JP7073264B2 - 遺伝子構築物 - Google Patents
遺伝子構築物 Download PDFInfo
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- JP7073264B2 JP7073264B2 JP2018540229A JP2018540229A JP7073264B2 JP 7073264 B2 JP7073264 B2 JP 7073264B2 JP 2018540229 A JP2018540229 A JP 2018540229A JP 2018540229 A JP2018540229 A JP 2018540229A JP 7073264 B2 JP7073264 B2 JP 7073264B2
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Description
AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG
[配列番号57]
AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGT
[配列番号58]
AGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA
[配列番号59]
CTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG
[配列番号1]
CTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTG
[配列番号2]
CTAGATCTGAATTCGGTACCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG
[配列番号3]
GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG
[配列番号48]
QAGDVEENPGP
[配列番号4]
GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGGCTGGAGACGTGGAGGAGAACCCTGGACCT
[配列番号5]
GSGATNFSLLQAGDVEENPGP
[配列番号6]
AGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT
[配列番号7]
SGATNFSLLKQAGDVEENPGP
[配列番号8]
MSSWIRWHGPAMARLWGFCWLVVGFWRAAFACPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPENITEIFIANQKRLEIINEDDVEAYVGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPFTCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSANLAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDYGTAANDIGDTTNRSNEIPSTDVTDKTGREHLSVYAVVVIASVVGFCLLVMLFLLKLARHSKFGMKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDAVIIGMTKIPVIENPQYFGITNSQLKPDTFVQHIKRHNIVLKRELGEGAFGKVFLAECYNLCPEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHIVKFYGVCVEGDPLIMVFEYMKHGDLNKFLRAHGPDAVLMAEGNPPTELTQSQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLVKIGDFGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTESDVWSLGVVLWEIFTYGKQPWYQLSNNEVIECITQGRVLQRPRTCPQEVYELMLGCWQREPHMRKNIKGIHTLLQNLAKASPVYLDILG
[配列番号9]
ATGTCGTCCTGGATAAGGTGGCATGGACCCGCCATGGCGCGGCTCTGGGGCTTCTGCTGGCTGGTTGTGGGCTTCTGGAGGGCCGCTTTCGCCTGTCCCACGTCCTGCAAATGCAGTGCCTCTCGGATCTGGTGCAGCGACCCTTCTCCTGGCATCGTGGCATTTCCGAGATTGGAGCCTAACAGTGTAGATCCTGAGAACATCACCGAAATTTTCATCGCAAACCAGAAAAGGTTAGAAATCATCAACGAAGATGATGTTGAAGCTTATGTGGGACTGAGAAATCTGACAATTGTGGATTCTGGATTAAAATTTGTGGCTCATAAAGCATTTCTGAAAAACAGCAACCTGCAGCACATCAATTTTACCCGAAACAAACTGACGAGTTTGTCTAGGAAACATTTCCGTCACCTTGACTTGTCTGAACTGATCCTGGTGGGCAATCCATTTACATGCTCCTGTGACATTATGTGGATCAAGACTCTCCAAGAGGCTAAATCCAGTCCAGACACTCAGGATTTGTACTGCCTGAATGAAAGCAGCAAGAATATTCCCCTGGCAAACCTGCAGATACCCAATTGTGGTTTGCCATCTGCAAATCTGGCCGCACCTAACCTCACTGTGGAGGAAGGAAAGTCTATCACATTATCCTGTAGTGTGGCAGGTGATCCGGTTCCTAATATGTATTGGGATGTTGGTAACCTGGTTTCCAAACATATGAATGAAACAAGCCACACACAGGGCTCCTTAAGGATAACTAACATTTCATCCGATGACAGTGGGAAGCAGATCTCTTGTGTGGCGGAAAATCTTGTAGGAGAAGATCAAGATTCTGTCAACCTCACTGTGCATTTTGCACCAACTATCACATTTCTCGAATCTCCAACCTCAGACCACCACTGGTGCATTCCATTCACTGTGAAAGGCAACCCCAAACCAGCGCTTCAGTGGTTCTATAACGGGGCAATATTGAATGAGTCCAAATACATCTGTACTAAAATACATGTTACCAATCACACGGAGTACCACGGCTGCCTCCAGCTGGATAATCCCACTCACATGAACAATGGGGACTACACTCTAATAGCCAAGAATGAGTATGGGAAGGATGAGAAACAGATTTCTGCTCACTTCATGGGCTGGCCTGGAATTGACGATGGTGCAAACCCAAATTATCCTGATGTAATTTATGAAGATTATGGAACTGCAGCGAATGACATCGGGGACACCACGAACAGAAGTAATGAAATCCCTTCCACAGACGTCACTGATAAAACCGGTCGGGAACATCTCTCGGTCTATGCTGTGGTGGTGATTGCGTCTGTGGTGGGATTTTGCCTTTTGGTAATGCTGTTTCTGCTTAAGTTGGCAAGACACTCCAAGTTTGGCATGAAAGGCCCAGCCTCCGTTATCAGCAATGATGATGACTCTGCCAGCCCACTCCATCACATCTCCAATGGGAGTAACACTCCATCTTCTTCGGAAGGTGGCCCAGATGCTGTCATTATTGGAATGACCAAGATCCCTGTCATTGAAAATCCCCAGTACTTTGGCATCACCAACAGTCAGCTCAAGCCAGACACATTTGTTCAGCACATCAAGCGACATAACATTGTTCTGAAAAGGGAGCTAGGCGAAGGAGCCTTTGGAAAAGTGTTCCTAGCTGAATGCTATAACCTCTGTCCTGAGCAGGACAAGATCTTGGTGGCAGTGAAGACCCTGAAGGATGCCAGTGACAATGCACGCAAGGACTTCCACCGTGAGGCCGAGCTCCTGACCAACCTCCAGCATGAGCACATCGTCAAGTTCTATGGCGTCTGCGTGGAGGGCGACCCCCTCATCATGGTCTTTGAGTACATGAAGCATGGGGACCTCAACAAGTTCCTCAGGGCACACGGCCCTGATGCCGTGCTGATGGCTGAGGGCAACCCGCCCACGGAACTGACGCAGTCGCAGATGCTGCATATAGCCCAGCAGATCGCCGCGGGCATGGTCTACCTGGCGTCCCAGCACTTCGTGCACCGCGATTTGGCCACCAGGAACTGCCTGGTCGGGGAGAACTTGCTGGTGAAAATCGGGGACTTTGGGATGTCCCGGGACGTGTACAGCACTGACTACTACAGGGTCGGTGGCCACACAATGCTGCCCATTCGCTGGATGCCTCCAGAGAGCATCATGTACAGGAAATTCACGACGGAAAGCGACGTCTGGAGCCTGGGGGTCGTGTTGTGGGAGATTTTCACCTATGGCAAACAGCCCTGGTACCAGCTGTCAAACAATGAGGTGATAGAGTGTATCACTCAGGGCCGAGTCCTGCAGCGACCCCGCACGTGCCCCCAGGAGGTGTATGAGCTGATGCTGGGGTGCTGGCAGCGAGAGCCCCACATGAGGAAGAACATCAAGGGCATCCATACCCTCCTTCAGAACTTGGCCAAGGCATCTCCGGTCTACCTGGACATTCTAGGC
[配列番号10]
MSSWIRWHGPAMARLWGFCWLVVGFWRAAFACPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPENITEIFIANQKRLEIINEDDVEAYVGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPFTCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSANLAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDYGTAANDIGDTTNRSNEIPSTDVTDKTGREHLSVYAVVVIASVVGFCLLVMLFLLKLARHSKFGMKDFSWFGFGKVKSRQGVGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDAVIIGMTKIPVIENPQYFGITNSQLKPDTFVQHIKRHNIVLKRELGEGAFGKVFLAECYNLCPEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHIVKFYGVCVEGDPLIMVFEYMKHGDLNKFLRAHGPDAVLMAEGNPPTELTQSQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLVKIGDFGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTESDVWSLGVVLWEIFTYGKQPWYQLSNNEVIECITQGRVLQRPRTCPQEVYELMLGCWQREPHMRKNIKGIHTLLQNLAKASPVYLDILG
[配列番号11]
