Patent Application: US-83685497-A

Abstract:
a method is disclosed for the efficient production of a transfected cell which comprises a step of culturing transfected cells in the presence of a cell - adhesive substance , after injection of a foreign gene into target cells , upon production of the transfected cells by transfer of a foreign gene into the target cells through cell perforation . also provided are transfected cells produced by the method , and a kit for the production of transfected cells that includes a cell - adhesive substance as an essential component , which kit is suitable for use with the method for the efficient production of transfected cells by cell perforation .

Description:
the method of the present invention is characterized by culturing the cell in the presence of a substance having the cell - adhesive activity after a foreign gene is transferred into target cells using a perforation method . as used herein , the perforation method means a method for injection of a gene by perforating a cell wall , including an electroporation method , a microinjection method , a particle gun method and the like . the electroporation method is as described in , for example , tanpakushitsu , kakusan , koso , volume 31 , pages 1591 - 1603 ( 1986 ). the microinjection method is as described in , for example , cell , volume 22 , pages 479 - 488 ( 1980 ). the particle gun method is as described in , for example , technique , volume 3 , pages 3 - 16 ( 1991 ). these methods include the known methods used for transferring a gene into cells . as cells to be used in these perforation methods , for example , there are animal cells which are prepared according to a known method &# 34 ; shin - seikagaku jikkenkoza 18 , saibobaiyogijyutsu &# 34 ;, 1st edition ( 1990 ), edited by nippon seikagakugakkai , published by tokyo kagakudojin ! and cultured animal cells may be used . as used herein , a cell - adhesive substance refers to a substance having the cell - adhesive , that is , the activity to make target cells adhere to a cell , to an extracellular matrix which is a substance filling a space between cells in the tissue , or to a material such as plastic , glass and the like . in the present invention , any substances having the activity can be used as long as they give no adverse effects on transfection of target cells . such the activity is to fix cells , for example , to culture ware covered with a cell - adhesive substance while maintaining the cell in its form , or in a spreaded form , that is , in the changed form after the cell has been spreaded in one or more directions . attachment between the cell - adhesive substance and the target cell can be assayed using a conventional method . the method includes , for example , a method described in nature , 352 : 438 - 441 ( 1991 ). briefly , the cell - adhesive substance covers a plastic dish , and a population of cells to be assayed is put into a medium and allowing it to stand for 30 minutes to 2 hours . after this incubation period , non - adhered cells are recovered , counted and assayed for viability . cells adhered to the cell - adhesive substance are recovered using trypsin or a cell dissociation buffer ( for example , gibco ), counted and assayed for viability . then , a ratio of adhered cells is calculated and compared with a standard control such as a plastic dish covered with bovine serum albumin ( bsa ). a combination of cell - adhesive substance / cell can be determined by substantial adhesion of the target cell with the cell - adhesive substance assayed . in addition , the cell - spreading activity can be determined by observing a change in the form of the adhered cells under a microscope before dissociating cells using trypsin or a cell dissociation buffer , in the above procedures . examples of the cell - adhesive substance include , for example , a cell - adhesive polypeptide or a functional equivalent thereof and a cell - adhesive synthetic polymer . examples of the polypeptide to be used in the present invention , having the cell - adhering activity include a cell - adhesive polypeptide such as invasin , polylysine which is not derived from an extracellular matrix , a polypeptide showing the cell - spreading activity described in jp - a 2 - 311498 , components of an extracellular matrix such as fibronectin , laminin , collagen , vitronectin , osteopontin , thrombospondin , tenascin and the like . the extracellular matrix components can be prepared from a natural or cultured source by a known method international journal of cancer , volume 20 , pages 1 - 5 ( 1977 ); journal of biological chemistry , volume 254 , pages 9933 - 9937 , ( 1979 ); &# 34 ; zoku - seikagaku jikkenkoza &# 34 ;, volume 6 , saibokokkaku no kozo to kino ( structure and function of cell skeleton ) ( last volume ), ( 1st edition ) ( 1986 ) edited by nippon seikagakugakkai , published by tokyo kagakudojin ; cell structure and function , volume 13 , pages 281 - 292 ( 1988 ); journal of biological chemistry , volume 264 , pages 18202 - 18208 ( 1989 ); and journal of biological chemistry , volume 260 , pages 12240 - 12245 ( 1985 )!. the cell - adhesive polypeptide may be substantially purified extracellular matrices exhibiting the cell - adhering activity , substantially purified extracellular matrix fragments or a mixture thereof . more particularly , proteins and polypeptides having the cell - adhesive or the cell spreading activity , or a functional