Patent Application: US-46921704-A

Abstract:
the present invention relates to novel tri - hybrid melanoma antigens , including antigenic fragments or derivatives thereof , of a tyrosinase antigen , a tyrosinase - related protein 1 antigen , and a tyrosinase - related protein 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:
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 . “ 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 .). [ 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 . 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 . 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 .). 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 .). 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 . 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 . the present example demonstrates construction and production of a tri - hybrid melanoma antigen , recombinant htrpx3 protein . 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 ( fig1 ). 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 . induction of bl21 e . coli cells containing plasmids encoding human htrpx3 genes resulted in expression of 155 - kda - fusion proteins in insoluble forms ( fig2 a ). 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 ( fig2 b ). the present example demonstrates induced of a humoral immune response following immunization with a tri - hybrid melanoma antigen , htrpx3 protein . 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 ( fig3 a ). a majority of immunized mice developed antibody responses against individual proteins and htrpx3 ( 27 / 30 ). to determine the subtypes of antibodies involved in these responses , antibody subtyping was performed on the elisa plates ( fig3 b ). 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 . the present example demonstrates that immunization with a tri - hybrid melanoma antigen , htrpx3 protein , induced a t cell immune response . 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 . 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 . 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 . as shown in fig5 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 . the present example demonstrates that immunization with a tri - hybrid melanoma antigen , htprx3 protein , was useful in treating tumors in mammals . 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 . mice immunized with recombinant htrpx3 were significantly protected from lung metastases of b16bl16 melanoma ( fig6 a ). in comparison with control mice , htrpx3 protein immunized mice had a significant less lung metastases , with 80 % ( p = 0 . 001 ) fewer lung nodules . 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 ( fig6 b ). 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 . 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 . atg agt gct cct aaa ctc ctc tct ctg ggc tgt atc ttc ttc ccc ttg 48 cta ctt ttt cag cag gcc cgg gct caa ttc cca aga cag tgt gcc act 96 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 tct ggg cct ggg aca gac cgc tgt ggc tca tca tca ggg agg ggc aga 192 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 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 agg aca tgt cac tgc aac ggc aat ttc tca gga cac aac tgt ggg acg 336 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 gtc agg aga aat ctt ctg gac tta agt aaa gaa gaa aag aac cac ttt 432 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 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 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 tat tac tca gtc aaa aag act ttc ctt ggg gta gga cag gaa agc ttt 624 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 agg tac cac ctc ctg cgt ctg gag aaa gac atg cag gaa atg ttg caa 720 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 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 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 gtc tgt gac tcc ttg gaa gat tat gat acc ctg gga aca ctt tgt aac 912 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 aga cca atg gtg caa cgt ctt cct gaa cca cag gat gtc gct cag tgc 1008 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 aac agt ttc cga aac aca gtg gaa ggt tac agt gac ccc acg gga aag 1104 tat gac cct gct gtt