Abstract:
The present invention relates to novel tri-hybrid melanoma antigens, including antigenic fragments or derivatives thereof, of a tyrosinase (TYR) antigen, a tyrosinase-related protein 1 (TRP-1) antigen, and a tyrosinase-related protein 2 (TRP-2) antigen and nucleic acids encoding them. The novel tri-hybrid melanoma antigens of the present invention are useful in the diagnosis, treatment and prevention of human neoplasms, including malignant tumors, such as carcinomas, sarcomas, leukemia, and lymphomas, and pre-malignant lesions, such as adenomas and dysplastic lesions.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to novel tri-hybrid melanoma antigens, including antigenic fragments or derivatives thereof, of a tyrosinase (TYR) antigen, a tyrosinase related protein-1 (TRP-1) antigen, and a tyrosinase related protein-2 (TRP-2) antigen and nucleic acids encoding them, which are useful in the prevention and treatment of human neoplasms.  
         BACKGROUND OF THE INVENTION  
         [0002]    Cancer cells are not static in nature, but are changing constantly. To escape immunosurveillance and multiply, cancer cells have developed a number of different mechanisms, include the following: decreasing expression in MHC molecules (see Ferrone et al., Immunol. Today, 16:487-494 (1995)); deficiencies in antigen processing (see Kamarashev et al., Int. J. Cancer, 95:23-28 (2001)); secretion of immune suppressing cytokines (see Spellman et al., Surg Oncol., 5(5-6):237-44 (1996)); and loss of tumor antigen expression (see Ohmacht et al., J. Cell Physiol., 182: 332-338 (2000)).  
           [0003]    With respect to the loss of tumor antigen expression, a number of melanoma-specific antigens have been identified in recent years. One major group of such antigens, which are recognized by the immune system, consists of melanocyte differentiation antigens, such as tyrosinase, TRP-1 (also designated gp75), TRP-2, gp100 and MART-1/Melan-A. All of these antigens are present in melanosomes.  
           [0004]    Tyrosinase, TRP-1, and TRP-2, are melanocyte differentiation antigens, located in the melanosomes of melanocytes and melanomas, and involved in melanin synthesis. See Kawakami et al., Immunol. Res., 16: 313-339 (1997); Kawakami et al., J. Immunother., 21: 237-246 (1998). Human tyrosinase, a 529 amino acid melanosomal membrane protein, has tyrosine hydroxylase, DOPA oxidase and DHI activity, and is the principal enzyme involved in melanin synthesis. Human TRP-1 consists of 537 amino acids and has DHI-2-carboxylic acid oxidase activity. Human TRP-2 is a 519 amino acid melanosomal enzyme with DOPAchrome tautomerase activity. Antibodies and T cells to these antigens have been identified in melanoma patients. See Houghton et al., Ann. N.Y. Acad. Sci., 690: 59-68 (1993); Brichard et al., J. Exp. Med., 178: 489-495 (1993); Kang et al., J. Immunol., 155: 1443-1348 (1995); Wang et al., J. Exp. Med., 181: 799-804 (1995); Wang et al., J. Exp. Med., 184: 2207-2216 (1996). However, it remains unclear how tolerance to these differentiation antigens is broken in cancerous state.  
           [0005]    Animal models of cancer vaccines have demonstrated the feasibility of melanoma vaccines using the tyrosinase family of proteins. Vaccination of mice with recombinant vaccinia viruses expressing mTRP-1 (see Overwijk et al., Proc. Nat&#39;l Acad. Sci. USA., 96: 2982-2987 (1999)), or naked DNA encoding hTRP-1 (see Weber et al., J. Clin. Invest., 102: 1258-1264 (1998)), or insect cells containing mTRP-1 (see Naftger et al., Proc. Nat&#39;l Acad. Sci. USA., 93: 14809-14814 (1996)) have demonstrated the ability to break tolerance to TRP-1 and have shown induction of strong antitumor activities, which were dependent on TRP-1 specific antibodies and CD4 T cells. Immune tolerance to TRP-2 also has also been broken with naked DNA encoding human TRP-2, following induction of CD8 T cells (see Browne et al., J. Exp. Med., 190: 1717-1722 (1999)).  
           [0006]    Many of the clinical vaccine trials targeting the tyrosinase family of proteins are underway to treat melanoma. It has been reported that melanoma cells can lose the expression of TRP-1, TRP-2, or tyrosinase. See Id., Marincola et al., Adv. Immunol., 74: 181-273 (2000). However, the likelihood that a melanoma will lose all three antigens at same times is low; therefore, a chimeric protein containing all three of these melanoma antigens will have great advantage.  
           [0007]    Most of the vaccine approaches are based on defined peptide epitopes, plasmid DNA, and recombinant viruses, each of which suffers from various disadvantages. The identification of HLA-binding epitopes and HLA types of each patient are needed for peptide epitope-based vaccine. Plasmid DNAs in general are week immunogens. Recombinant viruses generate neutralizing antibodies, which limit the administration of vaccine. As such, use of a recombinant protein vaccine is an attractive alternative approach for a cancer vaccine.  
           [0008]    There are several advantages to use of a recombinant protein vaccine, including the fact that the vaccine can be administered repeatedly, it can induce a wise spectrum of immune responses, including production of antibodies, cytotoxic T-lymphocytes (CTLs) (with appropriate adjuvants), and CD4+ T cells (important in maintaining tumor immunity). Moreover, a protein vaccine can contain all possible epitopes of the antigen.  
         SUMMARY OF THE INVENTION  
         [0009]    The present invention is directed to tri-hybrid melanoma antigens, and isolated DNAs encoding such antigens, comprising tyrosinase or a fragment thereof, TRP-1 or a fragment thereof, or TRP-2 or a fragment thereof. The present invention is also directed to compositions for inhibiting melanosomal activity or tumor growth comprising such tri-hybrid melanoma antigens and isolated DNAs. In addition, the present invention is directed to methods of eliciting an immune response against a melanosomal antigen, methods of treating a tumor, or methods of vaccination using such tri-hybrid melanoma antigens and isolated DNAs. 
       
    
    
     DESCRIPTION OF THE FIGURES  
       [0010]    [0010]FIG. 1 shows construction of a tri-hybrid melanoma antigen, hTRPx3, containing human TRP-1, TRP-2, and tyrosinase. A tri-hybrid melanoma antigen DNA fragment was generated with primers and the resulting fragments were mixed and fused following 10 PCR cycles. The final chimeric DNA was generated with primers htyrF-1 (SEQ ID NO:1) and htyrF-6 (SEQ ID NO:6) and then cloned into bacterial expression vector pET28a(+).  
         [0011]    [0011]FIG. 2 shows purification and characterization of a recombinant tri-hybrid melanoma antigen, hTRPx3 protein, by SDS-PAGE and Western blotting. In FIG. 2A, an SDS-PAGE gel shows the expression and purification of the hTRPx3 protein and in FIG. 2B, a Western blot shows that the purified hTRPx3 protein is recognized by antibodies specific for human TRP-1, TRP-2, and tyrosinase.  
         [0012]    [0012]FIG. 3 graphically demonstrates the antibody responses in mice following immunization with a tri-hybrid melanoma antigen, hTRPx3 protein. In FIG. 3A, a graph shows that hTRPx3 protein immunization induced antibodies against TRP-1, TRP-2, and tyrosinase and hTRPx3. In FIG. 3B, a graph shows the isotypes of the antibodies specific to the hTRPx3 protein.  
         [0013]    [0013]FIG. 4 graphically demonstrates that immunization of mice with a tri-hybrid melanoma antigen, hTRPx3, resulted in production of IFNγ releasing T cells specific for TRP-1, TRP-2, and tyrosinase.  
         [0014]    [0014]FIG. 5 shows induction of a T cell immune response following immunization with a tri-hybrid melanoma antigen, hTRPx3 protein. In FIG. 5A, an ELISPOT blot shows induction of IFNγ-producing T cells, and in FIG. 5B, this IFNγ-producing T cell induction is represented graphically.  
         [0015]    [0015]FIG. 6 shows that immunization with a tri-hybrid melanoma antigen, hTPRx3 protein, was useful in treating tumors in mammals. In FIG. 6A, the number of lung surface metastases following immunization is represented graphically. In FIG. 6B, the number of lung surface metastases following immunization of MHC class I knock-out mice, MHC class II knock-out mice, and FcRγ knock-out mice is represented graphically. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0016]    The present invention relates to tri-hybrid melanoma antigens of a tyrosinase antigen (U.S. Pat. No. 4,898,814), a TRP-1 antigen (WO 91/14775) and a TRP-2 antigen (U.S. Pat. No. 5,831,016), including antigenic fragments and derivatives thereof. Derivatives include, for example, antigens that are mutational or allelic variants. The antigens can be of human or, more generally, of mammalian origin, and the components of the tri-hybrid melanoma antigen can be from different sources (e.g., homologous antigens from different species). Moreover, each component (i.e., antigen or antigenic fragment) can be a hybrid combining sequences from more than one source. The invention provides a tri-hybrid melanoma antigen that is more effective for immunization against melanoma than any single antigen from which it is derived.  
         [0017]    “Antigenic fragment,” as the term is used herein, means any antigenic segment of a protein or gene, usually having at least 5 or 6 amino acids in the case of a protein fragment and at least 15-18 nucleotides in the case of a gene. Thus, a fragment generally encompasses a segment of a protein, or the nucleotide sequence that encodes it, which comprises at least one B cell or T cell epitope.  
         [0018]    Tyrosinase, TRP-1 (also known as gp75) and TRP-2 are expressed primarily in melanomas, normal melanocytes, and in the retina. The three proteins are related in sequence, sharing (pairwise) greater than 40% amino acid sequence identity and greater than 50% amino acid sequence similarity, and have in common N-terminal signal peptides and a C-terminal sequence involved in intracellular retention and sorting to melanosomes along the endosomal/lysosomal pathway. Moreover, these three members of the tyrosinase family of proteins are highly conserved among species.  
         [0019]    The invention further relates to homologs of human tyrosinase, TRP-1 and TRP-2, that can be used to break tolerance in humans to the human proteins. Table 1 provides two examples of such homologs for each of these three tyrosinase family proteins. The value for percent identity of the homolog to the human protein is calculated over the entire length of the protein. Homologs that can be used according to the invention have at least 60% identity to the corresponding protein of the species in which tolerance is to be broken. Preferred homologs have at least 70% identity. More preferred homologs have at least 80% identity. It is noted that where less than a complete amino acid sequence is to be incorporated into a tri-hybrid melanoma antigen, percent identity is calculated only over the length of the protein fragment that is incorporated.  
         [0020]    Nucleotide sequences of mRNAs encoding human tyrosinase, TRP-1/gp75 and TRP-2 and the sequences of the proteins themselves are publicly available from GenBank (National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md.), as are homologous sequences from other species. Nucleotide and amino acid sequences referred to herein correspond to GenBank accession numbers as given in Table 1. The sequences give therein are meant as examples only, and do not limit the scope of the invention.  
                               TABLE 1                               Nucleotide   Amino Acid   Percent Identity           Source   Sequence   Sequence   to Human Protein                   Tyrosinase   Human   NM_000372   NP_000363   —           Mouse   NM_011661   NP_035791   86           Rabbit   AF210660   AAF43895   89       TRP-1   Human   NM_000550   NP_000541   —           Mouse   MMTYRR   TYR1_MOUSE   85           Goat   AF136926   AAD34802   88       TRP-2   Human   NM_001922   NP_001913   —           Mouse   NM_010024   NP_034154   83           Chicken   AF023471   AAC63434   69                  
 