equivalent thereof , may be used . as these cell - adhesive polypeptides , substantially purified natural polypeptides , polypeptides from enzymatical or chemical degradation of the natural polypeptides , or the similar polypeptides made by genetic engineering may be used . further , materials obtained by modifying these polypeptides without impairing the function , that is , the cell - adhering activity or the cell - spreading activity may be used . in the present invention , even if the polypeptide has a deletion , substitution , addition and / or insertion of amino acids in the amino acid sequence of a polypeptide from natural origin , as long as the polypeptide has the desired cell - adhering activity or the cell - spreading activity , it is referred to as a functional equivalent of a polypeptide having the natural amino acid sequence . that is , it is known that naturally occurring proteins include proteins of which amino acid sequences have mutations such as deletions , insertions , additions , substitutions and the like of amino acids , which are due to a modification reaction in the living body after production or during purification , in addition to polymorphism or mutation of genes encoding those naturally occurring proteins . regardless of these mutations , there are proteins exhibiting the physiological and biological activity substantially equivalent to that of proteins having no mutation . like this , even when there is a structural difference between polypeptides , as long as they share the common main functions , they are called polypeptides having the functionally equivalent activity . this is also true where the above mutations are artificially introduced into the amino acid sequence of proteins . in this case , a greater variety of mutants may be made . as long as these mutants exhibit the physiological activity substantially equivalent to that of proteins having no mutations , they are interpreted to be a polypeptide having the functionally equivalent activity . for example , in many cases , a methionine residue which is present at a n - terminus of a protein expressed in escherichia coli is said to be removed by action of methionine aminopeptidase , thus , generating both proteins having a methionine residue or those having no methionine residue depending upon the kind of proteins . however , whether or not a protein has a methionine residue does not affect the protein activity in many cases . in addition , it is known that a polypeptide , a certain cysteine residue of which is substituted with a serine residue in the amino acid sequence of human interleukin - 2 ( il - 2 ), retains the interleukin - 2 activity science , volume 224 , page 1431 ( 1984 )!. further , upon production of proteins by genetic engineering , proteins are frequently expressed as fused proteins . for example , in order to increase an amount of an expressed protein of interest , the protein is expressed by adding a n - terminal peptide chain derived from another protein to the n - terminal of the protein of interest , or adding a suitable peptide chain to the n - terminus or the c - terminus of the protein of interest to facilitate purification of the protein of interest by using a carrier having the affinity to the added peptide chain . in this respect , the related biotechnological 10 techniques have advanced to a state in which deletion , substitution , addition or other modification of amino acids in a functional region of the polypeptide can be routinely carried out . then , the resulting amino acid sequence may be routinely screened for the desired cell - adhering activity or the cell - spreading activity according to the above method . polypeptides having the cell - adhering activity may be an artificial polypeptide containing , in the molecule , the amino acid sequence necessary for the cell - adhering activity , for example , the amino acid sequence may be selected from the amino acid sequence represented by seq id no : 1 ( rgds ), the amino acid sequence represented by seq id no : 2 ( cs1 ) and the amino acid sequence represented by seq id no : 6 ( central sequence of laminin , yigsr ). these polypeptides can be prepared in a large amount by a genetic engineering method or a chemical synthesis method and may be used as a purified polypeptide . examples of the artificial polypeptide having , in the molecule , the amino acid sequence represented by seq id no : 1 include a polypeptide represented by seq id no : 7 described in jp - a 1 - 180900 . the polypeptide can be prepared using escherichia coli hb101 / ptf1409 ( ferm bp - 1939 ) according to a method described in jp - a 1 - 180900 . in addition polypeptides represented by their respective sequence id numbers in the sequence list shown in table 1 below can be prepared according to a genetic engineering method described in each specification . in addition , a plasmid pchv90 contained in escherichia coli hb101 / pchv90 in table 1 can be prepared using escherichia coli hb101 / phd101 ( ferm bp - 2264 ) and escherichia coli jm109 / ptf7021 ( ferm bp - 1941 ) according to a method described in jp - a 5 - 271291 . table 1______________________________________laid open seq id living bacteriumpublication no ( escherichia coli ) accession no . ______________________________________jp - a 1 - 206998 8 jm109 / ptf7021 ferm bp - 1941jp - a 1 - 261398 9 hb101 / ptf1801 ferm p - 9948jp - a 2 - 97397 3 jm109 / ptf7221 ferm bp - 1915jp - a 2 - 152990 10 jm109 / ptfb800 ferm bp - 2126jp - a 2 - 311498 11 hb101 / pch101 ferm bp - 2799jp - a 3 - 59000 12 jm109 / pcf406 ferm p - 10837jp - a 3 - 232898 13 hb101 / pce102 ferm p - 11226jp - a 4 - 54199 14 jm109 / ptf7520 + ferm p - 11526 vn - in . taa 15 jm109 / ptf7520 + ferm p - 11527 col . sup .