ATGTCGTCCTGGATAAGGTGGCATGGACCCGCCATGGCGCGGCTCTGGGGCTTCTGCTGGCTGGTTGTGGGCTTCTGGAGGGCCGCTTTCGCCTGTCCCACGTCCTGCAAATGCAGTGCCTCTCGGATCTGGTGCAGCGACCCTTCTCCTGGCATCGTGGCATTTCCGAGATTGGAGCCTAACAGTGTAGATCCTGAGAACATCACCGAAATTTTCATCGCAAACCAGAAAAGGTTAGAAATCATCAACGAAGATGATGTTGAAGCTTATGTGGGACTGAGAAATCTGACAATTGTGGATTCTGGATTAAAATTTGTGGCTCATAAAGCATTTCTGAAAAACAGCAACCTGCAGCACATCAATTTTACCCGAAACAAACTGACGAGTTTGTCTAGGAAACATTTCCGTCACCTTGACTTGTCTGAACTGATCCTGGTGGGCAATCCATTTACATGCTCCTGTGACATTATGTGGATCAAGACTCTCCAAGAGGCTAAATCCAGTCCAGACACTCAGGATTTGTACTGCCTGAATGAAAGCAGCAAGAATATTCCCCTGGCAAACCTGCAGATACCCAATTGTGGTTTGCCATCTGCAAATCTGGCCGCACCTAACCTCACTGTGGAGGAAGGAAAGTCTATCACATTATCCTGTAGTGTGGCAGGTGATCCGGTTCCTAATATGTATTGGGATGTTGGTAACCTGGTTTCCAAACATATGAATGAAACAAGCCACACACAGGGCTCCTTAAGGATAACTAACATTTCATCCGATGACAGTGGGAAGCAGATCTCTTGTGTGGCGGAAAATCTTGTAGGAGAAGATCAAGATTCTGTCAACCTCACTGTGCATTTTGCACCAACTATCACATTTCTCGAATCTCCAACCTCAGACCACCACTGGTGCATTCCATTCACTGTGAAAGGCAACCCCAAACCAGCGCTTCAGTGGTTCTATAACGGGGCAATATTGAATGAGTCCAAATACATCTGTACTAAAATACATGTTACCAATCACACGGAGTACCACGGCTGCCTCCAGCTGGATAATCCCACTCACATGAACAATGGGGACTACACTCTAATAGCCAAGAATGAGTATGGGAAGGATGAGAAACAGATTTCTGCTCACTTCATGGGCTGGCCTGGAATTGACGATGGTGCAAACCCAAATTATCCTGATGTAATTTATGAAGATTATGGAACTGCAGCGAATGACATCGGGGACACCACGAACAGAAGTAATGAAATCCCTTCCACAGACGTCACTGATAAAACCGGTCGGGAACATCTCTCGGTCTATGCTGTGGTGGTGATTGCGTCTGTGGTGGGATTTTGCCTTTTGGTAATGCTGTTTCTGCTTAAGTTGGCAAGACACTCCAAGTTTGGCATGAAAGATTTCTCATGGTTTGGATTTGGGAAAGTAAAATCAAGACAAGGTGTTGGCCCAGCCTCCGTTATCAGCAATGATGATGACTCTGCCAGCCCACTCCATCACATCTCCAATGGGAGTAACACTCCATCTTCTTCGGAAGGTGGCCCAGATGCTGTCATTATTGGAATGACCAAGATCCCTGTCATTGAAAATCCCCAGTACTTTGGCATCACCAACAGTCAGCTCAAGCCAGACACATTTGTTCAGCACATCAAGCGACATAACATTGTTCTGAAAAGGGAGCTAGGCGAAGGAGCCTTTGGAAAAGTGTTCCTAGCTGAATGCTATAACCTCTGTCCTGAGCAGGACAAGATCTTGGTGGCAGTGAAGACCCTGAAGGATGCCAGTGACAATGCACGCAAGGACTTCCACCGTGAGGCCGAGCTCCTGACCAACCTCCAGCATGAGCACATCGTCAAGTTCTATGGCGTCTGCGTGGAGGGCGACCCCCTCATCATGGTCTTTGAGTACATGAAGCATGGGGACCTCAACAAGTTCCTCAGGGCACACGGCCCTGATGCCGTGCTGATGGCTGAGGGCAACCCGCCCACGGAACTGACGCAGTCGCAGATGCTGCATATAGCCCAGCAGATCGCCGCGGGCATGGTCTACCTGGCGTCCCAGCACTTCGTGCACCGCGATTTGGCCACCAGGAACTGCCTGGTCGGGGAGAACTTGCTGGTGAAAATCGGGGACTTTGGGATGTCCCGGGACGTGTACAGCACTGACTACTACAGGGTCGGTGGCCACACAATGCTGCCCATTCGCTGGATGCCTCCAGAGAGCATCATGTACAGGAAATTCACGACGGAAAGCGACGTCTGGAGCCTGGGGGTCGTGTTGTGGGAGATTTTCACCTATGGCAAACAGCCCTGGTACCAGCTGTCAAACAATGAGGTGATAGAGTGTATCACTCAGGGCCGAGTCCTGCAGCGACCCCGCACGTGCCCCCAGGAGGTGTATGAGCTGATGCTGGGGTGCTGGCAGCGAGAGCCCCACATGAGGAAGAACATCAAGGGCATCCATACCCTCCTTCAGAACTTGGCCAAGGCATCTCCGGTCTACCTGGACATTCTAGGC
[配列番号12]
MSSWIRWHGPAMARLWGFCWLVVGFWRAAFACPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPENITEIFIANQKRLEIINEDDVEAYVGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPFTCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSANLAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDYGTAANDIGDTTNRSNEIPSTDVTDKTGREHLSVYAVVVIASVVGFCLLVMLFLLKLARHSKFGMKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDAVIIGMTKIPVIENPQEFGITNSQLKPDTFVQHIKRHNIVLKRELGEGAFGKVFLAECYNLCPEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHIVKFYGVCVEGDPLIMVFEYMKHGDLNKFLRAHGPDAVLMAEGNPPTELTQSQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLVKIGDFGMSRDVESTDEERVGGHTMLPIRWMPPESIMYRKFTTESDVWSLGVVLWEIFTYGKQPWYQLSNNEVIECITQGRVLQRPRTCPQEVYELMLGCWQREPHMRKNIKGIHTLLQNLAKASPVELDILG
[配列番号13]
ATGTCGTCCTGGATAAGGTGGCATGGACCCGCCATGGCGCGGCTCTGGGGCTTCTGCTGGCTGGTTGTGGGCTTCTGGAGGGCCGCTTTCGCCTGTCCCACGTCCTGCAAATGCAGTGCCTCTCGGATCTGGTGCAGCGACCCTTCTCCTGGCATCGTGGCATTTCCGAGATTGGAGCCTAACAGTGTAGATCCTGAGAACATCACCGAAATTTTCATCGCAAACCAGAAAAGGTTAGAAATCATCAACGAAGATGATGTTGAAGCTTATGTGGGACTGAGAAATCTGACAATTGTGGATTCTGGATTAAAATTTGTGGCTCATAAAGCATTTCTGAAAAACAGCAACCTGCAGCACATCAATTTTACCCGAAACAAACTGACGAGTTTGTCTAGGAAACATTTCCGTCACCTTGACTTGTCTGAACTGATCCTGGTGGGCAATCCATTTACATGCTCCTGTGACATTATGTGGATCAAGACTCTCCAAGAGGCTAAATCCAGTCCAGACACTCAGGATTTGTACTGCCTGAATGAAAGCAGCAAGAATATTCCCCTGGCAAACCTGCAGATACCCAATTGTGGTTTGCCATCTGCAAATCTGGCCGCACCTAACCTCACTGTGGAGGAAGGAAAGTCTATCACATTATCCTGTAGTGTGGCAGGTGATCCGGTTCCTAATATGTATTGGGATGTTGGTAACCTGGTTTCCAAACATATGAATGAAACAAGCCACACACAGGGCTCCTTAAGGATAACTAACATTTCATCCGATGACAGTGGGAAGCAGATCTCTTGTGTGGCGGAAAATCTTGTAGGAGAAGATCAAGATTCTGTCAACCTCACTGTGCATTTTGCACCAACTATCACATTTCTCGAATCTCCAACCTCAGACCACCACTGGTGCATTCCATTCACTGTGAAAGGCAACCCCAAACCAGCGCTTCAGTGGTTCTATAACGGGGCAATATTGAATGAGTCCAAATACATCTGTACTAAAATACATGTTACCAATCACACGGAGTACCACGGCTGCCTCCAGCTGGATAATCCCACTCACATGAACAATGGGGACTACACTCTAATAGCCAAGAATGAGTATGGGAAGGATGAGAAACAGATTTCTGCTCACTTCATGGGCTGGCCTGGAATTGACGATGGTGCAAACCCAAATTATCCTGATGTAATTTATGAAGATTATGGAACTGCAGCGAATGACATCGGGGACACCACGAACAGAAGTAATGAAATCCCTTCCACAGACGTCACTGATAAAACCGGTCGGGAACATCTCTCGGTCTATGCTGTGGTGGTGATTGCGTCTGTGGTGGGATTTTGCCTTTTGGTAATGCTGTTTCTGCTTAAGTTGGCAAGACACTCCAAGTTTGGCATGAAAGGCCCAGCCTCCGTTATCAGCAATGATGATGACTCTGCCAGCCCACTCCATCACATCTCCAATGGGAGTAACACTCCATCTTCTTCGGAAGGTGGCCCAGATGCTGTCATTATTGGAATGACCAAGATCCCTGTCATTGAAAATCCCCAGGAATTTGGCATCACCAACAGTCAGCTCAAGCCAGACACATTTGTTCAGCACATCAAGCGACATAACATTGTTCTGAAAAGGGAGCTAGGCGAAGGAGCCTTTGGAAAAGTGTTCCTAGCTGAATGCTATAACCTCTGTCCTGAGCAGGACAAGATCTTGGTGGCAGTGAAGACCCTGAAGGATGCCAGTGACAATGCACGCAAGGACTTCCACCGTGAGGCCGAGCTCCTGACCAACCTCCAGCATGAGCACATCGTCAAGTTCTATGGCGTCTGCGTGGAGGGCGACCCCCTCATCATGGTCTTTGAGTACATGAAGCATGGGGACCTCAACAAGTTCCTCAGGGCACACGGCCCTGATGCCGTGCTGATGGCTGAGGGCAACCCGCCCACGGAACTGACGCAGTCGCAGATGCTGCATATAGCCCAGCAGATCGCCGCGGGCATGGTCTACCTGGCGTCCCAGCACTTCGTGCACCGCGATTTGGCCACCAGGAACTGCCTGGTCGGGGAGAACTTGCTGGTGAAAATCGGGGACTTTGGGATGTCCCGGGACGTGGAAAGCACTGACGAAGAAAGGGTCGGTGGCCACACAATGCTGCCCATTCGCTGGATGCCTCCAGAGAGCATCATGTACAGGAAATTCACGACGGAAAGCGACGTCTGGAGCCTGGGGGTCGTGTTGTGGGAGATTTTCACCTATGGCAAACAGCCCTGGTACCAGCTGTCAAACAATGAGGTGATAGAGTGTATCACTCAGGGCCGAGTCCTGCAGCGACCCCGCACGTGCCCCCAGGAGGTGTATGAGCTGATGCTGGGGTGCTGGCAGCGAGAGCCCCACATGAGGAAGAACATCAAGGGCATCCATACCCTCCTTCAGAACTTGGCCAAGGCATCTCCGGTCGAACTGGACATTCTAGGC