         [0021]    A preferred tri-hybrid melanoma antigen of the present invention comprises the soluble portion of each of tyrosinase, TRP-1 and TRP-2. For example, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11 provide examples of DNA sequences encoding a tri-hybrid melanoma antigen in the context of the present invention. The corresponding protein sequence for the tri-hybrid melanoma antigen is set forth in SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. Accordingly, in one embodiment, an isolated DNA encoding the tri-hybrid melanoma antigen comprises SEQ ID NO:7 or a fragment thereof, SEQ ID NO:9 or a fragment thereof, or SEQ ID NO:11 or a fragment thereof and the tri-hybrid melanoma antigen itself comprises SEQ ID NO:8 or a fragment thereof, SEQ ID NO:10 or a fragment thereof, or SEQ ID NO:12 or a fragment thereof.  
         [0022]    An example of a more preferred tri-hybrid melanoma antigen comprises the sequence from about amino acid residue 25 to about amino acid residue 477 of human TRP-1 or a homolog thereof, the sequence from about amino acid residue 24 to about amino acid residue 472 of human TRP-2 or a homolog thereof, and the sequence from about amino acid residue 19 to about amino acid residue 476 of human tyrosinase or a homolog thereof. The individual fragments can be linked in any order and the tri-hybrid melanoma antigen can further comprise glycine residues by which the fragments are linked.  
         [0023]    Thus, a particularly preferred tri-hybrid melanoma antigen is represented by SEQ ID NO:8 from about amino acid residue number 25 to about amino acid residue number 1392 (containing glycine linkages) and SEQ ID NO:12 from about amino acid residue number 25 to about amino acid residue number 1384 (without glycine linkages). It will be apparent to one of ordinary skill in the art that there can be some variation in the extent of the protein fragments of which the tri-hybrid melanoma antigen is comprised, which will have little or no effect on its immunogenic properties.  
         [0024]    Where non-human mammalian proteins, mutational variants, hybrids, fragments, or derivatives are used, they can be selected such that they possess desirable properties such as increased immunogenicity, decreased side effects, and increased half-life. For example, fragments of the individual antigens can be selected for incorporation into a tri-hybrid melanoma antigen of the invention based on the presence of known or postulated B cell or T cell epitopes. As another example, mutational variants that exhibit improved binding to MHC molecules can be selected.  
         [0025]    Production of large quantities of tri-hybrid melanoma antigens can be accomplished by methods known in the art. For example, large amounts of tri-hybrid melanoma antigens can readily be synthesized in vitro. As another example, nucleic acid encoding tri-hybrid melanoma antigens can be transfected into bacterial, insect, or mammalian cells using appropriate vectors and methods as known in the art. Accordingly, the present invention encompasses cloning or expression vectors comprising a DNA encoding a tri-hybrid melanoma antigen and host cells comprising such cloning or expression vectors.  
         [0026]    Where needed or desired for expression of the tri-hybrid melanoma antigen in a given cell type, the first protein fragment of the tri-hybrid melanoma antigen will be preceded by a signal peptide, which can be the signal peptide that is native to the first protein fragment in the tri-hybrid melanoma antigen, or can be a signal peptide derived from another source. For example, a signal sequence signaling for secretion is useful where it is desired to provide for secretion of a tri-hybrid melanoma antigen into external mileau of a cell. For a review of secretion and signal peptide function, see, for example, Pugsley, Curr. Opin. Cell Biol., 2:609-616 (1990). Algorithms and computer implementations for secretory signal sequence prediction are available. See, e.g., von Heijne et al., Nucleic Acids Res., 14:4683-4690 (1986); Nielsen et al., Protein Eng., 10:1-6 (1997).  
         [0027]    For expression in bacterial cells, a bacterial signal sequence can be preferred. Alternatively, a signal sequence is not necessary to express the tri-hybrid melanoma antigen in a bacterial host in inclusion bodies. For example, a tri-hybrid melanoma antigen of the invention can comprise the amino acid sequence represented by SEQ ID NO:8 from about amino acid residue number 25 to about amino acid residue number 1392. Such a tri-hybrid melanoma antigen can be expressed with an N-terminal methionine residue, depending on the nature of the host bacteria. The methionine can be cleaved in vivo in the bacterial host cell or remain intact. Such a tri-hybrid melanoma antigen can be expressed in  E. coli  and obtained from inclusion bodies by methods well known in the art.  
         [0028]    To assist in purification, tri-hybrid proteins of the invention can be expressed with fused “tag” sequences. For example, the tri-hybrid melanoma antigen encoding DNA sequence can be cloned in an expression vector that provides for production of the tri-hybrid melanoma antigen linked to an N-terminal His tag sequence. The His tag allows purification by metal chelation chromatography. In certain embodiments, the His tag can be cleaved from the tri-hybrid melanoma antigen after purification. Alternatively, tag sequences that provide for affinity purification can be used. In a preferred embodiment, a tri-hybrid melanoma antigen is expressed from pET-28a(+), purified by metal chelation chromatography, and the His tag removed by specific proteolysis at the thrombin cleavage site.  
         [0029]    The invention provides novel tri-hybrid melanoma antigens that are particularly useful as vaccines for inducing immune responses effective for treating, inhibiting, and preventing cancers and precancers. Of particular interest are tri-hybrid melanoma antigens that induce anti-tumor immune responses in patients with melanoma. Accordingly, the present tri-hybrid melanoma antigens can be administered for prophylactic and/or therapeutic treatments of various conditions. Treatment, in the context of the present invention, is intended to encompass inhibiting, slowing, or reversing the progress of the underlying condition, ameliorating clinical symptoms of a condition or preventing the appearance of clinical symptoms of the condition.  
         [0030]    The term “melanoma” includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocyte related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma, invasive melanoma and familial atypical mole and melanoma (FAM-M) syndrome.  
         [0031]    Many methods suitable for administering the tri-hybrid melanoma antigen are known in the art. For example, the tri-hybrid melanoma antigen can be administered in soluble form. As another example, autologous mammalian cells capable of expressing the tri-hybrid melanoma antigen can be administered. As another example, virus having tri-hybrid melanoma antigen on its surface can be administered. As yet another example, virus carrying nucleic acid encoding the tri-hybrid melanoma antigen can be administered. As still another example, naked DNA or other nucleic acid encoding the tri-hybrid melanoma antigen can be administered.  
         [0032]    Tri-hybrid melanoma antigens can be administered alone, combined with adjuvants, linked to helper (carrier) peptides, proteins, lipids or liposomes, or pulsed onto purified antigen presenting cells (APCs) and the antigen presenting cells used for immunization. Adjuvants for use in immunization and other treatment methods include, for example, RIBI Detox (Ribi Immunochemical), QS2 1, CRIS-2 1, alum, BCG and incomplete Freund&#39;s adjuvant. For test animals, adjuvants further include complete Freund&#39;s adjuvant and others commonly used in the art. Tri-hybrid melanoma antigens can be also be complexed with heat shock binding proteins. APCs are generally eukaryotic cells with major histocompatibility complex (MHC), either class I or class II, gene products at their cell surface. Some examples of APCs that can be used in the present invention include DC, as well as macrophages, preferably MHC class II positive macrophages, monocytes, preferably MHC class II positive monocytes, and lymphocytes. See generally U.S. Pat. No. 5,597,563. It should be appreciated that such administration can be carried out before, simultaneously with, or after administration of the novel tri-hybrid melanoma antigens.  
         [0033]    Tri-hybrid melanoma antigens can be administered as DNA vaccines. DNA vaccines can comprise “naked” DNA encoding tri-hybrid melanoma antigens. Preferably, the tri-hybrid melanoma antigen-encoding DNA is taken up by host cells and expressed polypeptides are efficiently presented to the immune system. For example, naked DNA can be injected intradermally or intramuscularly or linked to lipids. Preferably, vaccines comprising tri-hybrid melanoma antigen injected directly into muscle or into the skin raise both cellular and humoral immune reactions to encoded antigens. See, for example, U.S. Pat. No. 5,831,016, Gregersen, Naturwissenschaften, 88(12):504-13 (December 2001), and Wang et al., Expert Opin. Biol. Ther., 1(2):277-90 (March 2001) for methods of preparation and use of DNA vaccines. Vaccines can comprise non-viable DNA vectors comprising DNA encoding tri-hybrid melanoma antigen of the present invention. Non-viable DNA vectors have the advantage of ease of preparation and safety of administration. DNA sequences encoding tri-hybrid melanoma antigens of the present invention can be administered using a gene gun in amounts to elicit a cellular response against a cancer cell. Nanogram quantities can be useful for such purposes.  
         [0034]    DNA encoding tri-hybrid melanoma antigens can also be expressed by bacteria or from recombinant viruses upon infection of host cells of the patient. Such bacterial or viral vectors can be designed to also express co-immunostimulatory molecules which enhance an immune response. Co-immunostimulatory molecules include, for example, IL-2, IL-6, IL-10, IL-12, and γ-interferon (IFN-γ). Co-immunostimulatory molecules can be selected so as to favor humoral immune responses or cytotoxic immune responses.  
         [0035]    DNA encoding tri-hybrid melanoma antigens of the present invention can also be used to create genetically modified immune cells capable of recognizing human tumor antigens. Such genetically modified immune cells can be particularly effective in, for example, mediating the regression of cancer in selected patients with metastatic melanoma. Techniques by which human lymphocytes are sensitized in vitro to tumor antigen peptides presented on antigen presenting cells are known in the art. By repetitive in vitro stimulation cells can be derived with a far greater capacity to recognize and respond to human tumor antigens. Thus by repeated in vitro sensitization with the tumor antigen of the present invention, lymphocytes can be derived with increased potency. The cells to be sensitized can be obtained from the subject to be treated or can be MHC matched cells from other sources. Adoptive transfer of these cells into the subject to be treated can result in increased effectiveness in mediating tumor regression in vivo.  
         [0036]    Tri-hybrid tumor antigens can be administered via one or more of several routes including, but not limited to, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, intrapleural, intrauterine, rectal, vaginal, topical, intratumor and the like.  
         [0037]    Administration can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be by nasal sprays, for example, or suppositories. For oral administration, the tumor antigen, cancer peptides or variants thereof are formulated into conventional oral administration forms such as capsules, tablets and tonics.  
         [0038]    It is understood that the tri-hybrid melanoma antigens of the present invention, where used in an animal for the purpose of prophylaxis or treatment, can be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins. The compositions of the injection can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.  
         [0039]    The compositions of this invention can be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.  
         [0040]    Such compositions of the present invention are prepared in a manner well known in the pharmaceutical art. In making the composition the active ingredient will usually be mixed with a carrier, or diluted by a carrier and/or enclosed within a carrier which can, for example, be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, excipient or medium for the active ingredient. Thus, the composition can be in the form of tablets, lozenges, sachets, cachets, elixirs, suspensions, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, injection solutions, suspensions, sterile packaged powders and as a topical patch.  
         [0041]    It should be appreciated that the immunogen of the present invention can be administered to any suitable animal. For example, the animal is preferably a mammal, such as a rabbit, rat, or mouse. More preferably, the animal is a human.  
         [0042]    The tri-hybrid tumor antigen according to the present invention is preferably provided in a therapeutically effective amount. Preferably, the dose is effective to prime, stimulate and/or cause the clonal expansion of cancer antigen specific B and T lymphocytes, which in turn are capable of preventing, inhibiting, or treating cancer in the recipient.  
         [0043]    Therapeutically effective doses can be determined by those skilled in the relevant arts via clinical studies. Therapeutically effective doses can also be determined by in appropriate animal models, then extrapolated to humans using known techniques. For example, for systemic administration, the amount of compound administered per unit body weight determined in rats can easily be applied to humans.  
         [0044]    Therapeutically effective doses will vary, depending on such factors as the weight and condition of the patient, the type of melanoma or other cancer to be treated, inhibited, or prevented, and the method of administration.  
         [0045]    The dosage of tri-hybrid tumor antigen for a human can be at least about 1 pg per Kg of body weight. A range of from about 1 ng per Kg body weight to about 100 mg per Kg body weight is preferred. More preferably, the amount administered can be at least about 1 □g per Kg body weight to about 1 mg body weight.  
         [0046]    The dose is administered at least once and can be provided as a bolus or a continuous administration. Multiple administrations of the dose over a period of several weeks to months can be preferable. Where multiple doses are provided, the dosage amount and formulation can be the same or can differ among doses.  
         [0047]    Effective treatment of melanoma or other cancer or pre-malignant lesion can be measured by many parameters known in the art, including, but not limited to, the decrease of tumor burden, increase in humoral immune response, changes in serum tumor markers, and increase in cytotoxic or cell-mediated immune responses. As more specific examples, antibody levels and CTL levels can be measured. Still other examples of criteria that can be used to evaluate effective treatment include standard World Health Organization (WHO) criteria for tumor response. Effective prevention of melanoma can be measured by many parameters known in the art, including, but not limited to, the decrease of incidence in a treated population compared to an untreated population. Such criteria can be measured by methods known in the art.  
         [0048]    Accordingly, the present invention can be used in vivo and in vitro for investigative, diagnostic, prophylactic, or treatment methods, which are well known in the art.  
         [0049]    Of course, it is to be understood and expected that variations in the principles of the invention herein disclosed can be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention.  
         [0050]    All references mentioned herein are incorporated in their entirety.  
       EXAMPLES  
       [0051]    The examples that follow further illustrate the invention, but should not be construed to limit the scope in any way. Detailed descriptions of conventional methods, such as those employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids, the introduction of plasmids into host cells, and the expression and determination thereof of genes and gene products can be obtained from numerous publications, including Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press.  
         [0052]    General Methods  
         [0053]    Cells and Animals  
         [0054]    The mouse melanoma cell line B16BL6 was kindly provided by Dr. Isaiah Fidler (M. D. Anderson Cancer Center, Houston); B16BL6 expression of TRP-1, TRP-2, and tyrosinase was determined by RT-PCR and Western analysis. The EL4 cell line was obtained from American Type Culture Collection (Manassas, Va.). C57BL/6 and C57BL/6 with deficiencies in MHC class I, or MHC class II, or FcR were purchased from Jackson Laboratory (Bar Harbor, Me.). All cell lines were maintained in RPMI 1640 (Gibco BRL, Gaithersburg, Md.) with 10% heat-inactivated fetal bovine serum and without antibiotics and were routinely tested for Mycoplasma contamination and were negative.  
         [0055]    All experiments and procedures were performed in accordance with the United States Department of Agriculture and Human Services, and NIH policies regarding the use of laboratory animals.  
         [0056]    Generation of a DNA Fragment Containing hTRP-1, hTRP-2, and h-Tyrosinase  
         [0057]    To generate a chimeric DNA fragment, three pairs of primers were designed. Primer pair one used hgp75 as template, primer pair two used hTRP-2 as template, and primer pair three used h-tyrosinase as template. Pair one, termed htyrF-1 and htyrF-2, generated a DNA fragment containing a soluble hgp75, four-glycines linker and the 5′ portion of hTRP-2 (htyrF-1 (SEQ ID NO:1): 5′ gccgaatcca tgagtgctcc taaactcctc 3′ and htyrF-2 (SEQ ID NO:2): 5′ cgtcatgcag actcggggga actgccctcc gccaccctca ggtacactaa actcccgact tgg 3′). Pair two, termed htyr-3 and htyr-4, cover the 3′ portion of hgp75, the soluble portion of hTRP-2, four glycines linker, and the 5′ portion of h-tyrosinase (htyrF-3 (SEQ ID NO:3): 5′ ccaagtcggg agtttagtgt acctgagggt ggcggagggc agttcccccg agtctgcatg acg 3′ and htyrF-4 (SEQ ID NO:4): 5′ ggagacacag gctctaggga aatgtccacc cccgccagtt gtgggccaac ctggagtttc 3′). Pair three, termed htyr-5 and htyr-6, PCR-cloned a fragment containing the 3′ portion of h-tyrosinase, the soluble portion of h-tyrosinase, and the stop codon (htyrF-5 (SEQ ID NO:5): 5′ gaaactccag gttggcccac aactggcggg ggtggacatt tccctagagc ctgtgtctcc 3′ and htyr-6 (SEQ ID NO:6): 5′ gcggcgctcg agctatgacc agatccgact cgcttg 3′). Each PCR reaction resulted in a 1.4 kb fragment (PCR product one). The resulting three PCR products were then mixed and underwent 10 cycle PCR reactions (PCR product two). Finally, chimeric DNA was generated with primers htyr-1 and htyr-6 (PCR product three). PCR product three was about 4.1 kb and digested with EcoR I and Xho I before cloning into pET28a(+) (Novogen, Madison, Wis.).  
         [0058]    Expression and Purification of Recombinant hTRPx3  
         [0059]    [0059] E. coli  BL21 (Novogen, Madison, Wis.) transformed with pET28a(+) containing hTRPx3 cDNA were grown to a cell density of 0.6 (A 600  nm) at 37° C. in shake flasks. The lac promoter was induced by addition of IPTG to a final concentration of 1 mM, and cells were grown for an additional 4 hrs at 37° C. The cells were harvested by centrifugation, resuspended in PBS and disrupted with a Cell Disrupter (Constant Systems Ltd, Warwick, UK). The insoluble fraction of the  E. coli  homogenate, which contained recombinant inclusion bodies, was harvested by centrifugation (10,000 g, 4° C., 30 min). The pellets were dissolved in a solution containing 8M urea, 50 mM Tris, and 5 mM imidazole at pH 8.0. The recombinant hTRPx3 were affinity purified using Toyopearl His binding resin according to manufacturer&#39;s instruction (TosoHaas, Montgomeryville, Pa.). The recombinant human TRP-1, TRP-2, tyrosinase was expressed and purified in a similar manner.  
         [0060]    Western blots were performed to ascertain that the recombinant hTRPx3 was recognized by antibodies specific for hTRP-1, hTRP-2, and h-tyrosinase. The purified hTRPx3 protein was run on 12% SDS-PAGE and transferred to PVDF membranes (Novex, San Diego, Calif.). The membranes were blocked overnight at 4° C. in 5% non-fat dry milk (blocking buffer) and probed with mouse sera specifically for individual antigens diluted 1:500 in PBS-0.2% Tween-20 at room temperature for 1 hr with gentle agitation. Blots were then washed with PBS-0.2%Tween-20 solution and incubated with peroxidase-conjugated goat anti-mouse IgG (Biosource, Camarillo, Calif.) diluted in PBS-0.2% Tween-20 (1:5000) for 1 hr. Blots were washed extensively as above and detected via ELC Western blotting reagents (Amersham Pharmacia Biotech, Little Chalfont, UK). Mouse sera specific for hTRP-1, hTRP-2, and h-tyrosinase were generated from mice immunized with individual proteins.  
         [0061]    Enzyme-Linked Immunosorbent Assay (ELISA)  
         [0062]    An indirect ELISA was developed to detect antibody responses. Purified human protein antigens, 50 ng in 50 μl PBS buffer, was added to each well of a 96-well Immunolon-2 plate (Dynex Technologies, Chantilly, Va.) and incubated overnight at 4° C. Plates were washed twice with PBS-0.2% Tween-20 and then incubated in blocking buffer (PBS containing 5% non-fat dry milk) at room temperature for 2 hrs. Mouse sera diluted at different concentrations in PBS-0.2% Tween-20 were added to plates and incubated at room temperature for 2 hrs. Wells were washed three times as above and then 100 μl peroxidase conjugated goat anti-mouse IgG (Biosource, Camarillo, Calif.) diluted 1:5000 in PBS-0.2% Tween-20 was added and incubated for 1 hr at room temperature. Wells then were washed three times and the peroxidase substrate TMB (KLP, Gaithersburg, Md.) was added. The absorbance at 450 nm was read on a kinetic microplate plate reader (Molecular Devices, Sunnyvale, Calif.).  
         [0063]    IFNγ Release Assay  
         [0064]    Splenocytes (4×10 5 ) were harvested one week after the last immunization and cultured with 10 mg/ml recombinant proteins in a total volume of 2 ml of RPMI 1640 with 10% fetal bovine serum in a 24 well plate for 72 hrs. The supernatant were harvested and tested for IFNγ using ELISA kits (Research Diagnostics Inc., Flanders, N.J.).  
         [0065]    ELISPOT Assay  
         [0066]    To conduct ELISPOT assay, 1×10 4  fresh isolated spleen cells from each vaccinated mice group were added to each well of 96 well plate along with 20 IU/ml IL-2. Cells were incubated at 37° C. for 24 hrs either with B16 or control EL4 cells. After culture, the plate was washed and then followed by incubation with 5 mg/ml biotinylated IFNγ antibody (clone XMG1.2, PharMingen) in 50 ml in PBS at 4° C. overnight. After washing six times, 1.25 mg/ml avidin-alkalinephosphatase (Sigma, St. Louis, Mo.) in 50 ml of PBS were added and incubated for 2 hrs at room temperature. After washing, spots were developed by adding 50 ml of 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution (Boehringer Mannheim, Indianapolis, Ind.) and incubated at room temperature for 1 hr. The spots were counted using a dissecting microscope.  
         [0067]    Immunization and Tumor Protection Assay  
         [0068]    One hundred micrograms of purified hTRPx3 was emulsified in 100 □l complete Freund&#39;s adjuvant (CFA) for each injection. C57BL/6 mice were vaccinated subcutaneously every 10 days for five times and bled one week after boosting. To compare different adjuvant, some mice were immunized with hTRPx3/BCG or hTRPx3/GMCSF intradermally. Unless stated, all results were from mice vaccinated with hTRPx3/CFA. For melanoma protection assay, mice were injected intravenously through the tail vein with 5×10 4  B16BL6 cells 10 days after last immunization. Mice were sacrificed and lungs were removed 20 days after B16 melanoma challenge. The surface lung metastases were scored and counted under a dissecting microscope. Statistical analysis of surface lung metastases was performed using student t test.  
       Example 1  
       [0069]    The present example demonstrates construction and production of a tri-hybrid melanoma antigen, recombinant hTRPx3 protein.  
         [0070]    To generate a chimeric hTRPx3 molecule, a DNA fragment encoding human TRP-1, TRP-2, and tyrosinase were made using PCR primer pairs as shown above (FIG. 1). Each PCR resulted a 1.4 kb fragment (PCR product one). The resulting three PCR products were then mixed and underwent a 10-cycle PCR reaction (PCR product two). Finally, PCR product two was amplified with primers htyr-1 and htyr-6 (PCR product three). PCR product three was about 4.1 kb and was digested with EcoR I and Xho I before cloning into pET28 (Novogen, bacteria expression vector). The final product was confirmed by sequencing. The coding sequence of the cloned tri-hybrid nucleotide sequences was determined by standard techniques and is given by SEQ ID NO:7. The amino acid sequence of the translation product is given by SEQ ID NO:8.  
         [0071]    Induction of BL21  E. coli  cells containing plasmids encoding human hTRPx3 genes resulted in expression of 155-kDa-fusion proteins in insoluble forms (FIG. 2A). The supernatant fraction (i.e. the soluble protein) from cell lysates showed little amounts of hTRPx3 proteins in soluble form. No inhibition of cell growth during the induction was observed, indicating that human TRPx3 was not toxic when expressed in the fusion proteins. The level of expressed proteins could be increased if cells were induced at 37° C. due to the formation of inclusion bodies. The level of the expressed TRP1 protein was about 5 mg/L. The expressed hTRPx3 proteins were conformed by Western analysis using anti-sera specific for either TRP-1, or TRP-2 or tyrosinase (FIG. 2B).  
       Example 2  
       [0072]    The present example demonstrates induced of a humoral immune response following immunization with a tri-hybrid melanoma antigen, hTRPx3 protein.  
         [0073]    Protein immunization with hTRPx3 protein was performed on 10 mice per group. One week after last boost, serum samples were tested for their ability to react with the purified individual antigens on ELISA plates (FIG. 3A). A majority of immunized mice developed antibody responses against individual proteins and hTRPx3 (27/30).  
         [0074]    To determine the subtypes of antibodies involved in these responses, antibody subtyping was performed on the ELISA plates (FIG. 3B). Briefly, the immune plates were coded with recombinant hTRPx3 and then incubated with sera from immunized mice. The bound antibodies were determined by second antibodies specific for mouse immunoglobulin IgG 1 , IgG 2a , IgG 2b , IgG 3 , and IgM. Antibodies against hTRPx3 were IgG subclass; predominantly IgG 1  and IgG 2a  (IgG 2a /IgG 1 =0.56) suggesting that Th1 and Th2 responses were induced.  
       Example 3  
       [0075]    The present example demonstrates that immunization with a tri-hybrid melanoma antigen, hTRPx3 protein, induced a T cell immune response.  
         [0076]    To determine if T cells specific for individual antigens were induced in mice, splenocytes from hTRPx3 immunized mice were incubated with hTRP-1, hTRP-2, and tyrosinase protein respectively. The IFNγ released by the T cells in the supernatant was determined by standard IFNγ kits.  
         [0077]    The splenocytes from hTRPx3 immunized mice were found to release significantly higher amounts of IFNγ as compared to control mice following stimulation by antigens (8, 4, and 4-fold increase in IFNγ release upon hTRP-1, hTRP-2, and h-tyrosinase stimulation, respectively). Splenocytes from saline control mice also released IFNγ when stimulated with individual antigens, suggesting these recombinant proteins can have induced some T cell activation in vitro.  
         [0078]    B16 melanoma cells express all tyrosinase family members and MHC class I molecules. T cells specific for hTRPx3 should also react with syngeneic B16 cells. To confirm this, ELISPOT assays were conducted. Briefly, different concentrations of fresh isolated spleen cells from each vaccinated mice group were added to the well of ELISPOT plate. Cells were incubated either with B16 or EL4 control cells. The IFNγ released were captured by mAb to mouse IFNγ on ELISPOT plate.  
         [0079]    As shown in FIG. 5, splenocytes from hTRPx3 immunized mice release IFNγ upon B16 cell stimulation. Since B16 cell did not express MHC class II molecule, it is highly possible that CD8 T cells were responsible for these IFNγ spots.  
       Example 4  
       [0080]    The present example demonstrates that immunization with a tri-hybrid melanoma antigen, hTPRx3 protein, was useful in treating tumors in mammals.  
         [0081]    To determine the effects of autoimmunity to hTRPx3 on melanoma in vivo, a syngeneic mouse model was used. Ten days after the last booster, immunized C57BL/6 mice were injected intravenously (i.v.) with B16BL6 melanoma cells, which is a spontaneously occurring melanoma from C57BL/6 mouse.  
         [0082]    Mice immunized with recombinant hTRPx3 were significantly protected from lung metastases of B16BL16 melanoma (FIG. 6A). In comparison with control mice, hTRPx3 protein immunized mice had a significant less lung metastases, with 80% (p=0.001) fewer lung nodules.  
         [0083]    To determine the mechanisms of antitumor activity, tumor protection was evaluated following hTRPx3 immunization in MHC class I, class II, and FcR knock-out mice (FIG. 6B). Mice with deficiency in MHC class I have lost tumor protection. Deficiency in MHC class II molecules has no effect on antitumor activity in mice, suggesting that CD8 cells can play a key role in hTRPx3 mediated antitumor activity.  
         [0084]    hTRPx3 immunization was further tested using different adjuvants. Both BCG and GM-CSF have been reported as Th1 driven adjuvants (see, e.g., Disis et al., Blood, 88(1):202-10 (July 1996); Kumar et al., Immunology, 97(3):515-21 (July 1999)). Mice vaccinated with hTRPx3/BCG were protected from melanoma challenge similar to mice immunized with hTRPx3/CFA. Mice vaccinated with hTRPx3/GM-CSF were not protected from tumor challenge.  
     