× 1jp - a 5 - 271291 16 hb101 / pchv179 ferm p - 12183 17 hb101 / pchv90 18 hb101 / pchv89 ferm p - 182jp - a 5 - 97698 19 jm109 / ptf7520colv ferm bp - 5277jp - a 5 - 178897 20 jm109 / pymh - cf . a ferm bp - 5278______________________________________ alternatively , artificial polypeptides having , in the molecule , the amino acid sequence represented by seq id no : 1 can be polyrgds described , described in jp - a 3 - 173828 , can be synthesized and used . examples of artificial polypeptides having , in the molecule , the amino acid sequence represented by seq id no : 2 include a polypeptide represented by seq id no : 4 , described in jp - a 2 - 311498 , and the polypeptide can be prepared by genetic engineering using escherichia coli hb101 / phd102 ( ferm p - 10721 ) according to a method described in jp - a 2 - 311498 . in addition , a polypeptide represented by seq id no : 2 may be chemically synthesized according to a method described in jp - a 3 - 284700 . further , examples of artificial polypeptides having , in the molecule , the amino acid sequence represented by seq id no : 2 and the amino acid sequence represented by seq id no : 3 include a polypeptide represented by seq id no : 21 described in jp - a 2 - 311498 and the polypeptide can be prepared by genetic engineering using escherichia coli hb101 / pch102 ( ferm bp - 2800 ) according to a method described in jp - a 2 - 311498 . in addition , a polypeptide represented by seq id no : 5 described in jp - a 3 - 284700 is a polypeptide containing , in the molecule , the amino acid sequences of seq id nos : 1 and 2 and the polypeptide can be prepared by genetic engineering using escherichia coli hb101 / pcs25 ( ferm p - 11339 ) according to a method described in jp - a 3 - 284700 . as described above , examples of the polypeptides used in the present invention are cell - adhesive polypeptides containing , in the molecule , the amino acid sequence represented by seq id no : 1 and / or the amino acid sequence represented by seq id no : 2 . as the polypeptide , a polypeptide obtained by covalently binding a polypeptide derived from a cell adhesion domain of human fibronectin &# 34 ; fibronectin &# 34 ;, pages 47 - 121 ( 1989 ), edited by mosher , d . f ., published by academic press ! with a cs1 polypeptide derived from the same ( ibid ), a polypeptide derived from a heparin binding domain ( ibid ) containing a cs1 polypeptide , or a polypeptide derived from a cell adhesion domain can be used , and they can be made by genetic engineering , respectively . for example , respective necessary regions are taken out from a vector containing a dna encoding a cell adhesion domain - derived polypeptide , a vector containing a dna encoding a cs1 polypeptide , and a vector containing a dna encoding a heparin binding domain - derived peptide containing a cs1 polypeptide , respectively , and they can be used alone or in combination thereof to make a vector expressing a polypeptide containing , in the molecule , the amino acid sequence represented by seq id no : 1 and / or the amino acid sequence represented by seq id no : 2 . when a polypeptide , where a polypeptide containing , in the molecule , the amino acid sequence represented by seq id no : 1 and a polypeptide containing , in the molecule , the amino acid sequence represented by seq id no : 2 are covalently bound , is made , a covalent bonding between polypeptides may be a direct bonding or an indirect bonding , for example , an indirect bonding via a spacer . a spacer is an insertion sequence for adjusting an intermolecular distance in each region . as the spacer , an arbitrary peptide chain can be used , for example , a sequence upstream of a cs1 region in the fibronectin molecule . the spacer sequence can be easily introduced therein by genetic engineering . in the present invention , the target cell include , but is not limited to , hematopoietic stem cell , peripheral blood stem cell , umbilical blood cell , es cell , lymphocyte , cancer cell and the like . examples of the foreign gene include , but are not limited to , nucleic acid selected from nucleic acids encoding proteins , nucleic acids encoding polypeptides , antisense dnas , antisense rnas , ribozymes , nucleic acids encoding intracellular antibodies and pseudogenes ( decoy genes ). in the present invention , the foreign gene may be inserted into a vector . examples of the vector are retrovirus vector , adenovirus vector , vacciniavirus vector , herpesvirus vector and the like . according to the present invention a transfected cell with a foreign gene can be obtained with high efficiency by culturing a target cell , into which a foreign gene has been transferred by a perforation method according to a conventional method , in the presence of a cell - adhesive substance . a cell culture method may be selected from the known methods depending upon the cell used . for example , when cell culturing is performed in the presence of a cell - adhesive polypeptide , 250 to 2000 μg / ml of the cell - adhesive polypeptide may be used in a culture medium to culture it according to a conventional method . particularly , culturing is preferably