cga agt ctt cac aat ttg gct cat cta ttc ctg 1152 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 gtc ctc ctg cac acc ttc aca gat gca gtc ttt gat gaa tgg ctg agg 1248 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 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 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 gaa att caa tgg cca agt cgg gag ttt agt gta cct gag ggt ggc gga 1440 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 gag tgc tgc cca cgc ctg ggt gca gag tcg gcc aat gtc tgt ggc tct 1536 cag caa ggc cgg ggg cag tgc aca gag gtg cga gcc gac aca agg ccc 1584 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 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 ggc tat aat tgt gga gac tgc aag ttt ggc tgg acc ggt ccc aac tgc 1728 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 cct cag gaa aga gag cag ttc ttg ggc gcc tta gat ctc gcg aag aag 1824 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 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 gat ttt ttt gtg tgg ctc cat tat tat tct gtt aga gat aca tta tta 1968 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 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 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 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 ggg gca gcg aga cca gac gat ccg act ctg att agt cgg aac tca aga 2208 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 ctg gtc acc ttg tgc aat gga acc tat gaa ggt ttg ctg aga aga aat 2304 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 gat tgc ctg tct ctc cag aag ttt gac aat cct ccc ttc ttc cag aac 2400 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 ggg act ctg gat tct caa gtg atg agc ctt cat aat ttg gtt cat tcc 2496 ttc ctg aac ggg aca aac gct ttg cca cat tca gcc gcc aat gat ccc 2544 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 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 cct att ggt cac aat cgg atg tac aac atg gtt cct ttc ttc cct cca 2688 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 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 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 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 ccc tgt ggc cag ctt tca ggc aga ggt tcc tgt cag aat atc ctt ctg 2928 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 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 ggc aac ttc atg gga ttc aac tgt gga aac tgc aag ttt ggc ttt tgg 3072 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 gat ttg agt gcc cca gag aag gac aaa ttt ttt gcc tac ctc act tta 3168 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 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 tat gac ctc ttt gtc tgg atg cat tat tat gtg tca atg gat gca ctg 3312 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 cca gct ttt ctg cct tgg cat aga ctc ttc ttg ttg cgg tgg gaa caa 3408 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 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 gga ggt cag cac ccc aca aat cct aac tta ctc agc cca gca tca ttc 3552 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 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 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 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 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 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 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 gcc aac gat cct atc ttc ctt ctt cac cat gca ttt gtt gac agt att 3936 ttt gag cag tgg ctc cga agg cac cgt cct ctt caa gaa gtt tat cca 3984 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 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 ctg ggc tat gac tat agc tat cta caa gat tca gac cca gac tct ttt 4128 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 val glu ala leu arg ser gly met cys cys pro asp leu ser pro val cys glu ala val thr ala asp ser arg pro his ser pro gln tyr pro his asp gly arg asp asp arg glu val trp pro leu arg phe phe asn cys arg pro gly trp arg gly ala ala cys asp gln arg val leu ile val arg ala leu asp met ala lys arg thr thr his pro leu phe val ile ala thr arg arg ser glu glu ile leu gly pro asp gly asn thr pro gln phe glu asn ile ser ile tyr asn tyr phe val trp thr