       
       
         1 
         
           
             16  
           
           
             1  
             30  
             DNA  
             Artificial Sequence  
             
               Amplification Primer  
             
           
            1 

gccgaatcca tgagtgctcc taaactcctc                                      30 

 
           
             2  
             63  
             DNA  
             Artificial Sequence  
             
               Amplification primer  
             
           
            2 

cgtcatgcag actcggggga actgccctcc gccaccctca ggtacactaa actcccgact     60 

tgg                                                                   63 

 
           
             3  
             63  
             DNA  
             Artificial Sequence  
             
               Amplification primer  
             
           
            3 

ccaagtcggg agtttagtgt acctgagggt ggcggagggc agttcccccg agtctgcatg     60 

acg                                                                   63 

 
           
             4  
             60  
             DNA  
             Artificial Sequence  
             
               Amplification primer  
             
           
            4 

ggagacacag gctctaggga aatgtccacc cccgccagtt gtgggccaac ctggagtttc     60 

 
           
             5  
             60  
             DNA  
             Artificial Sequence  
             
               Amplification primer  
             
           
            5 

gaaactccag gttggcccac aactggcggg ggtggacatt tccctagagc ctgtgtctcc     60 

 
           
             6  
             36  
             DNA  
             Artificial Sequence  
             
               Amplification primer  
             
           
            6 

gcggcgctcg agctatgacc agatccgact cgcttg                               36 

 
           
             7  
             4176  
             DNA  
             Artificial Sequence  
             
               Tri-hybrid antigen encoding sequence  
             
           
            7 

atg agt gct cct aaa ctc ctc tct ctg ggc tgt atc ttc ttc ccc ttg       48 
Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 
                  5                  10                  15 

cta ctt ttt cag cag gcc cgg gct caa ttc cca aga cag tgt gcc act       96 
Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 
             20                  25                  30 

gtt gag gct ttg aga agt ggt atg tgt tgc cca gac ctg tcc cct gtg      144 
Val Glu Ala Leu Arg Ser Gly Met Cys Cys Pro Asp Leu Ser Pro Val 
         35                  40                  45 

tct ggg cct ggg aca gac cgc tgt ggc tca tca tca ggg agg ggc aga      192 
Ser Gly Pro Gly Thr Asp Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 
     50                  55                  60 

tgt gag gca gtg act gca gac tcc cgg ccc cac agc cct cag tat ccc      240 
Cys Glu Ala Val Thr Ala Asp Ser Arg Pro His Ser Pro Gln Tyr Pro 
 65                  70                  75                  80 

cat gat ggc aga gat gat cgg gag gtc tgg ccc ttg cgc ttc ttc aat      288 
His Asp Gly Arg Asp Asp Arg Glu Val Trp Pro Leu Arg Phe Phe Asn 
                 85                  90                  95 

agg aca tgt cac tgc aac ggc aat ttc tca gga cac aac tgt ggg acg      336 
Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 
            100                 105                 110 

tgc cgt cct ggc tgg aga gga gct gcc tgt gac cag agg gtt ctc ata      384 
Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Asp Gln Arg Val Leu Ile 
        115                 120                 125 

gtc agg aga aat ctt ctg gac tta agt aaa gaa gaa aag aac cac ttt      432 
Val Arg Arg Asn Leu Leu Asp Leu Ser Lys Glu Glu Lys Asn His Phe 
    130                 135                 140 

gtc cgg gcc ctg gat atg gca aag cgc aca act cac cct tta ttt gtc      480 
Val Arg Ala Leu Asp Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 
145                 150                 155                 160 

att gcc acc agg aga tca gaa gaa ata ctg ggg cca gat ggc aac acg      528 
Ile Ala Thr Arg Arg Ser Glu Glu Ile Leu Gly Pro Asp Gly Asn Thr 
                165                 170                 175 

cca caa ttt gag aac att tcc att tat aac tac ttt gtt tgg aca cac      576 
Pro Gln Phe Glu Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 
            180                 185                 190 

tat tac tca gtc aaa aag act ttc ctt ggg gta gga cag gaa agc ttt      624 
Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Glu Ser Phe 
        195                 200                 205 

ggt gaa gtg gat ttc tct cat gag gga cca gct ttt ctc aca tgg cac      672 
Gly Glu Val Asp Phe Ser His Glu Gly Pro Ala Phe Leu Thr Trp His 
    210                 215                 220 

agg tac cac ctc ctg cgt ctg gag aaa gac atg cag gaa atg ttg caa      720 
Arg Tyr His Leu Leu Arg Leu Glu Lys Asp Met Gln Glu Met Leu Gln 
225                 230                 235                 240 

gag cct tct ttc tcc ctt cct tac tgg aat ttt gca acg ggg aaa aat      768 
Glu Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 
                245                 250                 255 

gtc tgt gat atc tgc acg gat gac ttg atg gga tcc aga agc aac ttt      816 
Val Cys Asp Ile Cys Thr Asp Asp Leu Met Gly Ser Arg Ser Asn Phe 
            260                 265                 270 

gat tcc act cta ata agc cca aac tct gtc ttt tct caa tgg cga gtg      864 
Asp Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 
        275                 280                 285 

gtc tgt gac tcc ttg gaa gat tat gat acc ctg gga aca ctt tgt aac      912 
Val Cys Asp Ser Leu Glu Asp Tyr Asp Thr Leu Gly Thr Leu Cys Asn 
    290                 295                 300 

agc acc gag gat ggg cca att agg aga aat cca gct gga aat gtg gcc      960 
Ser Thr Glu Asp Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 
305                 310                 315                 320 

aga cca atg gtg caa cgt ctt cct gaa cca cag gat gtc gct cag tgc     1008 
Arg Pro Met Val Gln Arg Leu Pro Glu Pro Gln Asp Val Ala Gln Cys 
                325                 330                 335 

ttg gaa gtt ggt tta ttt gac acg cct cct ttt tat tcc aac tct aca     1056 
Leu Glu Val Gly Leu Phe Asp Thr Pro Pro Phe Tyr Ser Asn Ser Thr 
            340                 345                 350 

aac agt ttc cga aac aca gtg gaa ggt tac agt gac ccc acg gga aag     1104 
Asn Ser Phe Arg Asn Thr Val Glu His Asn Leu Ala His Leu Phe Leu 
        355                 360                 365 

tat gac cct gct gtt cga agt ctt cac aat ttg gct cat cta ttc ctg     1152 
Asn Gly Thr Gly Gly Gln Thr His Gly Tyr Ser Asp Pro Thr Gly Lys 
    370                 375                 380 

aat gga aca ggg gga caa acc cat ttg tct cca aat gat cct att ttt     1200 
Tyr Asp Pro Ala Val Arg Ser Leu Leu Ser Pro Asn Asp Pro Ile Phe 
385                 390                 395                 400 

gtc ctc ctg cac acc ttc aca gat gca gtc ttt gat gaa tgg ctg agg     1248 
Val Leu Leu His Thr Phe Thr Asp Ala Val Phe Asp Glu Trp Leu Arg 
                405                 410                 415 

aga tac aat gct gat ata tcc aca ttt cca ttg gaa aat gcc cct att     1296 
Arg Tyr Asn Ala Asp Ile Ser Thr Phe Pro Leu Glu Asn Ala Pro Ile 
            420                 425                 430 

gga cat aat aga caa tac aac atg gtg cca ttc tgg ccc cca gtc acc     1344 
Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 
        435                 440                 445 

aac aca gaa atg ttt gtt act gct cca gac aac ctg gga tac act tat     1392 
Asn Thr Glu Met Phe Val Thr Ala Pro Asp Asn Leu Gly Tyr Thr Tyr 
    450                 455                 460 

gaa att caa tgg cca agt cgg gag ttt agt gta cct gag ggt ggc gga     1440 
Glu Ile Gln Trp Pro Ser Arg Glu Phe Ser Val Pro Glu Gly Gly Gly 
465                 470                 475                 480 

ggg cag ttc ccc cga gtc tgc atg acg gtg gac agc cta gtg aac aag     1488 
Gly Gln Phe Pro Arg Val Cys Met Thr Val Asp Ser Leu Val Asn Lys 
                485                 490                 495 

gag tgc tgc cca cgc ctg ggt gca gag tcg gcc aat gtc tgt ggc tct     1536 
Glu Cys Cys Pro Arg Leu Gly Ala Glu Ser Ala Asn Val Cys Gly Ser 
            500                 505                 510 

cag caa ggc cgg ggg cag tgc aca gag gtg cga gcc gac aca agg ccc     1584 
Gln Gln Gly Arg Gly Gln Cys Thr Glu Val Arg Ala Asp Thr Arg Pro 
        515                 520                 525 

tgg agt ggt ccc tac atc cta cga aac cag gat gac cgt gag ctg tgg     1632 
Trp Ser Gly Pro Tyr Ile Leu Arg Cys Lys Cys Thr Gly Asn Phe Ala 
    530                 535                 540 

cca aga aaa ttc ttc cac cgg acc tgc aag tgc aca gga aac ttt gcc     1680 
Gly Tyr Asn Cys Gly Asp Cys Lys Asn Gln Asp Asp Arg Glu Leu Trp 
545                 550                 555                 560 

ggc tat aat tgt gga gac tgc aag ttt ggc tgg acc ggt ccc aac tgc     1728 
Pro Arg Lys Phe Phe His Arg Thr Phe Gly Trp Thr Gly Pro Asn Cys 
                565                 570                 575 

gag cgg aag aaa cca cca gtg att cgg cag aac atc cat tcc ttg agt     1776 
Glu Arg Lys Lys Pro Pro Val Ile Arg Gln Asn Ile His Ser Leu Ser 
            580                 585                 590 

cct cag gaa aga gag cag ttc ttg ggc gcc tta gat ctc gcg aag aag     1824 
Pro Gln Glu Arg Glu Gln Phe Leu Gly Ala Leu Asp Leu Ala Lys Lys 
        595                 600                 605 

aga gta cac ccc gac tac gtg atc acc aca caa cac tgg ctg ggc ctg     1872 
Arg Val His Pro Asp Tyr Val Ile Thr Thr Gln His Trp Leu Gly Leu 
    610                 615                 620 

ctt ggg ccc aat gga acc cag ccg cag ttt gcc aac tgc agt gtt tat     1920 
Leu Gly Pro Asn Gly Thr Gln Pro Gln Phe Ala Asn Cys Ser Val Tyr 
625                 630                 635                 640 

gat ttt ttt gtg tgg ctc cat tat tat tct gtt aga gat aca tta tta     1968 
Asp Phe Phe Val Trp Leu His Tyr Tyr Ser Val Arg Asp Thr Leu Leu 
                645                 650                 655 

gga cca gga cgc ccc tac agg gcc ata gat ttc tca cat caa gga cct     2016 
Gly Pro Gly Arg Pro Tyr Arg Ala Ile Asp Phe Ser His Gln Gly Pro 
            660                 665                 670 

gca ttt gtt acc tgg cac cgg tac cat ttg ttg tgt ctg gaa aga gat     2064 
Ala Phe Val Thr Trp His Arg Tyr His Leu Leu Cys Leu Glu Arg Asp 
        675                 680                 685 

ctc cag cga ctc att ggc aat gag tct ttt gct ttg ccc tac tgg aac     2112 
Leu Gln Arg Leu Ile Gly Asn Glu Ser Phe Ala Leu Pro Tyr Trp Asn 
    690                 695                 700 