[配列番号14]
MLPLPSCSLPILLLFLLPSVPIESQPPPSTLPPFLAPEWDLLSPRVVLSRGAPAGPPLLFLLEAGAFRESAGAPANRSRRGVSETAPASRRGELAVCDAVSGWVTDRRTAVDLRGREVEVLGEVPAAGGSPLRQYFFETRCKADNAEEGGPGAGGGGCRGVDRRHWVSECKAKQSYVRALTADAQGRVGWRWIRIDTACVCTLLSRTGRA
[配列番号49]
ATGCTCCCTCTCCCCTCATGCTCCCTCCCCATCCTCCTCCTTTTCCTCCTCCCCAGTGTGCCAATTGAGTCCCAACCCCCACCCTCAACATTGCCCCCTTTTCTGGCCCCTGAGTGGGACCTTCTCTCCCCCCGAGTAGTCCTGTCTAGGGGTGCCCCTGCTGGGCCCCCTCTGCTCTTCCTGCTGGAGGCTGGGGCCTTTCGGGAGTCAGCAGGTGCCCCGGCCAACCGCAGCCGGCGTGGGGTGAGCGAAACTGCACCAGCGAGTCGTCGGGGTGAGCTGGCTGTGTGCGATGCAGTCAGTGGCTGGGTGACAGACCGCCGGACCGCTGTGGACTTGCGTGGGCGCGAGGTGGAGGTGTTGGGCGAGGTGCCTGCAGCTGGCGGCAGTCCCCTCCGCCAGTACTTCTTTGAAACCCGCTGCAAGGCTGATAACGCTGAGGAAGGTGGCCCGGGGGCAGGTGGAGGGGGCTGCCGGGGAGTGGACAGGAGGCACTGGGTATCTGAGTGCAAGGCCAAGCAGTCCTATGTGCGGGCATTGACCGCTGATGCCCAGGGCCGTGTGGGCTGGCGATGGATTCGAATTGACACTGCCTGCGTCTGCACACTCCTCAGCCGGACTGGCCGGGCC
[配列番号50]
MLPLPSCSLPILLLFLLPSVPIES
[配列番号51]
ATGCTCCCTCTCCCCTCATGCTCCCTCCCCATCCTCCTCCTTTTCCTCCTCCCCAGTGTGCCAATTGAGTCC
[配列番号52]
QPPPSTLPPFLAPEWDLLSPRVVLSRGAPAGPPLLFLLEAGAFRESAGAPANRSRR
[配列番号53]
CAACCCCCACCCTCAACATTGCCCCCTTTTCTGGCCCCTGAGTGGGACCTTCTCTCCCCCCGAGTAGTCCTGTCTAGGGGTGCCCCTGCTGGGCCCCCTCTGCTCTTCCTGCTGGAGGCTGGGGCCTTTCGGGAGTCAGCAGGTGCCCCGGCCAACCGCAGCCGGCGT
[配列番号54]
GVSETAPASRRGELAVCDAVSGWVTDRRTAVDLRGREVEVLGEVPAAGGSPLRQYFFETRCKADNAEEGGPGAGGGGCRGVDRRHWVSECKAKQSYVRALTADAQGRVGWRWIRIDTACVCTLLSRTGRA
[配列番号55]
GGGGTGAGCGAAACTGCACCAGCGAGTCGTCGGGGTGAGCTGGCTGTGTGCGATGCAGTCAGTGGCTGGGTGACAGACCGCCGGACCGCTGTGGACTTGCGTGGGCGCGAGGTGGAGGTGTTGGGCGAGGTGCCTGCAGCTGGCGGCAGTCCCCTCCGCCAGTACTTCTTTGAAACCCGCTGCAAGGCTGATAACGCTGAGGAAGGTGGCCCGGGGGCAGGTGGAGGGGGCTGCCGGGGAGTGGACAGGAGGCACTGGGTATCTGAGTGCAAGGCCAAGCAGTCCTATGTGCGGGCATTGACCGCTGATGCCCAGGGCCGTGTGGGCTGGCGATGGATTCGAATTGACACTGCCTGCGTCTGCACACTCCTCAGCCGGACTGGCCGGGCC
[配列番号56]
APMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADTFEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR
[配列番号15]
GCCCCCATGAAAGAAGCAAACATCCGAGGACAAGGTGGCTTGGCCTACCCAGGTGTGCGGACCCATGGGACTCTGGAGAGCGTGAATGGGCCCAAGGCAGGTTCAAGAGGCTTGACATCATTGGCTGACACTTTCGAACACGTGATAGAAGAGCTGTTGGATGAGGACCAGAAAGTTCGGCCCAATGAAGAAAACAATAAGGACGCAGACTTGTACACGTCCAGGGTGATGCTCAGTAGTCAAGTGCCTTTGGAGCCTCCTCTTCTCTTTCTGCTGGAGGAATACAAAAATTACCTAGATGCTGCAAACATGTCCATGAGGGTCCGGCGCCACTCTGACCCTGCCCGCCGAGGGGAGCTGAGCGTGTGTGACAGTATTAGTGAGTGGGTAACGGCGGCAGACAAAAAGACTGCAGTGGACATGTCGGGCGGGACGGTCACAGTCCTTGAAAAGGTCCCTGTATCAAAAGGCCAACTGAAGCAATACTTCTACGAGACCAAGTGCAATCCCATGGGTTACACAAAAGAAGGCTGCAGGGGCATAGACAAAAGGCATTGGAACTCCCAGTGCCGAACTACCCAGTCGTACGTGCGGGCCCTTACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACACTTCTTGTGTATGTACATTGACCATTAAAAGGGGAAGATAG
[配列番号16]
APMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADTFEHMIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR
[配列番号17]
HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR
[配列番号18]
ATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTTGGTTGCATGAAGGCTGCCCCCATGAAAGAAGCAAACATCCGAGGACAAGGTGGCTTGGCCTACCCAGGTGTGCGGACCCATGGGACTCTGGAGAGCGTGAATGGGCCCAAGGCAGGTTCAAGAGGCTTGACATCATTGGCTGACACTTTCGAACACGTGATAGAAGAGCTGTTGGATGAGGACCAGAAAGTTCGGCCCAATGAAGAAAACAATAAGGACGCAGACTTGTACACGTCCAGGGTGATGCTCAGTAGTCAAGTGCCTTTGGAGCCTCCTCTTCTCTTTCTGCTGGAGGAATACAAAAATTACCTAGATGCTGCAAACATGTCCATGAGGGTCCGGCGCCACTCTGACCCTGCCCGCCGAGGGGAGCTGAGCGTGTGTGACAGTATTAGTGAGTGGGTAACGGCGGCAGACAAAAAGACTGCAGTGGACATGTCGGGCGGGACGGTCACAGTCCTTGAAAAGGTCCCTGTATCAAAAGGCCAACTGAAGCAATACTTCTACGAGACCAAGTGCAATCCCATGGGTTACACAAAAGAAGGCTGCAGGGGCATAGACAAAAGGCATTGGAACTCCCAGTGCCGAACTACCCAGTCGTACGTGCGGGCCCTTACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACACTTCTTGTGTATGTACATTGACCATTAAAAGGGGAAGATAG
[配列番号19]
MTILFLTMVISYFGCMKA
[配列番号20]
ATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG
[配列番号21]
MFHQVRRVMTILFLTMVISYFGCMKA
[配列番号22]
ATGTTCCACCAGGTGAGAAGAGTGATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG
[配列番号23]
MQSREEEWFHQVRRVMTILFLTMVISYFGCMKA
[配列番号24]
ATGCAGAGCCGGGAAGAGGAATGGTTCCACCAGGTGAGAAGAGTGATGACCATCCTTTTCCTT ACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG
[配列番号25]
MLCAISLCARVRKLRSAGRCGKFHQVRRVMTILFLTMVISYFGCMKA
[配列番号26]
ATGCTCTGTGCGATTTCATTGTGTGCTCGCGTTCGCAAGCTCCGTAGTGCAGGAAGGTGCGGGAAGTTCCACCAGGTGAGAAGAGTGATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG
[配列番号27]
MCGATSFLHECTRLILVTTQNAEFLQKGLQVHTCFGVYPHASVWHDCASQKKGCAVYLHVSVEFNKLIPENGFIKFHQVRRVMTILFLTMVISYFGCMKA
[配列番号28]
ATGTGTGGAGCCACCAGTTTTCTCCATGAGTGCACAAGGTTAATCCTTGTTACTACTCAGAATGCTGAGTTTCTACAGAAAGGGTTGCAGGTCCACACATGTTTTGGCGTCTACCCACACGCTTCTGTATGGCATGACTGTGCATCCCAGAAGAAGGGCTGTGCTGTGTACCTCCACGTTTCAGTGGAATTTAACAAACTGATCCCTGAAAATGGTTTCATAAAGTTCCACCAGGTGAGAAGAGTGATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG
[配列番号29]
MYRMQLLSCIALSLALVTNS
[配列番号30]
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT
[配列番号31]
MKRRVMIILFLTMVISYFGCMK
[配列番号32]
ATGAAAAGAAGAGTGATGATCATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGAGCG
[配列番号33]
MRRMQLLLLIALSLALVTNS
[配列番号34]
ATGAGGAGGATGCAACTCCTGCTCCTGATTGCACTAAGTCTTGCACTTGTCACAAACAGT
[配列番号35]
MRRMQLLLLTMVISYFGCMKA
[配列番号36]
ATGAGGAGGATGCAACTCCTGCTCCTGACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG
[配列番号37]
MRILLLTMVISYFGCMKA
[配列番号38]
ATGAGAATCCTTCTTCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG
[配列番号39]
MRRILFLTMVISYFGCMKA
[配列番号40]
ATGAGAAGAATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG
[配列番号41]
MRRFLFLLVISYFGCMKA
[配列番号42]
ATGAGGAGGTTCCTTTTCCTTCTTGTTATTTCATACTTCGGTTGCATGAAGGCG
[配列番号43]
MRRFLFLLYFGCMKA
[配列番号44]
ATGAGGAGGTTCCTTTTCCTTCTTTACTTCGGTTGCATGAAGGCG
[配列番号45]
CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT
[配列番号46]
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG
[配列番号47]
[配列番号107]
ATGTCATCTTGGATCCGCTGGCACGGGCCAGCGATGGCCCGATTGTGGGGCTTCTGCTGGCTTGTTGTAGGCTTCTGGCGCGCGGCGTTCGCGTGTCCGACCTCTTGCAAATGCTCAGCAAGCCGAATTTGGTGCTCAGACCCTAGTCCAGGAATTGTTGCATTCCCCCGACTGGAACCAAACTCCGTCGACCCGGAGAATATAACTGAGATATTTATTGCAAATCAAAAACGCCTTGAAATCATTAACGAGGATGACGTGGAGGCCTACGTTGGTTTGAGAAATCTTACTATTGTCGACTCCGGACTTAAATTTGTAGCTCATAAAGCCTTCCTGAAGAACTCTAATCTGCAGCACATTAATTTCACGAGAAATAAGCTGACCAGCTTGTCCCGGAAGCATTTCCGCCATCTCGACCTGAGCGAGCTCATACTGGTCGGAAACCCATTTACGTGCTCCTGTGACATCATGTGGATCAAAACTCTGCAAGAGGCGAAAAGTAGTCCGGATACCCAAGACCTTTACTGTCTTAATGAAAGCTCAAAAAATATCCCGCTGGCCAACCTGCAGATACCGAACTGCGGACTTCCTAGTGCGAATTTGGCTGCCCCAAATCTTACCGTCGAAGAAGGCAAATCAATCACGCTTTCTTGTTCTGTAGCTGGAGATCCAGTGCCTAATATGTATTGGGACGTGGGTAACCTCGTCTCAAAACATATGAACGAAACGAGCCACACCCAGGGCTCTTTGCGGATAACAAACATCTCCTCTGATGATTCTGGAAAGCAAATCAGTTGCGTAGCTGAAAATCTGGTTGGCGAAGATCAAGATTCAGTCAATCTGACAGTCCATTTCGCCCCAACGATCACCTTTCTGGAGAGCCCAACTAGCGATCACCACTGGTGTATTCCGTTTACGGTAAAAGGAAATCCAAAACCTGCACTCCAATGGTTTTATAATGGAGCCATCTTGAATGAAAGCAAATATATCTGTACTAAAATCCATGTGACGAATCACACCGAGTATCACGGGTGTCTTCAATTGGATAATCCAACCCATATGAATAATGGTGATTATACTTTGATAGCGAAGAACGAATACGGCAAAGACGAAAAGCAAATATCCGCACATTTCATGGGTTGGCCTGGCATCGACGACGGTGCGAACCCGAACTACCCAGATGTTATTTACGAGGATTATGGGACTGCGGCAAACGACATTGGCGACACCACAAACCGAAGCAACGAGATACCAAGTACTGACGTCACTGACAAAACGGGTCGAGAGCATTTGTCTGTTTACGCCGTTGTTGTTATCGCCTCAGTTGTCGGATTTTGCCTGTTGGTCATGCTTTTCCTCCTGAAGCTCGCGCGACATTCCAAGTTTGGCATGAAGGGG CCAGCAAGTGTTATATCCAATGATGATGATAGCGCTTCTCCATTGCACCACATAAGTAACGGCTCAAACACGCCGTCATCTAGTGAAGGTGGACCAGACGCGGTCATTATAGGGATGACTAAAATTCCCGTAATCGAAAACCCTCAGTACTTCGGCATAACCAACAGTCAGCTTAAACCCGATACTTTCGTGCAGCACATCAAAAGGCACAACATAGTCCTCAAGCGCGAACTCGGGGAGGGAGCCTTCGGAAAGGTCTTTCTTGCTGAGTGCTATAATTTGTGTCCTGAGCAGGATAAAATTCTTGTGGCTGTAAAAACTCTCAAAGATGCTTCCGACAACGCACGGAAGGATTTTCATCGGGAGGCCGAACTGTTGACGAATTTGCAGCACGAGCATATAGTAAAGTTCTACGGGGTATGTGTTGAGGGGGACCCGTTGATTATGGTCTTCGAGTATATGAAGCACGGGGACCTGAACAAATTTTTGCGCGCCCATGGGCCTGATGCCGTCCTTATGGCAGAAGGGAACCCTCCAACAGAACTCACCCAGAGTCAGATGTTGCACATAGCGCAACAGATCGCGGCCGGCATGGTTTACCTGGCCAGTCAACACTTCGTGCATAGAGATCTTGCCACTCGCAACTGTTTGGTCGGGGAGAACCTTCTGGTTAAGATTGGTGACTTTGGTATGTCACGAGATGTGTATTCCACTGACTATTACAGAGTTGGGGGTCATACAATGCTTCCTATTCGGTGGATGCCCCCCGAATCCATCATGTACAGAAAGTTCACGACAGAGAGTGATGTT TGG AGT CTCGGCGTGGTGCTCTGGGAAATTTTCACATACGGAAAGCAGCCGTGGTATCAACTTAGCAACAATGAGGTGATAGAGTGTATTACACAGGGTCGGGTGTTGCAGCGCCCTCGAACGTGCCCACAAGAAGTATATGAACTTATGCTCGGGTGCTGGCAAAGAGAACCACATATGAGAAAAAATATCAAGGGGATACATACATTGCTTCAGAACTTGGCCAAGGCATCACCCGTCTACCTCGATATACTGGGCAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGAGAATCCTTCTTCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCGCACTCCGACCCTGCCCGCCGTGGGGAGCTGAGCGTGTGTGACAGTATTAGCGAGTGGGTCACAGCGGCAGATAAAAAGACTGCAGTGGACATGTCTGGCGGGACGGTCACAGTCCTAGAGAAAGTCCCGGTATCCAAAGGCCAACTGAAGCAGTATTTCTACGAGACCAAGTGTAATCCCATGGGTTACACCAAGGAAGGCTGCAGGGGCATAGACAAAAGGCACTGGAACTCGCAATGCCGAACTACCCAATCGTATGTTCGGGCCCTTACTATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACACTTCCTGTGTATGTACACTGACCATTAAAAGGGGAAGATAG
[配列番号108]
方法と材料
分子クローニングおよびプラスミド構築物
DNA配列のコドン最適化を、オンラインツール(http://www.idtdna.com/CodonOpt)を用いて実施し、DNAブロックを、Integrated DNA technologies, Inc.(IDT; 9180 N. McCormick Boulevard, Skokie, IL 60076-2920, USA)またはGenScript(860 Centennial Ave, Piscataway, NJ 08854, USA)によって合成した。マスタープラスミドQTA001PAと、次のプラスミドを作製するためのクローニングは、標準の分子生物学およびクローニング技術を利用して実施した。
DNAプラスミドを、SURE競合細胞(Agilent Technologies; cat.#200238)中で一晩、拡大し、Maxi-Prep精製後に2.29μg/μlのプラスミドを提供した。残りのプラスミドは、500μgスケールおよび最小限の内毒素が存在する形質導入品質に拡大した。
HEK293細胞(400,000個)を、ポリ-L-リシン(10μg/mL、Sigma-Aldrich; cat.#P1274)コーティング6ウェルプレートにおいて、10%ウシ胎児血清(FBS)、1%ペニシリンおよび1%ストレプトマイシン(1%Pen/Strep)を含有するダルベッコ最小必須培地(DMEM)1.5mL中で80%コンフルエントになるまで培養した。その後、培地をDMEM(添加剤不含)2mLと交換した。2~3時間後に、プラスミドDNA 4μg+リポフェクタミン10μL(4μL/mL;Thermo Fisher Scientific; cat.#12566014)を含有するトランスフェクション培地のさらなる0.5mlを各ウェルに添加して、トランスフェクション期間全体および上清採取のために全容量2.5mlを得た。
HEK293細胞から分泌されたBDNFの量を、トランスフェクションの24時間後に細胞培地中で測定した。培地を遠心分離して、細胞破片を除去し、市販のヒトBDNF ELISAキット(Sigma-Aldrich、製品# RAB0026)を用いて測定した。試料を新たに作製されたBDNF標準と比較することにより、BDNF濃度を決定した。
DMEMインキュベーション培地を除去し、低温リン酸緩衝生理食塩水で細胞を洗浄し、新たに調製された溶解緩衝液350μLをウェルに添加することにより、HEK293細胞内のBDNFおよびTrkB免疫反応性の量を測定した(Lysis-M試薬10ml+cOmplete Miniプロテアーゼインヒビターカクテル、Roche; cat.#04719964001 1錠+Haltホスファターゼインヒビターカクテル(100倍)、Thermo Scientific; cat.#78428 100μl)。細胞のホモジナイズ後に、タンパク質懸濁液を、BCAアッセイ(Pierce BCA タンパク質アッセイキット、Thermo Scientific; cat.#23227)を用いて定量した。1レーンにHEK293細胞溶解物タンパク質6μg~15μgの間を、Bis-Trisゲル(12%NuPAGE Novex; cat.#NP0342BOX、Thermo Scientific)でランダウン(run down)して、一晩インキュベートされた、一次ウサギポリクローナル抗BDNF抗体(Santa Cruz Biotechnology Inc; 製品# sc-546;1:500希釈)、ウサギポリクローナル抗TrkB抗体(Abcam; cat.#ab33655、1:2000希釈で使用)またはeGFP抗体(Abcam 製品#ab-290、1:500で使用)を用いたウェスタンブロットにより検査した。一次抗体を、HRPコンジュゲート化抗ウサギ抗体(Vector Laboratories; cat.#PI-1000、1:8000)、ならびにECL Prime(Amersham, GE Healthcare, UK)およびAllianceウェスタンブロット画像システム(UVItec Ltd, Cambridge, UK)を用いたシグナル検出で視覚化した。マウス網膜のウェスタンブロットでは、ベクター処置された動物の目を、新たに調製された溶解緩衝液(Lysis-M試薬10ml+cOmplete Miniプロテアーゼインヒビターカクテル、Roche; 製品#04719964001 1錠+Halt ホスファターゼインヒビターカクテル(100倍)、Thermo Scientific;#78428 100μl)500μL中でホモジナイズした。組織を1分間破壊し(Qiagen, TissueRuptor 製品#9001273)、その後、さらに15分間、氷上で保持した。その後、タンパク質を上記の通りウェスタンブロットにより分析した。