his gly glu val asp phe ser his glu gly pro ala phe leu thr trp his glu pro ser phe ser leu pro tyr trp asn phe ala thr gly lys asn val cys asp ile cys thr asp asp leu met gly ser arg ser asn phe asp ser thr leu ile ser pro asn ser val phe ser gln trp arg val ser thr glu asp gly pro ile arg arg asn pro ala gly asn val ala leu glu val gly leu phe asp thr pro pro phe tyr ser asn ser thr asn ser phe arg asn thr val glu gly tyr ser asp pro thr gly lys tyr asp pro ala val arg ser leu his asn leu ala his leu phe leu asn gly thr gly gly gln thr his leu ser pro asn asp pro ile phe arg tyr asn ala asp ile ser thr phe pro leu glu asn ala pro ile gly his asn arg gln tyr asn met val pro phe trp pro pro val thr asn thr glu met phe val thr ala pro asp asn leu gly tyr thr tyr gly gln phe pro arg val cys met thr val asp ser leu val asn lys trp ser gly pro tyr ile leu arg asn gln asp asp arg glu leu trp glu arg lys lys pro pro val ile arg gln asn ile his ser leu ser arg val his pro asp tyr val ile thr thr gln his trp leu gly leu leu gly pro asn gly thr gln pro gln phe ala asn cys ser val tyr gly pro gly arg pro tyr arg ala ile asp phe ser his gln gly pro ala phe val thr trp his arg tyr his leu leu cys leu glu arg asp leu gln arg leu ile gly asn glu ser phe ala leu pro tyr trp asn phe ala thr gly arg asn glu cys asp val cys thr asp gln leu phe phe ser ser trp glu thr val cys asp ser leu asp asp tyr asn his gln met gly arg asn ser met lys leu pro thr leu lys asp ile arg ser thr phe ser phe arg asn ala leu glu gly phe asp lys ala asp ile phe val val leu his ser phe thr asp ala ile phe asp glu trp met lys arg phe asn pro pro ala asp ala trp pro gln glu leu ala val thr asn glu glu leu phe leu thr ser asp gln leu gly tyr ser tyr ala ile asp leu pro val ser val glu glu thr pro gly trp pro thr thr gly gly gly gly his phe pro arg ala cys val ser ser lys asn leu met glu lys glu cys cys pro pro trp ser gly asp arg ser ser asn ala pro leu gly pro gln phe pro phe thr gly val asp asp arg glu ser trp pro ser val phe tyr asn arg thr cys gln cys ser gly pro asn cys thr glu arg arg leu leu val arg arg asn ile phe ala lys his thr ile ser ser asp tyr val ile pro ile gly thr tyr gly gln met lys asn gly ser thr pro met phe asn asp ile asn ile leu gly gly ser glu ile trp arg asp ile asp phe ala his glu ala glu ile gln lys leu thr gly asp glu asn phe thr ile pro tyr trp asp trp arg asp ala glu lys cys asp ile cys thr asp glu tyr met phe ser ser trp gln ile val cys ser arg leu glu glu tyr asn ser his gln ser leu cys asn gly thr pro glu gly pro leu arg arg asn pro gly asn his asp lys ser arg thr pro arg leu pro ser ser ala asp val glu phe cys leu ser leu thr gln tyr glu ser gly ser met asp lys ala ala asn phe ser phe arg asn thr leu glu gly phe ala ser pro leu thr gly ile ala asp ala ser gln ser ser met his asn ala leu his ile tyr met asn gly thr met ser gln val gln gly ser glu ala asn ala pro ile gly his asn arg glu ser tyr met val pro phe ile pro leu tyr arg asn gly asp phe phe ile ser ser lys asp gln asp tyr ile lys ser tyr leu glu gln ala ser arg ile trp ser 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 tgt tgc cca gac ctg tcc cct gtg tct ggg cct ggg aca gac cgc tgt 96 ggc tca tca tca ggg agg ggc aga tgt gag gca gtg act gca gac tcc 144 cgg ccc cac agc cct cag tat ccc cat gat ggc aga gat gat cgg gag 192 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 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 gcc tgt gac cag agg gtt ctc ata gtc agg aga aat ctt ctg gac tta 336 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 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 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 tat aac tac ttt gtt tgg aca cac tat tac tca gtc aaa aag act ttc 528 ctt ggg gta gga cag gaa agc ttt ggt gaa gtg gat ttc tct cat gag 576 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 aaa gac atg cag gaa atg ttg caa gag cct tct ttc tcc ctt cct tac 672 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 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 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 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 aga aat cca gct gga aat gtg gcc aga cca atg gtg caa cgt ctt cct 912 