ttt gcc act ggg agg aac gag tgt gat gtg tgt aca gac cag ctg ttt     2160 
Phe Ala Thr Gly Arg Asn Glu Cys Asp Val Cys Thr Asp Gln Leu Phe 
705                 710                 715                 720 

ggg gca gcg aga cca gac gat ccg act ctg att agt cgg aac tca aga     2208 
Gly Ala Ala Arg Pro Asp Asp Pro Thr Leu Ile Ser Arg Asn Ser Arg 
                725                 730                 735 

ttc tcc agc tgg gaa act gtc tgt gat agc ttg gat gac tac aac cac     2256 
Phe Ser Ser Trp Glu Thr Val Cys Asp Ser Leu Asp Asp Tyr Asn His 
            740                 745                 750 

ctg gtc acc ttg tgc aat gga acc tat gaa ggt ttg ctg aga aga aat     2304 
Leu Val Thr Leu Cys Asn Gly Thr Tyr Glu Gly Leu Leu Arg Arg Asn 
        755                 760                 765 

caa atg gga aga aac agc atg aaa ttg cca acc tta aaa gac ata cga     2352 
Gln Met Gly Arg Asn Ser Met Lys Leu Pro Thr Leu Lys Asp Ile Arg 
    770                 775                 780 

gat tgc ctg tct ctc cag aag ttt gac aat cct ccc ttc ttc cag aac     2400 
Asp Cys Leu Ser Leu Gln Lys Phe Asp Asn Pro Pro Phe Phe Gln Asn 
785                 790                 795                 800 

tct acc ttc agt ttc agg aat gct ttg gaa ggg ttt gat aaa gca gat     2448 
Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu Gly Phe Asp Lys Ala Asp 
                805                 810                 815 

ggg act ctg gat tct caa gtg atg agc ctt cat aat ttg gtt cat tcc     2496 
Gly Thr Leu Asp Ser Gln Val Met Ser Leu His Asn Leu Val His Ser 
            820                 825                 830 

ttc ctg aac ggg aca aac gct ttg cca cat tca gcc gcc aat gat ccc     2544 
Phe Leu Asn Gly Thr Asn Ala Leu Pro His Ser Ala Ala Asn Asp Pro 
        835                 840                 845 

att ttt gtg gtt ctt cat tcc ttt act gat gcc atc ttt gat gag tgg     2592 
Ile Phe Val Val Leu His Ser Phe Thr Asp Ala Ile Phe Asp Glu Trp 
    850                 855                 860 

atg aaa aga ttt aat cct cct gca gat gcc tgg cct cag gag ctg gcc     2640 
Met Lys Arg Phe Asn Pro Pro Ala Asp Ala Trp Pro Gln Glu Leu Ala 
865                 870                 875                 880 

cct att ggt cac aat cgg atg tac aac atg gtt cct ttc ttc cct cca     2688 
Pro Ile Gly His Asn Arg Met Tyr Asn Met Val Pro Phe Phe Pro Pro 
                885                 890                 895 

gtg act aat gaa gaa ctc ttt tta acc tca gac caa ctt ggc tac agc     2736 
Val Thr Asn Glu Glu Leu Phe Leu Thr Ser Asp Gln Leu Gly Tyr Ser 
            900                 905                 910 

tat gcc atc gat ctg cca gtt tca gtt gaa gaa act cca ggt tgg ccc     2784 
Tyr Ala Ile Asp Leu Pro Val Ser Val Glu Glu Thr Pro Gly Trp Pro 
        915                 920                 925 

aca act ggc ggg ggt gga cat ttc cct aga gcc tgt gtc tcc tct aag     2832 
Thr Thr Gly Gly Gly Gly His Phe Pro Arg Ala Cys Val Ser Ser Lys 
    930                 935                 940 

aac ctg atg gag aag gaa tgc tgt cca ccg tgg agc ggg gac agg agt     2880 
Asn Leu Met Glu Lys Glu Cys Cys Pro Pro Trp Ser Gly Asp Arg Ser 
945                 950                 955                 960 

ccc tgt ggc cag ctt tca ggc aga ggt tcc tgt cag aat atc ctt ctg     2928 
Pro Cys Gly Gln Leu Ser Gly Arg Gly Ser Cys Gln Asn Ile Leu Leu 
                965                 970                 975 

tcc aat gca cca ctt ggg cct caa ttt ccc ttc aca ggg gtg gat gac     2976 
Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro Phe Thr Gly Val Asp Asp 
            980                 985                 990 

cgg gag tcg tgg cct tcc gtc ttt tat aat agg acc tgc cag tgc tct     3024 
Arg Glu Ser Trp Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Ser 
        995                 1000                1005 

ggc aac ttc atg gga ttc aac tgt gga aac tgc aag ttt ggc ttt tgg     3072 
Gly Asn Phe Met Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp 
    1010                1015                1020 

gga cca aac tgc aca gag aga cga ctc ttg gtg aga aga aac atc ttc     3120 
Gly Pro Asn Cys Thr Glu Arg Arg Leu Leu Val Arg Arg Asn Ile Phe 
1025                1030                1035                1040 

gat ttg agt gcc cca gag aag gac aaa ttt ttt gcc tac ctc act tta     3168 
Asp Leu Ser Ala Pro Glu Lys Asp Lys Phe Phe Ala Tyr Leu Thr Leu 
                1045                1050                1055 

gca aag cat acc atc agc tca gac tat gtc atc ccc ata ggg acc tat     3216 
Ala Lys His Thr Ile Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr 
            1060                1065                1070 

ggc caa atg aaa aat gga tca aca ccc atg ttt aac gac atc aat att     3264 
Gly Gln Met Lys Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile 
        1075                1080                1085 

tat gac ctc ttt gtc tgg atg cat tat tat gtg tca atg gat gca ctg     3312 
Tyr Asp Leu Phe Val Trp Met His Tyr Tyr Val Ser Met Asp Ala Leu 
    1090                1095                1100 

ctt ggg gga tct gaa atc tgg aga gac att gat ttt gcc cat gaa gca     3360 
Leu Gly Gly Ser Glu Ile Trp Arg Asp Ile Asp Phe Ala His Glu Ala 
1105                1110                1115                1120 

cca gct ttt ctg cct tgg cat aga ctc ttc ttg ttg cgg tgg gaa caa     3408 
Pro Ala Phe Leu Pro Trp His Arg Leu Phe Leu Leu Arg Trp Glu Gln 
                1125                1130                1135 

gaa atc cag aag ctg aca gga gat gaa aac ttc act att cca tat tgg     3456 
Glu Ile Gln Lys Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr Trp 
            1140                1145                1150 

gac tgg cgg gat gca gaa aag tgt gac att tgc aca gat gag tac atg     3504 
Asp Trp Arg Asp Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr Met 
        1155                1160                1165 

gga ggt cag cac ccc aca aat cct aac tta ctc agc cca gca tca ttc     3552 
Gly Gly Gln His Pro Thr Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe 
    1170                1175                1180 

ttc tcc tct tgg cag att gtc tgt agc cga ttg gag gag tac aac agc     3600 
Phe Ser Ser Trp Gln Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser 
1185                1190                1195                1200 

cat cag tct tta tgc aat gga acg ccc gag gga cct tta cgg cgt aat     3648 
His Gln Ser Leu Cys Asn Gly Thr Pro Glu Gly Pro Leu Arg Arg Asn 
                1205                1210                1215 

cct gga aac cat gac aaa tcc aga acc cca agg ctc ccc tct tca gct     3696 
Pro Gly Asn His Asp Lys Ser Arg Thr Pro Arg Leu Pro Ser Ser Ala 
            1220                1225                1230 

gat gta gaa ttt tgc ctg agt ttg acc caa tat gaa tct ggt tcc atg     3744 
Asp Val Glu Phe Cys Leu Ser Leu Thr Gln Tyr Glu Ser Gly Ser Met 
        1235                1240                1245 

gat aaa gct gcc aat ttc agc ttt aga aat aca ctg gaa gga ttt gct     3792 
Asp Lys Ala Ala Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly Phe Ala 
    1250                1255                1260 

agt cca ctt act ggg ata gcg gat gcc tct caa agc agc atg cac aat     3840 
Ser Pro Leu Thr Gly Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn 
1265                1270                1275                1280 

gcc ttg cac atc tat atg aat gga aca atg tcc cag gta cag gga tct     3888 
Ala Leu His Ile Tyr Met Asn Gly Thr Met Ser Gln Val Gln Gly Ser 
                1285                1290                1295 

gcc aac gat cct atc ttc ctt ctt cac cat gca ttt gtt gac agt att     3936 
Ala Asn Asp Pro Ile Phe Leu Leu His His Ala Phe Val Asp Ser Ile 
            1300                1305                1310 

ttt gag cag tgg ctc cga agg cac cgt cct ctt caa gaa gtt tat cca     3984 
Phe Glu Gln Trp Leu Arg Arg His Arg Pro Leu Gln Glu Val Tyr Pro 
        1315                1320                1325 

gaa gcc aat gca ccc att gga cat aac cgg gaa tcc tac atg gtt cct     4032 
Glu Ala Asn Ala Pro Ile Gly His Asn Arg Glu Ser Tyr Met Val Pro 
    1330                1335                1340 

ttt ata cca ctg tac aga aat ggt gat ttc ttt att tca tcc aaa gat     4080 
Phe Ile Pro Leu Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp 
1345                1350                1355                1360 

ctg ggc tat gac tat agc tat cta caa gat tca gac cca gac tct ttt     4128 
Leu Gly Tyr Asp Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe 
                1365                1370                1375 

caa gac tac att aag tcc tat ttg gaa caa gcg agt cgg atc tgg tca    4176 
Gln Asp Tyr Ile Lys Ser Tyr Leu Glu Gln Ala Ser Arg Ile Trp Ser 
            1380                1385                1390 

 
           
             8  
             1392  
             PRT  
             Artificial Sequence  
             
               Tri-hybrid antigen  
             
           
            8 

Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 
                  5                  10                  15 

Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 
             20                  25                  30 

Val Glu Ala Leu Arg Ser Gly Met Cys Cys Pro Asp Leu Ser Pro Val 
         35                  40                  45 

Ser Gly Pro Gly Thr Asp Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 
     50                  55                  60 

Cys Glu Ala Val Thr Ala Asp Ser Arg Pro His Ser Pro Gln Tyr Pro 
 65                  70                  75                  80 

His Asp Gly Arg Asp Asp Arg Glu Val Trp Pro Leu Arg Phe Phe Asn 
                 85                  90                  95 

Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 
            100                 105                 110 

Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Asp Gln Arg Val Leu Ile 
        115                 120                 125 

Val Arg Arg Asn Leu Leu Asp Leu Ser Lys Glu Glu Lys Asn His Phe 
    130                 135                 140 

Val Arg Ala Leu Asp Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 
145                 150                 155                 160 

Ile Ala Thr Arg Arg Ser Glu Glu Ile Leu Gly Pro Asp Gly Asn Thr 
                165                 170                 175 

Pro Gln Phe Glu Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 
            180                 185                 190 

Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Glu Ser Phe 
        195                 200                 205 

Gly Glu Val Asp Phe Ser His Glu Gly Pro Ala Phe Leu Thr Trp His 
    210                 215                 220 

Arg Tyr His Leu Leu Arg Leu Glu Lys Asp Met Gln Glu Met Leu Gln 
225                 230                 235                 240 

Glu Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 
                245                 250                 255 

Val Cys Asp Ile Cys Thr Asp Asp Leu Met Gly Ser Arg Ser Asn Phe 
            260                 265                 270 

Asp Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 
        275                 280                 285 

Val Cys Asp Ser Leu Glu Asp Tyr Asp Thr Leu Gly Thr Leu Cys Asn 
    290                 295                 300 

Ser Thr Glu Asp Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 
305                 310                 315                 320 

Arg Pro Met Val Gln Arg Leu Pro Glu Pro Gln Asp Val Ala Gln Cys 
                325                 330                 335 

Leu Glu Val Gly Leu Phe Asp Thr Pro Pro Phe Tyr Ser Asn Ser Thr 
            340                 345                 350 

Asn Ser Phe Arg Asn Thr Val Glu Gly Tyr Ser Asp Pro Thr Gly Lys 
        355                 360                 365 

Tyr Asp Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu 
    370                 375                 380 

Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Asp Pro Ile Phe 
385                 390                 395                 400 

Val Leu Leu His Thr Phe Thr Asp Ala Val Phe Asp Glu Trp Leu Arg 
                405                 410                 415 

Arg Tyr Asn Ala Asp Ile Ser Thr Phe Pro Leu Glu Asn Ala Pro Ile 
            420                 425                 430 

Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 
        435                 440                 445 

Asn Thr Glu Met Phe Val Thr Ala Pro Asp Asn Leu Gly Tyr Thr Tyr 
    450                 455                 460 

Glu Ile Gln Trp Pro Ser Arg Glu Phe Ser Val Pro Glu Gly Gly Gly 
465                 470                 475                 480 

Gly Gln Phe Pro Arg Val Cys Met Thr Val Asp Ser Leu Val Asn Lys 
                485                 490                 495 

Glu Cys Cys Pro Arg Leu Gly Ala Glu Ser Ala Asn Val Cys Gly Ser 
            500                 505                 510 

Gln Gln Gly Arg Gly Gln Cys Thr Glu Val Arg Ala Asp Thr Arg Pro 
        515                 520                 525 

Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln Asp Asp Arg Glu Leu Trp 
    530                 535                 540 

Pro Arg Lys Phe Phe His Arg Thr Cys Lys Cys Thr Gly Asn Phe Ala 
545                 550                 555                 560 

Gly Tyr Asn Cys Gly Asp Cys Lys Phe Gly Trp Thr Gly Pro Asn Cys 
                565                 570                 575 

Glu Arg Lys Lys Pro Pro Val Ile Arg Gln Asn Ile His Ser Leu Ser 
            580                 585                 590 

Pro Gln Glu Arg Glu Gln Phe Leu Gly Ala Leu Asp Leu Ala Lys Lys 
        595                 600                 605 

Arg Val His Pro Asp Tyr Val Ile Thr Thr Gln His Trp Leu Gly Leu 
    610                 615                 620 

Leu Gly Pro Asn Gly Thr Gln Pro Gln Phe Ala Asn Cys Ser Val Tyr 
625                 630                 635                 640 

Asp Phe Phe Val Trp Leu His Tyr Tyr Ser Val Arg Asp Thr Leu Leu 
                645                 650                 655 

Gly Pro Gly Arg Pro Tyr Arg Ala Ile Asp Phe Ser His Gln Gly Pro 
            660                 665                 670 

Ala Phe Val Thr Trp His Arg Tyr His Leu Leu Cys Leu Glu Arg Asp 
        675                 680                 685 

Leu Gln Arg Leu Ile Gly Asn Glu Ser Phe Ala Leu Pro Tyr Trp Asn 
    690                 695                 700 

Phe Ala Thr Gly Arg Asn Glu Cys Asp Val Cys Thr Asp Gln Leu Phe 
705                 710                 715                 720 

Gly Ala Ala Arg Pro Asp Asp Pro Thr Leu Ile Ser Arg Asn Ser Arg 
                725                 730                 735 

Phe Ser Ser Trp Glu Thr Val Cys Asp Ser Leu Asp Asp Tyr Asn His 
            740                 745                 750 

Leu Val Thr Leu Cys Asn Gly Thr Tyr Glu Gly Leu Leu Arg Arg Asn 
        755                 760                 765 

Gln Met Gly Arg Asn Ser Met Lys Leu Pro Thr Leu Lys Asp Ile Arg 
    770                 775                 780 

Asp Cys Leu Ser Leu Gln Lys Phe Asp Asn Pro Pro Phe Phe Gln Asn 
785                 790                 795                 800 

Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu Gly Phe Asp Lys Ala Asp 
                805                 810                 815 

Gly Thr Leu Asp Ser Gln Val Met Ser Leu His Asn Leu Val His Ser 
            820                 825                 830 

Phe Leu Asn Gly Thr Asn Ala Leu Pro His Ser Ala Ala Asn Asp Pro 
        835                 840                 845 

Ile Phe Val Val Leu His Ser Phe Thr Asp Ala Ile Phe Asp Glu Trp 
    850                 855                 860 

Met Lys Arg Phe Asn Pro Pro Ala Asp Ala Trp Pro Gln Glu Leu Ala 
865                 870                 875                 880 

Pro Ile Gly His Asn Arg Met Tyr Asn Met Val Pro Phe Phe Pro Pro 
                885                 890                 895 

Val Thr Asn Glu Glu Leu Phe Leu Thr Ser Asp Gln Leu Gly Tyr Ser 
            900                 905                 910 

Tyr Ala Ile Asp Leu Pro Val Ser Val Glu Glu Thr Pro Gly Trp Pro 
        915                 920                 925 

Thr Thr Gly Gly Gly Gly His Phe Pro Arg Ala Cys Val Ser Ser Lys 
    930                 935                 940 

Asn Leu Met Glu Lys Glu Cys Cys Pro Pro Trp Ser Gly Asp Arg Ser 
945                 950                 955                 960 

