CN116715752A - 凝血因子fviii蛋白变体、表达载体和用途 - Google Patents
凝血因子fviii蛋白变体、表达载体和用途 Download PDFInfo
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Abstract
本发明提供了一种凝血因子FVIII蛋白变体、表达载体和用途。本发明的凝血因子FVIII蛋白变体的编码核酸序列比野生型凝血因子FVIII蛋白的更短，可以容易地实现病毒载体的正常包装，并表现出增强的蛋白表达活性，在转移至细胞中时，具有较高的凝血因子FVIII蛋白的表达量。
Description
技术领域
本发明涉及重组凝血因子FVIII技术领域，尤其涉及一种改进的凝血因子FVIII蛋白变体及其编码核酸、相应的表达载体和用途。
背景技术
A型血友病(HA)是一种X连锁隐性遗传病，由于X染色体上的FVIII基因突变导致凝血功能异常，影响大约1/5000的男性，主要表现为出血倾向，出血部位广泛，常反复发生，可形成血肿、关节变形。临床上，A型血友病患者的严重程度取决于患者血液中FVIII蛋白的含量。严重血友病患者的血液中FVIII水平通常低于正常水平的1％，存在受伤后出血，并且关节经常自发出血的症状。中度患者的FVIII水平为正常的1-5％，而轻度患者的FVIII水平为正常的6％或更高，仅在严重损伤、创伤或手术后才会出血。
A型血友病患者通常需要静脉输注人血浆或重组FVIII蛋白酶替代治疗，其治疗频率基于血友病的严重程度。轻度的患者通常在受伤后根据需要接受治疗，而严重的血友病患者需要终生输注人血浆或重组FVIII蛋白，预防出血的发生。FVIII蛋白的半衰期很短(8-18小时)，患者需要频繁的输注。然而，由于费用高昂，全球只有不到30％的A型血友病患者接受这种治疗。此外，在接受FVIII蛋白酶替代治疗的患者中，约25％的患者会产生针对FVIII的中和抗体，导致治疗无效。相比之下，基因治疗提供了治愈的潜力，通过一次输注表达FVIII蛋白的腺相关病毒(AAV)，可在肝脏中持续表达FVIII蛋白，并且可以避免FVIII中和抗体的产生。
虽然基因治疗相对于酶替代治疗提供了治愈的潜力，但是A型血友病的基因治疗也面临一些其他的挑战：首先，FVIII蛋白编码序列的大小为7.0kb，远远超过AAV载体的正常包装容量；其次，与其他大小相似的蛋白相比，FVIII蛋白的表达效率低下，这导致通常需要较高的给药剂量来实现预期的FVIII蛋白表达量。通过对FVIII蛋白的生物工程改造，使用一个14个氨基酸的短连接肽(SQ)，来替代不影响FVIII蛋白活性的B结构域，可以大幅缩短FVIII蛋白的编码序列至4.4kb，实现AAV载体的正常包装。目前已有多家公司A型血友病基因治疗产品上市或申请临床研究(BioMarin-BMN270-已上市；Sangamo-SB-525-临床3期；Spark-SPK-8011-临床3期；Bayer-BAY2599023-临床1/2期)，他们均使用FVIII-SQ连接肽来代替FVIII蛋白B结构域，实现了FVIII蛋白的有效表达和临床症状的改善。但是由于FVIII蛋白的表达效率低下，很多产品均需要使用很高的给药剂量(BMN270-6e13vg/kg；SB-525-3e13 vg/kg；BAY2599023-2e13 vg/kg),这增加了肝脏损伤的风险和病毒生产的压力。
发明内容
本发明的目的旨在通过提供一种改进的凝血因子FVIII蛋白变体及其编码核酸、相应的表达载体和用途，旨在对凝血因子FVIII蛋白进行优化，进而提高其表达量，有效降低给药剂量。
本发明一方面提供了一种凝血因子FVIII蛋白变体，其氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
在另一个方面，本发明提供了一种编码凝血因子FVIII蛋白变体的核酸，其核苷酸序列如SEQ ID NO:3～15、SEQ ID NO:60～68中的任一序列所示。
在另一个方面，本发明提供了一种表达载体，其包含本发明提供的所述核酸。
在另一个方面，本发明提供了一种宿主细胞，其包含本发明提供的所述核酸或所述表达载体。
在另一个方面，本发明提供了一种药物组合物，其包含本发明提供的所述凝血因子FVIII蛋白变体、所述核酸和/或所述表达载体，以及药学上可接受的辅料。
在另一个方面，本发明提供了所述凝血因子FVIII蛋白变体、所述核酸和/或所述表达载体在表达凝血因子FVIII蛋白中的用途。
在另一个方面，本发明提供了所述凝血因子FVIII蛋白变体、所述核酸和/或所述表达载体在制备治疗需要凝血因子FVIII蛋白的疾病的药物中的用途。
在另一个方面，本发明提供了一种生产凝血因子FVIII蛋白的方法，其包括：
在细胞中表达本发明提供的所述核酸和/或所述表达载体；
回收所述细胞产生的凝血因子FVIII蛋白。
本发明提供的凝血因子FVIII蛋白变体的编码核酸序列比野生型凝血因子FVIII蛋白的更短，可以容易地实现病毒载体的正常包装，并表现出增强的蛋白表达活性，在转移至细胞中时，具有较高的凝血因子FVIII蛋白的表达量。本发明还进一步对上述编码核酸进行了密码子优化以进一步提升凝血因子FVIII蛋白的表达量和表达活性，进而显著降低病毒载体的给药剂量，具有更高的安全性和有效性。
附图说明
图1为本发明一个具体实施方案中提供的hFVIII基因表达盒的结构示意图；
图2为本发明实施例1中，在HepG2-AAVR细胞中不同hFVIII结构表达hFVIII蛋白量的比较结果；
图3为本发明实施例1中，在HepG2-AAVR细胞中不同hFVIII结构表达hFVIII蛋白活性的比较结果；
图4-5为本发明实施例2中，在野生型C57BL/6小鼠体内比较不同hFVIII优化基因表达hFVIII蛋白量的比较结果；其中，图4为AAV8-0TE-hFVIII-L1-WT\1\2\3\9\10-pA和AAV8-0TE-hFVIII-L1-pA病毒尾静脉给药野生型C57BL/6小鼠4周后，小鼠血浆中hFVIII蛋白表达量；图5为AAV8-0TE-hFVIII-L1-WT\4\5\6\8-pA和AAV8-0TE-hFVIII-L1-pA病毒尾静脉给药野生型C57BL/6小鼠4周后，小鼠血浆中hFVIII蛋白表达量；
图6为本发明实施例3中，在野生型C57BL/6小鼠体内不同元件组合表达hFVIII的比较结果；
图7为本发明的实施例4中，不同hFVIII结构病毒在给药FVIII KO小鼠后，1周～6周时间点小鼠血浆中hFVIII蛋白活性结果；
图8为本发明的实施例4中，不同hFVIII结构病毒在给药FVIII KO小鼠后，在8周实验终点小鼠断尾后流血时间；
图9为本发明的实施例5中，AAV8-0TE-hFVIII-L1-pA病毒按4e12，8e11 vg/kg剂量给药FVIII KO小鼠后，2，4，6周时间点小鼠血浆中hFVIII蛋白表达量结果；
图10为本发明的实施例5中，AAV8-0TE-hFVIII-L1-pA病毒按4e12，8e11 vg/kg剂量给药FVIII KO小鼠后，2，4，6周时间点小鼠血浆中hFVIII蛋白活性结果；
图11为本发明的实施例5中，AAV8-0TE-hFVIII-L1-pA病毒按4e12，8e11 vg/kg剂量给药FVIII KO小鼠后，6周实验终点小鼠断尾后流血时间。
具体实施方式
下面通过具体实施例对本发明做进一步说明，本发明所述实施例只是作为对本发明的说明，不限制本发明的范围。
根据野生型人源FVIII基因(GeneID：2157)，可知其核酸序列及氨基酸序列。野生型人源FVIII蛋白结构包括信号肽，A1-a1结构域，A2-a2结构域，B结构域，a3-A3结构域，C1-C2结构域。hFVIII-SQ(其氨基酸序列如SEQ ID NO:59所示)是SQ连接肽代替B结构域连接A2-a2结构域和a3-A3结构域，其结构为信号肽，A1-a1结构域，A2-a2结构域，SQ连接肽，a3-A3结构域，C1-C2结构域。hFVIII-SQ在血友病A的基因治疗中广泛使用。然而，尽管hFVIII-SQ通过缩短编码序列实现了FVIII蛋白的有效表达，但仍存在蛋白表达效率低下的问题，进而增加了肝脏损伤的风险和病毒生产的压力。基于此，本申请的发明人通过研究，获得了凝血因子FVIII蛋白的核酸及其编码的凝血因子FVIII蛋白变体，以进一步提高FVIII蛋白的表达效率。
hFVIII-SQ(SEQ ID NO:59)：
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT
SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV
GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSY
LSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQ
DRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFL
VRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRM
KNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYA
PLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGE
VGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVE
DGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVF
DENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI
LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRN
RGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTT
LQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSS
PHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT
FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK
AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTE
NMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNE
NIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS
TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWI
KVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD
SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS
YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSL
LTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ
SWVHQIALRMEVLGCEAQDLY
本发明提供了编码人凝血因子FVIII蛋白的核酸及其编码的凝血因子FVIII蛋白变体，所述核酸与编码野生型人凝血因子FVIII蛋白的核酸不同。本发明提供的编码核酸在细胞和/或动物中表达水平较高，因而能够提供较高的FVIII蛋白表达量和表达效率。
具体地，本发明实施例提供了一种凝血因子FVIII蛋白变体，具体将其命名为hFVIII-L1和hFVIII-L2，其氨基酸序列分别如SEQ ID NO:1或SEQ ID NO:2所示。
hFVIII-L1(SEQ ID NO:1)：
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT
SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV
GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSY
LSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQ
DRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFL
VRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRM
KNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYA
PLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGE
VGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVE
DGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVF
DENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI
LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRN
RGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKGGGGSEITRT
TLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSS
SPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMV
TFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDC
KAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT
ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNE
NIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS
TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWI
KVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD
SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS
YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSL
LTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ
SWVHQIALRMEVLGCEAQDLY
hFVIII-L2(SEQ ID NO:2)：
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT
SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV
GVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSY
LSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQ
DRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFL
VRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRM
KNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYA
PLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGE
VGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVE
DGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVF
DENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI
LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRN
RGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKAAAAEITRTT
LQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSS
PHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT
FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCK
AWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTE
NMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNE
NIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS
TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWI
KVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD
SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS
YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSL
LTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ
SWVHQIALRMEVLGCEAQDLY
相应地，本发明还提供了编码上述凝血因子FVIII蛋白变体的核酸，其核苷酸序列如SEQ ID NO:3～15、SEQ ID NO:60～68中的任一序列所示。
本发明实施例提供的编码核酸大幅缩短了hFVIII序列的长度，且不影响其编码的FVIII蛋白的功能。
核酸hFVIII-L1-WT(SEQ ID NO:3)
atgcaaatagagctctccacctgcttctttctgtgccttttgcgattctgctttagtgccaccagaagatactacctgggtgcagtggaactgtcat
gggactatatgcaaagtgatctcggtgagctgcctgtggacgcaagatttcctcctagagtgccaaaatcttttccattcaacacctcagtcgtg
tacaaaaagactctgtttgtagaattcacggatcaccttttcaacatcgctaagccaaggccaccctggatgggtctgctaggtcctaccatcca
ggctgaggtttatgatacagtggtcattacacttaagaacatggcttcccatcctgtcagtcttcatgctgttggtgtatcctactggaaagcttct
gagggagctgaatatgatgatcagaccagtcaaagggagaaagaagatgataaagtcttccctggtggaagccatacatatgtctggcagg
tcctgaaagagaatggtccaatggcctctgacccactgtgccttacctactcatatctttctcatgtggacctggtaaaagacttgaattcaggcc
tcattggagccctactagtatgtagagaagggagtctggccaaggaaaagacacagaccttgcacaaatttatactactttttgctgtatttgatg
aagggaaaagttggcactcagaaacaaagaactccttgatgcaggatagggatgctgcatctgctcgggcctggcctaaaatgcacacagt
caatggttatgtaaacaggtctctgccaggtctgattggatgccacaggaaatcagtctattggcatgtgattggaatgggcaccactcctgaa
gtgcactcaatattcctcgaaggtcacacatttcttgtgaggaaccatcgccaggcgtccttggaaatctcgccaataactttccttactgctcaa
acactcttgatggaccttggacagtttctactgttttgtcatatctcttcccaccaacatgatggcatggaagcttatgtcaaagtagacagctgtc
cagaggaaccccaactacgaatgaaaaataatgaagaagcggaagactatgatgatgatcttactgattctgaaatggatgtggtcaggtttg
atgatgacaactctccttcctttatccaaattcgctcagttgccaagaagcatcctaaaacttgggtacattacattgctgctgaagaggaggact
gggactatgctcccttagtcctcgcccccgatgacagaagttataaaagtcaatatttgaacaatggccctcagcggattggtaggaagtaca
aaaaagtccgatttatggcatacacagatgaaacctttaagactcgtgaagctattcagcatgaatcaggaatcttgggacctttactttatggg
gaagttggagacacactgttgattatatttaagaatcaagcaagcagaccatataacatctaccctcacggaatcactgatgtccgtcctttgtat
tcaaggagattaccaaaaggtgtaaaacatttgaaggattttccaattctgccaggagaaatattcaaatataaatggacagtgactgtagaag
atgggccaactaaatcagatcctcggtgcctgacccgctattactctagtttcgttaatatggagagagatctagcttcaggactcattggccct
ctcctcatctgctacaaagaatctgtagatcaaagaggaaaccagataatgtcagacaagaggaatgtcatcctgttttctgtatttgatgagaa
ccgaagctggtacctcacagagaatatacaacgctttctccccaatccagctggagtgcagcttgaggatccagagttccaagcctccaacat
catgcacagcatcaatggctatgtttttgatagtttgcagttgtcagtttgtttgcatgaggtggcatactggtacattctaagcattggagcacag
actgacttcctttctgtcttcttctctggatataccttcaaacacaaaatggtctatgaagacacactcaccctattcccattctcaggagaaactgt
cttcatgtcgatggaaaacccaggtctatggattctggggtgccacaactcagactttcggaacagaggcatgaccgccttactgaaggtttct
agttgtgacaagaacactggtgattattacgaggacagttatgaagatatttcagcatacttgctgagtaaaaacaatgccattgaaccaagaa
gcttctcccaaaacccaccagtcttgaaagggggagggggctctgaaataactcgtactactcttcagtcagatcaagaggaaattgactatg