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 cct cct ttt tat tcc aac tct aca aac agt ttc cga aac aca gtg gaa 1008 ggt tac agt gac ccc acg gga aag tat gac cct gct gtt cga agt ctt 1056 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 ttg tct cca aat gat cct att ttt gtc ctc ctg cac acc ttc aca gat 1152 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 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 gtg cca ttc tgg ccc cca gtc acc aac aca gaa atg ttt gtt act gct 1296 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 ttt agt gta cct gag ggt ggc gga ggg cag ttc ccc cga gtc tgc atg 1392 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 gag tcg gcc aat gtc tgt ggc tct cag caa ggc cgg ggg cag tgc aca 1488 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 aac cag gat gac cgt gag ctg tgg cca aga aaa ttc ttc cac cgg acc 1584 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 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 cgg cag aac atc cat tcc ttg agt cct cag gaa aga gag cag ttc ttg 1728 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 acc aca caa cac tgg ctg ggc ctg ctt ggg ccc aat gga acc cag ccg 1824 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 tat tct gtt aga gat aca tta tta gga cca gga cgc ccc tac agg gcc 1920 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 cat ttg ttg tgt ctg gaa aga gat ctc cag cga ctc att ggc aat gag 2016 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 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 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 gat agc ttg gat gac tac aac cac ctg gtc acc ttg tgc aat gga acc 2208 tat gaa ggt ttg ctg aga aga aat caa atg gga aga aac agc atg aaa 2256 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 gac aat cct ccc ttc ttc cag aac tct acc ttc agt ttc agg aat gct 2352 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 agc ctt cat aat ttg gtt cat tcc ttc ctg aac ggg aca aac gct ttg 2448 cca cat tca gcc gcc aat gat ccc att ttt gtg gtt ctt cat tcc ttt 2496 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 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 aac atg gtt cct ttc ttc cct cca gtg act aat gaa gaa ctc ttt tta 2640 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 gtt gaa gaa act cca ggt tgg ccc aca act ggc ggg ggt gga cat ttc 2736 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 cca ccg tgg agc ggg gac agg agt ccc tgt ggc cag ctt tca ggc aga 2832 ggt tcc tgt cag aat atc ctt ctg tcc aat gca cca ctt ggg cct caa 2880 ttt ccc ttc aca ggg gtg gat gac cgg gag tcg tgg cct tcc gtc ttt 2928 tat aat agg acc tgc cag tgc tct ggc aac ttc atg gga ttc aac tgt 2976 gga aac tgc aag ttt ggc ttt tgg gga cca aac tgc aca gag aga cga 3024 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 aaa ttt ttt gcc tac ctc act tta gca aag cat acc atc agc tca gac 3120 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 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 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 gac att gat ttt gcc cat gaa gca cca gct ttt ctg cct tgg cat aga 3312 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 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 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 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 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 ccc gag gga cct tta cgg cgt aat cct gga aac cat gac aaa tcc aga 3600 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 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 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 gcc tct caa agc agc atg cac aat gcc ttg cac atc tat atg aat gga 3792 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 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 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 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 gat ttc ttt att tca tcc aaa gat ctg ggc tat gac tat agc tat cta 4032 caa gat tca gac cca gac tct ttt caa gac tac att aag tcc tat ttg 4080 gln phe pro arg gln cys ala thr val glu ala leu arg ser gly met val trp pro leu arg phe phe asn arg thr cys his cys asn gly asn phe ser gly his asn cys gly thr cys arg pro gly trp arg gly ala ser lys glu glu lys asn his phe val arg ala leu