Pro Cys Gly Gln Leu Ser Gly Arg Gly Ser Cys Gln Asn Ile Leu Leu 
                965                 970                 975 

Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro Phe Thr Gly Val Asp Asp 
            980                 985                 990 

Arg Glu Ser Trp Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Ser 
        995                 1000                1005 

Gly Asn Phe Met Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp 
    1010                1015                1020 

Gly Pro Asn Cys Thr Glu Arg Arg Leu Leu Val Arg Arg Asn Ile Phe 
1025                1030                1035                1040 

Asp Leu Ser Ala Pro Glu Lys Asp Lys Phe Phe Ala Tyr Leu Thr Leu 
                1045                1050                1055 

Ala Lys His Thr Ile Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr 
            1060                1065                1070 

Gly Gln Met Lys Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile 
        1075                1080                1085 

Tyr Asp Leu Phe Val Trp Met His Tyr Tyr Val Ser Met Asp Ala Leu 
    1090                1095                1100 

Leu Gly Gly Ser Glu Ile Trp Arg Asp Ile Asp Phe Ala His Glu Ala 
1105                1110                1115                1120 

Pro Ala Phe Leu Pro Trp His Arg Leu Phe Leu Leu Arg Trp Glu Gln 
                1125                1130                1135 

Glu Ile Gln Lys Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr Trp 
            1140                1145                1150 

Asp Trp Arg Asp Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr Met 
        1155                1160                1165 

Gly Gly Gln His Pro Thr Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe 
    1170                1175                1180 

Phe Ser Ser Trp Gln Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser 
1185                1190                1195                1200 

His Gln Ser Leu Cys Asn Gly Thr Pro Glu Gly Pro Leu Arg Arg Asn 
                1205                1210                1215 

Pro Gly Asn His Asp Lys Ser Arg Thr Pro Arg Leu Pro Ser Ser Ala 
            1220                1225                1230 

Asp Val Glu Phe Cys Leu Ser Leu Thr Gln Tyr Glu Ser Gly Ser Met 
        1235                1240                1245 

Asp Lys Ala Ala Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly Phe Ala 
   1 250                1255                1260 

Ser Pro Leu Thr Gly Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn 
1265                1270                1275                1280 

Ala Leu His Ile Tyr Met Asn Gly Thr Met Ser Gln Val Gln Gly Ser 
                1285                1290                1295 

Ala Asn Asp Pro Ile Phe Leu Leu His His Ala Phe Val Asp Ser Ile 
            1300                1305                1310 

Phe Glu Gln Trp Leu Arg Arg His Arg Pro Leu Gln Glu Val Tyr Pro 
        1315                1320                1325 

Glu Ala Asn Ala Pro Ile Gly His Asn Arg Glu Ser Tyr Met Val Pro 
    1330                1335                1340 

Phe Ile Pro Leu Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp 
1345                1350                1355                1360 

Leu Gly Tyr Asp Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe 
                1365                1370                1375 

Gln Asp Tyr Ile Lys Ser Tyr Leu Glu Gln Ala Ser Arg Ile Trp Ser 
            1380                1385                1390 

 
           
             9  
             4104  
             DNA  
             Artificial Sequence  
             
               Tri-hybrid antigen coding sequence  
             
           
            9 

caa ttc cca aga cag tgt gcc act gtt gag gct ttg aga agt ggt atg       48 
Gln Phe Pro Arg Gln Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met 
                  5                  10                  15 

tgt tgc cca gac ctg tcc cct gtg tct ggg cct ggg aca gac cgc tgt       96 
Cys Cys Pro Asp Leu Ser Pro Val Ser Gly Pro Gly Thr Asp Arg Cys 
             20                  25                  30 

ggc tca tca tca ggg agg ggc aga tgt gag gca gtg act gca gac tcc      144 
Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser 
         35                  40                  45 

cgg ccc cac agc cct cag tat ccc cat gat ggc aga gat gat cgg gag      192 
Arg Pro His Ser Pro Gln Tyr Pro His Asp Gly Arg Asp Asp Arg Glu 
     50                  55                  60 

gtc tgg ccc ttg cgc ttc ttc aat agg aca tgt cac tgc aac ggc aat      240 
Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn 
 65                  70                  75                  80 

ttc tca gga cac aac tgt ggg acg tgc cgt cct ggc tgg aga gga gct      288 
Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala 
                 85                  90                  95 

gcc tgt gac cag agg gtt ctc ata gtc agg aga aat ctt ctg gac tta      336 
Ala Cys Asp Gln Arg Val Leu Ile Val Arg Arg Asn Leu Leu Asp Leu 
            100                 105                 110 

agt aaa gaa gaa aag aac cac ttt gtc cgg gcc ctg gat atg gca aag      384 
Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys 
        115                 120                 125 

cgc aca act cac cct tta ttt gtc att gcc acc agg aga tca gaa gaa      432 
Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu 
    130                 135                 140 

ata ctg ggg cca gat ggc aac acg cca caa ttt gag aac att tcc att      480 
Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile 
145                 150                 155                 160 

tat aac tac ttt gtt tgg aca cac tat tac tca gtc aaa aag act ttc      528 
Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe 
                165                 170                 175 

ctt ggg gta gga cag gaa agc ttt ggt gaa gtg gat ttc tct cat gag      576 
Leu Gly Val Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu 
            180                 185                 190 

gga cca gct ttt ctc aca tgg cac agg tac cac ctc ctg cgt ctg gag      624 
Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu 
        195                 200                 205 

aaa gac atg cag gaa atg ttg caa gag cct tct ttc tcc ctt cct tac      672 
Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr 
    210                 215                 220 

tgg aat ttt gca acg ggg aaa aat gtc tgt gat atc tgc acg gat gac      720 
Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp 
225                 230                 235                 240 

ttg atg gga tcc aga agc aac ttt gat tcc act cta ata agc cca aac      768 
Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn 
                245                 250                 255 

tct gtc ttt tct caa tgg cga gtg gtc tgt gac tcc ttg gaa gat tat      816 
Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr 
            260                 265                 270 

gat acc ctg gga aca ctt tgt aac agc acc gag gat ggg cca att agg      864 
Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Asp Gly Pro Ile Arg 
        275                 280                 285 

aga aat cca gct gga aat gtg gcc aga cca atg gtg caa cgt ctt cct      912 
Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro 
    290                 295                 300 

gaa cca cag gat gtc gct cag tgc ttg gaa gtt ggt tta ttt gac acg      960 
Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr 
305                 310                 315                 320 

cct cct ttt tat tcc aac tct aca aac agt ttc cga aac aca gtg gaa     1008 
Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu 
                325                 330                 335 

ggt tac agt gac ccc acg gga aag tat gac cct gct gtt cga agt ctt     1056 
His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His 
            340                 345                 350 

cac aat ttg gct cat cta ttc ctg aat gga aca ggg gga caa acc cat     1104 
Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu 
        355                 360                 365 

ttg tct cca aat gat cct att ttt gtc ctc ctg cac acc ttc aca gat     1152 
Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp 
    370                 375                 380 

gca gtc ttt gat gaa tgg ctg agg aga tac aat gct gat ata tcc aca     1200 
Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr 
385                 390                 395                 400 

ttt cca ttg gaa aat gcc cct att gga cat aat aga caa tac aac atg     1248 
Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met 
                405                 410                 415 

gtg cca ttc tgg ccc cca gtc acc aac aca gaa atg ttt gtt act gct     1296 
Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala 
            420                 425                 430 

cca gac aac ctg gga tac act tat gaa att caa tgg cca agt cgg gag     1344 
Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Ile Gln Trp Pro Ser Arg Glu 
        435                 440                 445 

ttt agt gta cct gag ggt ggc gga ggg cag ttc ccc cga gtc tgc atg     1392 
Phe Ser Val Pro Glu Gly Gly Gly Gly Gln Phe Pro Arg Val Cys Met 
    450                 455                 460 

acg gtg gac agc cta gtg aac aag gag tgc tgc cca cgc ctg ggt gca     1440 
Thr Val Asp Ser Leu Val Asn Lys Glu Cys Cys Pro Arg Leu Gly Ala 
465                 470                 475                 480 

gag tcg gcc aat gtc tgt ggc tct cag caa ggc cgg ggg cag tgc aca     1488 
Glu Ser Ala Asn Val Cys Gly Ser Gln Gln Gly Arg Gly Gln Cys Thr 
                485                 490                 495 

gag gtg cga gcc gac aca agg ccc tgg agt ggt ccc tac atc cta cga     1536 
Glu Val Arg Ala Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg 
            500                 505                 510 

aac cag gat gac cgt gag ctg tgg cca aga aaa ttc ttc cac cgg acc     1584 
Cys Lys Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Asp Cys Lys 
        515                 520                 525 

tgc aag tgc aca gga aac ttt gcc ggc tat aat tgt gga gac tgc aag     1632 
Asn Gln Asp Asp Arg Glu Leu Trp Pro Arg Lys Phe Phe His Arg Thr 
    530                 535                 540 

ttt ggc tgg acc ggt ccc aac tgc gag cgg aag aaa cca cca gtg att     1680 
Phe Gly Trp Thr Gly Pro Asn Cys Glu Arg Lys Lys Pro Pro Val Ile 
545                 550                 555                 560 

cgg cag aac atc cat tcc ttg agt cct cag gaa aga gag cag ttc ttg     1728 
Arg Gln Asn Ile His Ser Leu Ser Pro Gln Glu Arg Glu Gln Phe Leu 
                565                 570                 575 

ggc gcc tta gat ctc gcg aag aag aga gta cac ccc gac tac gtg atc     1776 
Gly Ala Leu Asp Leu Ala Lys Lys Arg Val His Pro Asp Tyr Val Ile 
            580                 585                 590 

acc aca caa cac tgg ctg ggc ctg ctt ggg ccc aat gga acc cag ccg     1824 
Thr Thr Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro 
        595                 600                 605 

cag ttt gcc aac tgc agt gtt tat gat ttt ttt gtg tgg ctc cat tat     1872 
Gln Phe Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr 
    610                 615                 620 

tat tct gtt aga gat aca tta tta gga cca gga cgc ccc tac agg gcc     1920 
Tyr Ser Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Arg Ala 
625                 630                 635                 640 

ata gat ttc tca cat caa gga cct gca ttt gtt acc tgg cac cgg tac     1968 
Ile Asp Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr 
                645                 650                 655 

cat ttg ttg tgt ctg gaa aga gat ctc cag cga ctc att ggc aat gag     2016 
His Leu Leu Cys Leu Glu Arg Asp Leu Gln Arg Leu Ile Gly Asn Glu 
            660                 665                 670 

tct ttt gct ttg ccc tac tgg aac ttt gcc act ggg agg aac gag tgt     2064 
Ser Phe Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Arg Asn Glu Cys 
        675                 680                 685 

gat gtg tgt aca gac cag ctg ttt ggg gca gcg aga cca gac gat ccg     2112 
Asp Val Cys Thr Asp Gln Leu Phe Gly Ala Ala Arg Pro Asp Asp Pro 
    690                 695                 700 

act ctg att agt cgg aac tca aga ttc tcc agc tgg gaa act gtc tgt     2160 
Thr Leu Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp Glu Thr Val Cys 
705                 710                 715                 720 

gat agc ttg gat gac tac aac cac ctg gtc acc ttg tgc aat gga acc     2208 
Asp Ser Leu Asp Asp Tyr Asn His Leu Val Thr Leu Cys Asn Gly Thr 
                725                 730                 735 

tat gaa ggt ttg ctg aga aga aat caa atg gga aga aac agc atg aaa     2256 
Tyr Glu Gly Leu Leu Arg Arg Asn Gln Met Gly Arg Asn Ser Met Lys 
            740                 745                 750 

ttg cca acc tta aaa gac ata cga gat tgc ctg tct ctc cag aag ttt     2304 
Leu Pro Thr Leu Lys Asp Ile Arg Asp Cys Leu Ser Leu Gln Lys Phe 
        755                 760                 765 

gac aat cct ccc ttc ttc cag aac tct acc ttc agt ttc agg aat gct     2352 
Asp Asn Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala 
    770                 775                 780 

ttg gaa ggg ttt gat aaa gca gat ggg act ctg gat tct caa gtg atg     2400 
Leu Glu Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met 
785                 790                 795                 800 

agc ctt cat aat ttg gtt cat tcc ttc ctg aac ggg aca aac gct ttg     2448 
Ser Leu His Asn Leu Val His Ser Phe Leu Asn Gly Thr Asn Ala Leu 
                805                 810                 815 

cca cat tca gcc gcc aat gat ccc att ttt gtg gtt ctt cat tcc ttt     2496 
Pro His Ser Ala Ala Asn Asp Pro Ile Phe Val Val Leu His Ser Phe 
            820                 825                 830 

act gat gcc atc ttt gat gag tgg atg aaa aga ttt aat cct cct gca     2544 
Thr Asp Ala Ile Phe Asp Glu Trp Met Lys Arg Phe Asn Pro Pro Ala 
        835                 840                 845 

gat gcc tgg cct cag gag ctg gcc cct att ggt cac aat cgg atg tac     2592 
Asp Ala Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr 
    850                 855                 860 

aac atg gtt cct ttc ttc cct cca gtg act aat gaa gaa ctc ttt tta     2640 
Asn Met Val Pro Phe Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu 
865                 870                 875                 880 

acc tca gac caa ctt ggc tac agc tat gcc atc gat ctg cca gtt tca     2688 
Thr Ser Asp Gln Leu Gly Tyr Ser Tyr Ala Ile Asp Leu Pro Val Ser 
                885                 890                 895 

gtt gaa gaa act cca ggt tgg ccc aca act ggc ggg ggt gga cat ttc     2736 
Val Glu Glu Thr Pro Gly Trp Pro Thr Thr Gly Gly Gly Gly His Phe 
            900                 905                 910 

cct aga gcc tgt gtc tcc tct aag aac ctg atg gag aag gaa tgc tgt     2784 
Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Glu Lys Glu Cys Cys 
        915                 920                 925 

cca ccg tgg agc ggg gac agg agt ccc tgt ggc cag ctt tca ggc aga     2832 
Pro Pro Trp Ser Gly Asp Arg Ser Pro Cys Gly Gln Leu Ser Gly Arg 
    930                 935                 940 

ggt tcc tgt cag aat atc ctt ctg tcc aat gca cca ctt ggg cct caa     2880 
Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln 
945                 950                 955                 960 

ttt ccc ttc aca ggg gtg gat gac cgg gag tcg tgg cct tcc gtc ttt     2928 
Phe Pro Phe Thr Gly Val Asp Asp Arg Glu Ser Trp Pro Ser Val Phe 
                965                 970                 975 

tat aat agg acc tgc cag tgc tct ggc aac ttc atg gga ttc aac tgt     2976 
Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met Gly Phe Asn Cys 
            980                 985                 990 

gga aac tgc aag ttt ggc ttt tgg gga cca aac tgc aca gag aga cga     3024 
Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr Glu Arg Arg 
        995                 1000                1005 

ctc ttg gtg aga aga aac atc ttc gat ttg agt gcc cca gag aag gac     3072 
Leu Leu Val Arg Arg Asn Ile Phe Asp Leu Ser Ala Pro Glu Lys Asp 
    1010                1015                1020 

aaa ttt ttt gcc tac ctc act tta gca aag cat acc atc agc tca gac     3120 
Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr Ile Ser Ser Asp 
1025                1030                1035                1040 

tat gtc atc ccc ata ggg acc tat ggc caa atg aaa aat gga tca aca     3168 
Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys Asn Gly Ser Thr 
                1045                1050                1055 

ccc atg ttt aac gac atc aat att tat gac ctc ttt gtc tgg atg cat     3216 
Pro Met Phe Asn Asp Ile Asn Ile Tyr Asp Leu Phe Val Trp Met His 
            1060                1065                1070 

tat tat gtg tca atg gat gca ctg ctt ggg gga tct gaa atc tgg aga     3264 
Tyr Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Ser Glu Ile Trp Arg 
        1075                1080                1085 

gac att gat ttt gcc cat gaa gca cca gct ttt ctg cct tgg cat aga     3312 
Asp Ile Asp Phe Ala His Glu Ala Pro Ala Phe Leu Pro Trp His Arg 
    1090                1095                1100 

ctc ttc ttg ttg cgg tgg gaa caa gaa atc cag aag ctg aca gga gat     3360 
Leu Phe Leu Leu Arg Trp Glu Gln Glu Ile Gln Lys Leu Thr Gly Asp 
1105                1110                1115                1120 

gaa aac ttc act att cca tat tgg gac tgg cgg gat gca gaa aag tgt     3408 
Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp Ala Glu Lys Cys 
                1125                1130                1135 

gac att tgc aca gat gag tac atg gga ggt cag cac ccc aca aat cct     3456 
Asp Ile Cys Thr Asp Glu Tyr Met Gly Gly Gln His Pro Thr Asn Pro 
            1140                1145                1150 