carried out using a culture ware covered with a cell - adhesive substance . the culture ware refers to any ware normally used for cell culture , for example , a culture dish , a culture ware using a microcarrier , and a culture ware using fibrous hollow fibers . the culture ware may be covered with the substance by coating or spraying . for example , the culture may be easily covered with the polypeptide by dissolving it in a suitable solution such as a phosphate buffered saline ( pbs ), adding the solution to the culture ware and allowing it to stand for a suitable period of time . an amount of the polypeptide with which the culture ware is covered may be selected from a range of 50 to 1000 pmol / cm 2 , suitably 150 to 600 pmol / cm 2 . transfected cells which have been cultured in the presence of the cell - adhesive substance can be obtained from a culture according to a conventional method . thus , transfected cells can be produced with high efficiency . the resulting transfected cells are useful for the production of useful substances by cells using recombinant dna techniques , development of disease models , gene therapy and the like . thus , transfected cells can be produced with high efficiency according to the present invention . in addition , the present invention can be simply carried out by using a kit containing a cell - adhesive substance . the cell - adhesive substance to be contained in the kit may be in a form of solutions or lyophilized powders . the kit may contain a buffer for dissolving or diluting the cell - adhesive substance , a cell culture medium , a cell culture ware and the like . for example , a transfected cell can be simply produced by preparing a kit combining polypeptides , pbs for diluting the polypeptide , a cell culture ware and the like which are used for the method of the present invention . a reagent contained in the kit may be in liquid form or in lyophilized form . a perforation method in the present invention can be used by appropriately selecting from an electroporation method , a microinjection method , a particle gun method and the like depending upon the purpose . a polypeptide represented by seq id no : 3 ( hereinafter referred to as &# 34 ; c274 &# 34 ;), a polypeptide represented by seq id no : 4 ( hereinafter referred to as h296 ) and a polypeptide represented by seq id no : 5 ( hereinafter referred to as &# 34 ; c · cs1 &# 34 ;) were each dissolved in a phosphate buffered saline ( pbs ) to 1 μm , respectively , which were then sterilized using a 0 . 22 μm filter ( millex - gv , millipore ). each 1 ml / well of these solutions was added to a 24 - well polystyrene culture dish ( manufactured by corning ), respectively , to coat the dish at 4 ° c . overnight . these dishes were rinsed with a 500μl / well of a dulbecco &# 39 ; s modified minimum basal medium not containing bovine fetal serum prior to the addition of a transformed cell described below . two culture dishes ( diameter : 100 mm ) of human epidermoid cancer cell a - 431 which had been cultured in a dulbecco &# 39 ; s modified minimum basal medium containing 10 % bovine fetal serum were rinsed with 10 ml of a dulbecco &# 39 ; s modified minimum basal medium not containing bovine fetal serum , respectively , and 3 ml of pbs containing 0 . 25 % bovine trypsin and 0 . 02 % edta was added thereto to detach cells from the culture dish . to these , 7 ml of a dulbecco &# 39 ; s modified minimum basal medium not containing bovine fetal serum was added , followed by centrifugation at 800 rpm for 3 minutes to collect cells . the resulting cells were suspended in 10 ml of a dulbecco &# 39 ; s modified minimum basal medium containing bovine fetal serum , followed by centrifugation at 800 rpm for 3 minutes to collect cells . the resulting cells were combined , suspended in 10 ml of pbs , a 3 / 10 aliquot of the suspension was taken and divided into two equal aliquots , which were centrifuged at 800 rpm for 3 minutes to collect cells , respectively . the resulting cells were suspended again in 10 ml of pbs , followed by centrifugation at 800 rpm for 3 minutes to collect two batches of cells . one batch of the resulting cells were suspended in 1 ml of pbs containing 15 μg of pcat - control vector ( promega ) which had been aseptically prepared , and placed in an electroporation cuvette for gene pulser ( biorad ), which was allowed to stand in ice for 10 minutes . the other batch of the resulting cells were suspended in 1 ml of pbs , and placed in an electroporation cuvette for gene pulser ( biorad ), which was allowed to 15 stand in ice for 10 minutes . each batch of cells was allowed to stand in ice for 10 minutes , and voltage was applied thereto at 250 v and 960 μf . after application , the cells were allowed to stand in a cuvette in ice for 10 minutes . thereafter , the cells were recovered into 15 ml 20 of a dulbecco &# 39 ; s modified minimum basal medium containing 10 % bovine fetal serum , 1 ml / well of which were added to a 24 - well polystyrene culture dish covered with the above polypeptide . these cells were cultured at 37 ° c . in the presence of 5 % co 2 gas overnight , the medium was removed by aspiration , and 1 ml / well of a fresh dulbecco &# 39 ; s modified minimum basal medium containing 10 % bovine fetal serum was added thereto , followed by culturing at 38 ° c . in the presence of 5 % co 2 gas overnight . the cultured cells were rinsed three times with 1 . 