asp met ala lys arg thr thr his pro leu phe val ile ala thr arg arg ser glu glu ile leu gly pro asp gly asn thr pro gln phe glu asn ile ser ile gly pro ala phe leu thr trp his arg tyr his leu leu arg leu glu trp asn phe ala thr gly lys asn val cys asp ile cys thr asp asp leu met gly ser arg ser asn phe asp ser thr leu ile ser pro asn ser val phe ser gln trp arg val val cys asp ser leu glu asp tyr asp thr leu gly thr leu cys asn ser thr glu asp gly pro ile arg glu pro gln asp val ala gln cys leu glu val gly leu phe asp thr gly tyr ser asp pro thr gly lys tyr asp pro ala val arg ser leu ala val phe asp glu trp leu arg arg tyr asn ala asp ile ser thr phe pro leu glu asn ala pro ile gly his asn arg gln tyr asn met pro asp asn leu gly tyr thr tyr glu ile gln trp pro ser arg glu thr val asp ser leu val asn lys glu cys cys pro arg leu gly ala glu val arg ala asp thr arg pro trp ser gly pro tyr ile leu arg asn gln asp asp arg glu leu trp pro arg lys phe phe his arg thr phe gly trp thr gly pro asn cys glu arg lys lys pro pro val ile gly ala leu asp leu ala lys lys arg val his pro asp tyr val ile gln phe ala asn cys ser val tyr asp phe phe val trp leu his tyr ile asp phe ser his gln gly pro ala phe val thr trp his arg tyr ser phe ala leu pro tyr trp asn phe ala thr gly arg asn glu cys asp val cys thr asp gln leu phe gly ala ala arg pro asp asp pro thr leu ile ser arg asn ser arg phe ser ser trp glu thr val cys leu pro thr leu lys asp ile arg asp cys leu ser leu gln lys phe leu glu gly phe asp lys ala asp gly thr leu asp ser gln val met thr asp ala ile phe asp glu trp met lys arg phe asn pro pro ala asp ala trp pro gln glu leu ala pro ile gly his asn arg met tyr thr ser asp gln leu gly tyr ser tyr ala ile asp leu pro val ser pro arg ala cys val ser ser lys asn leu met glu lys glu cys cys leu leu val arg arg asn ile phe asp leu ser ala pro glu lys asp tyr val ile pro ile gly thr tyr gly gln met lys asn gly ser thr pro met phe asn asp ile asn ile tyr asp leu phe val trp met his tyr tyr val ser met asp ala leu leu gly gly ser glu ile trp arg leu phe leu leu arg trp glu gln glu ile gln lys leu thr gly asp glu asn phe thr ile pro tyr trp asp trp arg asp ala glu lys cys asp ile cys thr asp glu tyr met gly gly gln his pro thr asn pro asn leu leu ser pro ala ser phe phe ser ser trp gln ile val cys ser arg leu glu glu tyr asn ser his gln ser leu cys asn gly thr thr pro arg leu pro ser ser ala asp val glu phe cys leu ser leu thr gln tyr glu ser gly ser met asp lys ala ala asn phe ser phe arg asn thr leu glu gly phe ala ser pro leu thr gly ile ala asp thr met ser gln val gln gly ser ala asn asp pro ile phe leu leu his his ala phe val asp ser ile phe glu gln trp leu arg arg his arg pro leu gln glu val tyr pro glu ala asn ala pro ile gly his asn arg glu ser tyr met val pro phe ile pro leu tyr arg asn gly atg agt gct cct aaa ctc ctc tct ctg ggc tgt atc ttc ttc ccc ttg 48 cta ctt ttt cag cag gcc cgg gct caa ttc cca aga cag tgt gcc act 96 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 tct ggg cct ggg aca gac cgc tgt ggc tca tca tca ggg agg ggc aga 192 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 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 agg aca tgt cac tgc aac ggc aat ttc tca gga cac aac tgt ggg acg 336 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 gtc agg aga aat ctt ctg gac tta agt aaa gaa gaa aag aac cac ttt 432 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 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 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 tat tac tca gtc aaa aag act ttc ctt ggg gta gga cag gaa agc ttt 624 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 agg tac cac ctc ctg cgt ctg gag aaa gac atg cag gaa atg ttg caa 720 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 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 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 gtc tgt gac tcc ttg gaa gat tat gat acc ctg gga aca ctt tgt aac 912 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 aga cca atg gtg caa cgt ctt cct gaa cca cag gat gtc gct cag tgc 1008 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 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 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 