aac tta ctc agc cca gca tca ttc ttc tcc tct tgg cag att gtc tgt     3504 
Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln Ile Val Cys 
        1155                1160                1165 

agc cga ttg gag gag tac aac agc cat cag tct tta tgc aat gga acg     3552 
Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr 
    1170                1175                1180 

ccc gag gga cct tta cgg cgt aat cct gga aac cat gac aaa tcc aga     3600 
Pro Glu Gly Pro Leu Arg Arg Asn Pro Gly Asn His Asp Lys Ser Arg 
1185                1190                1195                1200 

acc cca agg ctc ccc tct tca gct gat gta gaa ttt tgc ctg agt ttg     3648 
Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe Cys Leu Ser Leu 
                1205                1210                1215 

acc caa tat gaa tct ggt tcc atg gat aaa gct gcc aat ttc agc ttt     3696 
Thr Gln Tyr Glu Ser Gly Ser Met Asp Lys Ala Ala Asn Phe Ser Phe 
            1220                1225                1230 

aga aat aca ctg gaa gga ttt gct agt cca ctt act ggg ata gcg gat     3744 
Arg Asn Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr Gly Ile Ala Asp 
        1235                1240                1245 

gcc tct caa agc agc atg cac aat gcc ttg cac atc tat atg aat gga     3792 
Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn Gly 
    1250                1255                1260 

aca atg tcc cag gta cag gga tct gcc aac gat cct atc ttc ctt ctt     3840 
Thr Met Ser Gln Val Gln Gly Ser Ala Asn Asp Pro Ile Phe Leu Leu 
1265                1270                1275                1280 

cac cat gca ttt gtt gac agt att ttt gag cag tgg ctc cga agg cac     3888 
His His Ala Phe Val Asp Ser Ile Phe Glu Gln Trp Leu Arg Arg His 
                1285                1290                1295 

cgt cct ctt caa gaa gtt tat cca gaa gcc aat gca ccc att gga cat     3936 
Arg Pro Leu Gln Glu Val Tyr Pro Glu Ala Asn Ala Pro Ile Gly His 
            1300                1305                1310 

aac cgg gaa tcc tac atg gtt cct ttt ata cca ctg tac aga aat ggt     3984 
Asn Arg Glu Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr Arg Asn Gly 
        1315                1320                1325 

gat ttc ttt att tca tcc aaa gat ctg ggc tat gac tat agc tat cta     4032 
Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr Leu 
    1330                1335                1340 

caa gat tca gac cca gac tct ttt caa gac tac att aag tcc tat ttg     4080 
Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr Ile Lys Ser Tyr Leu 
1345                1350                1355                1360 

gaa caa gcg agt cgg atc tgg tca                                     4104 
Glu Gln Ala Ser Arg Ile Trp Ser 
                1365 

 
           
             10  
             1368  
             PRT  
             Artificial Sequence  
             
               Tri-hybrid antigen  
             
           
            10 

Gln Phe Pro Arg Gln Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met 
                  5                  10                  15 

Cys Cys Pro Asp Leu Ser Pro Val Ser Gly Pro Gly Thr Asp Arg Cys 
             20                  25                  30 

Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser 
         35                  40                  45 

Arg Pro His Ser Pro Gln Tyr Pro His Asp Gly Arg Asp Asp Arg Glu 
     50                  55                  60 

Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn 
 65                  70                  75                  80 

Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala 
                 85                  90                  95 

Ala Cys Asp Gln Arg Val Leu Ile Val Arg Arg Asn Leu Leu Asp Leu 
            100                 105                 110 

Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys 
        115                 120                 125 

Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu 
    130                 135                 140 

Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile 
145                 150                 155                 160 

Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe 
                165                 170                 175 

Leu Gly Val Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu 
            180                 185                 190 

Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu 
        195                 200                 205 

Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr 
    210                 215                 220 

Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp 
225                 230                 235                 240 

Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn 
                245                 250                 255 

Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr 
            260                 265                 270 

Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Asp Gly Pro Ile Arg 
        275                 280                 285 

Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro 
    290                 295                 300 

Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr 
305                 310                 315                 320 

Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu 
                325                 330                 335 

Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu 
            340                 345                 350 

His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His 
        355                 360                 365 

Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp 
    370                 375                 380 

Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr 
385                 390                 395                 400 

Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met 
                405                 410                 415 

Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala 
            420                 425                 430 

Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Ile Gln Trp Pro Ser Arg Glu 
        435                 440                 445 

Phe Ser Val Pro Glu Gly Gly Gly Gly Gln Phe Pro Arg Val Cys Met 
    450                 455                 460 

Thr Val Asp Ser Leu Val Asn Lys Glu Cys Cys Pro Arg Leu Gly Ala 
465                 470                 475                 480 

Glu Ser Ala Asn Val Cys Gly Ser Gln Gln Gly Arg Gly Gln Cys Thr 
                485                 490                 495 

Glu Val Arg Ala Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg 
            500                 505                 510 

Asn Gln Asp Asp Arg Glu Leu Trp Pro Arg Lys Phe Phe His Arg Thr 
        515                 520                 525 

Cys Lys Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Asp Cys Lys 
    530                 535                 540 

Phe Gly Trp Thr Gly Pro Asn Cys Glu Arg Lys Lys Pro Pro Val Ile 
545                 550                 555                 560 

Arg Gln Asn Ile His Ser Leu Ser Pro Gln Glu Arg Glu Gln Phe Leu 
                565                 570                 575 

Gly Ala Leu Asp Leu Ala Lys Lys Arg Val His Pro Asp Tyr Val Ile 
            580                 585                 590 

Thr Thr Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro 
        595                 600                 605 

Gln Phe Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr 
    610                 615                 620 

Tyr Ser Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Arg Ala 
625                 630                 635                 640 

Ile Asp Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr 
                645                 650                 655 

His Leu Leu Cys Leu Glu Arg Asp Leu Gln Arg Leu Ile Gly Asn Glu 
            660                 665                 670 

Ser Phe Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Arg Asn Glu Cys 
        675                 680                 685 

Asp Val Cys Thr Asp Gln Leu Phe Gly Ala Ala Arg Pro Asp Asp Pro 
    690                 695                 700 

Thr Leu Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp Glu Thr Val Cys 
705                 710                 715                 720 

Asp Ser Leu Asp Asp Tyr Asn His Leu Val Thr Leu Cys Asn Gly Thr 
                725                 730                 735 

Tyr Glu Gly Leu Leu Arg Arg Asn Gln Met Gly Arg Asn Ser Met Lys 
            740                 745                 750 

Leu Pro Thr Leu Lys Asp Ile Arg Asp Cys Leu Ser Leu Gln Lys Phe 
        755                 760                 765 

Asp Asn Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala 
    770                 775                 780 

Leu Glu Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met 
785                 790                 795                 800 

Ser Leu His Asn Leu Val His Ser Phe Leu Asn Gly Thr Asn Ala Leu 
                805                 810                 815 

Pro His Ser Ala Ala Asn Asp Pro Ile Phe Val Val Leu His Ser Phe 
            820                 825                 830 

Thr Asp Ala Ile Phe Asp Glu Trp Met Lys Arg Phe Asn Pro Pro Ala 
        835                 840                 845 

Asp Ala Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr 
    850                 855                 860 

Asn Met Val Pro Phe Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu 
865                 870                 875                 880 

Thr Ser Asp Gln Leu Gly Tyr Ser Tyr Ala Ile Asp Leu Pro Val Ser 
                885                 890                 895 

Val Glu Glu Thr Pro Gly Trp Pro Thr Thr Gly Gly Gly Gly His Phe 
            900                 905                 910 

Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Glu Lys Glu Cys Cys 
        915                 920                 925 

Pro Pro Trp Ser Gly Asp Arg Ser Pro Cys Gly Gln Leu Ser Gly Arg 
    930                 935                 940 

Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln 
945                 950                 955                 960 

Phe Pro Phe Thr Gly Val Asp Asp Arg Glu Ser Trp Pro Ser Val Phe 
                965                 970                 975 

Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met Gly Phe Asn Cys 
            980                 985                 990 

Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr Glu Arg Arg 
        995                 1000                1005 

Leu Leu Val Arg Arg Asn Ile Phe Asp Leu Ser Ala Pro Glu Lys Asp 
    1010                1015                1020 

Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr Ile Ser Ser Asp 
1025                1030                1035                1040 

Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys Asn Gly Ser Thr 
                1045                1050                1055 

Pro Met Phe Asn Asp Ile Asn Ile Tyr Asp Leu Phe Val Trp Met His 
            1060                1065                1070 

Tyr Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Ser Glu Ile Trp Arg 
        1075                1080                1085 

Asp Ile Asp Phe Ala His Glu Ala Pro Ala Phe Leu Pro Trp His Arg 
    1090                1095                1100 

Leu Phe Leu Leu Arg Trp Glu Gln Glu Ile Gln Lys Leu Thr Gly Asp 
1105                1110                1115                1120 

Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp Ala Glu Lys Cys 
                1125                1130                1135 

Asp Ile Cys Thr Asp Glu Tyr Met Gly Gly Gln His Pro Thr Asn Pro 
            1140                1145                1150 

Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln Ile Val Cys 
        1155                1160                1165 

Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr 
    1170                1175                1180 

Pro Glu Gly Pro Leu Arg Arg Asn Pro Gly Asn His Asp Lys Ser Arg 
1185                1190                1195                1200 

Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe Cys Leu Ser Leu 
                1205                1210                1215 

Thr Gln Tyr Glu Ser Gly Ser Met Asp Lys Ala Ala Asn Phe Ser Phe 
            1220                1225                1230 

Arg Asn Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr Gly Ile Ala Asp 
        1235                1240                1245 

Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn Gly 
    1250                1255                1260 

Thr Met Ser Gln Val Gln Gly Ser Ala Asn Asp Pro Ile Phe Leu Leu 
1265                1270                1275                1280 

His His Ala Phe Val Asp Ser Ile Phe Glu Gln Trp Leu Arg Arg His 
                1285                1290                1295 

Arg Pro Leu Gln Glu Val Tyr Pro Glu Ala Asn Ala Pro Ile Gly His 
            1300                1305                1310 

Asn Arg Glu Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr Arg Asn Gly 
        1315                1320                1325 

Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr Leu 
    1330                1335                1340 

Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr Ile Lys Ser Tyr Leu 
1345                1350                1355                1360 

Glu Gln Ala Ser Arg Ile Trp Ser 
                1365 

 
           
             11  
             4152  
             DNA  
             Artificial Sequence  
             
               Tri-hybrid antigen coding sequence  
             
           
            11 

atg agt gct cct aaa ctc ctc tct ctg ggc tgt atc ttc ttc ccc ttg       48 
Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 
                  5                  10                  15 

cta ctt ttt cag cag gcc cgg gct caa ttc cca aga cag tgt gcc act       96 
Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 
             20                  25                  30 

gtt gag gct ttg aga agt ggt atg tgt tgc cca gac ctg tcc cct gtg      144 
Val Gln Ala Leu Arg Ser Gly Met Cys Cys Pro Glu Leu Ser Pro Val 
         35                  40                  45 

tct ggg cct ggg aca gac cgc tgt ggc tca tca tca ggg agg ggc aga      192 
Ser Gly Pro Gly Thr Glu Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 
     50                  55                  60 

tgt gag gca gtg act gca gac tcc cgg ccc cac agc cct cag tat ccc      240 
Cys Gln Ala Val Thr Ala Glu Ser Arg Pro His Ser Pro Gln Tyr Pro 
65                  70                  75                  80 

cat gat ggc aga gat gat cgg gag gtc tgg ccc ttg cgc ttc ttc aat      288 
His Glu Gly Arg Glu Glu Arg Gln Val Trp Pro Leu Arg Phe Phe Asn 
                 85                  90                  95 

agg aca tgt cac tgc aac ggc aat ttc tca gga cac aac tgt ggg acg      336 
Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 
            100                 105                 110 

tgc cgt cct ggc tgg aga gga gct gcc tgt gac cag agg gtt ctc ata      384 
Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Glu Gln Arg Val Leu Ile 
        115                 120                 125 

gtc agg aga aat ctt ctg gac tta agt aaa gaa gaa aag aac cac ttt      432 
Val Arg Arg Asn Leu Leu Glu Leu Ser Lys Gln Gln Lys Asn His Phe 
    130                 135                 140 

gtc cgg gcc ctg gat atg gca aag cgc aca act cac cct tta ttt gtc      480 
Val Arg Ala Leu Glu Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 
145                 150                 155                 160 

att gcc acc agg aga tca gaa gaa ata ctg ggg cca gat ggc aac acg      528 
Ile Ala Thr Arg Arg Ser Gln Gln Ile Leu Gly Pro Glu Gly Asn Thr 
                165                 170                 175 

cca caa ttt gag aac att tcc att tat aac tac ttt gtt tgg aca cac      576 
Pro Gln Phe Gln Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 
            180                 185                 190 

tat tac tca gtc aaa aag act ttc ctt ggg gta gga cag gaa agc ttt      624 
Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Gln Ser Phe 
        195                 200                 205 

ggt gaa gtg gat ttc tct cat gag gga cca gct ttt ctc aca tgg cac      672 
Gly Gln Val Glu Phe Ser His Gln Gly Pro Ala Phe Leu Thr Trp His 
    210                 215                 220 

agg tac cac ctc ctg cgt ctg gag aaa gac atg cag gaa atg ttg caa      720 
Arg Tyr His Leu Leu Arg Leu Gln Lys Glu Met Gln Gln Met Leu Gln 
225                 230                 235                 240 

gag cct tct ttc tcc ctt cct tac tgg aat ttt gca acg ggg aaa aat      768 
Gln Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 
                245                 250                 255 

gtc tgt gat atc tgc acg gat gac ttg atg gga tcc aga agc aac ttt      816 
Val Cys Glu Ile Cys Thr Glu Glu Leu Met Gly Ser Arg Ser Asn Phe 
            260                 265                 270 

gat tcc act cta ata agc cca aac tct gtc ttt tct caa tgg cga gtg      864 
Glu Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 
        275                 280                 285 

gtc tgt gac tcc ttg gaa gat tat gat acc ctg gga aca ctt tgt aac      912 
Val Cys Glu Ser Leu Gln Glu Tyr Glu Thr Leu Gly Thr Leu Cys Asn 
    290                 295                 300 

agc acc gag gat ggg cca att agg aga aat cca gct gga aat gtg gcc      960 
Ser Thr Gln Glu Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 
305                 310                 315                 320 

aga cca atg gtg caa cgt ctt cct gaa cca cag gat gtc gct cag tgc     1008 
Arg Pro Met Val Gln Arg Leu Pro Gln Pro Gln Glu Val Ala Gln Cys 
                325                 330                 335 

ttg gaa gtt ggt tta ttt gac acg cct cct ttt tat tcc aac tct aca     1056 
Leu Gln Val Gly Leu Phe Glu Thr Pro Pro Phe Tyr Ser Asn Ser Thr 
            340                 345                 350 

aac agt ttc cga aac aca gtg gaa ggt tac agt gac ccc acg gga aag     1104 
Asn Ser Phe Arg Asn Thr Val Gln Gly Tyr Ser Glu Pro Thr Gly Lys 
        355                 360                 365 

tat gac cct gct gtt cga agt ctt cac aat ttg gct cat cta ttc ctg     1152 
Tyr Glu Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu 
    370                 375                 380 

aat gga aca ggg gga caa acc cat ttg tct cca aat gat cct att ttt     1200 
Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Glu Pro Ile Phe 
385                 390                 395                 400 

gtc ctc ctg cac acc ttc aca gat gca gtc ttt gat gaa tgg ctg agg     1248 
Val Leu Leu His Thr Phe Thr Glu Ala Val Phe Glu Gln Trp Leu Arg 
                405                 410                 415 

aga tac aat gct gat ata tcc aca ttt cca ttg gaa aat gcc cct att     1296 
Arg Tyr Asn Ala Glu Ile Ser Thr Phe Pro Leu Gln Asn Ala Pro Ile 
            420                 425                 430 

gga cat aat aga caa tac aac atg gtg cca ttc tgg ccc cca gtc acc     1344 
Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 
        435                 440                 445 

aac aca gaa atg ttt gtt act gct cca gac aac ctg gga tac act tat     1392 
Asn Thr Gln Met Phe Val Thr Ala Pro Glu Asn Leu Gly Tyr Thr Tyr 
    450                 455                 460 

gaa att caa tgg cca agt cgg gag ttt agt gta cct gag cag ttc ccc     1440 
Gln Ile Gln Trp Pro Ser Arg Gln Phe Ser Val Pro Gln Gln Phe Pro 
465                 470                 475                 480 

cga gtc tgc atg acg gtg gac agc cta gtg aac aag gag tgc tgc cca     1488 
Arg Val Cys Met Thr Val Glu Ser Leu Val Asn Lys Gln Cys Cys Pro 
                485                 490                 495 

cgc ctg ggt gca gag tcg gcc aat gtc tgt ggc tct cag caa ggc cgg     1536 
Arg Leu Gly Ala Gln Ser Ala Asn Val Cys Gly Ser Gln Gln Gly Arg 
            500                 505                 510 