25 ml of pbs per well , a lysed cell solution was prepared , and detection of expressed cat was carried out using cat - elisa kit ( manufactured by boehringer mannheim ) according to a method for using the present kit . since the present kit used a horseradish peroxidase - labelled secondary antibody and abts as a substrate , a ratio of absorbance at 405 nm / 490 nm was determined . a value obtained by subtracting a blank value from a value for each group in a case of addition of pcat - control vector , using a value for the group in a case of no addition of pcat - control vector upon electroporation as a blank , was adopted as an amount of expressed cat . the results thereof are shown in fig1 . that is , fig1 is a figure showing efficiency of gene transfer into a cell in each polypeptide - treatment group , where the ordinate shows non - treated group and each polypeptide - treatment group and the abscissa shows gene transfer efficiency expressed as a ratio of absorbance at 405 nm relative to that at 490 nm . as shown in fig1 an amount of expressed cat in the culture dish in the c274 , h296 or c · cs1 - treatment group is higher as compared with that in a non - treatment group , demonstrating that efficiency of transfer of pcat - control vector into a cell is higher . a polypeptide represented by seq id no : 3 ( hereinafter referred to as &# 34 ; c274 &# 34 ;), a polypeptide represented by seq id no : 4 ( hereinafter referred to as &# 34 ; h296 &# 34 ;) and a polypeptide represented by seq id no : 5 ( hereinafter referred to as &# 34 ; c · cs1 &# 34 ;) were each dissolved in a phosphate buffered saline ( pbs ) to 1 μm , respectively , which were then sterilized using a 0 . 22 μm filter ( millex - gv , millipore ). 1 ml / well of these solutions were added to a 24 - well polystyrene culture dish ( manufactured by corning ) to coat the dish at 4 ° c . overnight , respectively . these dishes were rinsed with 500 μl / well of a dulbecco &# 39 ; s modified minimum basal medium not containing bovine fetal serum prior to addition of a transformed cell described below . two culture dishes ( diameter : 100 mm ) of african green monkey kidney cell cos - 7 which had been cultured in a dulbecco &# 39 ; s modified minimum basal medium containing 10 % bovine fetal serum were rinsed with 10 ml of a dulbecco &# 39 ; s modified minimum basal medium not containing bovine fetal serum , respectively , and 3 ml of pbs containing 0 . 25 % bovine trypsin and 0 . 02 % edta was added thereto to detach cells from the culture dish . to these , 7 ml of a dulbecco &# 39 ; s modified minimum basal medium not containing bovine fetal serum was added , respectively , followed by centrifugation at 800 rpm for 3 minutes to collect cells . the resulting cells were suspended in 10 ml of a dulbecco &# 39 ; s modified minimum basal medium containing bovine fetal serum , followed by centrifugation at 800 rpm for 3 minutes to collect cells . the resulting cells were combined , suspended in 12 ml of pbs , a 5 / 6 aliquot of the suspension was taken and divided into two equal aliquots , which were centrifuged at 800 rpm for 3 minutes to collect cells , respectively . the resulting cells were suspended in 6 ml of pbs , followed by centrifugation at 800 rpm for 3 minutes to collect two batches of cells . one batch of the resulting cells were suspended in 1 ml of pbs containing 15 μg of pcat - control vector ( promega ) which had been aseptically prepared , and placed in an electroporation cuvette for gene pulser ( biorad ), which was allowed to stand in ice for 10 minutes . the other batch of the resulting cells were suspended in 1 ml of pbs , and placed in an electroporation cuvette for gene pulser ( biorad ), which was allowed to stand in ice for 10 minutes . each batch of cells was allowed to stand in ice for 10 minutes , and voltage was applied thereto at 250 v and 960 μf . after application , the cells were allowed to stand in a cuvette in ice for 10 minutes . thereafter , the cells were recovered into 15 ml of a dulbeccol &# 39 ; s modified minimum basal medium containing 10 % bovine fetal serum , 1 ml / well of the cells were added to a 24 - well polystyrene culture dish covered with the above polypeptide . these cells were cultured at 37 ° c . in the presence of 5 % co 2 gas overnight , the medium was removed by aspiration , and 1 ml / well of a fresh dulbecco &# 39 ; s modified minimum basal medium containing 10 % bovine fetal serum was added , followed by culturing at 37 ° c . in the presence of 5 % co 2 gas overnight . the cultured cells were rinsed three times with 1 . 25 ml of pbs per well , a lysed cell solution was prepared , and detection of expressed cat was carried out using cat - elisa kit ( manufactured by boehringer mannheim ) according to a method for using the present kit . since the present kit used a horseradish peroxidase - labelled secondary antibody and abts as a substrate , a ratio of absorbance at 405 nm / 490 nm was determined . a value obtained by subtracting a blank value from a value for each group in a case of addition of pcat - control vector , using a value for the group in a case of no addition of pcat - control vector upon electroporation as a blank , was adopted as an amount of expressed cat . the results thereof are shown in fig2 . that is , fig2 is a figure showing efficiency of gene transfer into a cell in each polypeptide - treatment group , where the ordinate shows non - treated group and each