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 gtc ctc ctg cac acc ttc aca gat gca gtc ttt gat gaa tgg ctg agg 1248 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 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 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 gaa att caa tgg cca agt cgg gag ttt agt gta cct gag cag ttc ccc 1440 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 cgc ctg ggt gca gag tcg gcc aat gtc tgt ggc tct cag caa ggc cgg 1536 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 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 ttc cac cgg acc tgc aag tgc aca gga aac ttt gcc ggc tat aat tgt 1680 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 cca cca gtg att cgg cag aac atc cat tcc ttg agt cct cag gaa aga 1776 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 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 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 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 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 tgg cac cgg tac cat ttg ttg tgt ctg gaa aga gat ctc cag cga ctc 2064 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 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 cca gac gat ccg act ctg att agt cgg aac tca aga ttc tcc agc tgg 2208 gaa act gtc tgt gat agc ttg gat gac tac aac cac ctg gtc acc ttg 2256 tgc aat gga acc tat gaa ggt ttg ctg aga aga aat caa atg gga aga 2304 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 ctc cag aag ttt gac aat cct ccc ttc ttc cag aac tct acc ttc agt 2400 ttc agg aat gct ttg gaa ggg ttt gat aaa gca gat ggg act ctg gat 2448 tct caa gtg atg agc ctt cat aat ttg gtt cat tcc ttc ctg aac ggg 2496 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 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 aat cct cct gca gat gcc tgg cct cag gag ctg gcc cct att ggt cac 2640 aat cgg atg tac aac atg gtt cct ttc ttc cct cca gtg act aat gaa 2688 gaa ctc ttt tta acc tca gac caa ctt ggc tac agc tat gcc atc gat 2736 ctg cca gtt tca gtt gaa gaa act cca ggt tgg ccc aca act cat ttc 2784 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 cca ccg tgg agc ggg gac agg agt ccc tgt ggc cag ctt tca ggc aga 2880 ggt tcc tgt cag aat atc ctt ctg tcc aat gca cca ctt ggg cct caa 2928 ttt ccc ttc aca ggg gtg gat gac cgg gag tcg tgg cct tcc gtc ttt 2976 tat aat agg acc tgc cag tgc tct ggc aac ttc atg gga ttc aac tgt 3024 gga aac tgc aag ttt ggc ttt tgg gga cca aac tgc aca gag aga cga 3072 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 aaa ttt ttt gcc tac ctc act tta gca aag cat acc atc agc tca gac 3168 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 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 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 gac att gat ttt gcc cat gaa gca cca gct ttt ctg cct tgg cat aga 3360 ctc ttc ttg ttg cgg tgg gaa caa gaa atc cag aag ctg aca gga gat 3408 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 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 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 agc cga ttg gag gag tac aac agc cat cag tct tta tgc aat gga acg 3600 ccc gag gga cct tta cgg cgt aat cct gga aac cat gac aaa tcc aga 3648 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 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 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 gcc tct caa agc agc atg cac aat gcc ttg cac atc tat atg aat gga 3840 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 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 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 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 gat ttc ttt att tca tcc aaa gat ctg ggc tat gac tat agc tat cta 4080 caa gat tca gac cca gac tct ttt caa gac tac att aag tcc tat ttg 4128 val gln ala leu arg ser gly met cys cys pro glu leu ser pro val cys gln ala val thr ala glu ser arg pro his ser pro gln tyr pro his glu gly arg glu glu arg gln val trp pro leu arg phe phe asn cys arg pro gly trp arg gly ala ala cys glu gln arg val leu ile val arg ala leu glu met ala lys arg thr thr his pro leu phe val ile ala thr arg arg ser gln gln ile leu gly pro glu gly asn thr pro gln phe gln asn ile ser ile