atgataccatatcagttgaaatgaagaaggaagattttgacatttatgatgaggatgaaaatcagagcccccgcagctttcaaaagaaaacac
gacactattttattgctgcagtggagaggctctgggattatgggatgagtagctccccacatgttctaagaaacagggctcagagtggcagtgt
ccctcagttcaagaaagttgttttccaggaatttactgatggctcctttactcagcccttataccgtggagaactaaatgaacatttgggactcctg
gggccatatataagagcagaagttgaagataatatcatggtaactttcagaaatcaggcctctcgtccctattccttctattctagccttatttcttat
gaggaagatcagaggcaaggagcagaacctagaaaaaactttgtcaagcctaatgaaaccaaaacttacttttggaaagtgcaacatcatat
ggcacccactaaagatgagtttgactgcaaagcctgggcttatttctctgatgttgacctggaaaaagatgtgcactcaggcctgattggaccc
cttctggtctgccacactaacacactgaaccctgctcatgggagacaagtgacagtacaggaatttgctctgtttttcaccatctttgatgagacc
aaaagctggtacttcactgaaaatatggaaagaaactgcagggctccctgcaatatccagatggaagatcccacttttaaagagaattatcgct
tccatgcaatcaatggctacataatggatacactacctggcttagtaatggctcaggatcaaaggattcgatggtatctgctcagcatgggcag
caatgaaaacatccattctattcatttcagtggacatgtgttcactgtacgaaaaaaagaggagtataaaatggcactgtacaatctctatccag
gtgtttttgagacagtggaaatgttaccatccaaagctggaatttggcgggtggaatgccttattggcgagcatctacatgctgggatgagcac
actttttctggtgtacagcaataagtgtcagactcccctgggaatggcttctggacacattagagattttcagattacagcttcaggacaatatgg
acagtgggccccaaagctggccagacttcattattccggatcaatcaatgcctggagcaccaaggagcccttttcttggatcaaggtggatct
gttggcaccaatgattattcacggcatcaagacccagggtgcccgtcagaagttctccagcctctacatctctcagtttatcatcatgtatagtct
tgatgggaagaagtggcagacttatcgaggaaattccactggaaccttaatggtcttctttggcaatgtggattcatctgggataaaacacaata
tttttaaccctccaattattgctcgatacatccgtttgcacccaactcattatagcattcgcagcactcttcgcatggagttgatgggctgtgattta
aatagttgcagcatgccattgggaatggagagtaaagcaatatcagatgcacagattactgcttcatcctactttaccaatatgtttgccacctg
gtctccttcaaaagctcgacttcacctccaagggaggagtaatgcctggagacctcaggtgaataatccaaaagagtggctgcaagtggact
tccagaagacaatgaaagtcacaggagtaactactcagggagtaaaatctctgcttaccagcatgtatgtgaaggagttcctcatctccagca
gtcaagatggccatcagtggactctcttttttcagaatggcaaagtaaaggtttttcagggaaatcaagactccttcacacctgtggtgaactctc
tagacccaccgttactgactcgctaccttcgaattcacccccagagttgggtgcaccagattgccctgaggatggaggttctgggctgcgag
gcacaggacctctactga
核酸hFVIII-L1-1(SEQ ID NO:4)
atgcagattgagctgtctacatgcttcttcttgtgtctgctcagattttgcttctctgccactaggagatactacttgggagcagtggagctgtcct
gggactacatgcagagtgaccttggggaactccctgtagatgcaagatttcctcccagagtccccaaaagtttcccctttaacacatcagttgt
gtacaagaagaccctgtttgtggagtttacagaccacctgttcaacattgcaaagcccagacctccatggatgggactcctaggcccaactat
ccaggcagaggtctatgatacagttgtcataaccttgaagaatatggcatctcatcctgtctccctgcatgctgttggagtcagctattggaagg
cctctgaaggtgctgagtatgatgaccagactagccaaagagaaaaggaggatgacaaagtgttccctggagggtctcacacatatgtttgg
caggtgctgaaggagaatggacccatggccagtgaccctctgtgcttgacctacagttatctgtctcatgtagacctagtgaaggacctcaac
tcaggactcataggagcattacttgtgtgtagagaggggagcttagctaaggaaaaaacccagacacttcataagttcatccttctgtttgcagt
ttttgatgaggggaaaagttggcactcagagacaaaaaactctctgatgcaggacagggatgctgccagtgctagggcctggcctaagatg
cacacagtaaatggatatgtgaacagatctctgccaggcctcataggttgccatagaaagtctgtttattggcatgtcataggaatggggacta
ccccagaggtgcactcaatctttctagaaggccacacattcttagtaagaaaccacaggcaagcatctttggagatcagtccaatcacattcct
cacagcacaaactctcctgatggacctgggccagttcctcttgttctgccacattagctctcaccagcatgatggaatggaggcatatgttaaa
gtagactcatgtccagaagagcctcagttaagaatgaaaaacaatgaagaagcagaagactatgatgatgacctgactgactctgagatgga
tgttgtgaggtttgatgatgataattctccctcctttatacaaataagatctgtagccaaaaagcatcccaaaacttgggttcactacatagctgct
gaggaagaagactgggattatgctcctctggtcctagcccctgatgacagatcctacaaaagtcagtacctcaataatggtcctcaaagaatt
ggcaggaagtacaagaaagtcaggttcatggcttacactgatgagactttcaaaaccagggaggctatacaacatgagtctggtattctggg
accactgttatatggtgaggtgggagatacactgctgatcatctttaagaatcaggcctcaagaccatacaacatctatcctcatggtatcactg
atgtaagacctctgtatagcagaagactgcccaaaggagttaagcaccttaaagattttcctatccttcctggagaaatcttcaaatacaaatgg
actgtcactgttgaggatggtcctactaaatcagaccccaggtgcctaaccagatactacagcagttttgtgaatatggagagggacttagctt
ctggactgattgggccattgctgatctgctataaggagtctgtagatcagaggggcaatcagatcatgagtgacaagagaaatgtgattctgtt
ctctgtgtttgatgagaataggagctggtatctgacagagaacatccagagattcctacccaatcctgctggggtacaattagaagaccctga
gttccaggctagcaatatcatgcactctatcaatgggtatgtgtttgattctcttcagctgtctgtttgcctacatgaggtagcctactggtacatcc
tttcaataggggctcagactgacttcctctcagtatttttctctggctacacttttaaacataagatggtgtatgaggacactctcacactgttcccc
ttctcaggtgagactgtttttatgagtatggagaacccaggcctctggattttaggctgtcacaacagtgatttcaggaacagggggatgactg
cactcctgaaggtgtcttcatgtgataagaacactggggactattatgaagattcctatgaggatatttctgcatatttgctctctaagaacaatgc
tattgaacctaggtctttttctcagaacccaccagtattaaagggtgggggaggatctgaaatcactaggacaaccctgcaatcagaccaaga
ggaaattgactatgatgacacaatctctgtagagatgaagaaagaagattttgatatctatgatgaagatgagaatcagagtcctaggtcttttca
gaaaaaaaccaggcactactttatagctgcagtagaaaggctgtgggattatggtatgtctagcagtccacatgtgctgagaaacagagccc
agtcaggcagtgtcccccagtttaagaaggtagttttccaggagttcactgatggcagtttcacccaacctctgtacagaggggagctgaatg
aacacctgggactgttgggcccttatatcagagctgaggttgaggacaacatcatggtgaccttcaggaatcaagcatctagaccctattcttt
ctactcttccctcatctcctatgaagaggaccagagacaaggagcagaacctaggaaaaactttgtcaaacctaatgagaccaagacttacttt
tggaaagttcagcaccatatggcccctacaaaggatgagtttgattgcaaggcatgggcttacttctcagatgtagatttagaaaaagatgtgc
atagtggtttaataggtccattgctggtttgccacacaaacacactgaaccctgcccatggcaggcaggtcacagtgcaagagtttgctctctt
ctttaccatatttgatgaaactaaatcttggtacttcacagaaaacatggaaagaaactgcagagctccctgcaacatacagatggaggatccc
actttcaaggaaaactacaggtttcatgccatcaatggttacataatggacacactccctggcctggtaatggctcaggaccaaaggataagg
tggtaccttttaagcatgggatccaatgaaaatatccactcaatccacttttctggtcatgtgttcacagtcagaaagaaagaagagtataagatg
gccctctataacctctacccaggggtatttgaaacagtggagatgctcccatctaaggcaggaatttggagagtggaatgcttgattggtgaac
acctgcatgcaggtatgtcaactctattcctggtgtactctaataagtgccagactccactgggcatggcctctggtcacatcagagacttccag
atcactgcctcaggccaatatggtcagtgggcccctaagctagccagattgcattattctgggtcaatcaatgcctggagcaccaaagagcct
ttctcttggatcaaagttgatcttctggctcctatgatcatccatgggatcaagactcagggagccaggcaaaagtttagctccctatacatttctc
agtttataatcatgtattctctggatggcaaaaaatggcaaacatacaggggtaattctacaggaaccctcatggtgttctttgggaatgtagact
cctcaggcataaagcataatatattcaaccctccaattatagcaaggtacatcagactgcatccaacacattatagcattagatctaccctgaga
atggaactaatggggtgtgacctgaactcctgttctatgcctctgggtatggagtctaaggctatctctgatgctcaaatcactgcctcttcctatt
tcactaatatgtttgctacctggagcccaagcaaagccaggctgcacctgcagggcaggtctaatgcctggagaccccaagttaacaacccc
aaggaatggctgcaagttgacttccaaaagaccatgaaagtgacaggtgtcactacccagggagtaaaatccttgttgactagcatgtatgta
aaggagttcctcatctcttcttctcaagatggtcaccaatggactctgtttttccagaatggtaaggtcaaggtatttcaaggtaaccaagacagc
ttcaccccagtggtaaattccttagaccctcctctgttgactaggtatctcagaattcacccacagagctgggtccaccagattgctttgagaatg
gaagttctgggttgtgaggcccaagacctgtactga
核酸hFVIII-L1-2(SEQ ID NO:5)
atgcagattgagctttccacctgttttttcctctgcctattgagattctgttttagtgccaccagaagatactacctaggagctgtagaactgagctg
ggactatatgcagagtgacctgggagaactgcctgtagatgctagatttcctcctagagtacctaaaagcttcccatttaacactagtgtggttta
caaaaaaactctctttgtagagtttactgaccacctcttcaacattgctaagcctaggcccccttggatgggactgttgggacccacaatccag
gctgaagtctatgacactgtagtaatcacactaaagaacatggcctctcatccagtttctcttcatgctgttggtgtgagttattggaaggctagtg
aaggggctgagtatgatgaccaaacctcacagagagaaaaggaggatgacaaggtgtttcctggggggtctcacacatatgtttggcaggt
cttaaaagagaatggcccaatggcttctgatcccctgtgcttgacctacagctacttatcacatgttgatttggtcaaggatctgaactctggcct
cataggtgccctactggtatgtagggagggctccctggccaaagagaagactcagaccttgcacaagttcatactactgtttgctgtgtttgat
gagggtaaaagctggcattctgagaccaaaaactctctgatgcaagatagagatgcagcctctgccagagcctggcctaagatgcacacag
ttaatgggtatgtaaacagatccttaccaggattgataggctgccataggaaaagtgtgtactggcatgtcattgggatgggcaccaccccag
aggtacactctattttccttgagggccacacattcttagtcagaaaccatagacaagcttcattggagatttctcctattacttttctaacagcacag
accttactcatggacctagggcaatttttactgttctgccacatcagttcccaccagcatgatggaatggaggcttatgtgaaggttgactcttgc
cctgaagaacctcagctgagaatgaagaacaatgaagaggctgaggattatgatgatgacctgactgattcagagatggatgtagtcaggttt
gatgatgataacagcccctccttcatacaaatcagatcagtagcaaagaagcatccaaagacatgggtacactatatagcagctgaggagga
agattgggactatgctcctttagtgctggcccctgatgatagatcctacaagagccaatacctgaacaatggcccccagaggattggtagaaa
atacaaaaaggttaggttcatggcctatacagatgagacatttaagacaagggaggctatccagcatgagtcaggaatcctaggtcctttatta
tatggggaagtgggggatactcttctcatcattttcaagaaccaggcttcaagaccttacaacatctacccacatggcatcactgatgttaggcc
cctgtattctaggagactgccaaagggagtgaagcatctaaaggactttccaatcttaccaggtgagatattcaagtataaatggactgtgaca
gtagaggatggcccaactaaatcagaccctagatgtttgactagatactacagttcctttgtcaacatggaaagagacttagcctctggtctcat
aggaccactcctcatctgctataaagagtctgtggatcagagaggcaatcaaattatgagtgataagagaaatgtgatcctgttttctgtctttga
tgagaataggtcttggtacttgacagaaaacatacagaggttccttcctaaccctgctggggtacagctagaagacccagagtttcaggcctc
caatattatgcatagtattaatggctatgtgtttgactccctgcagctatcagtgtgccttcatgaagtggcctattggtatatactttctattggtgc
ccagacagacttcttatctgtcttcttttctggctatactttcaaacacaaaatggtttatgaggacactctgacactttttcccttctctggtgagac
agtttttatgtctatggaaaaccctggcttatggatcctgggctgccacaacagtgattttagaaacagaggcatgactgcactgctaaaggttt
cttcttgtgacaaaaatactggggactattatgaggattcctatgaagatatctctgcttacctgctgagcaaaaataatgccattgaacctagaa
gcttctctcagaatcccccagtactcaaaggtggggggggctctgagatcactaggacaactctccagagtgaccaagaggagattgattat
gatgataccattagtgttgagatgaaaaaggaggactttgatatttatgatgaggatgaaaaccagtctcccagatctttccagaaaaaaacaa
gacattactttattgcagcagtagaaagattatgggactatggcatgtctagtagccctcatgttctgaggaacagggcccagagtggttcagt
accccaattcaagaaagtagtctttcaggaattcacagatggctcattcacacagcctctgtataggggtgagcttaatgaacacctgggcctt
ctaggcccctacatcagagctgaggtagaggacaatataatggtgactttcaggaatcaggcttctaggccttactctttctactcatctctgatc
agctatgaggaggaccaaagacaaggagcagagcctagaaagaactttgtgaagcccaatgaaactaaaacctacttttggaaggttcagc
atcacatggctcccacaaaggatgaatttgattgtaaggcctgggcttatttttcagatgtggatttggaaaaggatgttcactctgggttaatag
gtcctctgctggtgtgccacacaaatactcttaatcctgcacatggtagacaagtgactgtgcaggaatttgcactatttttcacaatttttgatga
aactaagagttggtattttacagaaaacatggagagaaattgcagggctccatgcaatatccaaatggaagatcctactttcaaggaaaattat
aggttccatgctattaatggttatataatggatacactgccaggactggtgatggcccaagatcaaagaattagatggtacctcttgtccatggg
ttctaatgaaaacattcactccatccacttcagtggacatgtatttacagtaagaaagaaagaggaatacaaaatggcactctacaatctatacc
caggagtgtttgagacagttgaaatgttgcccagtaaggcaggaatctggagggtagagtgcctcattggtgaacacctgcatgctggcatg
agtacccttttcttagtttacagtaacaagtgtcagacccccctggggatggccagtggacacataagagatttccagataacagcatctggtc
agtatggtcagtgggctccaaaactggccagattgcactactcaggttcaatcaatgcatggtctactaaggaacctttcagttggataaaggt
ggacttgttagctcctatgatcattcatggcatcaaaacccagggagcaaggcagaagtttagtagtctgtacatcagccaattcatcataatgt
actctctggatggcaaaaagtggcagacatacagggggaattctactgggactctgatggtgttctttggcaatgttgactctagtggtatcaa
gcacaacatcttcaaccctcccattatagcaagatacatcagacttcatccaacccactactctatcagatcaacattaaggatggaattgatgg
gctgtgaccttaattcttgctctatgcccctgggaatggaatccaaggcaatctcagatgcccagatcacagctagcagctacttcaccaatat
gtttgcaacttggtccccatctaaggccagactgcacctgcaaggcagatctaatgcctggaggccacaagtgaataatcctaaagaatggct