ggg cag tgc aca gag gtg cga gcc gac aca agg ccc tgg agt ggt ccc     1584 
Gly Gln Cys Thr Gln Val Arg Ala Glu Thr Arg Pro Trp Ser Gly Pro 
        515                 520                 525 

tac atc cta cga aac cag gat gac cgt gag ctg tgg cca aga aaa ttc     1632 
Tyr Ile Leu Arg Asn Gln Glu Glu Arg Gln Leu Trp Pro Arg Lys Phe 
    530                 535                 540 

ttc cac cgg acc tgc aag tgc aca gga aac ttt gcc ggc tat aat tgt     1680 
Phe His Arg Thr Cys Lys Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys 
545                 550                 555                 560 

gga gac tgc aag ttt ggc tgg acc ggt ccc aac tgc gag cgg aag aaa     1728 
Gly Glu Cys Lys Phe Gly Trp Thr Gly Pro Asn Cys Gln Arg Lys Lys 
                565                 570                 575 

cca cca gtg att cgg cag aac atc cat tcc ttg agt cct cag gaa aga     1776 
Pro Pro Val Ile Arg Gln Asn Ile His Ser Leu Ser Pro Gln Gln Arg 
            580                 585                 590 

gag cag ttc ttg ggc gcc tta gat ctc gcg aag aag aga gta cac ccc     1824 
Gln Gln Phe Leu Gly Ala Leu Glu Leu Ala Lys Lys Arg Val His Pro 
        595                 600                 605 

gac tac gtg atc acc aca caa cac tgg ctg ggc ctg ctt ggg ccc aat     1872 
Glu Tyr Val Ile Thr Thr Gln His Trp Leu Gly Leu Leu Gly Pro Asn 
    610                 615                 620 

gga acc cag ccg cag ttt gcc aac tgc agt gtt tat gat ttt ttt gtg     1920 
Gly Thr Gln Pro Gln Phe Ala Asn Cys Ser Val Tyr Glu Phe Phe Val 
625                 630                 635                 640 

tgg ctc cat tat tat tct gtt aga gat aca tta tta gga cca gga cgc     1968 
Trp Leu His Tyr Tyr Ser Val Arg Glu Thr Leu Leu Gly Pro Gly Arg 
                645                 650                 655 

ccc tac agg gcc ata gat ttc tca cat caa gga cct gca ttt gtt acc     2016 
Pro Tyr Arg Ala Ile Glu Phe Ser His Gln Gly Pro Ala Phe Val Thr 
            660                 665                 670 

tgg cac cgg tac cat ttg ttg tgt ctg gaa aga gat ctc cag cga ctc     2064 
Trp His Arg Tyr His Leu Leu Cys Leu Gln Arg Glu Leu Gln Arg Leu 
        675                 680                 685 

att ggc aat gag tct ttt gct ttg ccc tac tgg aac ttt gcc act ggg     2112 
Ile Gly Asn Gln Ser Phe Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly 
    690                 695                 700 

agg aac gag tgt gat gtg tgt aca gac cag ctg ttt ggg gca gcg aga     2160 
Arg Asn Gln Cys Glu Val Cys Thr Glu Gln Leu Phe Gly Ala Ala Arg 
705                 710                 715                 720 

cca gac gat ccg act ctg att agt cgg aac tca aga ttc tcc agc tgg     2208 
Pro Glu Glu Pro Thr Leu Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp 
                725                 730                735 

gaa act gtc tgt gat agc ttg gat gac tac aac cac ctg gtc acc ttg     2256 
Gln Thr Val Cys Glu Ser Leu Glu Glu Tyr Asn His Leu Val Thr Leu 
            740                 745                 750 

tgc aat gga acc tat gaa ggt ttg ctg aga aga aat caa atg gga aga     2304 
Cys Asn Gly Thr Tyr Gln Gly Leu Leu Arg Arg Asn Gln Met Gly Arg 
        755                 760                 765 

aac agc atg aaa ttg cca acc tta aaa gac ata cga gat tgc ctg tct     2352 
Asn Ser Met Lys Leu Pro Thr Leu Lys Glu Ile Arg Glu Cys Leu Ser 
    770                 775                 780 

ctc cag aag ttt gac aat cct ccc ttc ttc cag aac tct acc ttc agt     2400 
Leu Gln Lys Phe Glu Asn Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser 
785                 790                 795                 800 

ttc agg aat gct ttg gaa ggg ttt gat aaa gca gat ggg act ctg gat     2448 
Phe Arg Asn Ala Leu Gln Gly Phe Glu Lys Ala Glu Gly Thr Leu Glu 
                805                 810                 815 

tct caa gtg atg agc ctt cat aat ttg gtt cat tcc ttc ctg aac ggg     2496 
Ser Gln Val Met Ser Leu His Asn Leu Val His Ser Phe Leu Asn Gly 
            820                 825                 830 

aca aac gct ttg cca cat tca gcc gcc aat gat ccc att ttt gtg gtt     2544 
Thr Asn Ala Leu Pro His Ser Ala Ala Asn Glu Pro Ile Phe Val Val 
        835                 840                 845 

ctt cat tcc ttt act gat gcc atc ttt gat gag tgg atg aaa aga ttt     2592 
Leu His Ser Phe Thr Glu Ala Ile Phe Glu Gln Trp Met Lys Arg Phe 
    850                 855                 860 

aat cct cct gca gat gcc tgg cct cag gag ctg gcc cct att ggt cac     2640 
Asn Pro Pro Ala Glu Ala Trp Pro Gln Gln Leu Ala Pro Ile Gly His 
865                 870                 875                 880 

aat cgg atg tac aac atg gtt cct ttc ttc cct cca gtg act aat gaa     2688 
Asn Arg Met Tyr Asn Met Val Pro Phe Phe Pro Pro Val Thr Asn Gln 
                885                 890                 895 

gaa ctc ttt tta acc tca gac caa ctt ggc tac agc tat gcc atc gat     2736 
Gln Leu Phe Leu Thr Ser Glu Gln Leu Gly Tyr Ser Tyr Ala Ile Glu 
            900                 905                 910 

ctg cca gtt tca gtt gaa gaa act cca ggt tgg ccc aca act cat ttc     2784 
Leu Pro Val Ser Val Gln Gln Thr Pro Gly Trp Pro Thr Thr His Phe 
        915                 920                 925 

cct aga gcc tgt gtc tcc tct aag aac ctg atg gag aag gaa tgc tgt     2832 
Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Gln Lys Gln Cys Cys 
    930                 935                 940 

cca ccg tgg agc ggg gac agg agt ccc tgt ggc cag ctt tca ggc aga     2880 
Pro Pro Trp Ser Gly Glu Arg Ser Pro Cys Gly Gln Leu Ser Gly Arg 
945                 950                 955                 960 

ggt tcc tgt cag aat atc ctt ctg tcc aat gca cca ctt ggg cct caa     2928 
Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln 
                965                 970                 975 

ttt ccc ttc aca ggg gtg gat gac cgg gag tcg tgg cct tcc gtc ttt     2976 
Phe Pro Phe Thr Gly Val Glu Glu Arg Gln Ser Trp Pro Ser Val Phe 
            980                 985                 990 

tat aat agg acc tgc cag tgc tct ggc aac ttc atg gga ttc aac tgt     3024 
Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met Gly Phe Asn Cys 
        995                 1000                1005 

gga aac tgc aag ttt ggc ttt tgg gga cca aac tgc aca gag aga cga     3072 
Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr Gln Arg Arg 
    1010                1015                1020 

ctc ttg gtg aga aga aac atc ttc gat ttg agt gcc cca gag aag gac     3120 
Leu Leu Val Arg Arg Asn Ile Phe Glu Leu Ser Ala Pro Gln Lys Glu 
1025                1030                1035                1040 

aaa ttt ttt gcc tac ctc act tta gca aag cat acc atc agc tca gac     3168 
Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr Ile Ser Ser Glu 
                1045                1050                1055 

tat gtc atc ccc ata ggg acc tat ggc caa atg aaa aat gga tca aca     3216 
Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys Asn Gly Ser Thr 
            1060                1065                1070 

ccc atg ttt aac gac atc aat att tat gac ctc ttt gtc tgg atg cat     3264 
Pro Met Phe Asn Glu Ile Asn Ile Tyr Glu Leu Phe Val Trp Met His 
        1075                1080                1085 

tat tat gtg tca atg gat gca ctg ctt ggg gga tct gaa atc tgg aga     3312 
Tyr Tyr Val Ser Met Glu Ala Leu Leu Gly Gly Ser Gln Ile Trp Arg 
    1090                1095                1100 

gac att gat ttt gcc cat gaa gca cca gct ttt ctg cct tgg cat aga     3360 
Glu Ile Glu Phe Ala His Gln Ala Pro Ala Phe Leu Pro Trp His Arg 
1105                1110                1115                1120 

ctc ttc ttg ttg cgg tgg gaa caa gaa atc cag aag ctg aca gga gat     3408 
Leu Phe Leu Leu Arg Trp Gln Gln Gln Ile Gln Lys Leu Thr Gly Glu 
                1125                1130                1135 

gaa aac ttc act att cca tat tgg gac tgg cgg gat gca gaa aag tgt     3456 
Gln Asn Phe Thr Ile Pro Tyr Trp Glu Trp Arg Glu Ala Gln Lys Cys 
            1140                1145                1150 

gac att tgc aca gat gag tac atg gga ggt cag cac ccc aca aat cct     3504 
Glu Ile Cys Thr Glu Gln Tyr Met Gly Gly Gln His Pro Thr Asn Pro 
        1155                1160                1165 

aac tta ctc agc cca gca tca ttc ttc tcc tct tgg cag att gtc tgt     3552 
Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln Ile Val Cys 
    1170                1175                1180 

agc cga ttg gag gag tac aac agc cat cag tct tta tgc aat gga acg     3600 
Ser Arg Leu Gln Gln Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr 
1185                1190                1195                1200 

ccc gag gga cct tta cgg cgt aat cct gga aac cat gac aaa tcc aga     3648 
Pro Gln Gly Pro Leu Arg Arg Asn Pro Gly Asn His Glu Lys Ser Arg 
                1205                1210                1215 

acc cca agg ctc ccc tct tca gct gat gta gaa ttt tgc ctg agt ttg     3696 
Thr Pro Arg Leu Pro Ser Ser Ala Glu Val Gln Phe Cys Leu Ser Leu 
            1220                1225                1230 

acc caa tat gaa tct ggt tcc atg gat aaa gct gcc aat ttc agc ttt     3744 
Thr Gln Tyr Gln Ser Gly Ser Met Glu Lys Ala Ala Asn Phe Ser Phe 
        1235                1240                1245 

aga aat aca ctg gaa gga ttt gct agt cca ctt act ggg ata gcg gat     3792 
Arg Asn Thr Leu Gln Gly Phe Ala Ser Pro Leu Thr Gly Ile Ala Glu 
    1250                1255                1260 

gcc tct caa agc agc atg cac aat gcc ttg cac atc tat atg aat gga     3840 
Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn Gly 
1265                1270                1275                1280 

aca atg tcc cag gta cag gga tct gcc aac gat cct atc ttc ctt ctt     3888 
Thr Met Ser Gln Val Gln Gly Ser Ala Asn Glu Pro Ile Phe Leu Leu 
                1285                1290                1295 

cac cat gca ttt gtt gac agt att ttt gag cag tgg ctc cga agg cac     3936 
His His Ala Phe Val Glu Ser Ile Phe Gln Gln Trp Leu Arg Arg His 
            1300                1305                1310 

cgt cct ctt caa gaa gtt tat cca gaa gcc aat gca ccc att gga cat     3984 
Arg Pro Leu Gln Gln Val Tyr Pro Gln Ala Asn Ala Pro Ile Gly His 
        1315                1320                1325 

aac cgg gaa tcc tac atg gtt cct ttt ata cca ctg tac aga aat ggt     4032 
Asn Arg Gln Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr Arg Asn Gly 
    1330                1335                1340 

gat ttc ttt att tca tcc aaa gat ctg ggc tat gac tat agc tat cta     4080 
Glu Phe Phe Ile Ser Ser Lys Glu Leu Gly Tyr Glu Tyr Ser Tyr Leu 
1345                1350                1355                1360 

caa gat tca gac cca gac tct ttt caa gac tac att aag tcc tat ttg     4128 
Gln Glu Ser Glu Pro Glu Ser Phe Gln Glu Tyr Ile Lys Ser Tyr Leu 
                1365                1370                1375 

gaa caa gcg agt cgg atc tgg tca                                     4152 
Gln Gln Ala Ser Arg Ile Trp Ser 
            1380 

 
           
             12  
             1384  
             PRT  
             Artificial Sequence  
             
               Tri-hybrid antigen  
             
           
            12 

Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 
                  5                  10                  15 

Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 
             20                  25                  30 

Val Gln Ala Leu Arg Ser Gly Met Cys Cys Pro Glu Leu Ser Pro Val 
         35                  40                  45 

Ser Gly Pro Gly Thr Glu Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 
     50                  55                  60 

Cys Gln Ala Val Thr Ala Glu Ser Arg Pro His Ser Pro Gln Tyr Pro 
 65                  70                  75                  80 

His Glu Gly Arg Glu Glu Arg Gln Val Trp Pro Leu Arg Phe Phe Asn 
                 85                  90                  95 

Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 
            100                 105                 110 

Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Glu Gln Arg Val Leu Ile 
        115                 120                 125 

Val Arg Arg Asn Leu Leu Glu Leu Ser Lys Gln Gln Lys Asn His Phe 
    130                 135                 140 

Val Arg Ala Leu Glu Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 
145                 150                 155                 160 

Ile Ala Thr Arg Arg Ser Gln Gln Ile Leu Gly Pro Glu Gly Asn Thr 
                165                 170                 175 

Pro Gln Phe Gln Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 
            180                 185                 190 

Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Gln Ser Phe 
        195                 200                 205 

Gly Gln Val Glu Phe Ser His Gln Gly Pro Ala Phe Leu Thr Trp His 
    210                 215                 220 

Arg Tyr His Leu Leu Arg Leu Gln Lys Glu Met Gln Gln Met Leu Gln 
225                 230                 235                 240 

Gln Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 
                245                 250                 255 

Val Cys Glu Ile Cys Thr Glu Glu Leu Met Gly Ser Arg Ser Asn Phe 
            260                 265                 270 

Glu Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 
        275                 280                 285 

Val Cys Glu Ser Leu Gln Glu Tyr Glu Thr Leu Gly Thr Leu Cys Asn 
    290                 295                 300 

Ser Thr Gln Glu Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 
305                 310                 315                 320 

Arg Pro Met Val Gln Arg Leu Pro Gln Pro Gln Glu Val Ala Gln Cys 
                325                 330                 335 

Leu Gln Val Gly Leu Phe Glu Thr Pro Pro Phe Tyr Ser Asn Ser Thr 
            340                 345                 350 

Asn Ser Phe Arg Asn Thr Val Gln Gly Tyr Ser Glu Pro Thr Gly Lys 
        355                 360                 365 

Tyr Glu Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu 
    370                 375                 380 

Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Glu Pro Ile Phe 
385                 390                 395                 400 

Val Leu Leu His Thr Phe Thr Glu Ala Val Phe Glu Gln Trp Leu Arg 
                405                 410                 415 

Arg Tyr Asn Ala Glu Ile Ser Thr Phe Pro Leu Gln Asn Ala Pro Ile 
            420                 425                 430 

Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 
        435                 440                 445 

Asn Thr Gln Met Phe Val Thr Ala Pro Glu Asn Leu Gly Tyr Thr Tyr 
    450                 455                 460 

Gln Ile Gln Trp Pro Ser Arg Gln Phe Ser Val Pro Gln Gln Phe Pro 
465                 470                 475                 480 

Arg Val Cys Met Thr Val Glu Ser Leu Val Asn Lys Gln Cys Cys Pro 
                485                 490                 495 

Arg Leu Gly Ala Gln Ser Ala Asn Val Cys Gly Ser Gln Gln Gly Arg 
            500                 505                 510 

Gly Gln Cys Thr Gln Val Arg Ala Glu Thr Arg Pro Trp Ser Gly Pro 
        515                 520                 525 

Tyr Ile Leu Arg Asn Gln Glu Glu Arg Gln Leu Trp Pro Arg Lys Phe 
    530                 535                 540 

Phe His Arg Thr Cys Lys Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys 
545                 550                 555                 560 

Gly Glu Cys Lys Phe Gly Trp Thr Gly Pro Asn Cys Gln Arg Lys Lys 
                565                 570                 575 

Pro Pro Val Ile Arg Gln Asn Ile His Ser Leu Ser Pro Gln Gln Arg 
            580                 585                 590 

Gln Gln Phe Leu Gly Ala Leu Glu Leu Ala Lys Lys Arg Val His Pro 
        595                 600                 605 

