGCGCCG 3072 GTCTGCGACC TGAGCAACAA CATGAATGGT CTTCGGTTTC CGTG m CGT AAAGTCTGGA 3492 GCTACCCTGT GGA.~CACCTA CATCTGTATT AACGAAGCGC TTCTTCCGCT TCCTCGCTCA 3612 AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTT m CCAT AGGCTCCGCC 3792 TATAAAGATA CCAGGCG m CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC 3912 TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG C m CTCAAT 3972 CGAGGTATGT AGGCGGTGCT ACAGAGTTCT T~AAGTGGTG GCCTAACTAC GGCTACACTA g212 AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATC m TCTACGGGGT 4392 CTGACGCTCA GTGGAACGA~ AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA 4g52 GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAG m TAAATCAATC TAAAGTATAT 4512 ,~

PC~IJS 92/07188

6 ~O/US 23 0~C 1992 GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CA~TGATACC GCGAGACCCA CGCTCACCGG 4692 CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG ~752 CCCCCATGTT ~-TGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA 4992 GGATCTTACC GCTGTIG~GA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT 5292 ATTATTGAAG CA m ATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT 5~72 AGAAAAATAA ACAAAT~GGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT 5532 (2) INFORMATION FOR SEQ ID NO:9:
(~) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~81 amino acids ~) TYPE: amino acid (D) TOPOLOGY: linear ~ii) MOLECVLE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Ser Phe VA1 Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu 1 5 10 lS
Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala ` - , PCTIUS 92/0718 8 iJ~b BOJIS ~3 OEC 1992 Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu Val Val Glu Lys Cys Ala Phe Ser Asp Acp Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Lèu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile Pro TSp A~p Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arq Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met 195 200 ~05 Leu Glu Gln Leu Arg V~l Leu Trp Tyr Gly Glu Lys Ile Hi~ Val Ala Val Ala Gln Glu Val Pro Gly Ihr Gly Val Asp Thr Pro Glu Asp Leu Asp Pro S~r Thr Asn Ser Met Ala Val A~p Phe Ile Pro Val Glu Asn Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Val Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr 275 ~80 285 Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile A.~p Pro Asn Ile Arg Thr 325 330 ~335 Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr 3gO 345 350 Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile 355 360 36~
Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val 385 390 395 ~00 al Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro ~05 410 ~15 n Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr 420 425 g30 Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Arg His Leu Ile 435 ~gO 445 Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Al~ Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser al Ile Pro mr Ser Gly A~p Val Val Val Val Ala Thr Asp Ala Leu 485 g90 495 Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr ' 500 505 510 Cys Asn Ser Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu ~15 520 525 Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp alu Met Glu Glu Cy8 Ser Gln Hi~ Leu Pro Tyr Ile Glu Gln ly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu ln Thr Ala Ser Arg Gln Ala Glu Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Lys Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn ;~,113!J~ O/l~S 23 DEC t Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro e~ Thr Thr Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val 6~5 650 655 la Ala Gln Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu Gly Lys Val Leu lle A~p Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu VA1 Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu A~p Leu Val 705 710 ~15 720 sn 'eu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val al Cys Al~ Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Trp Asp Pro Leu Asp Cys Arg His Ala Lys (~) INFOKMATION FOR SEQ ID NO:10:
(i~ SEQU~NCE CHARACTFRISTICS:
(A) LENGTH: 1548 base pair~
(B) TYPE: nucleic acid (C) STR~NDEDNESS: ~ingle (D~ TOPOLOGY: circular (ii) MO~CULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1548 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
ATG AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CG~ CTG 4 8 Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His ~ i l J ~ ~ ii BO/ ~ S 2 3 OEC 1~2 Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 35 40 g5 Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Mot Thr Arg Ala Asp His Gln Ser Gly Thr Glu Ar~ Leu Ala Glu Val Val Glu Lys Cys Ala Phe Ser Asp A~p Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val GCT GAT AA~' CTC GCT CAG CGT CAG GTG GGT ATG GCG ACT CTG GCG GTG 384 A1A A~p A~l Leu Ala Gln Arq Gln Val Gly Met Ala Thr Leu Ala Val llS 120 1~5 Pro Il- Ni~ Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val V~l L~u Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile CCT Tr~G ~AT CGT GAT.CGT m GCA GAA GGC CTT GAA ACC GTT GGC GAT 528 Pro Txp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC m ATC 576 Asn Phe L-u Arg Hia Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile '. 180 185 190 , . .
CGT ~GT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624 Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met 195 200 ~05 Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile Hiq Val Ala GTT GCT CAG GAA GTT CCT GGC.ACA GGT GTG GAT ACC CCT GAA GAT CTC 720 Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser Pro Trp Thr His Tyr Val Pro Glu Ser Asp , ~ .

` - PCTIUS 92/071 8 8 .
~ l L J ~ ''li ~ 0 / ~ S 23DECl~

Ala Ala Ala Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu VA1 Leu Ser A~p Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln ~eu 305 310 ~15 320 Pro Gly Ile Pro Phe V~l Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp CGT GTT GAC GGT ATC ATG CAC ACC CGT TGC CA TGC GGT GCT GAA ATC ~ 1056 ~rg V~l Asp Gly Ile Met His Thr Arg Cy8 Hig Cy~ Gly Ala Glu Ile Thr Gly His Val Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr GGT CCG TGC ACC CCG CTG CCG GCT CCG AAC TAC ACC TTC GCT CTG TGG . 1200 ~ Gly Pro Cy~ Thr Pro Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp 3~ 385 390 395 ~00 CGT GTT 5C~ GCT GAA GAA ~AC GTT GAA ATC CGT CAG GTT GGT GAC TTC 12g8 Arg V~l ~ ff Ala Glu Glu Tyr Val C-lu Ile Arg Gln V~1 Gly Asp Phe ~- qO5 410 415 CAC TAC ~TT ACC GGT ATG ACC ACC GAC AAC CTG AAA TGC CCG TGC CAG 1296 His Tyr Val Thr Cly Met Thr Thr Asp Asn Leu Lys Cy~ Pro Cya Gln GTT ~CG TCT CCG GAG TTC TTC ACC GAA CTG GAC GGT GTT CGT CTG CAC 1344 Va1 PrO Ser Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe KO/US 2 3 ~EC 1992 Arg Val Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro Cy9 Glu Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser g85 490 495 His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu Asp Pro Leu Asp Cys Arg His Ala Lys (2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 516 amino acids (B) TYPE: amino acid lD) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N~:ll:
Met Ser Phe Val Val Ile Il~ Pro Ala Arg Tyr Ala Ser Thr Arg Leu ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val Hi~

V~l Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Asp VA1 Ala Arg Ala Yal Glu Ala Ala Gly Gly Glu ~ .
Val Cys ~ ~hr Arg Al~ Asp His 21n Ser Gly Thr Glu Arg Leu Ala 65 ~ 70 75 ~ 80 Glu Val Val Glu Ly~ Cys Ala Ph~ Ser A~p Asp Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile &TIIJS 92fO7188 ,~ S 2 3 OEC 1992 lqS 150 155 160 ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile rg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu sp Pro Ser Thr Asn Ser Pro Trp Thr His Tyr Val Pro Glu Ser Asp l~ Ala Al~ Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln eu Leu Arg Arg ~-eu Hi~ Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro ;Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val 2~0 295 300 Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala T-yc Leu Met Pro Gln Leu ro Gly Ile Pro Phe Val S~r Cy_ Gln Arg Gly Tyr Lys Gly Val Trp 3~5 330 335 r~ Val A~p Gly Ile ~e~ His Thr Arg Cyq His Cyq Gly Ala Glu Ile hr Gly ~ ~ ~1 Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr 3Ss 360 3~5 Cys Arg As~ Met Trp Ser Gly Thr Phe PrG Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp 385 390 395 gO0 rg Val Ser Ala Glu Glu Iyr Val Glu Ile Arg Gln Val Gly Asp Phe 405 410 415 .
is Tyr Val Thr Gly Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln A1 Pro Ser Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His g35 g40 445 JJ ~l3 l~O/lJS 23 DEC l992 Arg Phe Al~ Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe g50 455 460 Arg Val Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro cys Glu 465 g70 475 480 Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr A~p Pro Ser g85 490 495 His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu Asp Pro Leu Asp Cys Arg His Ala Lys (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1623 b~se pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
- (B) LOCATION: 1.... 1623 (xi) SEQUENCE DESC~IPTION: SEQ ID NO:12:
ATG A~T TTT GIG GTC ATT ATT ecc GCG CGC TAC GCG TCG ACG CGT CTG 48 Mst Ser Ph~ Val Val Ile Ile Pro Ala Arg Tyr Al~ Ser Thr Arg Leu CCC CGT Ah~ CCA T~G GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 96 Pro Gly Ly~ Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu ~rg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Aqp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu V~l Cys Met Thr Arg Ala Asp Hiq Gln Ser Gly Thr Glu Arg Leu Al~

GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC ACG GTG ATC GT~ AAT 288 PCT/U~ ~2/0718 8 26 R0/US 23 D~ 1992 Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val A~n Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile Pro Trp A~p Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp 165 170 . 175 A n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile CGT CGT TAC GTC AAC TGG CAG CCA A~T CCG TTA GAA CAC ATC GAA ATG 624 Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Ar~ Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu V~l Pro Gly Thr Gly Val Aqp Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser Met Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val `~` i~i PC~lUS 92/07 1 8 8 R0/IJS 23 DEC l9g.

Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Asp Pro Leu Val GCT GAA GAA GAC GAA CGT GAG ATC TCT GTT CCG GCT ~-AA ATC CTG CGT 1008 Ala Glu Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser Arg Arg Phe Ala Gln Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Trp Lys Lys Pro A~p Tyr Glu Pro CCG GTT GTT CAC GGT TGC CCG CTG CCG CCG ~CG AAA TCT CCG CCG GTT 1152 Pro Val Val His Gly Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu TC~ ACC GCT CTG 5CT GAA CTG GCT ACC CGT TCT TTC GGT TCT TCT TCT 1248 Ser Thr Ala Leu Ala Glu Leu A1A Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly Ile Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala 42~ 425 g30 CCG TCT GGT TGC CCG CCG GAC TCT GAC GCT GAA ICT TAC TCT ~CT ATG 13g4 Pro Ser Gly Cys Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro A8p Leu Ser Asp Gly Ser 450 455 ~60 Trp Ser Thr Val Ser Ser Glu Ala Asn Ala Glu Asp Val Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala GAA GAA CAG AAA CTG CCG ATC AAC GCT CTG TCT AAC TCT CTG CTG. CGT 1536 Glu Glu Gln Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Leu Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln Arg O/ljS 2 3 DEC 1992 Gln Ly~ Lys Val Thr Phe A~p Arg Leu Gln Val Leu Asp (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 541 amino acids (B~ TYPE: amino acid (D) TOPOLOGY: linear (,i) MOLECULE TYPE: pro~ein (xi) SEQUENCE DESCRIPTION:. SEQ ID NO:13:
et Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu ro Gly Lys Pro Lau ~al Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Ar~ Ala Arg Glu Ssr Gly Ala Glu Arg Ile Ile Val Ala mr Asp Hi3 Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cy~ Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala lu Val Val Glu Lys Cys Al~ Phe Ser Asp Asp Thr Val Ile Val A~n ~l Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp AJn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val 115 120 1~5 Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu A~p Ala Glu Gly Tyr-~la Leu Tyr Phe Ser Arg Ala Thr Ile ro Trp A~p Arg A~p Arg Phe Ala Glu Gly Leu Glu Thr Val Gly. Asp sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile rg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met PC~IUS 92/07188 2~ 120/US 23 OEC ~992 l9S 200 205 Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu L~s Ile His Val Ala 210 2~5 220 Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu sp Pro Ser Thr Asn Ser Met Arg Arg Leu Ala Arg Gly Ser Pro Pro er Val Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn Hiq Asp Ser Pro A~p Ala Glu Leu Ile Glu Al s A~n Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asns Lys Val Val Ile Leu Asp Ser Phe A~p Pro Leu Val las Glu Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg ys Ser Arg Arg Phe Alas Gl~s Ala Leu Pro Val Trp Al~ Arg Pro Asp Tyr A~s Pro Pro Leu Val Glu Thr Trp Ly~ Lys Pro Asp Tyr Glu Pro Pro Val Val Hi~ Gly Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro V~l 370 , 375 3ao Pro Pro Pro Asrg Ly3 Ly~ Arg Thr Val Vasl Leu Thsr Glu Ser Thr Leu er I~Sr Ala Leu Ala Glu Leu Ala Thr Arg Ser Phe Gly Ser Ser Ser hr Ser Gly Ile Thr Gly Asp Asn Thr l~.Sr Thr Ser Ser Glu Pro Ala Pro Ser Gly Cys Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser Met 435 440 4gS
Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser g50 455 460 Trp Ser Thr Val Ser Ser Glu Ala Asn Ala Glu Asp Val Val Cys Cys 465 470 475 ~80 Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala g85 g90 ~9~

"~.TlUS ~ Z l O 7 1 8 8 i ,j ~ v/ ~ S 2 3 OEC 1992 lu Glu Gln Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Leu Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Yal Leu Asp (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1488 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~-(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1488 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ATG AGT TTT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG ~8 Met S~r Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu CCC GGT AAA CCA T~G GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 96 Pro Gly Lys Pro Leu Val Acp Ile Asn Gly Ly~ Pro Met Ile Val His GTT CTT GAA CGC GCG CGT GAA TCA GGT GCC GAG CGC ATC ATC GT~ GCA 14~ -Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 35 40 ~5 Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp ~is Gln Ser Gly Thr Glu Arg Leu Ala 65 ~0 75 80 Glu Val Val Glu Lys Cys Ala-Phe Ser Asp Asp Thr Val Ile Val Asn VA1 Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val V~l Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp 165 . 170 175 AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 5?6 Asn Phe Lou Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met TTA GAG CAG CTT CGT GTT CTG TGG TAC G -C GAA AAA ATC CAT GTT GCT . 672 Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Ly~ Ile His Val Ala 21~ 215 220 YA l Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu 225 230 23~ 240 GAC CCG TCG ACG AAT -TCT CTA GAC TCC CAC TAC CAG GAC Gl~ CTG AAA 768 Asp Pro Ser Thr Asn Ser Leu Asp Ser His Tyr Gln Asp Val Leu Lys -~ 245 250 255 GAA GTT AAA ~CT GCT GCT TCT AbA GTT AAA GCT AAC CTG CTG TCT GTT 816 Glu Val Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val .
GAA GAA GCA TGC TCT CT& ACC CCG CCG CAC TCT GCT AAA TCT AAA TTC 864 Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Ly~ Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Thr His Ile A~n Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr Pro ATC GAC ACC ACC ATC ATG GCT AAA AAC GAA GTT TTC TGC GTT CAG CCG ~008 Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro .
' PCTIUs 92/07188 w~ 2~i ~0/lJS 23 OC l992 Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile V~l Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Lys Ser Lys Lys Thr 385 390 ~95 400 Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr g05 . 410 415 Glu Ser A~p Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp Leu GAC CCG CAG GCT CGT GTT GCT ATC AAA TCT CTG A~C GAA CGT CTG TAC 134g A~p Pro Gln Ala Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr 435 440 ~5 GTT GGT GGT CCG CTG ACC AAC TCT CGG GGT G~A A~C TGC GGT TAC CGT 1392 Val Gly Gly Pro Leu ThF Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg ~ Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cy~ Gly Asn Thr Leu F 465 470 ~75 480 ACC IGC ~AC ATC AAA GCT CGT GCT GCT TGC CGT GCT GCT GGT CTG CAG 1488 Thr Cy8 Tyr Ile Lys Ala Arg Ala Ala Cy Arg Ala Ala Gly Leu Gln 485 g90 ~95 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS: -(A) LENGTH: 496 amino acids (B~ TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

O/ ~ S 2 3 OEC Ig92 Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His al Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val A3n ~5 90 95 al Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala A p Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro A~n Ala Val Lys Val 130 135 lgO
Val Leu Asp Ala Glu Gly Iyr Ala Leu ~yr Phe Ser Arg Ala Thr Ile 145 i50 155 160 ro TSp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp 165 17~ 175 sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile 180 185 1~0 Arg Arg ~yr V~l Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu 225 230 235 2~0 sp Pro Ser Thr Asn Ser Leu Asp Ser His Tyr Gln Asp Val Leu Lys lu Val Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Thr ~`2~ ~O/US 23 DEC 1992 His Ile Asn Ser Val Trp Lys Asp L~u Leu Glu Asp Asn Val Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro 325 3~0 335 Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr Ly~