polypeptide - treatment group and the abscissa shows gene transfer efficiency expressed as a ratio of absorbance at 405 nm relative to that at 490 nm . as shown in fig2 an amount of expressed cat in the culture dish in the above c274 , h296 or c · cs1 - treatment group is higher as compared with that in a non - treatment group , demonstrating that efficiency of transfer of pcat - control vector into a cell is higher . a kit for production of gene - transferred cells was made from c274 , h296 , c · cs1 , pbs and a culturing dish as shown in table 2 below . reagents a , b and c were prepared so that the above polypeptides were adjusted with pbs to the indicated concentrations shown in the table . other components used are described in example 1 . in addition , all reagents a , b and c and a diluent for the reagents were aseptically prepared by pre - filtering with a 0 . 22 μm sterile filter . table 2______________________________________kit for production of transfected cell______________________________________reagent a . . . 100 μm c274 150 μlreagent b . . . 100 μm h296 150 μlreagent c . . . 100 μm c . cs1 150 μldiluent for reagents . . . pbs 45 ml24 - well polystyrene culture dish 3______________________________________ as described above , the present invention can overcome the problems of the previous methods for gene transfer into cells and provide a method , for production of transfected cells , having improved efficiency of gene transfer into target cells . the present invention can also provide a kit , for production of transfected cells , which is used for the method . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 21 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 4 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 1 : argglyaspser ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 25 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 2 : aspgluleuproglnleuvalthrleuprohisproasnleuhis151015glyprogluileleuaspvalproserthr2025 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 274 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 3 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasp ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 296 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 4 : alaileproalaprothraspleulysphethrglnvalthrpro51015thrserleuseralaglntrpthrproproasnvalglnleuthr202530glytyrargvalargvalthrprolysglulysthrglypromet354045lysgluileasnleualaproaspserserservalvalvalser505560glyleumetvalalathrlystyrgluvalservaltyralaleu657075lysaspthrleuthrserargproalaglnglyvalvalthrthr808590leugluasnvalserproproargargalaargvalthraspala95100105thrgluthrthrilethrilesertrpargthrlysthrgluthr110115120ilethrglypheglnvalaspalavalproalaasnglyglnthr125130135proileglnargthrilelysproaspvalargsertyrthrile140145150thrglyleuglnproglythrasptyrlysiletyrleutyrthr155160165leuasnaspasnalaargserserprovalvalileaspalaser170175180thralaileaspalaproserasnleuargpheleualathrthr185190195proasnserleuleuvalsertrpglnproproargalaargile200205210thrglytyrileilelystyrglulysproglyserproproarg215220225gluvalvalproargproargproglyvalthrglualathrile230235240thrglyleugluproglythrglutyrthriletyrvalileala245250255leulysasnasnglnlyssergluproleuileglyarglyslys260265270thraspgluleuproglnleuvalthrleuprohisproasnleu275280285hisglyprogluileleuaspvalproserthr290295 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 302 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 5 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysproseraspgluleuproglnleuvalthr275280285leuprohisproasnleuhisglyprogluileleuaspvalpro290295300serthr ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 5 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 6 : tyrileglyserarg15 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 283 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 7 : alavalproproprothraspleuargphethrasnileglypro151015aspthrmetargvalthrtrpalaproproproserileaspleu202530thrasnpheleuvalargtyrserprovallysasnglugluasp354045valalagluleuserileserproseraspasnalavalvalleu505560thrasnleuleuproglythrglutyrvalvalservalserser657075valtyrgluglnhisgluserthrproleuargglyargglnlys808590thrglyleuaspserprothrglyileasppheseraspilethr95100105alaasnserphethrvalhistrpilealaproargalathrile110115120thrglytyrargilearghishisprogluhispheserglyarg125130135proarggluaspargvalprohisserargasnserilethrleu140145150thrasnleuthrproglythrglutyrvalvalserilevalala155160165leuasnglyargglugluserproleuleuileglyglnglnser170175180thrvalseraspvalproargaspleugluvalvalalaalathr185190195prothrserleuleuilesertrpaspalaproalavalthrval200205210argtyrtyrargilethrtyrglygluthrglyglyasnserpro215220225valglngluphethrvalproglyserlysserthralathrile230235240serglyleulysproglyvalasptyrthrilethrvaltyrala245250255valthrglyargglyaspserproalaserserlysproileser260265270ileasntyrargthrgluileasplysproserglnmet275280 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 279 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 8 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysproserglnmet275 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 474 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 9 : alavalproproprothraspleuargphethrasnileglypro151015aspthrmetargvalthrtrpalaproproproserileaspleu202530thrasnpheleuvalargtyrserprovallysasnglugluasp354045valalagluleuserileserproseraspasnalavalvalleu505560thrasnleuleuproglythrglutyrvalvalservalserser657075valtyrgluglnhisgluserthrproleuargglyargglnlys808590thrglyleuaspserprothrglyileasppheseraspilethr95100105alaasnserphethrvalhistrpilealaproargalathrile110115120thrglytyrargilearghishisprogluhispheserglyarg125130135proarggluaspargvalprohisserargasnserilethrleu140145150thrasnleuthrproglythrglutyrvalvalserilevalala155160165leuasnglyargglugluserproleuleuileglyglnglnser170175180thrvalseraspvalproargaspleugluvalvalalaalathr185190195prothrserleuleuilesertrpaspalaproalavalthrval200205210argtyrtyrargilethrtyrglygluthrglyglyasnserpro215220225valglngluphethrvalproglyserlysserthralathrile230235240serglyleulysproglyvalasptyrthrilethrvaltyrala245250255valthrglyargglyaspserproalaserserlysproileser260265270ileasntyrargthrgluileasplysproserglnasnglugly275280285leuasnglnprothraspaspsercyspheaspprotyrthrval290295300serhistyralavalglyaspglutrpgluargmetsergluser305310315glyphelysleuleucysglncysleuglypheglyserglyhis320325330pheargcysaspserserargtrpcyshisaspasnglyvalasn335340345tyrlysileglyglulystrpaspargglnglygluasnglygln350355360metmetsercysthrcysleuglyasnglylysglygluphelys365370375cysaspprohisglualathrcystyraspaspglylysthrtyr380385390hisvalglygluglntrpglnlysglutyrleuglyalailecys395400405sercysthrcyspheglyglyglnargglytrpargcysaspasn410415420cysargargproglyglygluproserprogluglythrthrgly425430435glnsertyrasnglntyrserglnargtyrhisglnargthrasn440445450thrasnvalasncysproileglucysphemetproleuaspval455460465glnalaasparggluaspserargglu470 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 385 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 10 : alaproilevalasnlysvalvalthrproleuserproprothr151015asnleuhisleuglualaasnproaspthrglyvalleuthrval202530sertrpgluargserthrthrproaspilethrglytyrargile354045thrthrthrprothrasnglyglnglnglyasnserleugluglu505560valvalhisalaaspglnsersercysthrpheaspasnleuser657075proglyleuglutyrasnvalservaltyrthrvallysaspasp808590lysgluservalproileseraspthrileileproalavalpro95100105proprothraspleuargphethrasnileglyproaspthrmet110115120argvalthrtrpalaproproproserileaspleuthrasnphe125130135leuvalargtyrserprovallysasnglugluaspvalalaglu140145150leuserileserproseraspasnalavalvalleuthrasnleu155160165leuproglythrglutyrvalvalservalserservaltyrglu170175180glnhisgluserthrproleuargglyargglnlysthrglyleu185190195aspserprothrglyileasppheseraspilethralaasnser200205210phethrvalhistrpilealaproargalathrilethrglytyr215220225argilearghishisprogluhispheserglyargproargglu230235240aspargvalprohisserargasnserilethrleuthrasnleu245250255thrproglythrglutyrvalvalserilevalalaleuasngly260265270argglugluserproleuleuileglyglnglnserthrvalser275280285aspvalproargaspleugluvalvalalaalathrprothrser290295300leuleuilesertrpaspalaproalavalthrvalargtyrtyr305310315argilethrtyrglygluthrglyglyasnserprovalglnglu320325330phethrvalproglyserlysserthralathrileserglyleu335340345lysproglyvalasptyrthrilethrvaltyralavalthrgly350355360argglyaspserproalaserserlysproileserileasntyr365370375argthrgluileasplysproserglnmet380385 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 549 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 11 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysprosermetalaileproalaprothrasp275280285leulysphethrglnvalthrprothrserleuseralaglntrp290295300thrproproasnvalglnleuthrglytyrargvalargvalthr305310315prolysglulysthrglyprometlysgluileasnleualapro320325330aspserserservalvalvalserglyleumetvalalathrlys335340345tyrgluvalservaltyralaleulysaspthrleuthrserarg350355360proalaglnglyvalvalthrthrleugluasnvalserpropro365370375argargalaargvalthraspalathrgluthrthrilethrile380385390sertrpargthrlysthrgluthrilethrglypheglnvalasp395400405alavalproalaasnglyglnthrproileglnargthrilelys410415420proaspvalargsertyrthrilethrglyleuglnproglythr425430435asptyrlysiletyrleutyrthrleuasnaspasnalaargser440445450serprovalvalileaspalaserthralaileaspalaproser455460465asnleuargpheleualathrthrproasnserleuleuvalser470475480trpglnproproargalaargilethrglytyrileilelystyr485490495glulysproglyserproproarggluvalvalproargproarg500505510proglyvalthrglualathrilethrglyleugluproglythr515520525glutyrthriletyrvalilealaleulysasnasnglnlysser530535540gluproleuileglyarglyslysthr545 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 422 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 12 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysprosermetalaasngluglyleuasngln275280285prothraspaspsercyspheaspprotyrthrvalserhistyr290295300alavalglyaspglutrpgluargmetsergluserglyphelys305310315leuleucysglncysleuglypheglyserglyhispheargcys320325330aspserserargtrpcyshisaspasnglyvalasntyrlysile335340345glyglulystrpaspargglnglygluasnglyglnmetmetser350355360cysthrcysleuglyasnglylysglygluphelyscysasppro365370375hisglualathrcystyraspaspglylysthrtyrhisvalgly380385390gluglntrpglnlysglutyrleuglyalailecyssercysthr395400405cyspheglyglyglnargglytrpargcysaspasncysargarg410415420progly ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 332 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 13 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysprosermetalaasnseraspserglucys275280285proleuserhisaspglytyrcysleuhisaspglyvalcysmet290295300tyrileglualaleuasplystyralacysasncysvalvalgly305310315tyrileglygluargcysglntyrargaspleulystrptrpglu320325330leuarg ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 341 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 14 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysprosermetglyiletyrileserglymet275280285alaproargproserleuthrlyslysglnargphearghisarg290295300asnarglysglytyrargserglnargglyhisserargglyarg305310315asnglnasnserargargproserargalamettrpleuserleu320325330pheserserlysasnserserservalproala335340 