Glu Tyr Val Ile Thr Thr Gln His Trp Leu Gly Leu Leu Gly Pro Asn 
    610                 615                 620 

Gly Thr Gln Pro Gln Phe Ala Asn Cys Ser Val Tyr Glu Phe Phe Val 
625                 630                 635                 640 

Trp Leu His Tyr Tyr Ser Val Arg Glu Thr Leu Leu Gly Pro Gly Arg 
                645                 650                 655 

Pro Tyr Arg Ala Ile Glu Phe Ser His Gln Gly Pro Ala Phe Val Thr 
            660                 665                 670 

Trp His Arg Tyr His Leu Leu Cys Leu Gln Arg Glu Leu Gln Arg Leu 
        675                 680                 685 

Ile Gly Asn Gln Ser Phe Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly 
    690                 695                 700 

Arg Asn Gln Cys Glu Val Cys Thr Glu Gln Leu Phe Gly Ala Ala Arg 
705                 710                 715                 720 

Pro Glu Glu Pro Thr Leu Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp 
                725                 730                735 

Gln Thr Val Cys Glu Ser Leu Glu Glu Tyr Asn His Leu Val Thr Leu 
            740                 745                 750 

Cys Asn Gly Thr Tyr Gln Gly Leu Leu Arg Arg Asn Gln Met Gly Arg 
        755                 760                 765 

Asn Ser Met Lys Leu Pro Thr Leu Lys Glu Ile Arg Glu Cys Leu Ser 
    770                 775                 780 

Leu Gln Lys Phe Glu Asn Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser 
785                 790                 795                 800 

Phe Arg Asn Ala Leu Gln Gly Phe Glu Lys Ala Glu Gly Thr Leu Glu 
                805                 810                 815 

Ser Gln Val Met Ser Leu His Asn Leu Val His Ser Phe Leu Asn Gly 
            820                 825                 830 

Thr Asn Ala Leu Pro His Ser Ala Ala Asn Glu Pro Ile Phe Val Val 
        835                 840                 845 

Leu His Ser Phe Thr Glu Ala Ile Phe Glu Gln Trp Met Lys Arg Phe 
    850                 855                 860 

Asn Pro Pro Ala Glu Ala Trp Pro Gln Gln Leu Ala Pro Ile Gly His 
865                 870                 875                 880 

Asn Arg Met Tyr Asn Met Val Pro Phe Phe Pro Pro Val Thr Asn Gln 
                885                 890                 895 

Gln Leu Phe Leu Thr Ser Glu Gln Leu Gly Tyr Ser Tyr Ala Ile Glu 
            900                 905                 910 

Leu Pro Val Ser Val Gln Gln Thr Pro Gly Trp Pro Thr Thr His Phe 
        915                 920                 925 

Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Gln Lys Gln Cys Cys 
    930                 935                 940 

Pro Pro Trp Ser Gly Glu Arg Ser Pro Cys Gly Gln Leu Ser Gly Arg 
945                 950                 955                 960 

Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln 
                965                 970                 975 

Phe Pro Phe Thr Gly Val Glu Glu Arg Gln Ser Trp Pro Ser Val Phe 
            980                 985                 990 

Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met Gly Phe Asn Cys 
        995                 1000                1005 

Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr Gln Arg Arg 
    1010                1015                1020 

Leu Leu Val Arg Arg Asn Ile Phe Glu Leu Ser Ala Pro Gln Lys Glu 
1025                1030                1035                1040 

Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr Ile Ser Ser Glu 
                1045                1050                1055 

Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys Asn Gly Ser Thr 
            1060                1065                1070 

Pro Met Phe Asn Glu Ile Asn Ile Tyr Glu Leu Phe Val Trp Met His 
        1075                1080                1085 

Tyr Tyr Val Ser Met Glu Ala Leu Leu Gly Gly Ser Gln Ile Trp Arg 
    1090                1095                1100 

Glu Ile Glu Phe Ala His Gln Ala Pro Ala Phe Leu Pro Trp His Arg 
1105                1110                1115                1120 

Leu Phe Leu Leu Arg Trp Gln Gln Gln Ile Gln Lys Leu Thr Gly Glu 
                1125                1130                1135 

Gln Asn Phe Thr Ile Pro Tyr Trp Glu Trp Arg Glu Ala Gln Lys Cys 
            1140                1145                1150 

Glu Ile Cys Thr Glu Gln Tyr Met Gly Gly Gln His Pro Thr Asn Pro 
        1155                1160                1165 

Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln Ile Val Cys 
    1170                1175                1180 

Ser Arg Leu Gln Gln Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr 
1185                1190                1195                1200 

Pro Gln Gly Pro Leu Arg Arg Asn Pro Gly Asn His Glu Lys Ser Arg 
                1205                1210                1215 

Thr Pro Arg Leu Pro Ser Ser Ala Glu Val Gln Phe Cys Leu Ser Leu 
            1220                1225                1230 

Thr Gln Tyr Gln Ser Gly Ser Met Glu Lys Ala Ala Asn Phe Ser Phe 
        1235                1240                1245 

Arg Asn Thr Leu Gln Gly Phe Ala Ser Pro Leu Thr Gly Ile Ala Glu 
    1250                1255                1260 

Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn Gly 
1265                1270                1275                1280 

Thr Met Ser Gln Val Gln Gly Ser Ala Asn Glu Pro Ile Phe Leu Leu 
                1285                1290                1295 

His His Ala Phe Val Glu Ser Ile Phe Gln Gln Trp Leu Arg Arg His 
            1300                1305                1310 

Arg Pro Leu Gln Gln Val Tyr Pro Gln Ala Asn Ala Pro Ile Gly His 
        1315                1320                1325 

Asn Arg Gln Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr Arg Asn Gly 
    1330                1335                1340 

Glu Phe Phe Ile Ser Ser Lys Glu Leu Gly Tyr Glu Tyr Ser Tyr Leu 
1345                1350                1355                1360 

Gln Glu Ser Glu Pro Glu Ser Phe Gln Glu Tyr Ile Lys Ser Tyr Leu 
                1365                1370                1375 

Gln Gln Ala Ser Arg Ile Trp Ser 
            1380 

 
           
             13  
             529  
             PRT  
             human  
             
               Tyrosinase (GenBank Accession No. NP_000363)  
             
           
            13 

Met Leu Leu Ala Val Leu Tyr Cys Leu Leu Trp Ser Phe Gln Thr Ser 
                  5                  10                  15 

Ala Gly His Phe Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Glu 
             20                  25                  30 

Lys Glu Cys Cys Pro Pro Trp Ser Gly Asp Arg Ser Pro Cys Gly Gln 
         35                  40                  45 

Leu Ser Gly Arg Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro 
     50                  55                  60 

Leu Gly Pro Gln Phe Pro Phe Thr Gly Val Asp Asp Arg Glu Ser Trp 
 65                  70                  75                  80 

Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met 
                 85                  90                  95 

Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys 
            100                 105                 110 

Thr Glu Arg Arg Leu Leu Val Arg Arg Asn Ile Phe Asp Leu Ser Ala 
        115                 120                 125 

Pro Glu Lys Asp Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr 
    130                 135                 140 

Ile Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys 
145                 150                 155                 160 

Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile Tyr Asp Leu Phe 
                165                 170                 175 

Val Trp Met His Tyr Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Ser 
            180                 185                 190 

Glu Ile Trp Arg Asp Ile Asp Phe Ala His Glu Ala Pro Ala Phe Leu 
        195                 200                 205 

Pro Trp His Arg Leu Phe Leu Leu Arg Trp Glu Gln Glu Ile Gln Lys 
    210                 215                 220 

Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp 
225                 230                 235                 240 

Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr Met Gly Gly Gln His 
                245                 250                 255 

Pro Thr Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp 
            260                 265                 270 

Gln Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu 
        275                 280                 285 

Cys Asn Gly Thr Pro Glu Gly Pro Leu Arg Arg Asn Pro Gly Asn His 
    290                 295                 300 

Asp Lys Ser Arg Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe 
305                 310                 315                 320 

Cys Leu Ser Leu Thr Gln Tyr Glu Ser Gly Ser Met Asp Lys Ala Ala 
                325                 330                 335 

Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr 
            340                 345                 350 

Gly Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile 
        355                 360                 365 

Tyr Met Asn Gly Thr Met Ser Gln Val Gln Gly Ser Ala Asn Asp Pro 
    370                 375                 380 

Ile Phe Leu Leu His His Ala Phe Val Asp Ser Ile Phe Glu Gln Trp 
385                 390                 395                 400 

Leu Arg Arg His Arg Pro Leu Gln Glu Val Tyr Pro Glu Ala Asn Ala 
                405                 410                 415 

Pro Ile Gly His Asn Arg Glu Ser Tyr Met Val Pro Phe Ile Pro Leu 
            420                 425                 430 

Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp 
        435                 440                 445 

Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr Ile 
    450                 455                 460 

Lys Ser Tyr Leu Glu Gln Ala Ser Arg Ile Trp Ser Trp Leu Leu Gly 
465                 470                 475                 480 

Ala Ala Met Val Gly Ala Val Leu Thr Ala Leu Leu Ala Gly Leu Val 
                485                 490                 495 

Ser Leu Leu Cys Arg His Lys Arg Lys Gln Leu Pro Glu Glu Lys Gln 
            500                 505                 510 

Pro Leu Leu Met Glu Lys Glu Asp Tyr His Ser Leu Tyr Gln Ser His 
        515                 520                 525 

Leu 
529 

 
           
             14  
             537  
             PRT  
             human  
             
               TRP-1 (GenBank Accession No. NP_000541)  
             
           
            14 

Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 
                  5                  10                  15 

Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 
             20                  25                  30 

Val Glu Ala Leu Arg Ser Gly Met Cys Cys Pro Asp Leu Ser Pro Val 
         35                  40                  45 

Ser Gly Pro Gly Thr Asp Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 
     50                  55                  60 

Cys Glu Ala Val Thr Ala Asp Ser Arg Pro His Ser Pro Gln Tyr Pro 
 65                  70                  75                  80 

His Asp Gly Arg Asp Asp Arg Glu Val Trp Pro Leu Arg Phe Phe Asn 
                 85                  90                  95 

Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 
            100                 105                 110 

Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Asp Gln Arg Val Leu Ile 
        115                 120                 125 

Val Arg Arg Asn Leu Leu Asp Leu Ser Lys Glu Glu Lys Asn His Phe 
    130                 135                 140 

Val Arg Ala Leu Asp Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 
145                 150                 155                 160 

Ile Ala Thr Arg Arg Ser Glu Glu Ile Leu Gly Pro Asp Gly Asn Thr 
                165                 170                 175 

Pro Gln Phe Glu Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 
            180                 185                 190 

Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Glu Ser Phe 
        195                 200                 205 

Gly Glu Val Asp Phe Ser His Glu Gly Pro Ala Phe Leu Thr Trp His 
    210                 215                 220 

Arg Tyr His Leu Leu Arg Leu Glu Lys Asp Met Gln Glu Met Leu Gln 
225                 230                 235                 240 

Glu Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 
                245                 250                 255 

Val Cys Asp Ile Cys Thr Asp Asp Leu Met Gly Ser Arg Ser Asn Phe 
            260                 265                 270 

Asp Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 
        275                 280                 285 

Val Cys Asp Ser Leu Glu Asp Tyr Asp Thr Leu Gly Thr Leu Cys Asn 
    290                 295                 300 

Ser Thr Glu Asp Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 
305                 310                 315                 320 

Arg Pro Met Val Gln Arg Leu Pro Glu Pro Gln Asp Val Ala Gln Cys 
                325                 330                 335 

Leu Glu Val Gly Leu Phe Asp Thr Pro Pro Phe Tyr Ser Asn Ser Thr 
            340                 345                 350 

Asn Ser Phe Arg Asn Thr Val Glu Gly Tyr Ser Asp Pro Thr Gly Lys 
        355                 360                 365 

Tyr Asp Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu 
    370                 375                 380 

Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Asp Pro Ile Phe 
385                 390                 395                 400 

Val Leu Leu His Thr Phe Thr Asp Ala Val Phe Asp Glu Trp Leu Arg 
                405                 410                 415 

Arg Tyr Asn Ala Asp Ile Ser Thr Phe Pro Leu Glu Asn Ala Pro Ile 
            420                 425                 430 

Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 
        435                 440                 445 

Asn Thr Glu Met Phe Val Thr Ala Pro Asp Asn Leu Gly Tyr Thr Tyr 
    450                 455                 460 

Glu Ile Gln Trp Pro Ser Arg Glu Phe Ser Val Pro Glu Ile Ile Ala 
465                 470                 475                 480 

Ile Ala Val Val Gly Ala Leu Leu Leu Val Ala Leu Ile Phe Gly Thr 
                485                 490                 495 

Ala Ser Tyr Leu Ile Arg Ala Arg Arg Ser Met Asp Glu Ala Asn Gln 
            500                 505                 510 

Pro Leu Leu Thr Asp Gln Tyr Gln Cys Tyr Ala Glu Glu Tyr Glu Lys 
        515                 520                 525 

Leu Gln Asn Pro Asn Gln Ser Val Val 
    530                 535 

 
           
             15  
             519  
             PRT  
             human  
             
               TRP-2(GenBank Accession No. NP_001913)  
             
           
            15 

Met Ser Pro Leu Trp Trp Gly Phe Leu Leu Ser Cys Leu Gly Cys Lys 
                  5                  10                  15 

Ile Leu Pro Gly Ala Gln Gly Gln Phe Pro Arg Val Cys Met Thr Val 
             20                  25                  30 

Asp Ser Leu Val Asn Lys Glu Cys Cys Pro Arg Leu Gly Ala Glu Ser 
         35                  40                  45 

Ala Asn Val Cys Gly Ser Gln Gln Gly Arg Gly Gln Cys Thr Glu Val 
     50                  55                  60 

Arg Ala Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln 
 65                  70                  75                  80 

Asp Asp Arg Glu Leu Trp Pro Arg Lys Phe Phe His Arg Thr Cys Lys 
                 85                  90                  95 

Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Asp Cys Lys Phe Gly 
            100                 105                 110 

Trp Thr Gly Pro Asn Cys Glu Arg Lys Lys Pro Pro Val Ile Arg Gln 
        115                 120                 125 

Asn Ile His Ser Leu Ser Pro Gln Glu Arg Glu Gln Phe Leu Gly Ala 
    130                 135                 140 

Leu Asp Leu Ala Lys Lys Arg Val His Pro Asp Tyr Val Ile Thr Thr 
145                 150                 155                 160 

Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro Gln Phe 
                165                 170                 175 

Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser 
            180                 185                 190 

Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Arg Ala Ile Asp 
        195                 200                 205 

Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr His Leu 
    210                 215                 220 

Leu Cys Leu Glu Arg Asp Leu Gln Arg Leu Ile Gly Asn Glu Ser Phe 
225                 230                 235                 240 

Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Arg Asn Glu Cys Asp Val 
                245                 250                 255 

Cys Thr Asp Gln Leu Phe Gly Ala Ala Arg Pro Asp Asp Pro Thr Leu 
            260                 265                 270 

Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp Glu Thr Val Cys Asp Ser 
        275                 280                 285 

Leu Asp Asp Tyr Asn His Leu Val Thr Leu Cys Asn Gly Thr Tyr Glu 
    290                 295                 300 

Gly Leu Leu Arg Arg Asn Gln Met Gly Arg Asn Ser Met Lys Leu Pro 
305                 310                 315                 320 

Thr Leu Lys Asp Ile Arg Asp Cys Leu Ser Leu Gln Lys Phe Asp Asn 
                325                 330                 335 

Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu 
            340                 345                 350 

Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met Ser Leu 
        355                 360                 365 

His Asn Leu Val His Ser Phe Leu Asn Gly Thr Asn Ala Leu Pro His 
    370                 375                 380 

Ser Ala Ala Asn Asp Pro Ile Phe Val Val Leu His Ser Phe Thr Asp 
385                 390                 395                 400 

Ala Ile Phe Asp Glu Trp Met Lys Arg Phe Asn Pro Pro Ala Asp Ala 
                405                 410                 415 

Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr Asn Met 
            420                 425                 430 

Val Pro Phe Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu Thr Ser 
        435                 440                 445 

Asp Gln Leu Gly Tyr Ser Tyr Ala Ile Asp Leu Pro Val Ser Val Glu 
    450                 455                 460 

Glu Thr Pro Gly Trp Pro Thr Thr Leu Leu Val Val Met Gly Thr Leu 
465                 470                 475                 480 

Val Ala Leu Val Gly Leu Phe Val Leu Leu Ala Phe Leu Gln Tyr Arg 
                485                 490                 495 

Arg Leu Arg Lys Gly Tyr Thr Pro Leu Met Glu Thr His Leu Ser Ser 
            500                 505                 510 

Lys Arg Tyr Thr Glu Glu Ala 
        515 

 
           
             16  
             42  
             DNA  
             Artificial Sequence  
             
               Amplification primer  
             
           
            16 

gcggcggaat tccaattccc aagacagtgt gccactgttg ag                        42