tyr asn tyr phe val trp thr his gly gln val glu phe ser his gln gly pro ala phe leu thr trp his gln pro ser phe ser leu pro tyr trp asn phe ala thr gly lys asn val cys glu ile cys thr glu glu leu met gly ser arg ser asn phe glu ser thr leu ile ser pro asn ser val phe ser gln trp arg val ser thr gln glu gly pro ile arg arg asn pro ala gly asn val ala leu gln val gly leu phe glu thr pro pro phe tyr ser asn ser thr asn ser phe arg asn thr val gln gly tyr ser glu pro thr gly lys tyr glu pro ala 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phe gln gln trp leu arg arg his arg pro leu gln gln val tyr pro gln ala asn ala pro ile gly his asn arg gln ser tyr met val pro phe ile pro leu tyr arg asn gly met leu leu ala val leu tyr cys leu leu trp ser phe gln thr ser ala gly his phe pro arg ala cys val ser ser lys asn leu met glu leu gly pro gln phe pro phe thr gly val asp asp arg glu ser trp pro ser val phe tyr asn arg thr cys gln cys ser gly asn phe met thr glu arg arg leu leu val arg arg asn ile phe asp leu ser ala ile ser ser asp tyr val ile pro ile gly thr tyr gly gln met lys asn gly ser thr pro met phe asn asp ile asn ile tyr asp leu phe leu thr gly asp glu asn phe thr ile pro tyr trp asp trp arg asp ala glu lys cys asp ile cys thr asp glu tyr met gly gly gln his gln ile val cys ser arg leu glu glu tyr asn ser his gln ser leu asp lys ser arg thr pro arg leu pro ser ser ala asp val glu phe cys leu ser leu thr gln tyr glu ser gly ser met asp lys ala ala tyr met asn gly thr met ser gln val gln gly ser ala asn asp pro ile phe leu leu his his ala phe val asp ser ile phe glu gln trp pro ile gly his asn arg glu ser tyr met val pro phe ile pro leu lys ser tyr leu glu gln ala ser arg ile trp ser trp leu leu gly val glu ala leu arg ser gly met cys cys pro asp leu ser pro val cys glu ala val thr ala asp ser arg pro his ser pro gln tyr pro his asp gly arg asp asp arg glu val trp pro leu arg phe phe asn cys arg pro gly trp arg gly ala ala cys asp gln arg val leu ile val arg ala leu asp met ala lys arg thr thr his pro leu phe val ile ala thr arg arg ser glu glu ile leu gly pro asp gly asn thr pro gln phe glu asn ile ser ile tyr asn tyr phe val trp thr his gly glu val asp phe ser his glu gly pro ala phe leu thr trp his glu pro ser phe ser leu pro tyr trp asn phe ala thr gly lys asn val cys asp ile cys thr asp asp leu met gly ser arg ser asn phe asp ser thr leu ile ser pro asn ser val phe ser gln trp arg val ser thr glu asp gly pro ile arg arg asn pro ala gly asn val ala leu glu val gly leu phe asp thr pro pro phe tyr ser asn ser thr asn ser phe arg asn thr val glu gly tyr ser asp pro thr gly lys tyr asp pro ala val arg ser leu his asn leu ala his leu phe leu asn gly thr gly gly gln thr his leu ser pro asn asp pro ile phe arg tyr asn ala asp ile ser thr phe pro leu glu asn ala pro ile gly his asn arg gln tyr asn met val pro phe trp pro pro val thr asn thr glu met phe val thr ala pro asp asn leu gly tyr thr tyr ala ser tyr leu ile arg ala arg arg ser met asp glu ala asn gln ile leu pro gly ala gln gly gln phe pro arg val cys met thr val asp ser leu val asn lys glu cys cys pro arg leu gly ala glu ser arg ala asp thr arg pro trp ser gly pro tyr ile leu arg asn gln asp asp arg glu leu trp pro arg lys phe phe his arg thr cys lys trp thr gly pro asn cys glu arg lys lys pro pro val ile arg gln asn ile his ser leu ser pro gln glu arg glu gln phe leu gly ala leu asp leu ala lys lys arg val his pro asp tyr val ile thr thr ala asn cys ser val tyr asp phe phe val trp leu his tyr tyr ser phe ser his gln gly pro ala phe val thr trp his arg tyr his leu leu cys leu glu arg asp leu gln arg leu ile gly asn glu ser phe ala leu pro tyr trp asn phe ala thr gly arg asn glu cys asp val ile ser arg asn ser arg phe ser ser trp glu thr val cys asp ser thr leu lys asp ile arg asp cys leu ser leu gln lys phe asp asn gly phe asp lys ala asp gly thr leu asp ser gln val met ser leu ser ala ala asn asp pro ile phe val val leu his ser phe thr asp ala ile phe asp glu trp met lys arg phe asn pro pro ala asp ala trp pro gln glu leu ala pro ile gly his asn arg met tyr asn met asp gln leu gly tyr ser tyr ala ile asp leu pro val ser val glu arg leu arg lys gly tyr thr pro leu met glu thr his leu ser ser