HEK293細胞(70,000個)を、4ウェルプレート内の13mmのポリ-L-リシンコーティングカバースリップに播種して、培地0.5ml中に10%FBSおよび1%Pen/Strepを含有するDMEMでインキュベートした。細胞が80%コンフルエントまで発育したら、2~3時間の間、培地をDMEM 0.4ml(添加剤不含)と交換し、その後、トランスフェクション培地(プラスミドDNA 0.8μg+リポフェクタミン2μl)のさらなる0.1mLを添加して、最終容量を0.5mlにした。カバースリップをPBSで2回洗浄して、室温の1Mリン酸緩衝生理食塩水(PBS)中の4%パラホルムアルデヒドで30分間固定した。細胞をPBSでさらに3回洗浄した後、5%正常ヤギ血清(NGS)、3%ウシ血清アルブミン(BSA)およびPBS中の0.3%TritonX-100中、室温で60分間インキュベートすることによりブロックして、浸透させた。その後、細胞を、ブロッキング溶液で希釈されたBDNF用(Santa Cruz Biotechnology Inc; 製品# sc-546;1:300希釈)またはTrkB用(Abcam 製品# ab33655、1:500希釈)の市販のウサギポリクローナル抗体と共に4℃で一晩インキュベートした。alexa fluor 647 (Invitrogen, 製品# A21248、1:1000)にコンジュゲート化された二次抗ウサギ抗体を用いて室温で2時間、染色を明らかにした。細胞核もまた、1μg/ml DAPI (Thermo Scientific, 製品# D1306 1:8000)で対比染色した。細胞をさらに3回洗浄して、fluorSave(商標)試薬(Calbiochem/EMD Chemicals Inc., Gibbstown, NJ, USA)を添加した後、撮像した。撮像は、3倍デジタルズームおよび0.5~0.8シーケンシャルスキャンZステップ間隔を利用し、20倍対物レンズと、63倍油浸対物レンズを備えたLeica DM6000落射蛍光顕微鏡(Leica Microsystems, Wetzlar, Germany)またはLeica SP5共焦点顕微鏡(Leica Microsystems, Wetzlar, Germany)を用いて実施した。
マウスを、7~10日の馴化期間の後、様々な試験群に無作為化した。それらをその後、ケタミン(50mg/kg)およびキシラジン(5g/kg)の腹腔内注射で麻酔した。1%局所テトラカイン点眼薬を、試験1日目に投与しした。1%トロピカミド点眼薬を用いて、瞳孔拡張(Pupillary dilation)を実行した。手術用顕微鏡を利用して、強膜部分層のパイロットホール(partial-thickness scleral pilot hole)を30ゲージの針で作製して、先端径30μmおよび先端長2.5mmの微細金属製マイクロピペットによる下部の強膜、脈絡膜および網膜への透過を容易にした。マイクロピペットをその後、10μlガラスシリンジ(Hamilton Co., Reno, NV)に連結させた後、群に応じてベクター懸濁液2μlをピペット内に引き上げた。硝子体内注射の間、レンズの透過または渦静脈の損傷を回避するように注意を払った。注射部位として、上側角膜輪部(supero-temporal limbus)の後部およそ3mmを目標に定めた。注射は1分間かけて緩徐に行い、ベクター懸濁液を拡散させた。右目は触れないまま、内部対側対照とした。
ベクター投与後3週間(21日)目に、マウスをONC処置に供するか、未処置とするか、または偽処置で挫滅させた。両眼手術用スコープの下、スプリングシザーで、結膜の、眼球に対して下位から開始して目の周囲に、一時的に小さな切開を作製した。これにより、眼球の後面を露出させて、視神経を視覚化させた。クロスアクションタイプの鉗子(Dumont #N7 cat.#RS-5027; Roboz)により、神経を圧迫するセルフクランピング作用からの圧力のみで、露出された視神経を眼球からおよそ1~3mmで10秒間つかんだ。10秒後に、視神経を解放し、鉗子を取り除いて、眼を回転させて適所に戻した。ONCの7日後に、動物を選別した。各群の両眼を、4%パラホルムアルデヒド/0.1%PBS(pH7.4)に一晩静置することにより固定した。その後、角膜からの後部眼構造の切除およびレンズの除去の後に、網膜フラットマウントを作製した。網膜フラットマウントを、4%パラホルムアルデヒド/0.1%PBSで30分間、後固定して、PBS中の0.5%Triton X-100で洗浄した。網膜を-80℃で10分間凍結し、核膜に浸透させて抗体の浸透性を改善した後、10%正常ロバ血清(NDS)、2%ウシ血清アルブミン(BSA)およびPBS中の2%Triton X-100の中、室温で60分間ブロッキングした。RGCをBrn3A(Santa Cruz, #sc-31984 1:200)に対する抗体で対比染色して、20倍対物レンズおよびLeica DM6000落射蛍光顕微鏡(Leica Microsystems, Wetzlar, Germany)を用いた蛍光顕微鏡測定により視覚化した。高解像度画像を、1.5倍デジタルズームおよび0.5~0.8シーケンシャルスキャンZステップ間隔を利用し、40倍油浸対物レンズを備えたLeica SP5共焦点顕微鏡(Leica Microsystems)を用いて得た。RGC細胞数を、核カウントのための画像ツール(image-based tool for counting nuclei)(ITCN)のプラグインを利用したImageJにより測定して、RGC/mm2の密度として表した。
本発明者らは、緑内障もしくは蝸牛の病気などの視神経病に罹患した対象を処置するため、または神経再生および/もしくは生存を促進するために用いられ得る、図1に示された遺伝子構築物を作製した。該構築物は、mBDNFの同時生成および局所放出により、RGCの細胞表面上のTrkB受容体の密度を維持または増大し、そしてTrkB受容体経路を通したシグナル伝達を維持または増進するように設計された。
ATGACTATCCTGTTTCTGACAATGGTTATTAGCTATTTCGGTTGCATGAAGGCTCACAGTGATCCCGCACGCCGCGGAGAACTTAGCGTGTGCGACAGCATCAGCGAGTGGGTCACCGCCGCCGATAAGAAGACCGCTGTGGATATGTCCGGCGGGACCGTCACTGTACTCGAAAAAGTTCCAGTGAGCAAAGGCCAACTGAAACAATATTTCTATGAAACTAAGTGCAACCCCATGGGGTACACCAAGGAGGGCTGCCGGGGAATCGACAAGAGACACTGGAATTCCCAGTGCCGGACCACTCAGAGCTACGTCCGCGCCTTGACGATGGATTCAAAGAAGCGCATCGGATGGCGGTTCATAAGAATCGACACCAGTTGTGTGTGCACGCTGACGATAAAACGGGGGCGGGCCCCCGTGAAGCAGACCCTGAACTTTGATTTGCTCAAGTTGGCGGGGGATGTGGAAAGCAATCCCGGGCCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTTGTGCCAATACTGGTTGAGTTGGATGGCGATGTCAACGGACACAAATTTAGCGTAAGCGGGGAGGGAGAGGGCGACGCCACATATGGCAAGCTGACCCTGAAGTTCATTTGCACGACCGGCAAATTGCCCGTCCCTTGGCCCACACTTGTGACGACCCTGACTTATGGCGTACAGTGCTTCAGCAGGTACCCTGATCATATGAAGCAACACGACTTCTTTAAGAGTGCCATGCCAGAGGGATACGTCCAGGAAAGAACCATATTCTTCAAAGATGATGGAAATTACAAAACCCGGGCAGAGGTCAAGTTTGAAGGCGACACCCTGGTGAACAGGATCGAACTCAAAGGCATCGATTTCAAAGAGGACGGAAACATCCTCGGACACAAACTGGAATACAATTACAACAGCCACAACGTCTACATCATGGCAGATAAACAAAAGAACGGTATTAAAGTGAACTTCAAGATCCGGCACAACATCGAAGACGGCTCCGTCCAGCTTGCCGACCACTACCAGCAAAATACCCCGATCGGCGACGGCCCCGTTCTCCTCCCCGATAATCACTACCTGAGTACACAGTCAGCCTTGAGCAAAGACCCTAATGAAAAGCGGGACCACATGGTTTTGCTGGAGTTCGTTACCGCAGCGGGTATTACGCTGGGTATGGACGAGCTTTACAAGTAA
[配列番号104]
ATGACTATCCTGTTTCTGACAATGGTTATTAGCTATTTCGGTTGCATGAAGGCTCACAGTGATCCCGCACGCCGCGGAGAACTTAGCGTGTGCGACAGCATCAGCGAGTGGGTCACCGCCGCCGATAAGAAGACCGCTGTGGATATGTCCGGCGGGACCGTCACTGTACTCGAAAAAGTTCCAGTGAGCAAAGGCCAACTGAAACAATATTTCTATGAAACTAAGTGCAACCCCATGGGGTACACCAAGGAGGGCTGCCGGGGAATCGACAAGAGACACTGGAATTCCCAGTGCCGGACCACTCAGAGCTACGTCCGCGCCTTGACGATGGATTCAAAGAAGCGCATCGGATGGCGGTTCATAAGAATCGACACCAGTTGTGTGTGCACGCTGACGATAAAACGGGGGCGGGCCCCTGTCAAACAAACCCTCAATTTTGACTTGCTGAAGCTTGCTGGGGATGTCGAGTCCGCTGCCGCGGCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTTGTGCCAATACTGGTTGAGTTGGATGGCGATGTCAACGGACACAAATTTAGCGTAAGCGGGGAGGGAGAGGGCGACGCCACATATGGCAAGCTGACCCTGAAGTTCATTTGCACGACCGGCAAATTGCCCGTCCCTTGGCCCACACTTGTGACGACCCTGACTTATGGCGTACAGTGCTTCAGCAGGTACCCTGATCATATGAAGCAACACGACTTCTTTAAGAGTGCCATGCCAGAGGGATACGTCCAGGAAAGAACCATATTCTTCAAAGATGATGGAAATTACAAAACCCGGGCAGAGGTCAAGTTTGAAGGCGACACCCTGGTGAACAGGATCGAACTCAAAGGCATCGATTTCAAAGAGGACGGAAACATCCTCGGACACAAACTGGAATACAATTACAACAGCCACAACGTCTACATCATGGCAGATAAACAAAAGAACGGTATTAAAGTGAACTTCAAGATCCGGCACAACATCGAAGACGGCTCCGTCCAGCTTGCCGACCACTACCAGCAAAATACCCCGATCGGCGACGGCCCCGTTCTCCTCCCCGATAATCACTACCTGAGTACACAGTCAGCCTTGAGCAAAGACCCTAATGAAAAGCGGGACCACATGGTTTTGCTGGAGTTCGTTACCGCAGCGGGTATTACGCTGGGTATGGACGAGCTTTACAAGTAA
[配列番号105]