Leu Pro Leu Ala Vai Met Gly Ser Ser l~r Gly Phe Gln ryr Ser Pro '-Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Ly~ Ser Ly~ Lys Thr Pro Met Gly Phe Ser Tyr A~p Thr Arg Cys Phe Asp Ser Thr Val Thr 405 410 gl5 Glu Ser Asp Ile Arg ~r Glu Glu Ala Ile l~r Glsl Cys Cys Asp Leu ~20 425 430 Asp Pro Gln Al~ Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu ryr 435 440 4g~
Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg 450 ~55 460 Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu 465 ~ 470 475 480 Thr Cys Tyr Ile Lys Ala Ars~ Ala Ala Cys Arg Ala Ala Gly Lou Gln (2) INFO~5ATION FOR SEQ ID NO: 16:
( i ) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 1161 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular ~ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
( A ~ NAME/KEY: CDS
(B) LOCATION: 1..1161 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

;'.L.l~ 6 B0/1~S 23 DEC ~992 ATG AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 4 8 Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu CCC GGT AAA CCA rrG GTT GAT ATT AAC GGC AAA CCC ATG ATT GTT CAT 9 6 Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Alzl Glu Arg Ile Ile Val Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala GAA GTT GTC GAA AAA TGC GCA TTC AGC GAC GAC AC~: GTG ATC GTT AAT 2 8 8 Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val ~le V~l A~n GTG CAG GGT GAT GAA CCG Aq G ATC CCT GCG ACA ATC ATT CGT CAG GTT 3 3 6 Val Gln Gly Asp Glu Pro Met Ile Pro Al~ Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val CCA ATC CAC AAT GCG GAA GAA GCG m AAC CCG AAT GCG GTG AAA Gl~ 4 3 2 Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro A~;n Al~ Val Ly~ Val GTT CTC GAC GCT GAA GGG TAT GrA CTG TAC TTC TCT CGC GCC ACC ATT 480 Val Leu A~p Ala Glu Gly Tyr Ala Leu Iyr Phe Ser Arg Ala Thr Ile CCT TGG GAT CGT GAT CGT m GCA GAA GGC crr GAA ACC GTT GGC GAT 5 2 8 Pro Trp Asp Arg A~p Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met TTA GAG CAG CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTr GCT 6 7 2 Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala PCTIUS 9 2/ 07 1 8 a 26 ~O/US 23 DEC 1992

7 9 Val Al~ Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu A~p Leu Asp Pro Ser Thr Asn Cys Met Leu Gln Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Val Gln Glu Asp Al~ Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala 275 2a0 ~ 285 Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr TCT ~GC TCT TCT AAC GTT TCT GTT GCT CAC GAC GGT GCT GGT AAA CGT 960 Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys Arg V~l Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Al~ Arg His Thr Pro V~l Asn Ser Trp Leu Gly Asn Ile - Ile Met Phe Al~ Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His J 355 36~ 365 Phe Phe S-r Val Leu Ile Ala Arg Asp Gln Leu Glu Gln Ala Leu Asp Cys Glu Ile ~2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 387 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein J~ O~S 2 3 DE~ 1992 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Ser Phe Val Val Ile Ile Pro Ala Ar~ Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser ~ly Thr Glu Arg Leu Ala Glu V~l Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val A~n . 90 95 Val Gln Gly Asp Glu Pro ~et Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ilo His A~n Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val ~ 130 135 140 Val Leu Asp Ala Glu Gly Tyr Ala Lou Tyr Phe Ser Arg Ala Thr Ile Pro Trp Asp Arg A~p Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile - ~0 185 190 Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Me~

Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala V~l Al~ Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu A~p Pro Ser Thr A~n Cys Met Leu Gln Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Val Gln Glu Asp P~ U~ ~ ~ / U 7 1 8 8 2G l~O/~S 23 DEC ~992 Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Aln Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr 290 2~5 300 Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys Ars~

al Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala rp Glu Thr Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro Thr Leu Trp Ala Axg Met Ile Leu Met Thr Hi~

Phe Phe Ser Val Leu Ile Ala Arg Asp Gln Leu Glu Gln Ala Leu Asp Cys Glu Ile (2~ INFORMI~TION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1179 base pairs (~) TYPE: nucleic acid ( C ~ STR~NDEDNESS: g ingle (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA ~genomic) (ix) FEATURE:
~A) NAME/~ CDS
( El ) LOCATION: 1. .1179 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
ATG AGT m GTG GTC Al~ Al~ CCC GCG CGC TAC GCG TCG ACG CGT CTG 48 Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu CCC GGT AAA CCA TTG GTT GAT Al~ A~C GGC AAA CCC ATG ATT GTT CAT 96 Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His GTT CTT GAA CGC GCG CGT GAA . TCA GGT GCC GAG CGC ATC ATC GTG GCA 1 ~ 4 Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 35 40 . ~5 i PCT/US 92/071a8 b ~O/~i 23 OEC 1992.

Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu So 55 60 Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala GAA GTT GTC GAA AAA T~C GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 288 Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val P,ro Il- Hi~ Asn Al~ Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val L-u Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile Pro ~rp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp AAC TTC CTG CGT CAT CTT GGT ~TT TAT GGC TAC CGT GCA GGC m ATC 576 Asn Pho Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arq Arg Tyr V 1 Asn TSp Gln Pro Ser Pro Leu Glu His Ile Glu Met TTA GAG C~C CTT CGT GTT CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 672 Leu Glu Gln L~u Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala 2I0 215 ~ 220 Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser Met Glu Ile Tyr Gly Ala Cys ~yr Ser Ile Glu Pro Leu Asp Leu Pro Pro Ile Ile Gln Arg Leu His GIy Leu Ser .

;lia~ O/US ~3 DE~ 1992 GCT TTC TCT CTG CAC TCT TAC TCC CCG GGT GAA ATC AAC CGT GTT GCT 86i Ala Phe S-r Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala GCT TBC CTG CGT AAA CTG.GGT GTT CCG CCG CTG CGT GCT TGG CGT CAC 912 Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg Ala Arg Leu Leu Ala Arg Gly Gly Arg Ala GC~ ATC T&C GGT AAA TAC CTG TTC AAC TGG GCT GTT CGT ACC AAA CTG 1008 Ala Ile Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Arg m r Lys Leu Lys Leu m r Pro Ile Ala Ala Ala Gly Gln Leu Asp Leu Ser Gly Trp TTC ACC GC. GGT TAC TCT GGT GGT GAC ATC TAC CAC TCT GTT TCT CAC 1104 Ph Thr Al~ Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val Ser His GCT CGT-CC6 OGT TGG ATC TGG TTC TGC CTG CTG CTG CTG GCT GCT GGT ' 1152 Al~ Arg Pro Arg Trp Ile Trp Phe Cys Leu Leu Leu Leu Ala Ala Gly GTT CGT AT~ TAC CTG CTG CCG AAC CGT 117g Val Gly Ile Tyr Leu Leu Pro Asn Arg :
l2) INFO~MATION FOR SEQ ID NO:19:
.
(i) SEQUENCE CHAR~CTERISTICS:
~A) LENGTH: 3g3 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Met Ser Phe Val Vai Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val- His - Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Aqp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu .. . _ . ... .. . . . . .. ..

: l~J~2~ V/~- 23 DEC 1992 Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val la Asp A~n Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val 115 120 ~25 Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys V~l Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile 1~0 185 190 rg Arg Tyr Val A~n Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val A1A Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu 225 .230 235 240 sp Pro Ser Thr Asn Ser Met Glu Ile Tyr Gly Ala Cy8 Tyr Ser Ile lu Pro L~u A~p L~u Pro Pro Ile Ile Gln Arg Leu Hi3 Gly Leu Ser ~60 265 270 Ala Phe Ssr Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg Ala Arg Leu Leu Ala Arg Gly Gly Arg Ala la Ile Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr Lys Leu ys Leu Thr Pro Ile Ala Ala Ala Gly Gln Leu Asp Leu Ser Gly Trp _ .L ~ 0/ U S 2 3 o~c 1992 Phe Thr Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser V~l Ser Hi~