gcaagtagactttcaaaaaaccatgaaggttactggtgttacaacacaaggtgtcaagtcactcttaacctctatgtatgtgaaagaatttctgat
tagttccagccaggatggtcaccagtggacccttttctttcagaatggcaaggtcaaggtctttcaaggaaaccaggatagttttacaccagtg
gttaattctctggatcctcctctccttacaagatacctaagaatccaccctcagagctgggtgcaccagattgccctcagaatggaagtacttgg
gtgtgaagctcaagatctatactga
核酸hFVIII-L1-3(SEQ ID NO:6)
atgcaaattgagctgagtacctgctttttcctatgcttgctaagattctgcttctctgcaacaaggagatactatttaggagcagttgaactctcttg
ggattatatgcagtctgaccttggagaattacctgtagatgcaaggttccctccaagagtgcccaagagcttcccttttaacacttctgttgtttac
aagaagacactctttgtagagtttactgaccatctgttcaacattgctaagcctaggcccccatggatggggctcctgggcccaaccattcaag
cagaggtctatgacacagtagtgattactttaaaaaacatggcttcccaccctgtcagcctgcatgcagtaggtgtgtcttattggaaggcctct
gagggtgctgaatatgatgatcagactagtcaaagagagaaagaagatgacaaagtctttccaggtggatcacacacatatgtgtggcaggt
gctgaaggagaatggtcctatggcttcagatcctttgtgtctgacttacagctacttatctcatgtggaccttgtcaaggacttaaacagtggact
gataggggctctgctggtgtgcagagaggggagcctggcaaaggagaagactcaaactttgcataaatttattctactgtttgctgtttttgatg
aggggaagagttggcactctgaaactaagaacagccttatgcaggacagagatgctgcttctgcaagagcctggcctaaaatgcacacagt
aaatggatatgtgaacagatcccttccagggctcataggctgtcacagaaagagtgtgtactggcatgtaatagggatgggcacaactcctg
aggtgcatagcatatttctagaaggccacacattcctagttagaaatcatagacaagcatctctagagatcagtcctatcacattcttaactgcac
agaccctattgatggacttaggccagtttcttcttttttgccacattagctcccaccagcatgatggcatggaggcctatgtcaaagtggactcat
gccctgaagaaccccaactcaggatgaaaaacaatgaggaggctgaggactatgatgatgacctgacagacagtgagatggatgttgtga
ggtttgatgatgataacagcccctctttcatccagatcagatcagttgcaaagaaacacccaaagacttgggtgcactacattgctgctgagga
agaggactgggactatgctcctttagtgttggcaccagatgacaggagttacaaatcccaatacctgaataatggtcctcagaggattggcag
aaagtataagaaggtcaggttcatggcttacacagatgagacattcaagaccagagaagctattcagcatgaatctggcattctggggccttta
ctctatggggaagtgggtgacacactactcattattttcaaaaatcaagcctccagaccttacaacatctacccccatggaatcactgatgttag
gcccctttacagcagaagactacccaagggggtgaaacaccttaaagattttccaatcctgcctggagagattttcaaatacaaatggacagt
cacagtggaagatggacctaccaagagtgatcctaggtgtttgactaggtactatagttcatttgtcaatatggagagagacttggcctcaggc
ctaatagggccacttctcatctgctataaggaatcagtagaccagagaggaaaccagatcatgtcagacaaaaggaatgttatcctcttttcag
tgtttgatgagaacaggtcttggtacctcacagaaaacatccagagatttttaccaaacccagcaggtgttcaattggaagaccctgaattcca
agcctccaacatcatgcactcaataaatgggtatgtatttgactctctccagctcagtgtgtgccttcatgaggtagcttattggtacatcttatcta
ttggagctcagactgactttttatctgtgttttttagtggttatacattcaaacacaagatggtgtatgaagataccctgactttattccctttctctgg
agaaactgtgttcatgtctatggagaacccaggcctgtggattctggggtgtcacaatagtgacttcagaaacagaggaatgacagcactgct
taaggtgagcagttgtgataagaatacaggagactactatgaggactcttatgaggatatcagtgcatatctgctaagcaagaataatgcaatt
gaaccaagatctttcagccagaaccctcctgttctcaaagggggaggaggatcagaaataaccaggacaactctccagtctgatcaggaag
aaattgactatgatgacacaatctcagtggagatgaaaaaagaggactttgacatctatgatgaggatgaaaatcagtctcctaggtcatttcag
aagaagaccaggcactactttatagcagctgtggaaagactatgggactatggtatgagctcctccccccatgtgctgaggaatagagctca
aagtggctcagtgcctcagttcaagaaggtggtcttccaagaatttactgatgggagcttcacacagcccctgtatagaggggagttaaatga
acatctgggactactgggaccatacattagagcagaggtggaggacaacatcatggttactttcagaaaccaggccagtagaccatacagtt
tctacagctctctaatatcttatgaagaagatcaaagacaaggagcagaacccagaaagaactttgtaaaacctaatgagacaaagacctattt
ctggaaggtccagcaccacatggccccaaccaaggatgaatttgactgtaaggcatgggcttacttttctgatgtagatctggagaaggatgt
gcacagtggcctaattggaccattactagtctgccataccaacacactcaatcctgcacatggtagacaggtcacagtgcaagaatttgctctg
tttttcaccatttttgatgaaactaaaagttggtactttactgagaacatggagaggaactgcagagccccatgcaatatccagatggaagatcc
aactttcaaagagaattatagattccatgccataaatggttacattatggacacactgcctggcctggtcatggctcaggaccagaggatcaga
tggtatctcttgagtatgggtagcaatgagaacatacactccatccacttttcaggacatgtctttacagtcagaaaaaaagaggagtataaaat
ggccttatacaacttatatccaggagtgtttgagacagtggagatgctccctagcaaggcaggtatctggagagtagaatgtctcatagggga
acacctccatgctggtatgtcaacactgttcctggtgtatagtaacaagtgccaaacaccactgggcatggccagtggccatattagagacttt
caaataactgcctctgggcaatatggtcagtgggcccctaaactggctaggctgcactactctgggtcaatcaatgcttggtcaaccaaagaa
ccattcagctggattaaggttgacctgctggctcctatgataattcatggcatcaaaacccagggagccagacaaaagttttcttccctgtacat
ctctcaatttataataatgtactctttggatggaaaaaaatggcagacttacagagggaactcaacagggaccctgatggtgttctttgggaatg
tagactcttcaggtatcaaacataatattttcaaccctcccatcatagctagatatataagacttcaccccacccactattctataaggtctactctc
aggatggagttaatggggtgtgacttaaatagctgcagtatgcctctggggatggaaagcaaggctatctctgatgcacagattacagctagc
agctacttcactaatatgtttgctacatggtctccatctaaggcaagactgcatctccagggaagatctaatgcatggaggcctcaagtgaaca
acccaaaagagtggctgcaagtggacttccaaaaaacaatgaaggttacaggagtcacaacacagggggtgaaatctttactgacatcaat
gtatgtaaaggagtttctcatttcaagtagtcaggatggacaccagtggactttattctttcagaatggtaaggtcaaggtcttccaaggaaatca
ggacagctttacaccagtggtcaacagcctggatcctcccttactcaccagatacctgaggatccacccacagtcttgggttcaccaaattgct
ctgagaatggaagtgctgggatgtgaggcacaggacctctactga
核酸hFVIII-L1-4(SEQ ID NO:7)
atgcagattgagctgagcacctgcttcttcctgtgcctgctgagattctgcttctctgccaccagaagatactacctgggggctgtggagctga
gctgggactacatgcagtctgacctgggggagctgcctgtggatgccagattcccccccagagtgcccaagagctttcccttcaacacctct
gtggtgtacaagaagaccctgtttgtggaattcacagaccatctgttcaacattgcaaagcccagacccccctggatgggcttgctgggcccc
accatccaggctgaggtctatgacacagtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtgggggtgagct
actggaaagcttctgagggggctgagtatgatgaccagacctcccagagagagaaggaggatgacaaggtgttccctgggggcagccat
acctatgtgtggcaggtgctgaaagagaatggccccatggcctctgaccccctgtgcctgacctacagctacctgagccatgtggacctggt
gaaagacctcaattctggcctgattggggccttgctggtgtgcagagagggcagcctggctaaggagaagacccagaccctgcacaagttc
atattgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggacagagatgctgcctctgccaga
gcctggcctaaaatgcatacagtgaatggctatgtgaacagaagcctgcctggcctgattggctgccacagaaagtctgtttactggcatgtg
attggcatgggcaccacccctgaggtgcacagcatcttcctggagggccacaccttcctggtgagaaaccacaggcaggccagcctggag
atcagccccatcaccttcctgacagcccagaccctgctgatggatctgggacagttcctgctgttctgtcacatctctagccaccagcatgatg
gcatggaggcctatgtgaaggtggactcctgccctgaggagcctcagctgagaatgaagaacaatgaggaggctgaggactatgatgatg
acctgacagactctgagatggatgtggtgagatttgatgatgacaacagccctagcttcatccagatcagatctgtggccaagaagcaccca
aagacctgggtgcactacattgctgctgaggaagaggactgggactatgcccccctggtgctggcccctgatgacagaagctacaaaagc
cagtacctgaataatggcccccagagaattggcagaaagtacaagaaggtgagattcatggcttacacagatgagaccttcaagaccagag
aggccatccagcatgagtctggcatcttgggccctctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaatcaagccagc
agaccttacaacatctacccacatggcatcacagatgtgagaccactgtacagcagaagactgcccaaaggggtgaagcacctgaaggac
ttcccaatcctgcctggggagatcttcaagtataagtggacagtgacagtggaggatggccccactaagtctgaccccagatgcctgaccag
atactacagcagctttgtgaacatggaaagagatctggcctctggcctgattggccccctcctgatctgctacaaggagtctgtggaccagag
aggcaatcagatcatgtctgacaagagaaatgtgatactgttctctgtgtttgatgagaacagaagctggtacctcacagagaacattcagaga
ttcctgcccaaccctgctggggtgcaactggaagaccctgagttccaggccagcaacatcatgcacagcatcaatggctatgtctttgactcc
ctgcagctgtctgtgtgtctgcatgaggtggcctactggtacatcttgagcattggggcccagacagacttcctgtctgtgttcttctctggctac
acattcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctctggggagacagtgttcatgagcatggagaacccaggcct
gtggatcctgggctgccacaactctgacttcaggaacagaggcatgacagccctcttgaaggtgtccagctgtgacaagaacacagggga
ctactatgaggacagctatgaggacatatctgcctacctgctgagcaagaacaatgccattgagcccagatcttttagccagaacccacctgt
gctgaaggggggggggggctctgagatcaccagaaccaccctccagtctgaccaggaggagattgactatgatgacaccatctctgtgga
gatgaaaaaggaggactttgacatctatgatgaggatgagaaccagagcccaagatctttccagaagaagaccagacactacttcattgctg
ctgtggagagactgtgggactatggcatgagctctagcccccatgtgctgagaaacagagcacagtctggctctgtgccccagttcaagaa
ggtggtgttccaggagttcacagatggcagcttcacccagcccctgtacagaggggagctgaatgagcacctgggcctcctgggcccctat
atcagggctgaggtggaggacaacatcatggtgaccttcagaaaccaggccagcagaccctacagcttctatagcagcctgataagctatg
aggaggatcagagacagggggctgagcctagaaagaactttgtgaagcccaatgagaccaaaacctacttctggaaggtgcagcaccac
atggcccccaccaaggatgagtttgactgtaaggcctgggcctacttctctgatgtggacctggagaaagatgtgcactctggcctgattggc
cctctgctggtgtgccacaccaacaccttgaaccctgcccatggcagacaggtgacagtgcaggagtttgccctgttcttcaccatctttgatg
agaccaagagctggtacttcacagagaatatggagagaaattgtagagccccctgcaatatccagatggaggaccccaccttcaaggagaa
ctacagattccatgccatcaatggctacatcatggacaccctccctggcctggtcatggcccaggaccagagaatcagatggtatctgctgag
catgggcagcaatgagaatatccactccatccacttctctggccatgtgttcacagtgagaaagaaggaggagtacaagatggctctgtaca
acctgtaccctggggtgtttgagacagtggagatgctccccagcaaggctggcatctggagagtggagtgcctgattggggaacacctgca
tgctggcatgagcaccctgttcctggtgtactccaacaagtgccaaacccccctgggcatggcctctggccacataagagacttccagatca
cagcctctggccagtatggccagtgggcccccaagctggccagactgcactactctggcagcatcaatgcctggagcactaaggagccctt
cagctggatcaaggtggatctgctggcccccatgatcatccatggcatcaagacccagggggccagacagaagttcagcagcctgtacata
agccagttcatcatcatgtacagcctggatggcaaaaagtggcagacctacagaggcaacagcacaggcaccctgatggtgttctttggcaa
tgtggacagctctggcatcaagcacaacatcttcaacccccccatcattgccagatacatcagactgcaccccacccactacagcatcagaa
gcaccctgagaatggagctcatgggatgtgacctgaactcctgcagcatgcctctgggcatggaatctaaggccatctctgatgcccagatta
cagccagctcttacttcaccaacatgtttgccacctggagccctagcaaggccagactccacctgcagggcagaagcaatgcctggagacc
ccaggtgaacaaccccaaggagtggctgcaggtggacttccagaagaccatgaaggtgacaggggtgaccacccagggggtgaagagc
ctgctgaccagcatgtatgtgaaggagttcctgatcagctctagccaggatggccaccagtggacactgttcttccagaatggcaaggtgaa
ggtgttccaaggcaaccaggacagcttcacccctgtggtgaacagcttggacccccccctgctgactaggtacctgagaatccacccccag
agctgggtgcaccagattgccctgagaatggaagtcctgggctgtgaggcacaggacctgtactga
核酸hFVIII-L1-5(SEQ ID NO:8)
atgcagattgagctgagcacctgcttcttcctgtgcctgctgagattctgcttctctgccaccagaagatactacctgggagctgtggagctga
gctgggattacatgcagtctgacctgggggagctgcctgtggatgccagattcccccccagagtgcccaagagcttccccttcaacacctct
gtggtctacaagaagaccctgtttgtggaattcacagaccacttgtttaacatagccaagcccagacctccctggatgggcctcctgggcccc
accatccaggctgaggtctatgacacagtggtgatcacactgaagaacatggccagccaccctgtgagcctccatgctgtgggggtgagct
actggaaggcctctgagggggctgagtatgatgaccagacctctcagagagagaaggaagatgacaaggtgttccctgggggcagccac
acctatgtgtggcaggtgctgaaggagaatggccccatggcttctgaccccctgtgcctcacctacagctacctgagccatgtggacctggt
gaaggacctgaactctggcctgataggggccctgctggtgtgcagagaaggcagcctggccaaggagaaaacccagaccctgcacaagt
tcatcctgctgtttgctgtgtttgatgaggggaagagctggcactctgagaccaagaacagcctgatgcaggacagagatgctgcctctgcca
gagcctggcccaagatgcacacagtgaatggctatgtgaacagaagcctgcctggcctcattggctgccacagaaaatctgtgtactggcat
gtgattggcatgggcaccacccctgaggtgcacagcatcttcctggagggccacaccttcctggtgagaaaccacagacaggccagcctg
gagatcagccccatcaccttcctgacagcccagactctgctgatggacctgggccagttcctgctgttctgccacatcagcagccaccaacat
gatggcatggaggcctatgtgaaggtggacagctgccctgaggagccccagctgagaatgaaaaacaatgaggaggctgaggactatga
tgatgacctgacagactctgagatggatgtggtgagatttgatgatgacaacagccccagcttcatccagatcagatctgtggccaagaagca
ccccaaaacctgggtgcactacattgctgctgaggaagaggactgggactatgcccccctggtgctggcccctgatgacagaagctataag