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGCTCCCGTTAAACAAACTCTGAACTTCGACCTGCTGAAGCTGGCTGGAGACGTGGAGTCCAACCCTGGACCTATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCGCACTCCGACCCTGCCCGCCGTGGGGAGCTGAGCGTGTGTGACAGTATTAGCGAGTGGGTCACAGCGGCAGATAAAAAGACTGCAGTGGACATGTCTGGCGGGACGGTCACAGTCCTAGAGAAAGTCCCGGTATCCAAAGGCCAACTGAAGCAGTATTTCTACGAGACCAAGTGTAATCCCATGGGTTACACCAAGGAAGGCTGCAGGGGCATAGACAAAAGGCACTGGAACTCGCAATGCCGAACTACCCAATCGTATGTTCGGGCCCTTACTATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACACTTCCTGTGTATGTACACTGACCATTAAAAGGGGAAGATAG
[配列番号106]
1. Quigley HA, Number of people with glaucoma worldwide. Brit. J. Ophthalmol. 1996, vol. 80, PP: 389-393.
2. www.preventblindness.org
3. Goldberg I, Relationship between intraocular pressure and preservation of visual field in glaucoma. Surv. Ophthalmol. 2003 vol. 48 Suppl. 1, PP: S3-S7.
4. Glaucoma, Merck Manual of Diagnosis and Therapy, 1999, Merck Research Laboratories; Whitehouse Station, NJ, PP: 733-738.
5. Alward WL, Medical Management of Glaucoma. New Eng. J. Med., 1998; vol. 339, PP: 1298-1307
6. Coleman AL, Glaucoma. Lancet, 1999; vol. 354, PP: 1803-1810.
7. Medeiros FA, and Weinreb RN, Medical Backgrounders: glaucoma. Drugs of Today 2002, vol. 38, PP: 563-570.
8. Bakalash S, Kipnis J, Yoles E, and Schwartz M, Resistance of retinal ganglion cells to an increase in intraocular pressure is immune-dependent. Invest. Ophthalmol. Vis. Sci., 2002, vol. 43, PP: 2648-2653.
9. Kipnis J, Yoles E, Porat Z, Cohen A, Mor F, Sela M, Cohen IR, and Schwartz M, T cell immunity to copolymer 1 confers neuroprotection on the damaged optic nerve: Possible therapy for optic neuropathies. Proc. Natl. Acad. Sci. 2000, vol. 97, PP: 7446-7451.
10. Quigley HA, Nickells RW, Kerrigan LA, Pease ME, Thibault DJ, and Zack DJ, Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest. Ophthalmol. Vis. Sci. 1995 vol. 36, PP: 774-786.
11. Weinreb RN, and Levin LA, Is neuroprotection a viable therapy for glaucoma? Arch. Ophthalmol. 1999, vol. 117, PP: 1540-1544.
12. Chao MV. Neurotrophins and their receptors: A convergence point for many signalling pathways. Nature Rev. Neurosci. 2003, vol. 4, PP: 299-309.
13. Dawbarn D, and Allen SJ, Neurotrophins and neurodegeneration. Neuropathol. Appl. Neurobiol. 2003, vol.29, PP: 211-230.
14. Barde Y-A, Leibrock J, Lottspeich F, Edgar D, Yancopoulos G, and Thoenen H, Brain-derived neurotrophic factor 1993, US patent 05229500.
15. Mey J, and Thanos S, Intravitreal injections of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult rats in vivo. Brain Res. 1993, 602: 304-317.
16. Mansour-Rabaey S, Clarke DB, Wang Y-C, Bray GM, and Aguayo AJ, Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells. Proc. Natl. Acad. Sci. USA. 1994, vol. 91, PP: 1632-1636.
17. Peinado-Ramon P, Salvador M, Viuegas-Perez MP, and Vidal-Sanz M, Effects of axotomy and intraocular administration of NT-4, NT-3 and brain-derived neurotrophic factor on the survival of adult rat retinal ganglion cells. A quantitative in vivo study. Invest Ophthalmol. Vis. Sci. 1996, vol. 37, PP: 489-500.
18. Di Polo A, Aigner LJ, Dunn RJ, Bray GM, and Aguayo AJ, Prolonged delivery of brain-derived neurotrophic factor by adenovirus- infected Muller cells temporarily rescues injured retinal ganglion cells. Proc. Natl. Acad. Sci. USA. 1998, vol. 95, PP: 3978-3983.
19. Klocker N, Kermer P, Weishaupt JH, Labes M, Ankerhold R, and Bahr M, Brain-derived neurotrophic factor-mediated neuroprotection of adult rat retinal ganglion cells in vivo does not exclusively depend on phosphatidyl-inositol-3'-kinase/protein kinase B signaling. J. Neurosci. 2000, vol. 20, PP: 6962-6967.
20. Ko ML, Hu DN, Ritch R, Sharma SC, and Chen CF, Patterns of retinal ganglion cell survival after brain-derived neurotrophic factor administration in hypertensive eyes of rats. Neurosci. Lett. 2001, vol. 305, PP: 139-142.
21. Chen H, and Weber AJ, BDNF enhances retinal ganglion cell survival in cats with optic nerve damage. Invest Opthamol. Vis. Sci. 2001, vol. 42, PP: 966-974.
22. Porez MTR, and Caminos E, Expression of brain-derived neurotrophic factor and its functional receptor in neonatal and adult rat retina. Neυrosci. Lett. 1995, vol. 183, PP: 96-99.
23. Vecino E, Ugarte M, Nash MS, and Osborne NN. NMDA induces BDNF expression in the albino rat retina in vivo. Neuroreport. 1999 vol.10, PP: 1103-1106.
24. Mowla SJ, Farhadi HF, Pareek S, Atwal JK, Morris SJ, Seidah NG, and Murphy RA. Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor. J. Biol. Chem. 2001 vol 276, PP: 12660-12666.
25. Gupta VK, You Y, Gupta VB, Klistorner A, and Graham SL. TrkB receptor signalling: Implications in neurodegenerative, psychiatric and proliferative disorders. Int. J. Mol. Sci. 2013, vol.14, PP: 10122-10142
26. Teng,H.K., Teng,K.K., Lee,R., Wright,S., Tevar,S., Almeida,R.D., Kermani,P., Torkin,R., Chen,Z.Y., Lee,F.S., Kraemer,R.T., Nykjaer,A. and Hempstead,B.L. ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. J. Neurosci. 2005, vol. 25, PP: 5455-5463.
27. Wei Y, Zhang F, Zao J, Jiang X, Lu Q, Gao E and Wand N. Enhanced protein expression of proBDNF and proNGF in elevated intraocular pressure-induced rat retinal ischemia. Chin. Med. J. 2012, vol. 125, PP: 3875-3879.
28. Woo NH, Teng HK, Siao C-J, Chiaruttini C, Pang PT, Milner TA, Hempstead BL and Lu B. Activation of p75NTR by proBDNF facilitates hippocampal long-term depression. Nature Neurosci. 2005, vol 8, PP: 1069-1077.