Ala Arg Pro Arg Trp Ile Trp Phe Cys Leu Leu Leu Leu Ala Ala Gly V~l Gly Ile Tyr Leu Leu Pro Asn Arg (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1791 b~se pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
~A3 N~ME~KEY: CDS
(B) LOCATION: 1..1791 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
ATG AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 48 Met Ser Ph~ Val Val Ile Ile Pro Ala Arg Tyr Al~ Ser Thr Arg Leu 5 10 1~
CCC GG~ AAA CCA TTG GTT GAT A~T AAC GGC AAA CCC ATG ATT GTT C~T 96 Pro Gly Lys Pro L~u Val A~p Ile A3n Gly Ly~ Pxo M~t Ile Val His GTT CTT GA~ CGC ~CG rGT GAA TCA GGT GCC GAG C~C ATC ATC GTG GCA 144 Val Leu Glu Arg Ala Arg G1U Ser Gly Ala Glu Arg Ile Ile Val Ala ACC GAT CAT GAG G~T GTT GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA 192 Thr Asp His Glu Asp Val Ala Arg Al~ Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu Val Val Glu Ly-~ Cys Ala Phe Ser Asp A~p Thr Val Ile Val Asn Val Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val w~32~j ~O/~S 23 DEC l992 Al~ Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 . 135 140 GTT CTC GAC GCT GAA GnG TAT GCA CTG TAC TTC TCT CGC GCC ACC ATT 480 Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile 1~5 150 ~55 160 Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp AAC T~C CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC TTT ATC 576 A n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile CGT CGT TAC GTC AAC TGG CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 62g Arg Arg ~yr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile ~-lu Met Leu Glu Gln ~eu Arg Val Leu T~ qyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu GAC CCG TCG ~CG AAT TCC ATG GAC GCT CAC TTC CTG TCT CAG GCG CCG 768 Asp Pro S~r Thr Asn Ser Met Asp Ala Hi~ Phe Leu Ser Gln Ala Pro CCG CCG TCT TGG GAT CAG ~TG TGG AAA TGC CTG ATC CGT CTG AAA CCG 816 Pro Pro S~r Trp A~p Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro ACC CTG CAC GGC CCG ACC CCG CTG CTG TAC CGT CTG GGT GCT GTT CAG 86g Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Ile Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys 2~0 295 300 Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly GGT GTT CrG GCT GCT CTG GCT GCT TAC TGC CTG TCG ACC GGT TGC GTT 1008 Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val PCrlUS 92/0718 8 0/US 2 3 DE~ 1992

8 7 Val Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro 340 3g5 350 Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gln Hi~ Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe Ly~ Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala Glu 385 390 . 395 400 GTT ATC GCT CCG GCT GTT CAG ACC AAC TGG CAG AAA CTC GAG ACC TTC 12~R
Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Lys Leu Glu Thr Phe TGG GCT AAA CAC ATG TGG AAC Tl~- ATC TCT GGT ATC C~G TAC CTG GCT 1296 Trp Ala Ly~ His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala 420 425 g30 GGT CTG TCT ACC CTG CCG GGT AAC CCG GCT ATC ~CA AGC TTG ATG GCT 1344 Gly Leu Ser Thr Leu Pro Gly A~n Pro Ala Ile Ala Ser Leu Met Ala Phe Thr A1~ Ala Val Thr Ser Pro Leu Ihr Thr Ser Gln ~hr Leu Leu 450 g55 g60 Phe Asn Ile Leu Gly 51y Trp Val Ala Ala Gln Leu Ala Al~ Pro Gly 465 470 47~ 480 Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly 485 490 g95 Ser Val Gly Leu Gly Lys Val Leu Ile Asp Ile Leu Ala Gly Tyr Gly Ala ~ly Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser PCTIUS 92/07188 .
32~i BO/~S 23 ~EC t992 Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg CAC GTT GGC CCG GGT GAA GGT GCT GT~ CAG TGG AIG AAC CGT CTG ATC 1728 His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Trp Asp Pro Leu Asp Cys Arg His Ala Lys (2) INFORMATION FOR SEQ ID NO:21-(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 597 amino acids (B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His ~5 30 Val Leu Glu Arg Ala Arg ~lu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr A3p Hi~ Glu A~p Val Ala Arg Ala ~al Glu Ala Ala Gly Gly Glu Val Cy~ M t Thr Arg Ala Asp His Gln Ser Gly Thr Glu Ar~ Leu Ala ` 70 75 ~0 Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg ~ln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val ,, aJ~i PCTAJS 92/071~ 8 8,0/~S 23DECt9~2 Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile lgS 150 155 160 ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile 180. 185 190 rg Arg ~yr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly V~l Asp Thr Pro Glu Asp Leu 225 230 . 235 240 sp Pro Ser Thr Asn Ser Met Asp Ala His Phe Leu Ser Gln Al~ Pro 2~5 250 255 ro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Il-e Arg Leu Ly~ Pro hr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Il~ Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu V~l Val Thr 5er Thr Trp Val Leu Val Gly 305 310 315 3~0 ly Val Leu Al~ Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val al Ile V~l Gly Arg V~l Val Leu Ser Gly Lys Pro Ala Ile Ile Pro sp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe 3~0 315 380 Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala Glu al Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Lys Leu Glu Thr Phe rp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala 420 g25 430 ly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala ;i ij 1~ 0/ U S 2 3 0~c 199;!

Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Ser Gln Thr Leu Leu 450 gS5 460 Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala Pro Gly 465 470 a,75 480 *
la Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly ~85 g90 495 er Val Gly Leu Gly Lys Val Leu Ile A~p Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lyc~ Ile Met Ser Gly Glu Val Pro Ser Thr Glu A~p Leu Val A~n Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cy~ Ala Ala Ile Leu Arg Arg i~ Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile la Phe Ala Ser Arg Gly Asn His Val Ser Pro Trp Asp Pro Leu A~p C~rs Arg His Ala Lys 2~ INFORMATION FOR SEQ ID NO-22:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENG~: 1797 ba~e pairs (B) TYPE: nucleic acid ( C ) S'rRANDEDNESS: s inS~le (D~ TOPS)LC)GY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix~ FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.. 1797 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
A~G AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 48 Met Ser Phe Val Val Ile Ile Pro Ala Arg ~r Ala Ser Thr Arg Leu r~ 0/ 1~ S 2 3 DEC 1992 ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His V~l Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 35 40 g5 Thr Asp Hi3 Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu Val Val Glu Lys Cys Ala Phe Ser Asp *9p Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val 100 105 ' 110 GCT GAT AAC CTC GCT CAG CGT CAG GTG G~T ATG GCG ACT CTG GCG GTG 384 Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG g32 Pro Ile His Asn Ala Glu Glu Ala Phe A~n Pro Asn Ala Val Lys Val - 130 135 lgO
GTT CTC GAC &CT GAA G G TAT GCA CT~ TAC TTC TCT CGC GCC ACC ATT 480 ~al Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile 145 15g 155 160 CCT TGG GAT CGT GAT CG. TTT GCA GAA GGC CTT GAA ACC GTT GGC GAT 528 Pro ~rp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp Asn Phe Lou Arg His Leu Gly Ile Tyr Gly ~yr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu ~`, . P~CTIUS 9 2 1 0 7 1 8 8 U/~TS 23 0EC ~992 Asp Pro Ser Thr Asn Ser Met Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Ile Thr Leu Thr His Pro Val Thr Ly~ Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cy~ Val Val Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala Ile 340 3~5 350 - Ile Pro A9p Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gln Hi~ Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu 370 375 3ao CAG TTC AAA CAG AAA GCT CTG GGT CTG CTG CAG ACC GCT ~CT CGT CAG 1200 Gln Phe Lys Gln Lys Ala Leu Gly Leu ~eu Gln Thr Ala Ser Arg Gln Ala Glu VA1 Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Ly~ Leu Glu gO5 glO 415 Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu 435 440 gg5 ATG ~-CT TTC ACC GCT GCT GTT ACC TCT CCG CTG ACC ACC TCT CAG ACC 1392 Met Ala Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Ser Gln Thr ~ g50 455 460 "~

i J ~ / ~ S 23 DEC 1~

CTG CTG TTC AAC A~T CTG GGT GGT TGG GTT GCT GCT CA~ CTG GCT GCT1440 Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala 465 470 g75 480 Pro Gly Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala 485 490 g95 ATC GGT T~T GTA GGC CTG GGT AAA GTT CTG ATC GAC ATT CTG GCT GGT1536 Ile Gly Ser Val Gly Leu Gly Lys Val Leu Ile Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser GG~ GAA GTT CCG TCT ACC GAA GAT CTG GTT AAC CTG CTG CCG GCT ATC1632 Gly Glu Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu V~l Val Gly Val Val Cys Ala Ala Ile Leu CGT CGT CAC GTT GGC CCG GGT GAA GGT GCT GTT CAG TGG ATG AAC CGT. 1728 Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg - C$G ATC GCT TTC GCT TCT CGT GGT AAC CAC GTT TCT CCA TGG GAT CCT 1776 Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val 5er Pro Trp Asp Pro ~eu A~p Cys Arg His Ala Lys ~ 595 J
(2) INFO~MATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 599 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His ' ~