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 446 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 15 : 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( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 457 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 16 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysprosermetasnvalserproproargarg275280285alaargvalthraspalathrgluthrthrilethrilesertrp290295300argthrlysthrgluthrilethrglypheglnvalaspalaval305310315proalaasnglyglnthrproileglnargthrilelysproasp320325330valargsertyrthrilethrglyleuglnproglythrasptyr335340345lysiletyrleutyrthrleuasnaspasnalaargserserpro350355360valvalileaspalaserthralaileaspalaproserasnleu365370375argpheleualathrthrproasnserleuleuvalsertrpgln380385390proproargalaargilethrglytyrileilelystyrglulys395400405proglyserproproarggluvalvalproargproargprogly410415420valthrglualathrilethrglyleugluproglythrglutyr425430435thriletyrvalilealaleulysasnasnglnlysserglupro440445450leuileglyarglyslysthr455 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 368 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 17 : 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( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 367 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 18 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysprosermetasnvalserproproargarg275280285alaargvalthraspalathrgluthrthrilethrilesertrp290295300argthrlysthrgluthrilethrglypheglnvalaspalaval305310315proalaasnglyglnthrproileglnargthrilelysproasp320325330valargsertyrthrilethrglyleuglnproglythrasptyr335340345lysiletyrleutyrthrleuasnaspasnalaargserserpro350355360valvalileaspalaserthr365 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 464 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 19 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysprosermetglyileargglyleulysgly275280285thrlysglyglulysglygluaspglypheproglyphelysgly290295300aspmetglyilelysglyaspargglygluileglyproprogly305310315proargglygluaspglyprogluglyprolysglyargglygly320325330proasnglyaspproglyproleuglyproproglyglulysgly335340345lysleuglyvalproglyleuproglytyrproglyargglngly350355360prolysglyserileglypheproglypheproglyalaasngly365370375glulysglyglyargglythrproglylysproglyproarggly380385390glnargglyprothrglyproargglygluargglyproarggly395400405ilethrglylysproglyprolysglyasnserglyglyaspgly410415420proalaglyproproglygluargglyproasnglyproglngly425430435prothrglypheproglyprolysglyproproglyproprogly440445450lysaspglyleuproglyhisproglyglnargglygluthr455460 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 432 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 20 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysprosermetalaalaglyserilethrthr275280285leuproalaleuprogluaspglyglyserglyalaphepropro290295300glyhisphelysaspprolysargleutyrcyslysasnglygly305310315phepheleuargilehisproaspglyargvalaspglyvalarg320325330glulysseraspprohisilelysleuglnleuglnalagluglu335340345argglyvalvalserilelysglyvalcysalaasnargtyrleu350355360alametlysgluaspglyargleuleualaserlyscysvalthr365370375aspglucysphephephegluargleugluserasnasntyrasn380385390thrtyrargserarglystyrthrsertrptyrvalalaleulys395400405argthrglyglntyrlysleuglyserlysthrglyproglygln410415420lysalaileleupheleuprometseralalysser425430 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 574 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 21 : prothraspleuargphethrasnileglyproaspthrmetarg151015valthrtrpalaproproproserileaspleuthrasnpheleu202530valargtyrserprovallysasnglugluaspvalalagluleu354045serileserproseraspasnalavalvalleuthrasnleuleu505560proglythrglutyrvalvalservalserservaltyrglugln657075hisgluserthrproleuargglyargglnlysthrglyleuasp808590serprothrglyileasppheseraspilethralaasnserphe95100105thrvalhistrpilealaproargalathrilethrglytyrarg110115120ilearghishisprogluhispheserglyargproarggluasp125130135argvalprohisserargasnserilethrleuthrasnleuthr140145150proglythrglutyrvalvalserilevalalaleuasnglyarg155160165glugluserproleuleuileglyglnglnserthrvalserasp170175180valproargaspleugluvalvalalaalathrprothrserleu185190195leuilesertrpaspalaproalavalthrvalargtyrtyrarg200205210ilethrtyrglygluthrglyglyasnserprovalglngluphe215220225thrvalproglyserlysserthralathrileserglyleulys230235240proglyvalasptyrthrilethrvaltyralavalthrglyarg245250255glyaspserproalaserserlysproileserileasntyrarg260265270thrgluileasplysprosermetalaileproalaprothrasp275280285leulysphethrglnvalthrprothrserleuseralaglntrp290295300thrproproasnvalglnleuthrglytyrargvalargvalthr305310315prolysglulysthrglyprometlysgluileasnleualapro320325330aspserserservalvalvalserglyleumetvalalathrlys335340345tyrgluvalservaltyralaleulysaspthrleuthrserarg350355360proalaglnglyvalvalthrthrleugluasnvalserpropro365370375argargalaargvalthraspalathrgluthrthrilethrile380385390sertrpargthrlysthrgluthrilethrglypheglnvalasp395400405alavalproalaasnglyglnthrproileglnargthrilelys410415420proaspvalargsertyrthrilethrglyleuglnproglythr425430435asptyrlysiletyrleutyrthrleuasnaspasnalaargser440445450serprovalvalileaspalaserthralaileaspalaproser455460465asnleuargpheleualathrthrproasnserleuleuvalser470475480trpglnproproargalaargilethrglytyrileilelystyr485490495glulysproglyserproproarggluvalvalproargproarg500505510proglyvalthrglualathrilethrglyleugluproglythr515520525glutyrthriletyrvalilealaleulysasnasnglnlysser530535540gluproleuileglyarglyslysthraspgluleuproglnleu545550555valthrleuprohisproasnleuhisglyprogluileleuasp560565570valproserthr__________________________________________________________________________