tcccagtacctcaacaatggcccccagagaataggcagaaagtacaagaaggtgagattcatggcctacacagatgagaccttcaaaacca
gagaagccatccagcatgagtctggcatcctgggccccctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaaccaagc
cagcagaccttacaacatctacccccatggcatcacagatgtgagacccctgtacagcagaagactgccaaagggggtgaaacacctgaa
ggactttcccatcctgcctggggagatcttcaagtacaagtggacagtgacagtggaggatggccccaccaagtctgaccctagatgcctga
ccagatactacagcagctttgtgaatatggagagagatctggcttctggcctgattggccccctgctgatctgctacaaggagtctgtggacca
aagaggcaaccaaatcatgtctgacaagagaaatgttatcttgttctctgtgtttgatgagaatagaagctggtacctgacagagaacatccag
agattcctgcccaaccctgctggggtgcagctggaggaccctgagttccaggccagcaacatcatgcacagcatcaatggctatgtgtttga
cagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagacagacttcctgtctgtgttcttctctg
gctacaccttcaagcacaagatggtgtatgaggacacactgaccctgttccccttctctggggagacagtgttcatgagcatggagaaccctg
gcctgtggatcctgggctgccacaactctgacttcagaaacagaggcatgacagccctgctgaaggtcagcagctgtgacaagaacacag
gggactactatgaggacagctatgaggacatttctgcctacctgctcagcaagaacaatgccattgagcccagaagctttagccagaacccc
cctgtgctgaaggggggggggggctctgagatcaccagaaccaccttgcaatctgaccaagaggagattgactatgatgacactatctctgt
ggagatgaagaaggaggactttgacatctatgatgaggatgagaaccaaagccccagaagcttccagaaaaagaccagacactacttcatt
gctgctgtggagagactgtgggactatggcatgtccagcagcccccatgtgctgagaaacagagcccagtctggctctgtgccccagttca
agaaggtggtgttccaggagttcacagatggcagcttcacccagcccctgtacagaggggagctgaatgagcacctgggcctgctgggac
cctacatcagagctgaggtggaagacaacattatggtgaccttcagaaatcaggccagcagaccctacagcttctacagcagcctgatcagc
tatgaggaagatcagagacagggggctgagcccagaaagaactttgtgaagcctaatgagaccaagacctacttctggaaggtgcagcat
cacatggcccccaccaaggatgagtttgactgcaaggcctgggcctacttttctgatgtggacctggagaaagatgtgcactctggcctgatt
ggccccctgctggtgtgccacaccaacaccctgaaccctgcccatggcagacaggtgacagtgcaggagtttgccctgttcttcactatttttg
atgagaccaagtcctggtacttcactgagaatatggaaagaaactgtagagccccctgcaacatccagatggaggaccccaccttcaagga
gaactacagattccatgcaataaatggctacatcatggacaccctgcctggcctggtgatggcccaagaccagagaatcagatggtacctgc
tgagcatggggtctaatgagaatatccacagcatccacttctctggccatgtgttcacagtgaggaagaaagaggaatacaagatggccctgt
acaacctctaccctggggtgtttgagacagtggagatgctgccctccaaggctggcatctggagagtggagtgtctgattggggagcacctg
catgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacccccctgggcatggcctctggccacataagagactttcaaatt
acagcctctggccagtatggccagtgggcccccaaactggccagactgcactactctggcagcatcaatgcctggagcaccaaggagccc
ttcagctggatcaaggtggatctgctggcccccatgatcatccatggcatcaagacccagggggccagacagaagttcagcagcctgtaca
taagccagttcatcatcatgtacagcctggatggcaagaagtggcagacctacagaggcaacagcactggcaccctcatggtgttctttggc
aatgtggacagctctggcattaagcacaacatcttcaacccccctatcattgccagatacatcagactgcatcccacccactacagcatcaga
agcactctgagaatggaactgatgggctgtgacctcaacagctgcagcatgcccctggggatggagagcaaggcaatctctgatgcccag
atcacagccagcagctacttcaccaacatgtttgccacctggagcccctccaaggctagactgcacctgcaaggaagaagcaatgcctgga
ggccccaggtgaacaaccccaaggagtggctgcaggtggacttccagaagaccatgaaggtgacaggggtgaccacccagggggtgaa
gagcctgctgaccagcatgtatgtgaaggagttcctgatcagctctagccaggatggccaccaatggaccctgttcttccagaatggcaagg
tcaaggtgttccagggcaaccaggatagcttcacccctgtggtgaacagcctggacccccccctgctgactagatacctgagaatccacccc
cagagctgggtgcaccagattgccctgagaatggaggtgctgggctgtgaggcccaggacttgtactga
核酸hFVIII-L1-6(SEQ ID NO:9)
atgcagattgagctgagcacctgcttctttctgtgcctgctgagattctgcttctctgccaccagaagatactacctgggggctgtggaactgag
ctgggattacatgcagtctgacctgggggagctgcctgtggatgccagattcccccccagagtgcccaagagcttccccttcaacacctctgt
ggtgtacaagaaaaccttgtttgtggagttcacagaccacctgttcaacattgccaagcccagacccccctggatgggcctgctgggcccca
ctatccaggctgaggtgtatgacacagtggtgatcaccctgaagaatatggccagccaccctgtgagcctgcatgctgtgggggtgagctat
tggaaggcctctgagggggctgagtatgatgaccagaccagccagagagagaaggaggatgacaaggtgttccctgggggcagccaca
cctatgtgtggcaggtgctgaaggaaaatggccccatggcctctgaccccctgtgcctgacctacagctacctgagccatgtggacctggtg
aaggacctgaactctgggctgattggggccctgctggtgtgtagagagggcagcctggccaaggagaagacccagaccctgcacaaattc
atattgctgtttgctgtgtttgatgagggcaagagctggcattctgagaccaagaacagcctcatgcaggacagagatgctgcctctgccaga
gcttggcccaaaatgcacacagtgaatggctatgtgaacagaagcctgccaggactgattggctgccacagaaagtctgtgtactggcatgt
gattggcatgggcacaacccctgaggtgcactctatcttcctggagggccataccttcctggtgagaaaccacagacaggccagcctggag
atctcccccatcaccttcctgacagcccagacactgctgatggatctgggccaattcctgctgttctgccacatcagcagccaccagcatgat
ggcatggaggcctatgtgaaggtggacagctgccctgaggagccccagctgagaatgaagaacaatgaggaggctgaggattatgatgat
gacctcacagactctgagatggatgtggtgagatttgatgatgacaactctcccagcttcatccagattagatctgtggccaagaagcacccc
aagacctgggtccactacattgctgctgaggaagaggactgggactatgcccccctggtgctggcacctgatgacagatcctataagagcc
agtacctgaacaatggcccccagagaattggcagaaagtacaagaaggtgagattcatggcctacacagatgagaccttcaagaccagag
aggccatccagcatgagtctggcatcctgggccccctgctgtatggggaggtgggggacaccctgctcatcatcttcaagaaccaggcctc
cagaccctataacatctacccccatggcatcacagatgtgagacccctgtacagcagaagactgcccaagggggtgaagcacctgaagga
cttccccattctgccaggggagatcttcaagtacaagtggacagtgacagtggaggatggccccaccaagtctgaccccagatgcctgacc
agatactacagcagctttgtgaacatggagagagacctggcctctggcctgattggccctctcctgatctgctacaaggagtctgtggaccag
agaggcaaccagatcatgtctgacaagagaaatgtgatcctgttctctgtgtttgatgagaatagaagctggtacctgacagagaacattcaaa
gattcctgcccaaccctgctggggtgcagctggaggaccctgagttccaggccagcaacatcatgcacagcatcaatggctatgtgtttgact
ccctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagacagacttcctgtctgtgtttttctctggct
ataccttcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctctggggagacagtgttcatgagcatggagaatcctggcc
tgtggatcctgggctgccacaactctgacttcagaaacagaggcatgacagccctgctgaaggtgagcagctgtgacaagaacacagggg
actactatgaggacagctatgaggacatctctgcctacttgctgagcaagaacaatgccattgagcccagaagcttcagccagaacccccct
gtgctgaaggggggggggggctctgagatcaccagaaccaccctgcaatctgaccaggaggaaattgactatgatgacaccatctctgtg
gaaatgaagaaggaagactttgacatctatgatgaggatgagaaccagagccccagaagcttccagaagaagaccagacactacttcattg
ctgctgtggagagactgtgggactatggcatgagctctagcccccatgtgctgagaaacagagcccagtctggctctgtgccccaattcaag
aaggtggtgttccaggagttcacagatggcagcttcacccagcccctgtacagaggggagctgaatgagcacctgggcctgctgggcccc
tatatcagagctgaggtggaggataacatcatggtgacctttagaaatcaggccagcagaccctacagcttttacagcagcctgatcagctat
gaggaggaccagagacagggggctgagcccagaaagaactttgtgaagccaaatgagaccaagacctacttctggaaggtgcagcacc
acatggcccccacaaaggatgagtttgactgcaaggcctgggcctacttctctgatgtggacctggagaaggatgtgcactctggcctgattg
gccccctgttggtgtgccacaccaacaccctgaaccctgcccatggcagacaggttacagtgcaggagtttgccctgtttttcaccatttttgat
gagaccaagtcttggtacttcacagagaatatggaaagaaactgtagagccccctgcaacatccagatggaggaccccaccttcaaggaga
actacagatttcatgccattaatggctacatcatggacaccctgcctggcctggttatggcccaggaccagagaatcagatggtacctgctga
gcatgggctctaatgagaacattcacagcatccacttctctggccatgtgttcacagtgagaaagaaggaagagtacaagatggcactgtac
aacttgtaccctggggtgtttgagacagtggaaatgctgcccagcaaggctggcatctggagagtggaatgtctcattggggagcacctgca
tgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacacccctgggcatggcttctggccacatcagagacttccagatca
cagcctctggccaatatggccagtgggcccccaagctggccagactccactattctggcagcatcaatgcctggagcaccaaggagccctt
cagctggatcaaggtggacctgctggcccccatgatcatccatggcatcaagacccagggggccagacagaagttcagcagcctgtacat
cagccagttcatcatcatgtacagcctggatggcaagaaatggcagacctacagaggcaacagcactggcaccctgatggtgttctttggca
atgtggacagctctggcatcaagcacaacatcttcaacccccccatcattgccagatatatcagactgcaccccacccactacagcattagaa
gcaccctgagaatggagctgatgggctgtgacctgaacagctgcagcatgcccctgggcatggagtccaaggccatctctgatgcccaaat
cacagccagcagctacttcaccaacatgtttgccacctggagccctagcaaggccagactgcacctgcagggcagaagcaatgcctggag
accccaggtgaacaaccccaaggagtggctgcaggtggacttccagaagaccatgaaggtgacaggggtgaccacacagggggtgaag
agcctcctgaccagcatgtatgtgaaggagttcctgattagctctagccaggatggccaccagtggaccctgttcttccagaatggcaaggtg
aaggtcttccagggcaaccaggacagcttcacccctgtggtgaacagcctggacccccctctcctgacaagatacctgagaatccaccccc
agagctgggtgcaccagattgccctgagaatggaggtgctgggctgtgaggcccaggacctctactga
核酸hFVIII-L1-7(SEQ ID NO:10)
atgcagattgagctgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgccaccaggaggtattacctgggggctgtggagctgag
ctgggactatatgcagtctgacctgggggagctgcctgtggatgctaggttcccccccagggtgcccaagagcttcccctttaacacttctgt
ggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacattgccaagcccaggcccccctggatggggctgctggggccca
ccatccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtgggggtgagcta
ctggaaggcttctgagggggctgagtatgatgaccagactagccagagggagaaggaggatgacaaggtgtttcctgggggcagccata
cctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacctacagctacctgtctcatgtggacctggtga
aggacctgaactctggcctgattggggctctgctggtgtgtagggagggcagcctggctaaggaaaagacccagaccctgcataagtttatc
ctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggatagggatgctgcctctgccagggc
ttggcctaagatgcacactgtgaatgggtatgtgaataggagcctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgatt
gggatgggcaccacccctgaggtccatagcatcttcctggagggccacactttcctggtgaggaaccacagacaggcctctctggagatct
ctcccatcaccttcctgactgctcagactctgctgatggacctgggccagttcctgctgttttgccatattagcagccaccagcatgatgggatg
gaggcctatgtgaaggtggatagctgccctgaggagcctcagctgaggatgaagaacaatgaggaggctgaagactatgatgatgacctg
actgattctgagatggatgtggtgaggtttgatgatgacaatagccccagcttcattcagatcaggtctgtggccaagaaacaccccaagacc
tgggtgcactacattgctgctgaggaagaggactgggactatgctcccctggtgctggcccctgatgataggtcttataagagccagtacctg
aacaatgggccccagaggattggcaggaagtacaagaaggtgaggttcatggcctacactgatgaaaccttcaaaaccagggaggccatt
cagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaaccaggccagcaggccct
acaacatctatcctcatggcatcactgatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacctgaaagacttccccat
cctgcctggggagatctttaagtataagtggactgtgactgtggaggatggccctaccaagtctgaccccaggtgtctgaccaggtactattct
agctttgtgaacatggagagggacctggcctctggcctgattgggcccctgctgatctgctacaaggagtctgtggaccagaggggcaacc
agatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgagaataggagctggtacctgactgagaacatccagaggtttctgccc
aatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcatgcatagcatcaatggctatgtgtttgacagcctgcagctg
tctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagactgactttctgtctgtgttcttttctggctataccttcaagc
acaagatggtgtatgaggataccctgaccctgttccccttctctggggagactgtgttcatgagcatggagaatcctgggctgtggatcctgg
ggtgccacaactctgattttaggaacagggggatgactgccctgctgaaggtgtctagctgtgataagaacactggggactactatgaggac
agctatgaggacatttctgcttatctgctgtctaagaataatgccattgagcccagaagcttcagccagaatccccctgtgctgaagggggga
gggggctctgagatcaccagaactaccctgcagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggagg
actttgacatctatgatgaggatgagaatcagtctcccaggagctttcagaagaagaccagacattacttcattgctgctgtggagaggctgtg
ggactatggcatgagctctagccctcatgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttccaggaat
tcactgatggcagcttcacccagcccctgtacaggggggagctgaatgagcacctgggcctgctggggccttatatcagggctgaggtgga
ggataatattatggtgactttcaggaaccaggccagcaggccctactctttctatagcagcctgatctcttatgaggaggatcagaggcaggg
ggctgagcctaggaagaactttgtgaagcccaatgagactaagacctacttctggaaggtccagcaccacatggcccctaccaaggatgag
tttgactgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtccattctgggctgattggccccctgctggtgtgccacactaa