'J~6 E~O~-S 23 DEC 199

9 4 al Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 4~
Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser ¢ly Thr Glu Arg Leu Ala Glu Val Val Glu Lys Cys Ala Phe Ser A p Asp Thr Val Ile Val Asn ~5 90 95 Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln V~l Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Mht Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 135 lgO
Val Leu A~p Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Acp As~ Phe Leu Ar~ His Leu Gly Ile ~yr Gly Tyr Arg Ala Gly Phe Ile - ~80 185 lgO
Arg Arg Tyr Val Asn Trp.Gln Pro Ser Pro Leu Glu Hi Ile Glu ~et Lsu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile ~i9 Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu A~p Leu Asp Pro Sar Thr A~n Ser Met Asp Ala His Phe Leu Ser Gln Thr Ly~

Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val ys Ala Arg Ala Gln Ala Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Ile Thr Leu -Thr H1s Pro Val Thr Lys Tyr Ile Met Thr Cy5 Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu ` - `. PCTIUS 't 2 / 0 7 1 8 8 b R0/~S 23 DEC 1992 Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cy~ Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala Glu Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Lya Leu Glu 405 . 410 ~ 415 Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Ser Gln Thr 450 455 ~60 Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala 465 470 475 ~80 Pro Gly Ala Ala Thr Al~ Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu Gly Lya Val Leu Ile Asp Ile Leu Ala Gly Tyr Gly Al~ Gly V~l Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu V~l Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Al~ Ser Arg Gly Asn His Val Ser Pro Trp Asp Pro Leu Asp Cys Arg His Ala Lys - - PCTIUS 92/07188 .
2~ R0/lJS 23 DEC 1992 ~2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1251 ba~e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1251 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly3 Pro Met Ile Val His 20 ~5 30 Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu V~l Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala GAA GTT GTC GAA AAA TCC GCA TTC AGC GAC GAC ACG GTG ~TC GTT AAT 288 Glu Val V~l Glu Ly3 Cy8 Ala Phe Ser Asp Asp Thr Val Ile Val Asn G~G CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 336 Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 135 . 140 Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile ` - PCT/US 92/0718 8 L;J~ 2~ ~0/~S 23 DEC l992 1~5 150 155 160 Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp A~n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile CGT CGT TAC GTC AAC T~G CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624 Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Al~ Gln Glu Val Pro Gly m r Gly V~l Asp m r Pro Glu Asp Leu 225 230 235 . 240 A~p Pro Ser Thr Arg Ile Arg Ala Arg Tyr Pro Glu Thr Ile Thr Leu 2~5 250 255 Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg m r Gly Arg GGG AAG CCA GGC ATC TAC AGA TTT GT~ GCA CCG GGG GAG CGC CCT TCC 864 Gly Lys Pro Gly Ile Tyr Arg Phe Val Al~ Pro Gly Glu Arg Pro Ser 2~5 280 285 Gly ~et Phe Asp Ser Ser Val Leu Cy~ Glu Cys Tyr Asp Ala Gly Trp Pro Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala 305 310 ~ 315 320 Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu : Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr ~: 355 360 365 2r, ~O/US 23 DEC ~g92 Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Gly Gly Ser Ser Arg Leu Gln Ala g05 410 415 ~GC 1251 Cys (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 417 amino acids (B) TYPE: amino acid (D) TOPOL~GY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SE~ ID NO:25:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His ~al Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala r Asp Hi~ Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cy~ Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr.Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pxo Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile r~ l l 3 ~ b l~ O / U S 2 3 DEC ~

ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile rg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met lg5 200 205 Leu Glu Gln Leu Arg Vai Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu sp Pro Ser Thr Arg Ile Arg Ala Arg Tyr Pro Glu Thr Ile Thr Leu ro G1~ Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg 260 265 2~0 ly Lys Pro Gly Ile Tyr Arg Phe V~l Ala Pro Gly Glu Arg Pro Ser 275 . 280 285 Gly het Phe Asp Ser Ser Val Leu Cys Glu Cy9 Tyr Asp Ala Gly Trp Pro Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala yr Met Asn Thr Pro Gly Leu Pro Val Cy~ Gln Asp Hi~ Leu Glu Phe ~p Glu Gly Val Phe Thr G~y Leu Thr His Ile Asp Ala His Phe Leu 340 345 ~50 er Gln Thr Ly3 Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Al~ Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cy5 Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro hr Pro Leu Leu Tyr Arg Leu Gly Gly Gly Ser Ser Arg Leu Gln Ala 40~ 410 415 ys 2) ~NFORMATION FOR SEQ ID NO:26:

- PCTIUS 92/07188~ ~ ~ J ~ 2 ~ BO/ U S 2 3 OEC 1992 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1275 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY:.circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1275 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
ATG AGT m GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG 48 Met Ser Phe Val Val Ile Ile Pro.Ala Arg Iyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly~ Pro Met Ile Val Hi~

Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 35 40 ~5 Tbr Asp His Glu Asp Val Ala Arg Ala Val Glu Al~ Ala Gly Gly Glu V~l Cys Met Thr Arg Al~ Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu Val Yal Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro:Met Ile Pro Ala Thr Ile Ile Ar~ Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val CCA ~TC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG 432 Pro Ile His Asn Ala Glu Glu Ala Phe A~n Pro Asn Ala Val Lys Val 130 135 lg0 Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala ~hr Ile P~TIUS 9 2 / 0 7 X ~ 1~0/ ~ S 2 3 OEC 1992 CCT TGG GAT CGT GAT CGT m GCA GAA GGC CTT GAA ACC GTT GGC GAT 528 Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp AAC TTC CTG CGT CAT CTT GGT ATT TAT GGC TAC CGT GCA GGC m ATC 576 A~n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile 180 lB5 190 CGT CGT TAC GTC AAC T~G CAG CCA AGT CCG TTA GAA CAC ATC GAA ATG 624 Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met TTA GAG CAG CTT CGT GTT CT~ TGG TAC GGC GAA AAA ATC CAT GTT GCT 672 Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala V~l Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Arg Ile Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu GrC GGC GCC CCT CTT GGA GGC GCT GCC AGG GCC CTG GGC CAT GGC GTC 86g Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Gly His Gly Val CGG Gl~ CTG GAA GAC GGC GTG AAC TAT GCG ACA GGG AAT Cl~ CCT GGT 912 ArS,~ Val Leu Glu Asp Gly V~l Asn Tyr Ala Thr Gly Asn Leu Pro Gly 2gO 295 300 TGC TCT TTC TCT ATC I~C CTT CTG GCC CTG CTC TCT TGC CTG ACC GTG 960 Cys Ser Ph~ Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val CC~C GCA TCA GCC TAC CAA GTA CGC AAC TCC TCG GGC CTT TAC CAT GTC 1008 Pro Ala Ser Al~ Tyr Gln Val Arg Asn Ser Ser Gly Leu Tyr His Val mr Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr Glu Thr Ala Asp Ala 340 3g5 ` 350 Ile Leu His m r Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ala Ser Arg Cys Trp Val Ala Val Ala Pro Thr Val Ala Thr Arg Asp Gly .~

0/ ~ S 2 3 OEC ~992 AAA CTC CCC GCA ACG CAG CTT CGA CGT CAC ATT GAT CTG CrT GTC GGG 12 0 0 Lys Leu Pro Ala Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly 385 390 395 d,00 Ser Ala Thr Leu Cys Ser Ala Leu Tyr Leu Arg Ser Ser Val Pro Gly Asp Pro Leu Asp Cys Arg His Ala Lys (2) INFORMATION FOR SEQ ID NO : 27:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~25 amino aclds (B) IrYPE: amino acid ( D ) TOPOLOGY: l inear ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu . 15 Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His ;25 30 ~al Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala ,35 40 45 Thr Asp His Glu Asp Val Ala Ar~7 Ala Val Glu Ala Ala (~ly Gly Glu Val Cys M0t Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu Val Val Glu Lys Cy9 Ala Phe Ser A~p Asp Thr Val Ile Val Asn ~0 95 Val Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile - - PCTIUS 't2/07188 .
26 BO/I~S 23 OEC t992 Pro Trp Asp Arg Aisp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly ryr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Arg Ile Arg Arg Ser Arg A~sn Leu Gly Lys Val Ile Asp m r Leu Thr Cys Gly Phe Ala Asp Leu Met Gly l~r Ile Pro Leu ~1 Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Gly His Gly Val 2'75 280 285 Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cy~ Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Gln Val Arg Asn Ser Ser Gly Leu l~r Hls Val :; 325 330 335 Thr Asn Asp Cys Pro Asn Ser Ser Ile Yal Tyr Glu Thr Ala Asp Ala Ile Leu His m r Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ala 'Ser Arg Cys Trp Val Ala Val Ala Pro Thr Val Ala Thr AL-g Asp Gly Lyis Leu Pro Ala Thr Gln Leu Arg Arg Xis Ile Asp Leu Leu Val Gly 3~5 390 395 400 Ser Ala Thr Leu Cys Ser Ala Leu Tyr Leu Arg Ser Ser Val Pro Gly :~ Asp Pro Leu Asp Cy5 Arg His Ala Lys (2) INFORMATION FOR SEQ ID NO:28:

` - PCTIUS 92/07188 ~2~ l~O/~iS 23 DEC 1992 ~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1401 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(~) LOCATION: 1..1401 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Arg AlA Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr A~p Ni~ Glu Asp Val Ala Arg Al~ Val Glu Ala Ala Gly Gly Glu , ~, Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu Val Val Glu Lys Cys Ala Phe Ser Asp A~p Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Ual . 130 135 }40 Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile ,.~;,, , ~L~ 6 ~0/1,S 23 DE~ 1992 Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Al~

Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Arg Ile Leu Leu Val Gly Ser Ala Thr Leu Cys Ser Ala Leu ~yr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Gly Gln CTG TTC ACT TTC TCC CCC AGG CAG CAC T~G ACA ACG CAA GAC TGC AAC 864 Leu Phe m r Phe Ser Pro Arg Gl~ His Trp Thr Thr Gln Asp Cys Asn TGT TCT ATC TAC CCC GGC CAC GTA ACG ~GT CAC CGC ATG GCA TGG GAT 912 Cy~ Ser Ile Tyr Pro Gly Hi~ Val Thr Gly His Arg Met Ala Trp Asp Met Met M~t Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ala Gln Leu Leu Arg Val Pro Gln Ala Ile Leu A~p Met Ile Al~ Gly Ala His Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val ~-ly Asn Trp Ala 3gO 345 350 AAG GTC CTG GTA GTG CTG CTG.CTA TTT GCC GGC GTT GAC GCG GAA ACC 1104 Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr His Val Thr Gly Gly Ser Ala Gly His Ile Thr Ala Gly Le~ Val Arg ` PCT/IJS 92/0718 8 2G B,O~ ~,S 2 3 OEC l992 Leu Leu Ser Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn GGC AGT TGG CAC ATC AAT AGC ACG GCC TTG AAC TGC AAT GAA AGC CTT 124 a Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr His His Lys Phe Asn Ser Ser Gly Cy9 Pro Glu Arg Val Ala Ser Cys Arg Arg Leu Thr Asp Phe 435 ~40 445 Asp Gln Gly Trp Glu Phe Glu Leu Gly Thr Arg Gly Ser Ser Arg Leu CAG GCA TGC 1~01 Gln Ala Cys 2) INFORMATION FOR SEQ ID NO:29:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids (P) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Met Ser Ph~ V~l Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly ~y~ Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala m r Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu Val Val Glu Ly~ Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn ,: .

- , ~ PCT/US 9 2 / 0 7 1 8 8 1~ 0 / ~ S 2 3 DEC 1992 Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val VA1 Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile ro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu Hi~ Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gl~ Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu 225 ~30 235 240 sp Pro Ser Thr Arg Ile Leu Leu Val Gly Ser Ala Thr Leu Cys Ser 24~ 250 255 Ala Leu Tyr V~l Gly A3p Leu Cys Gly Ser Val Phe Leu Val Gly Gln ~ 260 265 270 L~u Phe Thr Phe Ser Pro Arg Gln His Trp Thr Thr Gln Asp Cys Asn Cyq Ser Ils Tyr Pro Gly Hi~ Val Thr Gly His Arg Met Ala Trp A~p Met ~et Met A~n Trp Ser Pro Thr Thr Ala Leu Val Val Ala Gln Leu eu Arg Val Pro Gln Ala Ile Leu Asp Met Ile Ala Gly Ala His Trp ly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val Gly Asn Trp Ala Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val A~p Ala Glu Thr His Val Thr Gly Gly Ser Ala Gly His Ile Thr Ala Gly Leu Val Arg PCTlUS 92/0718 8 6 ~O/~ 23 DE~

Leu Leu Ser Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr His His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Val Ala Ser Cys Arg Arg Leu Thr Asp Phe Asp Gln Gly Trp Glu Phe Glu Leu Gly Thr Arg Gly Ser Ser Arg Leu Gln Ala Cys (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1422 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) - (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1422 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu CCC GGT AAA CCA TTG GTT GAT ATT AAC GGC:AAA CCC ATG ATT GTT CAT ~6 Pro Gly Lys Pro Leu V~l A~p Ile A~n Gly Lys Pro Met Ile Val Hi-~

Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala ACC GAT CAT GAG GAT G~T GCC CGC GCC GTT GAA GCC GCT GGC GGT GAA . 192 Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala ~Q~ 2 3 DEC ~992 os Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala A p Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val CCA ATC CAC AAT GCG GAA GAA GCG TTT AAC CCG AAT GCG GTG AAA GTG ~ 32 Pro Ile ~is Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Ars1 Ala Thr Ile 1~5 150 155 160 CCT T~G GAT CGT GAT CGT m GCA GAA GGC CTT GAA ACC GTT GGC GAT 528 Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp A~n Phe Lell Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr V~l A3n Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met 195 . 200 205 TTA GAG CAG CTT CGT G~T CTG TGG TAC GGC GAA AAA ATC CAT GTT GCT 6?2 Leu Glu Gln Leu Arg V~l Leu Trp Tyr Gly Glu Ly~ Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val A~3p Thr Pro Glu Asp Leu 225 230 235 2~0 Asp Pro Ser Thr Glu Phe Gly Asp Ile Ile Asn Gly Leu Pro Val Ser Al~ hrg Arg Gly Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Val TCC AAG GGG Tt; G AGG 1~ CTG GCG CCC ATC ACG GCG TAC GCC CAG CAG 864 Ser Lys Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys lle Ile Thr Ser Leu Thr Gly Arg A~p ~_3 PCTIUS 9 2 / 0 71 8 8 .BO/l~ 23 DEC 1992 1ys Asn Gln Ala Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Leu Ala S~r Pro Lys Gly Pro V~l Ile Gln ATG TAT ACC AAT GTA GAC CAA GAC CTT GTG GGC I~G CCC GCT CCT CAA 1104 Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln 355 360 ~ 365 Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly 405 410 gl5 TCC TCG GGG GGT CCG CTG ~G 5GC CCC GCG GGA CAC GCC GTG GGC ATA 1296 Sor Ser Gly Gly Pro L~u Leu Cys Pro Al~ Gly Hi~ Ala Val Gly Ile ~20 425 g30 ~IC AGG GCC GCG GIY~ TGT ACC C5T GGA GIY~ GCT AAG GCG GTG GAC l~T 1344 Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe GTC CCC G~G GAG AAC CTC GA~ ACA ACC ATG AAT TCG AGC TCG GTA CCC 1392 Val Pro Val Glu Asn Leu Glu Thr Thr Met Asn Ser Ser Ser Val PI~O

GGG GAT CCT CTA GAC TGC AGt; CAT GCT AAG 1422 Gly Asp Pro Leu Asp Cy4 Arg His Ala Lys (2) INFORMATION FOR SEQ ID NO: 31:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 474 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLEC'JLE TYPE: protein ~ J ~ 0 ~ ~ S 23 OEC ~2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
~et S~r Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu ro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu Val Cyq Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala ~0 1U VA1 Val Glu Lys Cys Ala Phe Ser Asp Asp Thr V~l Ile Val Asn . g5 al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gln ~al Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile ~45 lS0 155 160 ro Irp Asp Arg A3p Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp sn Phe Lau Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile 180 1~5 190 Arg Arg ~yr Val Asn Trp Gln Pro Ser Pro Leu Glu Hi~ Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu sp Pro Ser Thr Glu Phe Gly Asp Ile Ile Asn Gly Leu Pro Val Ser la Arg Arg Gly Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Met V~l !3~ ~O/US 23 DEC 1992 Ser Lys Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln 275 28~ 285 Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Ala Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln hr Phe Leu Ala Thr Cy~ Ile Asn Gly Val Cy3 Trp Thr Val Tyr His ly Ala Gly Thr Arg Thr Leu Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp L~u Tyr Leu Val m r Arg His Ala Asp Val Ile Pro Val Arg Arg ~rg Gly Asp er Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly 405 ~10 415 er Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile 420 425 ~30 Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe - 435 440 4~65 V~l Pro V~l Glu Asn Leu Glu Thr Thr Met Ac~ Ser Ser Ser Val Pro Gly A~p Pro L~u Asp Cys Arg His Ala Ly (2) INFORMATION FOR SEQ ID NO:32.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTB: 1401 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAMEtKEY: CDS
~B) LOCATION: l..lg01 ~Ll`~9~ O/~ S 23 DEC l992 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu S0 55 . 60 V~l Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala GAA GTT GTC GAA AAA T~C GCA TTC AGC GAC GAC ACG GTG ATC GTT AAT 2 8 8 Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn G$G CAG GGT GAT GAA CCG ATG ATC CCT GCG ACA ATC ATT CGT CAG GTT 3 3 6 Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val GCT GAT AA~: CTC GCT CAG CGT CAG GTG GGT ATG ACG ACT CTG GCG GTG 3 8 g Al~ A~p Asn Leu Ala Gln Arg Gln Val Gly M~t Thr Thr Leu Ala V~l CCA ATC CAC AAT GCG GAA GAA GCG m AAC CCG ~T GCG GTG AAA GTG 432 Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro A~n Ala Val Lys Val Val Leu Asp Ala Glu Gly q~r Ala Leu Tyr Phe Ser Arg Ala Thr Ile CCT TGG GAT CGT GAT CGT 1~ GCA GAA GGC CTT GAA ACC GTT GGC GAT 5 2 8 Pro Trp A~p Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp AAC 1~1 C CTG CGT CAT CTT GGT ATT TAT S~GC TAC CGT GCA GGC TTT ATC 57 6 Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile 180 185 lgO

Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met lg5 200 205 TTA GAG CAG CTT CGT Gl~ CTG TGG TAC GGC GAA AAA ATC CAT Gl~ GCT 67 2 ~ T/US ~ 2 / 0 7 1 8 8 y~f~ K~ S 2 3 OEC 1992 Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser Thr Met Gly His Tyr Pro Cys Thr Ile Asn Tyr Thr Leu Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Val Ala Cys Asn Trp Thr Arg Gly Glu Ars Cys Asp Leu Asp AJP Arg A~p Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln TGG CAG GTC CTT CCG TGT TCC TTC ACG ACC TTG CCA GCC TTG ACC ACC . 960 Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Thr Thr G~C CTC ATC CAC CTC CAC CAG AAC ATC GTG GAC GTG CAA TAC TTG TAC 1008 Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr G~G GTG GGG TCA AGC ATT GTG TCC TG5 GCC ATC AAG TGG GAG TAC GTC 1056 Gly Val Gly Ser Ser Ile Val Ser Irp Ala Ile Lys Trp Glu Tyr Val 340 345 350 . -~

Ile 1eu Lou Phs Leu Leu Lau Ala Asp Ala Arg Ile Cy~ Ser Cys Leu Trp Met ~et Leu Leu Ile Ser Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Leu Leu Asn Ala Ala Ser Leu Ala Gly Thr His Gly Leu Val Ser TTC CTC GTG m TTC TGC TTT GCA TGG TAT CTG AAG GGT AAG TGG GTG 1248 ~: Phe Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Lys Gly Lys Trp Val ; gOS 410 415 Pro Gly Val Ala Tyr Ala Phe Tyr Gly Met Trp Pro Phe Leu Leu Leu 326 BO/~S 23 OEC Im Lau Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Met Ala 435 g40 445 Ala Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr Leu TCA CCA TAT 14Ql Ser Pro Tyr (2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amIno acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu ro Gly Lys Pro Leu V~l Asp Ile Asn Gly Lys Pro Met Ile Val His Val Leu Glu Axg Ala Ar~ Glu Ser Gly Ala Glu Ars Ile Ile Val Ala Thr Asp His Glu A~p Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu 5~ 60 Val Cys Met m r Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala lu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn al Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Al~ Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val 115 120 12~
Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile 0/l,S 230ECl992 b Pro Trp A~p Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp ~n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val A!3n Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met l9S 200 20S
Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu sp Pro Ser Thr Asn Ser Thr Met Gly His Tyr Pro Cys Thr Ile Asn 2gS 250 ~ 255 yr Thr Leu Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Val Ala Cys As~ Trp Thr Arg Gly Glu Arg Cys Asp Leu A7sp Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln 29~ 295 30~
Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Thr Thr 30~ ' 310 315 320 ly L~u Ile His Leu Hi~ Gln A~n Ile Val Asp Val Gln 1~7r Leu l~rr ly Val Gly Ser Ser Ile Val Ser Trp Ala Ile Lys Trp Glu l~r Val 3~0 3~5 350 Ile Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Ile Cys Ser Cy~ Leu 355 360 ' 365 Trp Met Mot Leu Leu Ile Ser Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Leu Leu Asn Ala Ala Ser Leu Ala Gly Thr ~lis Gly Leu Val Ser 385 390 395 gO0 he Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Lys Gly Lys Trp Val gOS 410 glS
ro Gly Val Ala Tyr Ala Phe Tyr Gly Met Trp Pro Phe Leu Leu Leu 420 ' 425 430 Leu Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Met Ala Ala Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr Leu PCTIUS 9 2/ 07 1 8 ~ .
t ~ O/ ~ S 2 3 DEC 199~

Ser Pro Tyr (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1851 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1851 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3~:
ATG AGT ITT GTG GTC ATT ATT CCC GCG CGC TAC GCG TCG ACG CGT CTG . 48 Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Ly~ Pro Met Ile Val His Yal Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 3~ 40 45 Thr Asp His Glu Asp V~l Ala Arg Ala Val Glu Ala Ala Gly Gly Glu GTA TGT ATG ACG CGC GCC GAT CAT C~G TCA GGA ACA GAA CGT CTG GCG 240 Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala 6~ 70 75 80 Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val

10~ 105 110 Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Ala Thr Leu Ala Val 2 ~ lllS 9 2 / 0 7 1 8 8 ~0/l~S 23DEC199 CCA ATC CAC AAT GCG GAA GAA GCG m AAC CCG AAT GCG GTG AAA GTG 432 Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 135 lgO

Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile C'-T TGG GAT CGT GAT CGT TTT GCA GAA GGC CTI GAA ACC GTT GGC GAT 528 Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp A~n Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile Hi3 Val Ala ~al Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro Glu Asp Leu GAC CCG TCG ACT CGA ATT CGT AGG TCG CGC AAT $TG GGT AAG GTC ATC 7 68 Asp Pro Ser Thr Arg Ile Arg Arg 5er Arg Asn Leu Gly Ly3 Val Ile GAT ACC CTC ACG TGC GGC TTC GCC GAC CTC ATG GGG TAC Al~r CCG CTC 816 A~p Thr Leu Thr Cys Gly Phe Ala A~p Leu Met Gly l~r Ile Pro Leu Gl~ GGC GCC CCT CTT GGA GGC GCT GCC AGG GCC CTG GCG CAT GGC GTC 864 Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val CGG GTT csa GAA GAC GGC GTG AAC TAT GCA ACA GGG AAC CrT CCC GGT 912 Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Prs Gly Cy~ Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val CCC GCG TCA TCC TAC CAA GTA CGC AAC TCC TCG GGC CTT TAT CP.T GTC 1008 Pro hla Ser Ser Tyr Gln Val Arg Asn Ser Ser Gly Leu Tyr His Val Thr Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr Glu Thr Ala Asp Thr 340 345 350~