29. Lebrun-Julien F, Bertrand MJ, De Backer O, Stellwagen D, Morales CR, Di Polo A, and Barker PA. ProNGF induces TNFalpha-dependent death of retinal ganglion cells through a p75NTR non-cell-autonomous signaling pathway. Proc. Natl. Acad. Sci. U S A. 2010 vol. 107, PP: 3817-3822.
30. Quigley HA, McKinnon SJ, Zack DJ, Pease ME, Kerrigan-Baumrind LA, Kerrigan DF, and Mitchell RS, Retrograde axonal transport of BDNF in retinal ganglion cells is blocked by acute IOP elevation in rats. Invest. Ophthalmol. Vis. Sci., 2000 vol. 41, PP: 3460-3466.
31. Pease ME McKinnon SJ, Quigley HA, Kerrigan-Baumrind LA, and Zack DJ, Obstructed axonal transport of BDNF and its receptor TRKB in experimental glaucoma. Invest. Ophthalmol. Vis. Sci. 2000, vol. 41, PP: 764-774.
32. Wei Y, Wang N, Lu Q, Zhang N, Zheng D, and Li J. Enhanced protein expressions of sortilin and p75NTR in retina of rat following elevated intraocular pressure-induced retinal ischemia. Neurosci. Lett. 2007, vol. 429, PP: 169-174.
33. Martin KRG, Quigley HA, Zack DJ, Levkovitch-Verbin H, Kielczewski J, Valenta D, Baumrind L, Pease ME, Klein RL, and Hauswirth WW, Gene therapy with brain-derived neurotrophic factor as a protection: Retinal ganglion cells in a rat glaucoma model. Invest. Ophthalmol. Vis. Sci., 2003, vol. 44, PP: 4357-4365.
34. Ren R, Li Y, Liu Z, Liu K, and He S, Long-term rescue of rat retinal ganglion cells and visual function by AAV-mediated BDNF expression after acute elevation of intraocular pressure. Invest. Ophthamol. Vis. Sci., 2012, vol. 53, PP: 1003-1011.
35. Cheng L, Sapieha P, Kittlerova P, Hauswirth WW, Di Polo A, TrkB gene transfer protects retinal ganglion cells from axotomy-induced death in vivo. J. Neurosci., 2002, vol. 22, PP: 3977-3986.
36. Bai Y, Xu J, Brahimi F, Zhuo Y, Sarunic MV, and Saragovi HU, An agonistic TrkB mAb causes sustained TrkB activation, delays RGC death, and protects the retinal structure in optic nerve axotomy and in glaucoma. Invest. Ophthalmol. Vis. Sci. 2012, vol. 51, PP: 4722-4731.
37. Jelsma TN, Hyman Friedman H, Berkelaar M, Bra. GM, and Aguayo AJ, Different forms of the neurotrophin receptor trkB mRNA predominate in rat retina and optic nerve. J. Neurobiol. 1993, vol. 24, PP: 1207-1214.
38. Gomes JR, Costa JT, Melo CV, Felizzi F, Monteiro P, Pinto MJ, Inacio AR, Wieloch T, Almeida RD, Graos M, and Duarte CB, Excitotoxicity down regulates TrkB.Fl signaling and up regulates the neuroprotective truncated TrkB receptors in cultured hippocampal and striatal neurons J. Neurosci. 2012, vol. 32, PP: 4610-4622.
39. Gupta VK, You Y, Klistorner A, and Graham SL. Shp-2 regulates the TrkB receptor activity in the retinal ganglion cells under glaucomatous stress. Biochimica et Biophysica Acta 2012, vol. 1822, PP: 1643-1649.
40. Khalin I, Alyautdin R, Kocherga G, Bakar MA, Targeted delivery of brain-derived neurotrophic factor for the treatment of blindness and deafness. Int. J. Nanomedicine. 2015, vol. 10, PP: 3245-3267.
41. Budenz CL, Wong HT, Swiderski DL, Shibata SB, Pfingst BE, Raphael Y, Differential effects of AAV.BDNF and AAV.Ntf3 in the deafened adult guinea pig ear. Sci. Rep. 2015, vol. 5 PP: 8619.
42. Havenith S, Versnel H, Klis SF, Grolman W, Local delivery of brain-derived neurotrophic factor on the perforated round window membrane in Guinea pigs: a possible clinical application. Otol Neurotol. 2015, vol.36, PP:705-711.
43. Jian-Yi Zhang J-Y, Luo X-G, Xian CJ, Liu Z-H, Zhou X-F (2008) Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents. Eur. J. Neurosci. Vol. 12, PP: 4171-4180.
44. Lindsey RM (1988) Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons. J. Neurosci. vol. 8, PP: 2394-2405.
45. Martinez-Salas E. Internal ribosome entry site biology and its use in expression vectors. Curr. Opin. Biotechnol. 1999, vol. 10, PP: 458-464.
46. Harries M et al. Comparison of bicistronic retroviral vectors containing internal ribosome entry sites (IRES) using expression of human interleukin-12 (IL-12) as a readout. J. Gene Med. 200 vol. 2, PP: 243-249.
47. Furler S, Paterna J-C, Weibel M and Bueler H Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons Gene Ther. 2001, vol. 8, PP: 864-873.
Claims (35)
- チロシンキナーゼ受容体B(TrkB)をコードする第一のコード配列と、前記TrkB受容体のアゴニストをコードする第二のコード配列と、プロモーターとを含み、前記第一のコード配列と前記第二のコード配列とが、前記プロモーターに5’から3’方向で動作可能になるように連結された遺伝子構築物を含む組換えアデノ随伴ベクター(rAAV)であって、前記アゴニストが、成熟型BDNFであり、前記第二のコード配列が、前記TrkB受容体の前記アゴニストの分泌を増幅するシグナルペプチドをコードするヌクレオチド配列を含み、前記遺伝子構築物が、前記第一のコード配列と前記第二のコード配列との間に配置されたスペーサー配列を含み、前記スペーサー配列が、消化され、それにより前記TrkB受容体および前記アゴニストを別個の分子として生成するように構成されたペプチドスペーサーをコードする、組換えベクター。
- 前記遺伝子構築物が、ウッドチャック肝炎ウイルス転写後調節エレメント(WHPE)を表すヌクレオチド配列を含む、請求項1に記載の組換えベクター。
- 前記WHPEが、配列番号57もしくは58に表される核酸配列、または配列番号57もしくは58と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項2に記載の組換えベクター。
- 前記遺伝子構築物が、ポリAテールをコードするヌクレオチド配列を含む、請求項1~3のいずれか1項に記載の組換えベクター。
- 前記ポリAテールが、配列番号59に表される核酸配列、または配列番号59と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項4に記載の組換えベクター。
- 前記プロモーターが、ヒトシナプシンI(SYN I)プロモーターである、請求項1~5のいずれか1項に記載の組換えベクター。
- 前記プロモーターが、配列番号1に表されるヌクレオチド配列、または配列番号1と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項6に記載の組換えベクター。
- 前記プロモーターが、CAGプロモーターである、請求項1~5のいずれか1項に記載の組換えベクター。
- 前記プロモーターが、配列番号2、3もしくは48に表されるヌクレオチド配列、または配列番号2、3もしくは48と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項8に記載の組換えベクター。
- 前記スペーサー配列が、ウイルスペプチドスペーサー配列を含み、且つコードする、請求項1~9のいずれか1項に記載の組換えベクター。
- 前記スペーサー配列が、ウイルス2Aペプチドスペーサー配列を含み、且つコードする、請求項10に記載の組換えベクター。
- 前記ペプチドスペーサー配列が、配列番号4に表されるアミノ酸配列、または配列番号4と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、又は前記スペーサー配列が、配列番号5に表されるヌクレオチド配列、または配列番号5と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、又は前記ペプチドスペーサー配列が、配列番号6に表されるアミノ酸配列、または配列番号6と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、又は前記スペーサー配列が、配列番号7に表されるヌクレオチド配列、または配列番号7と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、又は前記ペプチドスペーサー配列が、配列番号8に表されるアミノ酸配列、または配列番号8と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項1~11のいずれか1項に記載の組換えベクター。
- 前記第一のコード配列が、TrkBのヒトカノニカルアイソフォームをコードするヌクレオチド配列を含み、前記TrkBのカノニカルアイソフォームが、配列番号9に表されるアミノ酸配列、または配列番号9と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、及び/又は前記第一のコード配列が、配列番号10に表されるヌクレオチド配列、または配列番号10と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、及び/又は前記第一のコード配列が、TrkBのアイソフォーム4をコードするヌクレオチド配列を含む、請求項1~12のいずれか1項に記載の組換えベクター。
- 前記TrkBのアイソフォーム4が、配列番号11に表されるアミノ酸配列、または配列番号11と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項13に記載の組換えベクター。
- 前記第一のコード配列が、配列番号12に表されるヌクレオチド配列、または配列番号12と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項13又は14に記載の組換えベクター。
- 前記第一のコード配列が、配列番号9で表されるアミノ酸配列をコードするヌクレオチド配列を含み、配列番号9の516位、701位、705位、706位および/または816位の1つまたは複数のチロシン残基が、異なるアミノ酸残基に修飾されている、請求項1~15のいずれか1項に記載の組換えベクター。
- 配列番号9の516位、701位、705位、706位および/または816位の少なくとも2つのチロシン残基が、異なるアミノ酸残基に修飾されている、請求項16に記載の組換えベクター。
- 配列番号9の516位、701位、705位、706位および/または816位の5つのチロシン残基全てが、異なるアミノ酸残基に修飾されている、請求項17に記載の組換えベクター。