cactctgaatcctgcccatggcaggcaggtgactgtccaggagtttgccctgttcttcactatctttgatgagaccaagagctggtactttactg
agaacatggagaggaactgcagagctccttgcaatattcagatggaggaccccaccttcaaggagaattacaggttccatgccattaatggg
tacatcatggacaccctgcctggcctggtgatggctcaggaccagaggatcaggtggtacctgctgagcatgggctctaatgagaatatcca
cagcatccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggctctgtataatctgtaccctggggtgtttgaaact
gtggagatgctgccctctaaggctggcatctggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctggt
gtacagcaacaagtgccagacccccctgggcatggcctctggccacatcagggacttccagatcactgcctctggccagtatggccagtgg
gcccccaagctggccaggctgcactattctggcagcatcaatgcctggagcaccaaggagcccttcagctggatcaaggtggacctgctg
gcccccatgatcattcatggcatcaagacccagggggccaggcagaagttcagctctctgtacatctctcagttcatcatcatgtactctctgg
atgggaagaagtggcagacctacaggggcaacagcactggcaccctgatggtgttctttgggaatgtggactcttctggcatcaagcacaa
catcttcaatccccccatcattgctaggtatattaggctgcatcccacccactacagcatcaggtctaccctgaggatggagctgatgggctgt
gacctgaactcttgcagcatgcccctgggcatggagtctaaggccatctctgatgcccagattactgccagcagctacttcaccaacatgtttg
ccacctggagcccctctaaggccaggctgcatctgcaggggaggagcaatgcctggaggcctcaggtgaacaaccccaaggagtggctg
caggtggatttccagaagaccatgaaggtgactggggtgaccacccagggggtcaagagcctgctgaccagcatgtatgtgaaggagttc
ctgatcagcagcagccaggatggccaccagtggactctgttctttcagaatgggaaggtgaaggtgtttcagggcaatcaggactctttcacc
cctgtggtgaacagcctggacccccccctgctgaccagatacctgaggatccacccccagtcttgggtgcatcagattgccctgaggatgg
aggtgctgggctgtgaggctcaggatctgtactga
核酸hFVIII-L1-8(SEQ ID NO:11)
atgcagattgagctgagcacctgcttcttcctctgcctgctgaggttctgcttctctgccactagaagatactacctgggggctgtggagctgag
ctgggactacatgcagtctgacctgggggagctgcctgtggatgccagattcccacccagggtgcccaagagcttccccttcaacacctctg
tggtgtacaagaagaccctgtttgtggagtttacagaccacctgttcaatattgccaagcccagacccccctggatggggctgctgggcccca
ccatccaggcagaggtgtatgacacagtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtgggggtgagcta
ctggaaggcctctgagggggctgagtatgatgaccagaccagccagagagagaaggaggatgacaaggtgttccctgggggcagccac
acctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacatacagctacctgagccatgtggacctggt
gaaggatctcaattctggcctgattggggccctgctggtgtgcagagagggcagcctggccaaggagaagacccagaccctgcacaagtt
catcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggacagagatgctgcctctgcca
gagcctggcccaagatgcacacagtgaatggctatgtgaacagaagcctgcctggcctgattggctgccatagaaagtctgtgtactggcat
gtgattggcatgggcaccacccctgaggtgcacagcatcttcctggaaggccacaccttcctggtgagaaaccacagacaggccagcctg
gagatcagccccatcaccttcctgacagcccaaaccctgttgatggacctgggccagttcctgctgttctgtcacatcagctctcaccagcatg
atggcatggaggcctatgtgaaggtggacagctgccctgaggagccccagctgagaatgaagaacaatgaggaggctgaggactatgat
gatgacctgacagactctgagatggatgtggtgagatttgatgatgacaacagccccagcttcatccagatcagatctgtggccaagaagca
ccccaagacctgggtgcattacattgctgctgaggaagaggactgggactatgcccccctggttctggcccctgatgacagaagctacaagt
ctcaatacctgaacaatggcccccagagaattggcagaaagtacaagaaggtgagattcatggcctacacagatgagacctttaagaccag
agaagccatccagcatgagtctggcatcctgggccccctcctgtatggggaggtgggggacaccttgctgatcatcttcaagaaccaggcc
agcagaccctacaacatctacccccatggcatcacagatgtgagacccctgtacagcagaagactccccaagggggtgaagcacctgaag
gacttccccatcctgcctggggagatcttcaagtataagtggacagtgacagtggaggatggccccaccaagtctgaccccagatgcctga
ccagatactacagcagctttgtgaacatggaaagagacctggcatctggcctcattggccctctgctgatttgctacaaggaatctgtggacca
gagaggcaatcagatcatgtctgacaagagaaatgtgatcctgttctctgtgtttgatgagaacagaagctggtacctgacagagaacatcca
aaggttcctgcccaaccctgctggggtgcagctggaggaccctgagttccaggcctctaatatcatgcacagcatcaatggctatgtgtttga
cagcctccagctgtctgtgtgcctgcatgaggtggcctattggtacatcctgagcattggggcccagacagacttcctgtctgtgttcttctctg
gctacaccttcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctctggggagacagtgttcatgagcatggagaaccctg
gcctgtggatcctgggctgccacaactctgacttcagaaacagagggatgactgccctgctgaaggtgagcagctgtgacaagaacacag
gggactactatgaggacagctatgaggacatctctgcttacctgctgagcaagaacaatgccattgagcccagaagcttcagccagaaccc
ccctgtgctgaaggggggggggggctctgagatcaccagaaccaccctgcaatctgaccaggaggagattgactatgatgacaccatctct
gtggagatgaagaaggaggactttgacatctatgatgaggatgagaaccaatcccccagatccttccagaaaaagaccagacactactttatt
gctgctgtggagagactgtgggactatggcatgtccagcagcccccatgtgctgagaaacagagcccagtctggctctgtgccccagttca
agaaggtggtgttccaggaattcacagatggcagcttcacccagcctctgtacagaggggagctgaatgagcacctgggcctgctgggcc
cctacatcagagctgaggtggaggacaacatcatggttaccttcagaaaccaagcctctagaccctacagcttctacagcagcctgatcagct
atgaggaagaccagagacagggggctgagcccagaaagaactttgtgaagcccaatgagaccaagacatacttctggaaggtgcagcac
cacatggcccccaccaaggatgagtttgactgcaaggcctgggcctacttctctgatgtggatctggaaaaggatgtgcattctggcctgatt
gggcctctgctggtgtgtcacaccaacaccctgaaccctgcccatggcagacaggtgacagtgcaggagtttgccctgttcttcaccatttttg
atgagaccaagagctggtacttcacagagaacatggagagaaactgcagagccccctgcaacatccagatggaggatcccaccttcaagg
agaactacaggttccatgccatcaatggctacatcatggacaccctccctggcctggttatggctcaggaccagagaatcagatggtatctgc
tgagcatgggcagcaatgagaatatccacagcatccacttctctggccatgtgttcacagtgagaaagaaggaggagtacaagatggccct
gtacaacctgtaccctggggtgtttgagacagtggagatgctgcccagcaaggctggcatctggagagtggagtgcctgattggggagcac
ctgcatgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacacccctgggcatggcctctggccatatcagagacttcca
gattacagcctctggccagtatggccagtgggcccccaagctggccagattgcactactctggctccataaatgcctggagcaccaaggag
cccttctcttggatcaaggtggacctgctggcccccatgatcatccatggcatcaagacccagggggccagacagaagttcagctccctgta
catcagccagttcatcatcatgtacagcctggatggcaagaagtggcagacctacagagggaactctacaggcaccctgatggtgttctttgg
caatgtggacagctctggcatcaagcacaacatcttcaacccccccatcattgccagatacattagactgcaccccacccactacagcatcag
aagcaccctgagaatggagctgatgggctgtgacctgaacagctgcagcatgcccctgggcatggagagcaaggccatctctgatgccca
gatcacagcctcctcttacttcaccaacatgtttgccacctggagcccctctaaggccagactgcacctgcagggcagaagcaatgcctgga
gaccccaggtcaacaaccccaaggagtggctgcaggtggacttccagaagactatgaaggtgacaggggtgaccacccaaggggtgaa
gagcctgctgaccagcatgtatgtgaaggagttcctgatcagctctagccaggatggccaccagtggaccctgttcttccagaatggcaagg
tgaaagttttccagggcaaccaggactccttcacccctgtggtgaacagcctggacccccccctgctgactagatacctgagaatccacccc
cagagctgggtgcatcagattgccctcagaatggaggtgctgggctgtgaggcccaggacctgtactga
核酸hFVIII-L1-9(SEQ ID NO:12)
atgcagattgagctgagcacatgcttcttcctgtgcctgctgaggttctgcttttcagctaccagaaggtactacctgggtgctgtggagctgtc
ctgggactacatgcagtctgatctgggagagctgcctgtggatgccaggttcccccctagggtgcctaagagcttccctttcaacacctctgtg
gtgtacaagaagacactgtttgttgagttcacagaccacctgttcaacatagctaagcccagacccccctggatggggctgctgggccccac
cattcaggctgaggtgtatgatactgttgtcatcaccctgaagaacatggcctcccaccctgtgtccctgcatgctgttggtgtgtcctactgga
aggcttcagagggggctgagtatgatgaccagacctcccagagagaaaaggaggatgataaggtgtttccaggtgggtcccacacctatgt
ctggcaggtgctgaaggagaatggtcccatggcttctgatcctctgtgcctgacctactcctacctctcccatgtggacctggttaaggacctg
aactctggcctgataggtgccctgctggtgtgcagagagggctccctggccaaggagaagacccagaccctccacaagttcatcttgctgtt
tgctgtgtttgatgagggcaagtcttggcacagtgagacaaagaactccctgatgcaggacagggatgctgcatcagctagagcttggccca
agatgcacactgtgaatggttatgtgaacaggtccctgcctggcctgattggttgccacagaaagtctgtctactggcatgttataggtatggg
caccaccccagaggtgcactccatctttctggaggggcacacattcctggtgagaaaccacaggcaggcctctctggagatcagccccatc
acctttctgacagctcagaccctgctgatggacctgggccagttcctgctcttctgccacatctcctcccaccagcatgatgggatggaggcct
atgtgaaggtggatagctgccctgaggaaccccagctgaggatgaagaacaatgaggaggcagaggactatgatgatgacctgactgatt
ctgagatggatgtagtgaggtttgatgatgataactccccctctttcatccagatcaggtctgtggccaagaagcaccctaagacctgggtgca
ctacattgctgctgaggaggaggactgggactatgctcccctggtgctggctccagatgacaggagctacaagagccagtacctgaacaat
ggtccccagaggattggtaggaagtacaagaaggtgagattcatggcttacacagatgagaccttcaagaccagggaggccatccagcat
gagagtggtatcctgggtcccctgctgtatggtgaggtgggtgacaccctgctgatcatcttcaagaaccaggcctccagaccctacaacatc
taccctcatggtatcactgatgtgagacctctgtactctagaagactccccaagggggtgaagcacctgaaggactttcccatcctccctggtg
aaatcttcaagtacaagtggactgtcactgttgaggatggccccaccaagagtgatcccaggtgcctgaccaggtactactcctcctttgttaa
catggagagagacctggcctctggtctgattggtcccctgctgatctgctacaaggagtctgttgatcagagagggaaccagatcatgtcaga
taagaggaatgtgatcctgttctctgtgtttgatgagaacaggagctggtacctgactgagaacatccagaggttcctgcccaaccctgctggt
gtgcagctggaggaccctgagttccaggcctccaacatcatgcactctatcaatggttatgtatttgatagcctgcagctgtctgtgtgcctgca
tgaggtggcctactggtacatcctgtccattggtgctcagactgatttcctgtctgtgttcttctctggttacaccttcaagcacaagatggtgtatg
aggacaccctgaccctgttccccttctcaggtgagacagtgttcatgtccatggagaacccaggtctgtggatcctgggctgccacaactcag
atttcaggaacagggggatgacagctctgctgaaggtgtcctcttgtgacaagaacacaggtgactactatgaggactcctatgaggacatct
ctgcctacctgctgagcaagaacaatgccattgagcccaggagcttcagccagaacccacctgtcctgaagggggggggtggcagtgag
atcactagaaccaccctgcagtcagatcaggaggagattgattatgatgacaccatctctgtggagatgaagaaggaggactttgatatttatg
atgaggatgagaaccagagccccagatccttccagaagaagactagacactacttcatagctgctgtggagaggctgtgggactatggtatg
tcctctagccctcatgtgctgaggaacagggcccagtctggttctgtgccccagttcaagaaggtggtgttccaggagttcactgatggcagc
ttcacacagccactctacaggggtgagctcaatgagcacctgggactgctgggcccctacatcagggctgaggtggaggacaacatcatg
gtcaccttcagaaaccaggcctccagaccctacagcttctactccagcctgatctcctatgaggaggaccagaggcagggtgctgagccca
gaaagaactttgttaagcccaatgagaccaagacctacttctggaaggtgcagcaccacatggctcccaccaaggatgagtttgattgcaag
gcctgggcctacttctctgatgtggacctggagaaggatgtgcacagtggtctgattggtcccctgctggtgtgccacaccaacaccctgaac
cctgcccatggtaggcaggtgactgtccaggagtttgctctgttcttcaccatctttgatgagacaaagagctggtatttcacagagaacatgg
agagaaactgcagagccccctgcaacatccagatggaggaccccaccttcaaggagaactacagattccatgccatcaatggttacatcat
ggacaccctgcctggcctggtcatggcccaggaccagagaatcaggtggtacctgctgtccatgggctctaatgagaacatccactccatcc
acttctctggtcatgtgttcacagtcagaaagaaggaggagtacaagatggccctgtacaacctgtaccctggtgtgtttgagactgtagagat
gctgccctccaaggctgggatctggagagtggagtgcctgataggtgagcacctccatgctgggatgtccaccctgttcctggtctactccaa
taagtgccagacacccctgggcatggccagtggtcacatcagagacttccagatcacagcttctggtcagtatggtcagtgggccccaaag
ctggccaggctccactactctggtagcatcaatgcatggagcacaaaggagcccttctcctggatcaaggtggacctgctggctcctatgatc
atccatggtatcaagacccagggtgccagacagaagttctcctccctgtatatctctcagttcatcatcatgtactccctggatggtaagaagtg
gcagacctacagggggaactccactggtaccctgatggtgttctttggtaatgtagactccagtggtatcaagcacaacatcttcaacccccc
catcattgctaggtacattaggctgcaccccacccactactccatcaggagcaccctgagaatggagctgatgggctgtgacctgaacagct
gctccatgcccctgggcatggagtccaaggccatctcagatgcccagatcactgcctcctcctacttcaccaacatgtttgctacctggtcccc
ctccaaggccaggctgcacctgcagggcagatccaatgcatggaggcctcaggtgaacaaccccaaggagtggctgcaggtggacttcc
agaagaccatgaaggtgacaggtgtgaccacccagggtgtcaagtcactgctgacctccatgtatgtgaaggagttcctcatctccagctcc
caggatggtcaccagtggaccctgttcttccagaatggtaaggtgaaggtgttccagggcaaccaggactctttcacccctgtagtgaactcc
ctggacccccccctgctgaccagatacctcaggatccatccccagtcttgggtgcaccagattgctctgagaatggaggtgcttggctgtgaa
gcccaggacctgtactga
核酸hFVIII-L1-10(SEQ ID NO:13)
atgcagattgagctgtccacctgcttcttcctgtgcctgctgaggttctgtttcagtgctaccaggaggtactacctgggtgctgtggagctgtcc
tgggactatatgcagtctgacctgggtgaactgcctgtggatgctagattccctcccagggtgcccaagtccttccccttcaatacctctgttgt