~ O/ ~TS 2 3 OEC ~

Ile Leu His Ser Pro Gly Cy~ Val Pro Cys Val Arg Glu Gly A~n Thr Ser Lys Cy8 Trp Val Ala Val Ala Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Ser Thr Gln Leu Arg Arg His Il~ Asp Leu Leu Val Gly Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser 405 glO ~15 Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp ACA ACG CAA GAC TGC ~C TGT TCT ATC TAC CCC GGC CAT ATA ACG GGT 1344 Thr m r Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg M~t Ala Trp Asp Met Met M~t Asn Trp Ser Pro Thr m r Ala CTG GTA G~A GCT CAG CTG CTC AGG GTC CCA CAA GCC ATC ~TG GAC ATG 1440 Leu Val V~l Ala Gln Leu Leu Arg Val Pro Gln Ala Ile Leu Asp Met 465 470 g75 480 Ile Al~ Gly Ala His Irp Gly V~l Leu Ala Gly Ile Al~ Tyr Phe Ser ~85 g90 ~95 ATG GTG GGG AAC T¢G GCG AAG GTC CTG GTA GTG CTG ~TG CTG m TCC 1536 Met V~l Gly Asn Trp Al~ Ly~ Val Leu Val Val Leu Leu Lel~ Phe Ser ~00 505 510 GGC GTC GAT GCG GCA ACC TAC ACC ACC GGG GGG AGC GTT GCT AGG ACC 158g Gly Val Asp Ala Ala Thr Tyr Thr Thr Gly Gly Ser Val Ala Arg Thr Thr His Gly Phe Ser Ser Leu Phe Ser Gln Gly Ala Lys Gln Asn Ile ~30 535 540 Gln Leu Ile Asn Thr A~n Gly Ser Trp His Ile Asn Arg Thr Ala Leu 5g5 550 555 560 Asn Cy~ Asn Ala Ser Leu Asp Thr Gly Trp Val Ala Gly Leu Phe Tyr - i i J ~ ~ S PCTNS 9 2 / 0 71 8 l Tyr His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser C ys AGA CCC CTT GCC GAT m GAC CAG GGC IY~G GAA TTC GAG CTC GGT ACC 18 2 4 Arg Pro Leu Ala Asp Phe Asp Gln Gly Trp Glu Phe Glu Leu Gly Thr Arg Gly Ser Ser Arg Leu Gln Ala Cys ( 2 ) INFORMATION FOR SEQ ID NO:35:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 617 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Mst Ile Val His VA1 LeU Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala Thr Asp Hi~ Glu Asp Val Ala Arg A1A Val Glu Ala Al~ Gly Gly Glu _~ 50 55 60 Val Cyi~ M~t Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala Glu Val V~l Glu Lys Cys Ala Phe Ser Asp A~p Thr Val Ile Val Asn Val Gln Gly A~p Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val Ala Asp Asn Leu Ala Gln Arg Gl,rl Val Gly Met Ala Thr Leu Ala Val Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile 9~6 1~0/1/S 2 3 DEC 1992 Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Glu Thr Val Gly Asp sn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val A~p Thr Pro Glu Aap Leu 2~5 230 235 2~0 sp Pro Ser Thr Arg Ile Arg Arg Ser Arg Asn Leu Gly Lys Val Ile sp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val L~u Glu Asp Gly Val Asn Tyr Ala Thr Gly A~n Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val ro Ala Ser ser Tyr Gln Val Arg Asn Ser Ser Gly Leu Tyr His Val hr As~ Asp Cys Pro A~n Ser Ser Ile Val Tyr Glu Thr Al~ A~p Thr 3~0 345 350 Ile Leu Hia Ser Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Thr Ser Lys cy9 Trp Val Ala Val Ala Pro Thr Val Ala Thr Arg Asp Gly 370 375 ~80 Lys Leu Pro Ser Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly er A~a Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser 405 410 415.
al Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp 420 g25 430 hr Thr Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly g35 440 445 1~0/ ~; S 2 3 DEC t992 1 2l~

His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ala Gln Leu Leu Arg Val Pro Gln Ala Ile Leu Asp Met le Ala Gly Ala His Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser 4~5 490 495 et Val Gly Asn Trp Ala Lys Val Leu Val Val Leu Leu Leu Phe Ser 500 505 SlO
Gly Val Asp Ala Ala Thr Tyr Thr Thr Gly Gly Ser Val Ala Arg Thr SlS 520 525 m r His Gly Phe Ser Ser Leu Phe Ser Gln Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr A~n Gly Ser Trp His Ile Asn Arg Thr Ala Leu sn Cys Asn Ala Ser Leu Aap Thr Gly Trp Val Ala Gly Leu Phe Tyr yr His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Leu Ala Asp Phe Asp Gln Gly Trp Glu Phe Glu Leu Gly Thr Arg Gly Ser Ssr Arg Leu Gln Ala Cys

Claims (19)

122

1. A recombinant fusion protein SEQ. ID. NO. 1.

2. A recombinant fusion protein SEQ. ID. NO. 2.

3. A recombinant fusion protein SEQ. ID. NO. 3.

4. A recombinant fusion protein SEQ. ID. NO. 4.

5. A recombinant fusion protein SEQ. ID. NO. 5.

6. A polypeptide SEQ. ID. NO. 1.

7. A polypeptide SEQ. ID. NO. 2.

8. A polypeptide SEQ. ID. NO. 3.

9. A polypeptide SEQ. ID. NO. 4.

10. A polypeptide SEQ. ID. NO. 5.

11. An assay for identifying the presence of an antibody immunologically reactive with an HCV antigen in a fluid sample comprising:
contacting the sample with at least one polypeptide selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID.
NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5 under conditions suitable for complexing the antibody with the polypeptide; and detecting the antibody-polypeptide complex.

12. In a confirmatory assay for identifying the presence of an antibody in a fluid sample immunologically reactive with an HCV antigen wherein the sample is used to prepare first and second immunologically equivalent aliquots and the first aliquot is contacted with at least one polypeptide selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ.
ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID.
NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5 under conditions suitable for complexing the antibody with the polypeptide and wherein the first antibody-antigen complex is detected, and:
contacting the second aliquot with a polypeptide selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID.
NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5 under conditions suitable to form a second antibody-antigen complex; and detecting the second antibody-antigen complex; wherein the polypeptide selected in the first aliquot is not the same as the polypeptide selected in the second aliquot.

13. In an immunodot assay for identifying the presence of an antibody immunologically reactive with an HCV antigen in a fluid sample wherein the sample is concurrently contacted with at least two polypeptides separately bound to distinct regions of the solid support, each containing distinct epitopes of an HCV antigen under conditions suitable for complexing the antibody with the polypeptide; and detecting the antibody-polypeptide complex, and wherein said polypeptides are selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID.
NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5.

14. In a competition assay for identifying the presence of an antibody immunologically reactive with an HCV antigen in a fluid sample wherein the sample is used to prepare first and second immunologically equivalent aliquots wherein the first aliquot is contacted with a polypeptide bound to a solid support under conditions suitable for complexing the antibody with the polypeptide to form a detectable antibody-polypeptide complex, and wherein the second aliquot is firstcontacted with unbound polypeptide and then contacted with said bound polypeptide wherein the polypeptide is selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO.
5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5.

15. In a competition assay for identifying the presence of an antibody immunologically reactive with an HCV antigen in a fluid sample wherein the sample is used to prepare first and second immunologically equivalent aliquots wherein the first aliquot is contacted with a polypeptide bound to a solid support under conditions suitable for complexing the antibody with the polypeptide to form a detectable antibody-polypeptide complex and wherein the second aliquot is first contacted with unbound polypeptide and then contacted with said bound polypeptide wherein the polypeptide is selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO.
5, and polypeptides SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5; wherein the second aliquot is contacted with unbound and bound polypeptide simultaneously.

16. In a neutralization assay for identifying the presence of an antibody immunologically reactive with an HCV antigen in a fluid sample wherein the sample is used to prepare first and second immunologically equivalent aliquots wherein the first aliquot is contacted with a polypeptide bound to a solid support under conditions suitable for complexing the antibody with the polypeptide to form a detectable antibody-polypeptide complex wherein the bound polypeptide is selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ.ID.NO.2,SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ.
SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 6;
and wherein the second aliquot is first contacted with unbound polypeptide and then contacted with said bound polypeptide wherein the unbound polypeptide is selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ.
ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3,SEQ. ID. NO.4, SEQ. ID. NO. 5 and wherein the bound polypeptide selected is not the same as the same as the unbound polypeptide selected.

17. In a neutralization assay for identifying the presence of an antibody immunologically reactive with an HCV antigen in a fluid sample wherein the sample is used to prepare first and second immunologically equivalent aliquots wherein the first aliquot is contacted with a polypeptide bound to a solid support under conditions suitable for complexing the antibody with the polypeptide to form a detectable antibody-polypeptide complex wherein the bound polypeptide is selected from the group consisting of recombinant fusion proteins SEQ. ID. NO.1, SEQ. ID.NO. 2 ,SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO.5, and polypeptides SEQ. ID. NO.1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO.5;
and wherein the second aliquot is first contacted with unbound polypeptide and then contacted with said bound polypeptide wherein the unbound polypeptide is selected from the group consisting of recombinant fusion proteins SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and polypeptides SEQ.
ID. NO. 1, SEQ. ID. NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID. NO.5;
and wherein the bound polypeptide selected is not the same as the unbound polypeptide selected;
and wherein the second aliquot is contacted with unbound and bound polypeptide simultaneously.

18. An immunoassay kit comprising:
a polypeptide containing at least one HCV antigen selected from the group consisting of recombinant fusion proteins SEQ. ID NO. 1, SEQ. ID. NO. 2, SEQ. ID.
NO. 3, SEO. ID. NO. 4, SEO. ID. NO. 5, and polypeptides SEQ. ID. NO. 1, SEO. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5;
one or more sample preparation reagents;

and one or more detection and signal producing reagents.

19. A kit of claim 18 wherein the polypeptides are bound to a solid support.