- 前記チロシン残基または各チロシン残基が、グルタミン酸に修飾されている、請求項16~18のいずれか1項に記載の組換えベクター。
- 前記TrkB受容体の前記修飾形態が、配列番号13に表されるアミノ酸配列、または配列番号13と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項16に記載の組換えベクター。
- 前記第一のコード配列が、配列番号14に表されるヌクレオチド配列、または配列番号14と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項20に記載の組換えベクター。
- 前記成熟型BDNFが、配列番号18に表されるアミノ酸配列、または配列番号18と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項1~21のいずれか1項に記載の組換えベクター。
- 前記第二のコード配列が、配列番号19に表されるヌクレオチド配列、または配列番号19と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項1~22のいずれか1項に記載の組換えベクター。
- 前記第二のコード配列が、前記TrkB受容体の前記アゴニストのためのシグナルペプチドを含む、請求項1~23のいずれか1項に記載の組換えベクター。
- 前記第二のコード配列が、BDNFのためのシグナルペプチドをコードするヌクレオチド配列を含む、請求項24に記載の組換えベクター。
- 前記ヌクレオチド配列が、BDNFのためのカノニカルなシグナルペプチドをコードし、前記第二のコード配列が、配列番号20に表されるアミノ酸配列、又は配列番号20に表されるアミノ酸配列と少なくとも90%の配列同一性を有する断片もしくは変異体を含むシグナルペプチドをコードするヌクレオチド配列を含むか、又は配列番号21に表されるヌクレオチド配列、または配列番号21に表されるヌクレオチド配列と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、請求項25に記載の組換えベクター。
- 前記第二のコード配列が、配列番号23、25、27もしくは29のいずれか1つに表されるシグナル配列ペプチドをコードするヌクレオチド配列を含むか、または前記シグナルペプチドが、配列番号22、24、26もしくは28のいずれか1つに表されるアミノ酸配列を含む、及び/又は前記第二のコード配列が、配列番号31、33、35、37、39、41、43、45、61、63、65、67、69、71、73、75、77、79、81、83、85、87、89、91、93、95、97、99、101もしくは103のいずれか1つに表されるシグナル配列ペプチドをコードするヌクレオチド配列を含むか、または前記シグナルペプチドが、配列番号30、32、34、36、38、40、42、44、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100もしくは102のいずれか1つに表されるアミノ酸配列を含む、及び/又は前記構築物が、配列番号107もしくは108に表されるヌクレオチド配列、または配列番号107もしくは108と少なくとも90%の配列同一性を有する断片もしくは変異体を含む、及び/又は前記rAAVベクターが、AAV-1ベクター、AAV-2ベクター、AAV-3Aベクター、AAV-3Bベクター、AAV-4ベクター、AAV-5ベクター、AAV-6ベクター、AAV-7ベクター、AAV-8ベクター、AAV-9ベクター、AAV-10ベクターまたはAAV-11ベクターである、請求項1~26のいずれかに記載の組換えベクター。
- 前記rAAVベクターが、rAAV血清型-2ベクターである、請求項1~27のいずれかに記載の組換えベクター。
- 薬剤としての使用または治療における使用のための、請求項1~28のいずれか1項に記載の組換えベクター。
- 視神経障害もしくは蝸牛障害を処置、予防もしくは改良することにおける使用のため、または神経再生および/もしくは生存を促進するための、請求項1~29のいずれか1項に記載の組換えベクター。
- 処置される前記視神経障害が、RGCの損失をもたらし得る任意の病理生理学的状態であり、形質転換されていない、または形質転換されている幹細胞を患者の目または視力に関連する領域に導入することによりRGCの置換を支援するのに用いられる、請求項30に記載の組換えベクター。
- 処置される前記視神経障害が、緑内障である、請求項31に記載の組換えベクター。
- 処置される前記蝸牛障害が、難聴または聴覚消失である、請求項30に記載の組換えベクター。
- 請求項1~28のいずれか1項に記載の組換えベクターと、医薬的に許容し得るビヒクルとを含む医薬組成物。
- 請求項1~28のいずれか1項に記載の組換えベクターを、医薬的に許容し得るビヒクルと接触させることを含む、請求項34に記載の医薬組成物を調製する方法。
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---|---|---|---|---|
BR112018006480A2 (pt) | 2015-10-01 | 2018-10-09 | Goleini Inc | ?vetor, método de tratamento de uma doença ou condição, composição, combinação de um vetor, kit, uso de um vetor, método para a modulação in vivo da atividade eletrofisiológica de uma célula |
GB201705484D0 (en) * | 2017-04-05 | 2017-05-17 | Quethera Ltd | Genetic construct |
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EP3720460A1 (en) * | 2017-12-05 | 2020-10-14 | University of Washington | Compositions and methods for enhancing functional expression of therapeutic genes in photoreceptors |
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CN109022487A (zh) * | 2018-08-15 | 2018-12-18 | 上海市第人民医院 | 一种表达p65的基因载体及其用于治疗视网膜神经节细胞变性的基因治疗药物 |
KR20210113160A (ko) * | 2018-10-11 | 2021-09-15 | 데시벨 테라퓨틱스, 인크. | Aav1 벡터 및 귀 적응증의 치료를 위한 이의 용도 |
CA3120638A1 (en) * | 2018-12-19 | 2020-06-25 | Versameb Ag | Rna encoding a protein and a signal peptide with a hydrophobic amino-terminal end |
AU2020231228A1 (en) * | 2019-03-06 | 2021-09-02 | Generation Bio Co. | Closed-ended DNA (ceDNA) and immune modulating compounds |
JP2022536692A (ja) | 2019-06-11 | 2022-08-18 | シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド | 調節不全の血漿カリクレインによって媒介される疾患の治療のための抗体のアデノ随伴ウイルスベクター送達 |
CA3146192A1 (en) * | 2019-07-18 | 2021-01-21 | Lysogene | Compositions and methods for the treatment of sanfilippo disease and other disorders |
RU2719013C1 (ru) * | 2019-09-25 | 2020-04-16 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) | Генно-инженерная конструкция для стимуляции посттравматической регенерации периферических нервов |
US20230043051A1 (en) | 2019-10-23 | 2023-02-09 | Shire Human Genetic Therapies, Inc. | Adeno-associated virus vectors based gene therapy for hereditary angioedema |
CN116829595A (zh) | 2020-12-16 | 2023-09-29 | 武田药品工业株式会社 | 用于治疗由失调的血浆激肽释放酶介导的疾病的抗体的腺相关病毒载体递送 |
CA3208818A1 (en) * | 2021-02-26 | 2022-09-01 | Bo Chen | A method for decreasing degeneration of retinal ganglion cells |
CN116179495A (zh) * | 2022-11-28 | 2023-05-30 | 上海恩凯细胞技术有限公司 | 转基因免疫细胞及其应用 |
CN118256562A (zh) * | 2022-12-26 | 2024-06-28 | 科辉智药(深圳)新药研究中心有限公司 | 一种用于中枢神经***疾病治疗的基因序列构建体及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040110711A1 (en) | 2001-02-22 | 2004-06-10 | University Of Maryland, Baltimore | Novel treatment of neurodegenerative diseases by altering levels of TrkB isoforms and/or TrkC isoforms |
US20110288160A1 (en) | 2008-03-27 | 2011-11-24 | The Ohio State University Research Foundation | Treatment of Metabolic-Related Disorders Using Hypothalamic Gene Transfer of BDNF and Compositions Therefor |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5229500A (en) | 1989-08-30 | 1993-07-20 | Regeneron Pharmaceuticals, Inc. | Brain derived neurotrophic factor |
US6225282B1 (en) * | 1996-01-05 | 2001-05-01 | Genentech, Inc. | Treatment of hearing impairments |
CA2269075A1 (en) * | 1996-10-16 | 1998-04-23 | Regeneron Pharmaceuticals, Inc. | Method of enhancing the delivery of growth factors |
AU3418199A (en) * | 1998-04-20 | 1999-11-08 | Koninklijke Nederlandse Akademie Van Wetenschappen | Use of viral vectors for treatment of the injured peripheral and central nervoussystem |
ES2422301T3 (es) * | 2000-12-19 | 2013-09-10 | Res Dev Foundation | Transferencia de gen mediada por vector lentiviral y usos de la misma |
FR2906533B1 (fr) * | 2006-09-28 | 2013-02-22 | Pf Medicament | Procede de generation d'anticorps actifs contre un antigene de resistance,anticorps obtenus par ledit procede et leurs utilisations |
WO2009048605A1 (en) * | 2007-10-11 | 2009-04-16 | Biogen Idec Ma Inc. | Methods for treating pressure induced optic neuropathy, preventing neuronal degeneration and promoting neuronal cell, survival via administration of lingo-1 antagonists and trkb agonists |
CN103614416B (zh) * | 2013-09-30 | 2016-09-07 | 中国人民解放军第二军医大学东方肝胆外科医院 | 一种携带人穿膜肽p53与GM-CSF基因的重组溶瘤腺病毒及其用途 |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040110711A1 (en) | 2001-02-22 | 2004-06-10 | University Of Maryland, Baltimore | Novel treatment of neurodegenerative diseases by altering levels of TrkB isoforms and/or TrkC isoforms |
US20110288160A1 (en) | 2008-03-27 | 2011-11-24 | The Ohio State University Research Foundation | Treatment of Metabolic-Related Disorders Using Hypothalamic Gene Transfer of BDNF and Compositions Therefor |
Non-Patent Citations (2)
Title |
---|
IOVS,2010年,Vol. 51, No. 9,pp. 4722-4731 |
J. Neurosci.,2002年,Vol. 22, No. 10,pp. 3977-3986 |
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