gtacaagaagaccctgtttgttgagttcactgatcacctgttcaacatagctaagcccagacctccctggatgggcctgctgggccccaccat
ccaggcagaggtgtatgatactgtggtgattaccctgaagaacatggcctcccatcctgtgagcctgcatgctgtgggggtgtcctactggaa
ggcctcagagggtgctgagtatgatgaccagacctcccagagagagaaggaggatgacaaggtgttccctggtggctcccacacctatgtc
tggcaggtgctgaaggagaatggtcccatggcctctgatcccctgtgtctgacatactcttacctgagccatgtggatctggtcaaggacctg
aactctggtcttataggtgctctgctggtgtgcagggaggggtctctggctaaggagaagactcagaccctccacaaattcatcctgctgtttg
ctgtctttgatgaggggaagtcttggcattctgagaccaagaactccctgatgcaggacagagatgctgcttcagctagagcctggcccaag
atgcacactgttaatggttatgtgaacaggtccctgcctggtctgattggctgccacaggaagtctgtctactggcatgtgattggtatgggcac
cacacctgaggtgcactccatcttcttggagggacacaccttcctggtgaggaaccacaggcaggcatcattggagatcagccccatcacct
tcctgacagctcagaccctgctcatggacctgggacagttcctgctgttctgccacattagctctcaccagcatgatggcatggaggcttatgt
caaggtggactcctgcccagaggagccccagctgaggatgaagaacaatgaggaggcagaggactatgatgatgatctgacagattctga
gatggatgtggtgaggtttgatgatgacaacagccccagcttcatccagatcaggtctgtggccaagaagcaccccaagacctgggtgcact
acatagctgctgaggaggaggactgggactatgccccactggtgctggccccagatgacaggagctacaagtcccagtacctgaacaatg
gccctcagaggataggtaggaagtacaagaaggtgaggttcatggcctacacagatgagaccttcaagaccagggaggccatccagcatg
agtcaggtatcctgggccccctgctgtatggtgaggtgggtgacaccctgctgatcatcttcaagaaccaggcctccagaccctacaacatct
accctcatggtatcacagatgtgaggcccctgtactccaggaggctgcccaagggggtgaagcatctgaaggacttccccatcctgcctggt
gagatcttcaagtacaagtggactgttactgtggaggatgggcctaccaagagtgatcccagatgcctgaccagatactactccagctttgtta
acatggagagggacctggcctcaggtctgattggtcccctcctgatctgctacaaagagtctgtggaccagaggggcaaccagatcatgag
tgataagaggaatgtaatcctgttctctgtctttgatgagaacaggtcctggtacctgacagagaacatccagagattcctgcccaacccagct
ggtgtacagctggaggaccctgagttccaggcctccaacatcatgcactccatcaatggttatgtctttgattccctgcagctttctgtatgcctg
catgaggtggcatactggtacatcctctccattggtgctcagacagatttcctctctgtgttcttcagtggttacaccttcaagcacaagatggtgt
atgaggacaccctgaccctgttccccttctctggtgagactgtcttcatgagtatggagaaccctggtctgtggatcctgggctgccacaactc
agatttcagaaacagggggatgacagctctgctgaaggtgtcttcatgtgacaagaacacaggtgactactatgaggactcctatgaggaca
tctctgcctacctgctgagcaagaacaatgccattgagccaaggagcttcagccagaacccccctgtgctgaagggggggggtggctctga
gatcaccagaaccaccctccagtctgatcaggaggagattgattatgatgataccatctctgtggagatgaagaaggaggactttgatatctat
gatgaggatgagaaccagagcccaaggagcttccagaagaagaccaggcactacttcattgctgctgtagagaggctctgggactatggta
tgtcctctagcccccatgtgctgagaaacagggcccagagtggttctgtgccccagttcaagaaggtggtgttccaggagttcactgatggg
agctttacccagcccctgtacaggggtgaactgaatgagcacctggggctgctgggcccctacatcagagctgaggtggaggacaacatc
atggtgaccttcagaaaccaggcttccaggccctacagcttctactcctcactgatctcctatgaggaggaccagaggcagggtgcagagcc
taggaagaactttgttaagcctaatgagaccaagacctacttctggaaggtgcagcaccacatggcccctaccaaggatgagtttgattgcaa
ggcctgggcctacttctcagatgtggacctggagaaggatgtgcactcaggcctcattggtcccctgctggtgtgccacaccaacaccctga
accctgctcatggtagacaggtgactgtgcaggagtttgctctgttctttacaatctttgatgagaccaagagctggtacttcactgagaacatg
gagaggaactgcagggctccatgcaacatccagatggaggaccccaccttcaaggagaactacagattccatgccatcaatggttacatcat
ggacacactgcctggccttgttatggcccaggaccagagaattaggtggtacctgctgtccatgggcagcaatgagaacatccactccatcc
acttctctggtcatgtcttcactgtgaggaagaaggaggagtacaagatggccctgtacaacctgtacccaggtgtgtttgagactgtggagat
gctgccctccaaggctggtatctggagggtggagtgcctgataggtgagcacctgcatgcaggaatgagcaccctgttcctggtttactcca
acaaatgccagacccccctgggcatggccagtggtcacatcagagacttccagatcactgcttctggtcagtatggtcagtgggcccccaag
ctggccaggctgcactactctggtagcatcaatgcttggtccaccaaggagcctttcagctggatcaaggtggacctgctggcccccatgatc
atccatggtatcaagacccagggggccaggcagaagttctccagcctgtacatctcccagttcatcattatgtactccctggatggcaagaag
tggcagacctacagaggcaacagcactggtaccctgatggtgttctttggtaatgtggactcaagtggtatcaagcacaacatcttcaacccc
cccatcatagctagatacattagactgcaccccacccactactcaatcagaagcacactgaggatggagctgatggggtgtgacctgaactc
ctgctccatgcccctgggcatggagagcaaggccatcagtgatgcccagatcacagctagctcctacttcaccaacatgtttgctacctggag
ccccagcaaggccaggctgcacctgcagggcaggagcaatgcttggagaccccaggtgaacaaccccaaggagtggctgcaggtgga
cttccagaagaccatgaaggtgacaggtgtgaccacccagggtgttaagagcctgctgaccagtatgtatgtgaaggagtttctgatctcctc
ctcacaggatggtcaccagtggaccctcttcttccagaatggtaaggtgaaggtgttccagggcaaccaggactccttcacccctgtggttaa
ctccctggacccccctctgctgaccaggtacctgaggatccacccccagtcctgggtgcaccagattgctctgaggatggaggtgctgggct
gtgaggctcaggacctctactga
核酸hFVIII-L2-WT(SEQ ID NO:14)
atgcaaatagagctctccacctgcttctttctgtgccttttgcgattctgctttagtgccaccagaagatactacctgggtgcagtggaactgtcat
gggactatatgcaaagtgatctcggtgagctgcctgtggacgcaagatttcctcctagagtgccaaaatcttttccattcaacacctcagtcgtg
tacaaaaagactctgtttgtagaattcacggatcaccttttcaacatcgctaagccaaggccaccctggatgggtctgctaggtcctaccatcca
ggctgaggtttatgatacagtggtcattacacttaagaacatggcttcccatcctgtcagtcttcatgctgttggtgtatcctactggaaagcttct
gagggagctgaatatgatgatcagaccagtcaaagggagaaagaagatgataaagtcttccctggtggaagccatacatatgtctggcagg
tcctgaaagagaatggtccaatggcctctgacccactgtgccttacctactcatatctttctcatgtggacctggtaaaagacttgaattcaggcc
tcattggagccctactagtatgtagagaagggagtctggccaaggaaaagacacagaccttgcacaaatttatactactttttgctgtatttgatg
aagggaaaagttggcactcagaaacaaagaactccttgatgcaggatagggatgctgcatctgctcgggcctggcctaaaatgcacacagt
caatggttatgtaaacaggtctctgccaggtctgattggatgccacaggaaatcagtctattggcatgtgattggaatgggcaccactcctgaa
gtgcactcaatattcctcgaaggtcacacatttcttgtgaggaaccatcgccaggcgtccttggaaatctcgccaataactttccttactgctcaa
acactcttgatggaccttggacagtttctactgttttgtcatatctcttcccaccaacatgatggcatggaagcttatgtcaaagtagacagctgtc
cagaggaaccccaactacgaatgaaaaataatgaagaagcggaagactatgatgatgatcttactgattctgaaatggatgtggtcaggtttg
atgatgacaactctccttcctttatccaaattcgctcagttgccaagaagcatcctaaaacttgggtacattacattgctgctgaagaggaggact
gggactatgctcccttagtcctcgcccccgatgacagaagttataaaagtcaatatttgaacaatggccctcagcggattggtaggaagtaca
aaaaagtccgatttatggcatacacagatgaaacctttaagactcgtgaagctattcagcatgaatcaggaatcttgggacctttactttatggg
gaagttggagacacactgttgattatatttaagaatcaagcaagcagaccatataacatctaccctcacggaatcactgatgtccgtcctttgtat
tcaaggagattaccaaaaggtgtaaaacatttgaaggattttccaattctgccaggagaaatattcaaatataaatggacagtgactgtagaag
atgggccaactaaatcagatcctcggtgcctgacccgctattactctagtttcgttaatatggagagagatctagcttcaggactcattggccct
ctcctcatctgctacaaagaatctgtagatcaaagaggaaaccagataatgtcagacaagaggaatgtcatcctgttttctgtatttgatgagaa
ccgaagctggtacctcacagagaatatacaacgctttctccccaatccagctggagtgcagcttgaggatccagagttccaagcctccaacat
catgcacagcatcaatggctatgtttttgatagtttgcagttgtcagtttgtttgcatgaggtggcatactggtacattctaagcattggagcacag
actgacttcctttctgtcttcttctctggatataccttcaaacacaaaatggtctatgaagacacactcaccctattcccattctcaggagaaactgt
cttcatgtcgatggaaaacccaggtctatggattctggggtgccacaactcagactttcggaacagaggcatgaccgccttactgaaggtttct
agttgtgacaagaacactggtgattattacgaggacagttatgaagatatttcagcatacttgctgagtaaaaacaatgccattgaaccaagaa
gcttctcccaaaacccaccagtcttgaaagccgccgctgccgaaataactcgtactactcttcagtcagatcaagaggaaattgactatgatga
taccatatcagttgaaatgaagaaggaagattttgacatttatgatgaggatgaaaatcagagcccccgcagctttcaaaagaaaacacgaca
ctattttattgctgcagtggagaggctctgggattatgggatgagtagctccccacatgttctaagaaacagggctcagagtggcagtgtccct
cagttcaagaaagttgttttccaggaatttactgatggctcctttactcagcccttataccgtggagaactaaatgaacatttgggactcctgggg
ccatatataagagcagaagttgaagataatatcatggtaactttcagaaatcaggcctctcgtccctattccttctattctagccttatttcttatgag
gaagatcagaggcaaggagcagaacctagaaaaaactttgtcaagcctaatgaaaccaaaacttacttttggaaagtgcaacatcatatggc
acccactaaagatgagtttgactgcaaagcctgggcttatttctctgatgttgacctggaaaaagatgtgcactcaggcctgattggaccccttc
tggtctgccacactaacacactgaaccctgctcatgggagacaagtgacagtacaggaatttgctctgtttttcaccatctttgatgagaccaaa
agctggtacttcactgaaaatatggaaagaaactgcagggctccctgcaatatccagatggaagatcccacttttaaagagaattatcgcttcc
atgcaatcaatggctacataatggatacactacctggcttagtaatggctcaggatcaaaggattcgatggtatctgctcagcatgggcagcaa
tgaaaacatccattctattcatttcagtggacatgtgttcactgtacgaaaaaaagaggagtataaaatggcactgtacaatctctatccaggtgt
ttttgagacagtggaaatgttaccatccaaagctggaatttggcgggtggaatgccttattggcgagcatctacatgctgggatgagcacacttt
ttctggtgtacagcaataagtgtcagactcccctgggaatggcttctggacacattagagattttcagattacagcttcaggacaatatggacag
tgggccccaaagctggccagacttcattattccggatcaatcaatgcctggagcaccaaggagcccttttcttggatcaaggtggatctgttgg
caccaatgattattcacggcatcaagacccagggtgcccgtcagaagttctccagcctctacatctctcagtttatcatcatgtatagtcttgatg
ggaagaagtggcagacttatcgaggaaattccactggaaccttaatggtcttctttggcaatgtggattcatctgggataaaacacaatattttta
accctccaattattgctcgatacatccgtttgcacccaactcattatagcattcgcagcactcttcgcatggagttgatgggctgtgatttaaatag
ttgcagcatgccattgggaatggagagtaaagcaatatcagatgcacagattactgcttcatcctactttaccaatatgtttgccacctggtctcc
ttcaaaagctcgacttcacctccaagggaggagtaatgcctggagacctcaggtgaataatccaaaagagtggctgcaagtggacttccaga
agacaatgaaagtcacaggagtaactactcagggagtaaaatctctgcttaccagcatgtatgtgaaggagttcctcatctccagcagtcaag
atggccatcagtggactctcttttttcagaatggcaaagtaaaggtttttcagggaaatcaagactccttcacacctgtggtgaactctctagacc
caccgttactgactcgctaccttcgaattcacccccagagttgggtgcaccagattgccctgaggatggaggttctgggctgcgaggcacag
gacctctactga
核酸hFVIII-L2-7(SEQ ID NO:15)
atgcagattgagctgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgccaccaggaggtattacctgggggctgtggagctgag
ctgggactatatgcagtctgacctgggggagctgcctgtggatgctaggttcccccccagggtgcccaagagcttcccctttaacacttctgt
ggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacattgccaagcccaggcccccctggatggggctgctggggccca
ccatccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtgggggtgagcta
ctggaaggcttctgagggggctgagtatgatgaccagactagccagagggagaaggaggatgacaaggtgtttcctgggggcagccata
cctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacctacagctacctgtctcatgtggacctggtga
aggacctgaactctggcctgattggggctctgctggtgtgtagggagggcagcctggctaaggaaaagacccagaccctgcataagtttatc
ctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggatagggatgctgcctctgccagggc
ttggcctaagatgcacactgtgaatgggtatgtgaataggagcctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgatt
gggatgggcaccacccctgaggtccatagcatcttcctggagggccacactttcctggtgaggaaccacagacaggcctctctggagatct
ctcccatcaccttcctgactgctcagactctgctgatggacctgggccagttcctgctgttttgccatattagcagccaccagcatgatgggatg
gaggcctatgtgaaggtggatagctgccctgaggagcctcagctgaggatgaagaacaatgaggaggctgaagactatgatgatgacctg
actgattctgagatggatgtggtgaggtttgatgatgacaatagccccagcttcattcagatcaggtctgtggccaagaaacaccccaagacc
tgggtgcactacattgctgctgaggaagaggactgggactatgctcccctggtgctggcccctgatgataggtcttataagagccagtacctg
aacaatgggccccagaggattggcaggaagtacaagaaggtgaggttcatggcctacactgatgaaaccttcaaaaccagggaggccatt
cagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaaccaggccagcaggccct
acaacatctatcctcatggcatcactgatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacctgaaagacttccccat
cctgcctggggagatctttaagtataagtggactgtgactgtggaggatggccctaccaagtctgaccccaggtgtctgaccaggtactattct
agctttgtgaacatggagagggacctggcctctggcctgattgggcccctgctgatctgctacaaggagtctgtggaccagaggggcaacc
agatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgagaataggagctggtacctgactgagaacatccagaggtttctgccc
aatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcatgcatagcatcaatggctatgtgtttgacagcctgcagctg
tctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagactgactttctgtctgtgttcttttctggctataccttcaagc
acaagatggtgtatgaggataccctgaccctgttccccttctctggggagactgtgttcatgagcatggagaatcctgggctgtggatcctgg
ggtgccacaactctgattttaggaacagggggatgactgccctgctgaaggtgtctagctgtgataagaacactggggactactatgaggac
agctatgaggacatttctgcttatctgctgtctaagaataatgccattgagcccagaagcttcagccagaatccccctgtgctgaaggccgccg
ctgccgagatcaccagaactaccctgcagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggaggacttt
gacatctatgatgaggatgagaatcagtctcccaggagctttcagaagaagaccagacattacttcattgctgctgtggagaggctgtgggac
tatggcatgagctctagccctcatgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttccaggaattcact
gatggcagcttcacccagcccctgtacaggggggagctgaatgagcacctgggcctgctggggccttatatcagggctgaggtggaggat
aatattatggtgactttcaggaaccaggccagcaggccctactctttctatagcagcctgatctcttatgaggaggatcagaggcagggggct
gagcctaggaagaactttgtgaagcccaatgagactaagacctacttctggaaggtccagcaccacatggcccctaccaaggatgagtttga
ctgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtccattctgggctgattggccccctgctggtgtgccacactaacact
ctgaatcctgcccatggcaggcaggtgactgtccaggagtttgccctgttcttcactatctttgatgagaccaagagctggtactttactgagaa
catggagaggaactgcagagctccttgcaatattcagatggaggaccccaccttcaaggagaattacaggttccatgccattaatgggtacat
catggacaccctgcctggcctggtgatggctcaggaccagaggatcaggtggtacctgctgagcatgggctctaatgagaatatccacagc
atccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggctctgtataatctgtaccctggggtgtttgaaactgtgg
agatgctgccctctaaggctggcatctggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctggtgtac
agcaacaagtgccagacccccctgggcatggcctctggccacatcagggacttccagatcactgcctctggccagtatggccagtgggcc
cccaagctggccaggctgcactattctggcagcatcaatgcctggagcaccaaggagcccttcagctggatcaaggtggacctgctggccc
ccatgatcattcatggcatcaagacccagggggccaggcagaagttcagctctctgtacatctctcagttcatcatcatgtactctctggatgg
gaagaagtggcagacctacaggggcaacagcactggcaccctgatggtgttctttgggaatgtggactcttctggcatcaagcacaacatct
tcaatccccccatcattgctaggtatattaggctgcatcccacccactacagcatcaggtctaccctgaggatggagctgatgggctgtgacct
gaactcttgcagcatgcccctgggcatggagtctaaggccatctctgatgcccagattactgccagcagctacttcaccaacatgtttgccacc
tggagcccctctaaggccaggctgcatctgcaggggaggagcaatgcctggaggcctcaggtgaacaaccccaaggagtggctgcaggt
ggatttccagaagaccatgaaggtgactggggtgaccacccagggggtcaagagcctgctgaccagcatgtatgtgaaggagttcctgatc
agcagcagccaggatggccaccagtggactctgttctttcagaatgggaaggtgaaggtgtttcagggcaatcaggactctttcacccctgtg
gtgaacagcctggacccccccctgctgaccagatacctgaggatccacccccagtcttgggtgcatcagattgccctgaggatggaggtgc
tgggctgtgaggctcaggatctgtactga
核酸hFVIII-L2-1(SEQ ID NO:60)
atgcagattgagctgtctacatgcttcttcttgtgtctgctcagattttgcttctctgccactaggagatactacttgggagcagtggagctgtcct
gggactacatgcagagtgaccttggggaactccctgtagatgcaagatttcctcccagagtccccaaaagtttcccctttaacacatcagttgt
gtacaagaagaccctgtttgtggagtttacagaccacctgttcaacattgcaaagcccagacctccatggatgggactcctaggcccaactat
ccaggcagaggtctatgatacagttgtcataaccttgaagaatatggcatctcatcctgtctccctgcatgctgttggagtcagctattggaagg
cctctgaaggtgctgagtatgatgaccagactagccaaagagaaaaggaggatgacaaagtgttccctggagggtctcacacatatgtttgg
caggtgctgaaggagaatggacccatggccagtgaccctctgtgcttgacctacagttatctgtctcatgtagacctagtgaaggacctcaac
tcaggactcataggagcattacttgtgtgtagagaggggagcttagctaaggaaaaaacccagacacttcataagttcatccttctgtttgcagt
ttttgatgaggggaaaagttggcactcagagacaaaaaactctctgatgcaggacagggatgctgccagtgctagggcctggcctaagatg
cacacagtaaatggatatgtgaacagatctctgccaggcctcataggttgccatagaaagtctgtttattggcatgtcataggaatggggacta
ccccagaggtgcactcaatctttctagaaggccacacattcttagtaagaaaccacaggcaagcatctttggagatcagtccaatcacattcct
cacagcacaaactctcctgatggacctgggccagttcctcttgttctgccacattagctctcaccagcatgatggaatggaggcatatgttaaa
gtagactcatgtccagaagagcctcagttaagaatgaaaaacaatgaagaagcagaagactatgatgatgacctgactgactctgagatgga
tgttgtgaggtttgatgatgataattctccctcctttatacaaataagatctgtagccaaaaagcatcccaaaacttgggttcactacatagctgct
gaggaagaagactgggattatgctcctctggtcctagcccctgatgacagatcctacaaaagtcagtacctcaataatggtcctcaaagaatt
ggcaggaagtacaagaaagtcaggttcatggcttacactgatgagactttcaaaaccagggaggctatacaacatgagtctggtattctggg
accactgttatatggtgaggtgggagatacactgctgatcatctttaagaatcaggcctcaagaccatacaacatctatcctcatggtatcactg
atgtaagacctctgtatagcagaagactgcccaaaggagttaagcaccttaaagattttcctatccttcctggagaaatcttcaaatacaaatgg
actgtcactgttgaggatggtcctactaaatcagaccccaggtgcctaaccagatactacagcagttttgtgaatatggagagggacttagctt
ctggactgattgggccattgctgatctgctataaggagtctgtagatcagaggggcaatcagatcatgagtgacaagagaaatgtgattctgtt
ctctgtgtttgatgagaataggagctggtatctgacagagaacatccagagattcctacccaatcctgctggggtacaattagaagaccctga
gttccaggctagcaatatcatgcactctatcaatgggtatgtgtttgattctcttcagctgtctgtttgcctacatgaggtagcctactggtacatcc
tttcaataggggctcagactgacttcctctcagtatttttctctggctacacttttaaacataagatggtgtatgaggacactctcacactgttcccc
ttctcaggtgagactgtttttatgagtatggagaacccaggcctctggattttaggctgtcacaacagtgatttcaggaacagggggatgactg
cactcctgaaggtgtcttcatgtgataagaacactggggactattatgaagattcctatgaggatatttctgcatatttgctctctaagaacaatgc
tattgaacctaggtctttttctcagaacccaccagtattaaaggccgccgctgccgaaatcactaggacaaccctgcaatcagaccaagagga
aattgactatgatgacacaatctctgtagagatgaagaaagaagattttgatatctatgatgaagatgagaatcagagtcctaggtcttttcagaa
aaaaaccaggcactactttatagctgcagtagaaaggctgtgggattatggtatgtctagcagtccacatgtgctgagaaacagagcccagtc
aggcagtgtcccccagtttaagaaggtagttttccaggagttcactgatggcagtttcacccaacctctgtacagaggggagctgaatgaaca
cctgggactgttgggcccttatatcagagctgaggttgaggacaacatcatggtgaccttcaggaatcaagcatctagaccctattctttctact
cttccctcatctcctatgaagaggaccagagacaaggagcagaacctaggaaaaactttgtcaaacctaatgagaccaagacttacttttgga
aagttcagcaccatatggcccctacaaaggatgagtttgattgcaaggcatgggcttacttctcagatgtagatttagaaaaagatgtgcatagt
ggtttaataggtccattgctggtttgccacacaaacacactgaaccctgcccatggcaggcaggtcacagtgcaagagtttgctctcttctttac
catatttgatgaaactaaatcttggtacttcacagaaaacatggaaagaaactgcagagctccctgcaacatacagatggaggatcccactttc
aaggaaaactacaggtttcatgccatcaatggttacataatggacacactccctggcctggtaatggctcaggaccaaaggataaggtggta
ccttttaagcatgggatccaatgaaaatatccactcaatccacttttctggtcatgtgttcacagtcagaaagaaagaagagtataagatggccc
tctataacctctacccaggggtatttgaaacagtggagatgctcccatctaaggcaggaatttggagagtggaatgcttgattggtgaacacct
gcatgcaggtatgtcaactctattcctggtgtactctaataagtgccagactccactgggcatggcctctggtcacatcagagacttccagatca
ctgcctcaggccaatatggtcagtgggcccctaagctagccagattgcattattctgggtcaatcaatgcctggagcaccaaagagcctttctc
ttggatcaaagttgatcttctggctcctatgatcatccatgggatcaagactcagggagccaggcaaaagtttagctccctatacatttctcagttt
ataatcatgtattctctggatggcaaaaaatggcaaacatacaggggtaattctacaggaaccctcatggtgttctttgggaatgtagactcctc
aggcataaagcataatatattcaaccctccaattatagcaaggtacatcagactgcatccaacacattatagcattagatctaccctgagaatgg
aactaatggggtgtgacctgaactcctgttctatgcctctgggtatggagtctaaggctatctctgatgctcaaatcactgcctcttcctatttcact
aatatgtttgctacctggagcccaagcaaagccaggctgcacctgcagggcaggtctaatgcctggagaccccaagttaacaaccccaagg
aatggctgcaagttgacttccaaaagaccatgaaagtgacaggtgtcactacccagggagtaaaatccttgttgactagcatgtatgtaaagg
agttcctcatctcttcttctcaagatggtcaccaatggactctgtttttccagaatggtaaggtcaaggtatttcaaggtaaccaagacagcttcac
cccagtggtaaattccttagaccctcctctgttgactaggtatctcagaattcacccacagagctgggtccaccagattgctttgagaatggaag
ttctgggttgtgaggcccaagacctgtactga
核酸hFVIII-L2-2(SEQ ID NO:61)
atgcagattgagctttccacctgttttttcctctgcctattgagattctgttttagtgccaccagaagatactacctaggagctgtagaactgagctg
ggactatatgcagagtgacctgggagaactgcctgtagatgctagatttcctcctagagtacctaaaagcttcccatttaacactagtgtggttta
caaaaaaactctctttgtagagtttactgaccacctcttcaacattgctaagcctaggcccccttggatgggactgttgggacccacaatccag
gctgaagtctatgacactgtagtaatcacactaaagaacatggcctctcatccagtttctcttcatgctgttggtgtgagttattggaaggctagtg
aaggggctgagtatgatgaccaaacctcacagagagaaaaggaggatgacaaggtgtttcctggggggtctcacacatatgtttggcaggt
cttaaaagagaatggcccaatggcttctgatcccctgtgcttgacctacagctacttatcacatgttgatttggtcaaggatctgaactctggcct
cataggtgccctactggtatgtagggagggctccctggccaaagagaagactcagaccttgcacaagttcatactactgtttgctgtgtttgat
gagggtaaaagctggcattctgagaccaaaaactctctgatgcaagatagagatgcagcctctgccagagcctggcctaagatgcacacag
ttaatgggtatgtaaacagatccttaccaggattgataggctgccataggaaaagtgtgtactggcatgtcattgggatgggcaccaccccag
aggtacactctattttccttgagggccacacattcttagtcagaaaccatagacaagcttcattggagatttctcctattacttttctaacagcacag
accttactcatggacctagggcaatttttactgttctgccacatcagttcccaccagcatgatggaatggaggcttatgtgaaggttgactcttgc
cctgaagaacctcagctgagaatgaagaacaatgaagaggctgaggattatgatgatgacctgactgattcagagatggatgtagtcaggttt
gatgatgataacagcccctccttcatacaaatcagatcagtagcaaagaagcatccaaagacatgggtacactatatagcagctgaggagga
agattgggactatgctcctttagtgctggcccctgatgatagatcctacaagagccaatacctgaacaatggcccccagaggattggtagaaa
atacaaaaaggttaggttcatggcctatacagatgagacatttaagacaagggaggctatccagcatgagtcaggaatcctaggtcctttatta
tatggggaagtgggggatactcttctcatcattttcaagaaccaggcttcaagaccttacaacatctacccacatggcatcactgatgttaggcc
cctgtattctaggagactgccaaagggagtgaagcatctaaaggactttccaatcttaccaggtgagatattcaagtataaatggactgtgaca
gtagaggatggcccaactaaatcagaccctagatgtttgactagatactacagttcctttgtcaacatggaaagagacttagcctctggtctcat
aggaccactcctcatctgctataaagagtctgtggatcagagaggcaatcaaattatgagtgataagagaaatgtgatcctgttttctgtctttga
tgagaataggtcttggtacttgacagaaaacatacagaggttccttcctaaccctgctggggtacagctagaagacccagagtttcaggcctc
caatattatgcatagtattaatggctatgtgtttgactccctgcagctatcagtgtgccttcatgaagtggcctattggtatatactttctattggtgc
ccagacagacttcttatctgtcttcttttctggctatactttcaaacacaaaatggtttatgaggacactctgacactttttcccttctctggtgagac
agtttttatgtctatggaaaaccctggcttatggatcctgggctgccacaacagtgattttagaaacagaggcatgactgcactgctaaaggttt
cttcttgtgacaaaaatactggggactattatgaggattcctatgaagatatctctgcttacctgctgagcaaaaataatgccattgaacctagaa
gcttctctcagaatcccccagtactcaaagccgccgctgccgagatcactaggacaactctccagagtgaccaagaggagattgattatgat
gataccattagtgttgagatgaaaaaggaggactttgatatttatgatgaggatgaaaaccagtctcccagatctttccagaaaaaaacaagac
attactttattgcagcagtagaaagattatgggactatggcatgtctagtagccctcatgttctgaggaacagggcccagagtggttcagtacc
ccaattcaagaaagtagtctttcaggaattcacagatggctcattcacacagcctctgtataggggtgagcttaatgaacacctgggccttctag
gcccctacatcagagctgaggtagaggacaatataatggtgactttcaggaatcaggcttctaggccttactctttctactcatctctgatcagct
atgaggaggaccaaagacaaggagcagagcctagaaagaactttgtgaagcccaatgaaactaaaacctacttttggaaggttcagcatca
catggctcccacaaaggatgaatttgattgtaaggcctgggcttatttttcagatgtggatttggaaaaggatgttcactctgggttaataggtcct
ctgctggtgtgccacacaaatactcttaatcctgcacatggtagacaagtgactgtgcaggaatttgcactatttttcacaatttttgatgaaacta
agagttggtattttacagaaaacatggagagaaattgcagggctccatgcaatatccaaatggaagatcctactttcaaggaaaattataggttc
catgctattaatggttatataatggatacactgccaggactggtgatggcccaagatcaaagaattagatggtacctcttgtccatgggttctaat
gaaaacattcactccatccacttcagtggacatgtatttacagtaagaaagaaagaggaatacaaaatggcactctacaatctatacccagga
gtgtttgagacagttgaaatgttgcccagtaaggcaggaatctggagggtagagtgcctcattggtgaacacctgcatgctggcatgagtacc
cttttcttagtttacagtaacaagtgtcagacccccctggggatggccagtggacacataagagatttccagataacagcatctggtcagtatg
gtcagtgggctccaaaactggccagattgcactactcaggttcaatcaatgcatggtctactaaggaacctttcagttggataaaggtggactt
gttagctcctatgatcattcatggcatcaaaacccagggagcaaggcagaagtttagtagtctgtacatcagccaattcatcataatgtactctct
ggatggcaaaaagtggcagacatacagggggaattctactgggactctgatggtgttctttggcaatgttgactctagtggtatcaagcacaa
catcttcaaccctcccattatagcaagatacatcagacttcatccaacccactactctatcagatcaacattaaggatggaattgatgggctgtg
accttaattcttgctctatgcccctgggaatggaatccaaggcaatctcagatgcccagatcacagctagcagctacttcaccaatatgtttgca
acttggtccccatctaaggccagactgcacctgcaaggcagatctaatgcctggaggccacaagtgaataatcctaaagaatggctgcaagt
agactttcaaaaaaccatgaaggttactggtgttacaacacaaggtgtcaagtcactcttaacctctatgtatgtgaaagaatttctgattagttcc
agccaggatggtcaccagtggacccttttctttcagaatggcaaggtcaaggtctttcaaggaaaccaggatagttttacaccagtggttaattc
tctggatcctcctctccttacaagatacctaagaatccaccctcagagctgggtgcaccagattgccctcagaatggaagtacttgggtgtgaa
gctcaagatctatactga
核酸hFVIII-L2-3(SEQ ID NO:62)
atgcaaattgagctgagtacctgctttttcctatgcttgctaagattctgcttctctgcaacaaggagatactatttaggagcagttgaactctcttg
ggattatatgcagtctgaccttggagaattacctgtagatgcaaggttccctccaagagtgcccaagagcttcccttttaacacttctgttgtttac
aagaagacactctttgtagagtttactgaccatctgttcaacattgctaagcctaggcccccatggatggggctcctgggcccaaccattcaag
cagaggtctatgacacagtagtgattactttaaaaaacatggcttcccaccctgtcagcctgcatgcagtaggtgtgtcttattggaaggcctct
gagggtgctgaatatgatgatcagactagtcaaagagagaaagaagatgacaaagtctttccaggtggatcacacacatatgtgtggcaggt
gctgaaggagaatggtcctatggcttcagatcctttgtgtctgacttacagctacttatctcatgtggaccttgtcaaggacttaaacagtggact
gataggggctctgctggtgtgcagagaggggagcctggcaaaggagaagactcaaactttgcataaatttattctactgtttgctgtttttgatg
aggggaagagttggcactctgaaactaagaacagccttatgcaggacagagatgctgcttctgcaagagcctggcctaaaatgcacacagt
aaatggatatgtgaacagatcccttccagggctcataggctgtcacagaaagagtgtgtactggcatgtaatagggatgggcacaactcctg
aggtgcatagcatatttctagaaggccacacattcctagttagaaatcatagacaagcatctctagagatcagtcctatcacattcttaactgcac
agaccctattgatggacttaggccagtttcttcttttttgccacattagctcccaccagcatgatggcatggaggcctatgtcaaagtggactcat
gccctgaagaaccccaactcaggatgaaaaacaatgaggaggctgaggactatgatgatgacctgacagacagtgagatggatgttgtga
ggtttgatgatgataacagcccctctttcatccagatcagatcagttgcaaagaaacacccaaagacttgggtgcactacattgctgctgagga
agaggactgggactatgctcctttagtgttggcaccagatgacaggagttacaaatcccaatacctgaataatggtcctcagaggattggcag
aaagtataagaaggtcaggttcatggcttacacagatgagacattcaagaccagagaagctattcagcatgaatctggcattctggggccttta
ctctatggggaagtgggtgacacactactcattattttcaaaaatcaagcctccagaccttacaacatctacccccatggaatcactgatgttag
gcccctttacagcagaagactacccaagggggtgaaacaccttaaagattttccaatcctgcctggagagattttcaaatacaaatggacagt
cacagtggaagatggacctaccaagagtgatcctaggtgtttgactaggtactatagttcatttgtcaatatggagagagacttggcctcaggc
ctaatagggccacttctcatctgctataaggaatcagtagaccagagaggaaaccagatcatgtcagacaaaaggaatgttatcctcttttcag
tgtttgatgagaacaggtcttggtacctcacagaaaacatccagagatttttaccaaacccagcaggtgttcaattggaagaccctgaattcca
agcctccaacatcatgcactcaataaatgggtatgtatttgactctctccagctcagtgtgtgccttcatgaggtagcttattggtacatcttatcta
ttggagctcagactgactttttatctgtgttttttagtggttatacattcaaacacaagatggtgtatgaagataccctgactttattccctttctctgg
agaaactgtgttcatgtctatggagaacccaggcctgtggattctggggtgtcacaatagtgacttcagaaacagaggaatgacagcactgct
taaggtgagcagttgtgataagaatacaggagactactatgaggactcttatgaggatatcagtgcatatctgctaagcaagaataatgcaatt
gaaccaagatctttcagccagaaccctcctgttctcaaagccgccgctgccgaaataaccaggacaactctccagtctgatcaggaagaaat
tgactatgatgacacaatctcagtggagatgaaaaaagaggactttgacatctatgatgaggatgaaaatcagtctcctaggtcatttcagaag
aagaccaggcactactttatagcagctgtggaaagactatgggactatggtatgagctcctccccccatgtgctgaggaatagagctcaaagt
ggctcagtgcctcagttcaagaaggtggtcttccaagaatttactgatgggagcttcacacagcccctgtatagaggggagttaaatgaacat
ctgggactactgggaccatacattagagcagaggtggaggacaacatcatggttactttcagaaaccaggccagtagaccatacagtttcta
cagctctctaatatcttatgaagaagatcaaagacaaggagcagaacccagaaagaactttgtaaaacctaatgagacaaagacctatttctg
gaaggtccagcaccacatggccccaaccaaggatgaatttgactgtaaggcatgggcttacttttctgatgtagatctggagaaggatgtgca
cagtggcctaattggaccattactagtctgccataccaacacactcaatcctgcacatggtagacaggtcacagtgcaagaatttgctctgttttt
caccatttttgatgaaactaaaagttggtactttactgagaacatggagaggaactgcagagccccatgcaatatccagatggaagatccaact
ttcaaagagaattatagattccatgccataaatggttacattatggacacactgcctggcctggtcatggctcaggaccagaggatcagatggt
atctcttgagtatgggtagcaatgagaacatacactccatccacttttcaggacatgtctttacagtcagaaaaaaagaggagtataaaatggc
cttatacaacttatatccaggagtgtttgagacagtggagatgctccctagcaaggcaggtatctggagagtagaatgtctcataggggaaca
cctccatgctggtatgtcaacactgttcctggtgtatagtaacaagtgccaaacaccactgggcatggccagtggccatattagagactttcaa
ataactgcctctgggcaatatggtcagtgggcccctaaactggctaggctgcactactctgggtcaatcaatgcttggtcaaccaaagaacca
ttcagctggattaaggttgacctgctggctcctatgataattcatggcatcaaaacccagggagccagacaaaagttttcttccctgtacatctct
caatttataataatgtactctttggatggaaaaaaatggcagacttacagagggaactcaacagggaccctgatggtgttctttgggaatgtaga
ctcttcaggtatcaaacataatattttcaaccctcccatcatagctagatatataagacttcaccccacccactattctataaggtctactctcagga
tggagttaatggggtgtgacttaaatagctgcagtatgcctctggggatggaaagcaaggctatctctgatgcacagattacagctagcagct
acttcactaatatgtttgctacatggtctccatctaaggcaagactgcatctccagggaagatctaatgcatggaggcctcaagtgaacaaccc
aaaagagtggctgcaagtggacttccaaaaaacaatgaaggttacaggagtcacaacacagggggtgaaatctttactgacatcaatgtatg
taaaggagtttctcatttcaagtagtcaggatggacaccagtggactttattctttcagaatggtaaggtcaaggtcttccaaggaaatcaggac
agctttacaccagtggtcaacagcctggatcctcccttactcaccagatacctgaggatccacccacagtcttgggttcaccaaattgctctga
gaatggaagtgctgggatgtgaggcacaggacctctactga
核酸hFVIII-L2-4(SEQ ID NO:63)
atgcagattgagctgagcacctgcttcttcctgtgcctgctgagattctgcttctctgccaccagaagatactacctgggggctgtggagctga
gctgggactacatgcagtctgacctgggggagctgcctgtggatgccagattcccccccagagtgcccaagagctttcccttcaacacctct
gtggtgtacaagaagaccctgtttgtggaattcacagaccatctgttcaacattgcaaagcccagacccccctggatgggcttgctgggcccc
accatccaggctgaggtctatgacacagtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtgggggtgagct
actggaaagcttctgagggggctgagtatgatgaccagacctcccagagagagaaggaggatgacaaggtgttccctgggggcagccat
acctatgtgtggcaggtgctgaaagagaatggccccatggcctctgaccccctgtgcctgacctacagctacctgagccatgtggacctggt
gaaagacctcaattctggcctgattggggccttgctggtgtgcagagagggcagcctggctaaggagaagacccagaccctgcacaagttc
atattgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggacagagatgctgcctctgccaga
gcctggcctaaaatgcatacagtgaatggctatgtgaacagaagcctgcctggcctgattggctgccacagaaagtctgtttactggcatgtg
attggcatgggcaccacccctgaggtgcacagcatcttcctggagggccacaccttcctggtgagaaaccacaggcaggccagcctggag
atcagccccatcaccttcctgacagcccagaccctgctgatggatctgggacagttcctgctgttctgtcacatctctagccaccagcatgatg
gcatggaggcctatgtgaaggtggactcctgccctgaggagcctcagctgagaatgaagaacaatgaggaggctgaggactatgatgatg
acctgacagactctgagatggatgtggtgagatttgatgatgacaacagccctagcttcatccagatcagatctgtggccaagaagcaccca
aagacctgggtgcactacattgctgctgaggaagaggactgggactatgcccccctggtgctggcccctgatgacagaagctacaaaagc
cagtacctgaataatggcccccagagaattggcagaaagtacaagaaggtgagattcatggcttacacagatgagaccttcaagaccagag
aggccatccagcatgagtctggcatcttgggccctctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaatcaagccagc
agaccttacaacatctacccacatggcatcacagatgtgagaccactgtacagcagaagactgcccaaaggggtgaagcacctgaaggac
ttcccaatcctgcctggggagatcttcaagtataagtggacagtgacagtggaggatggccccactaagtctgaccccagatgcctgaccag
atactacagcagctttgtgaacatggaaagagatctggcctctggcctgattggccccctcctgatctgctacaaggagtctgtggaccagag
aggcaatcagatcatgtctgacaagagaaatgtgatactgttctctgtgtttgatgagaacagaagctggtacctcacagagaacattcagaga
ttcctgcccaaccctgctggggtgcaactggaagaccctgagttccaggccagcaacatcatgcacagcatcaatggctatgtctttgactcc
ctgcagctgtctgtgtgtctgcatgaggtggcctactggtacatcttgagcattggggcccagacagacttcctgtctgtgttcttctctggctac
acattcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctctggggagacagtgttcatgagcatggagaacccaggcct
gtggatcctgggctgccacaactctgacttcaggaacagaggcatgacagccctcttgaaggtgtccagctgtgacaagaacacagggga
ctactatgaggacagctatgaggacatatctgcctacctgctgagcaagaacaatgccattgagcccagatcttttagccagaacccacctgt
gctgaaggccgccgctgccgagatcaccagaaccaccctccagtctgaccaggaggagattgactatgatgacaccatctctgtggagatg
aaaaaggaggactttgacatctatgatgaggatgagaaccagagcccaagatctttccagaagaagaccagacactacttcattgctgctgtg
gagagactgtgggactatggcatgagctctagcccccatgtgctgagaaacagagcacagtctggctctgtgccccagttcaagaaggtgg
tgttccaggagttcacagatggcagcttcacccagcccctgtacagaggggagctgaatgagcacctgggcctcctgggcccctatatcag
ggctgaggtggaggacaacatcatggtgaccttcagaaaccaggccagcagaccctacagcttctatagcagcctgataagctatgaggag
gatcagagacagggggctgagcctagaaagaactttgtgaagcccaatgagaccaaaacctacttctggaaggtgcagcaccacatggcc
cccaccaaggatgagtttgactgtaaggcctgggcctacttctctgatgtggacctggagaaagatgtgcactctggcctgattggccctctg
ctggtgtgccacaccaacaccttgaaccctgcccatggcagacaggtgacagtgcaggagtttgccctgttcttcaccatctttgatgagacc
aagagctggtacttcacagagaatatggagagaaattgtagagccccctgcaatatccagatggaggaccccaccttcaaggagaactaca
gattccatgccatcaatggctacatcatggacaccctccctggcctggtcatggcccaggaccagagaatcagatggtatctgctgagcatg
ggcagcaatgagaatatccactccatccacttctctggccatgtgttcacagtgagaaagaaggaggagtacaagatggctctgtacaacct
gtaccctggggtgtttgagacagtggagatgctccccagcaaggctggcatctggagagtggagtgcctgattggggaacacctgcatgct
ggcatgagcaccctgttcctggtgtactccaacaagtgccaaacccccctgggcatggcctctggccacataagagacttccagatcacagc
ctctggccagtatggccagtgggcccccaagctggccagactgcactactctggcagcatcaatgcctggagcactaaggagcccttcagc
tggatcaaggtggatctgctggcccccatgatcatccatggcatcaagacccagggggccagacagaagttcagcagcctgtacataagcc
agttcatcatcatgtacagcctggatggcaaaaagtggcagacctacagaggcaacagcacaggcaccctgatggtgttctttggcaatgtg
gacagctctggcatcaagcacaacatcttcaacccccccatcattgccagatacatcagactgcaccccacccactacagcatcagaagcac
cctgagaatggagctcatgggatgtgacctgaactcctgcagcatgcctctgggcatggaatctaaggccatctctgatgcccagattacagc
cagctcttacttcaccaacatgtttgccacctggagccctagcaaggccagactccacctgcagggcagaagcaatgcctggagaccccag
gtgaacaaccccaaggagtggctgcaggtggacttccagaagaccatgaaggtgacaggggtgaccacccagggggtgaagagcctgc
tgaccagcatgtatgtgaaggagttcctgatcagctctagccaggatggccaccagtggacactgttcttccagaatggcaaggtgaaggtgt
tccaaggcaaccaggacagcttcacccctgtggtgaacagcttggacccccccctgctgactaggtacctgagaatccacccccagagctg
ggtgcaccagattgccctgagaatggaagtcctgggctgtgaggcacaggacctgtactga
核酸hFVIII-L2-5(SEQ ID NO:64)
atgcagattgagctgagcacctgcttcttcctgtgcctgctgagattctgcttctctgccaccagaagatactacctgggagctgtggagctga
gctgggattacatgcagtctgacctgggggagctgcctgtggatgccagattcccccccagagtgcccaagagcttccccttcaacacctct
gtggtctacaagaagaccctgtttgtggaattcacagaccacttgtttaacatagccaagcccagacctccctggatgggcctcctgggcccc
accatccaggctgaggtctatgacacagtggtgatcacactgaagaacatggccagccaccctgtgagcctccatgctgtgggggtgagct
actggaaggcctctgagggggctgagtatgatgaccagacctctcagagagagaaggaagatgacaaggtgttccctgggggcagccac
acctatgtgtggcaggtgctgaaggagaatggccccatggcttctgaccccctgtgcctcacctacagctacctgagccatgtggacctggt
gaaggacctgaactctggcctgataggggccctgctggtgtgcagagaaggcagcctggccaaggagaaaacccagaccctgcacaagt
tcatcctgctgtttgctgtgtttgatgaggggaagagctggcactctgagaccaagaacagcctgatgcaggacagagatgctgcctctgcca
gagcctggcccaagatgcacacagtgaatggctatgtgaacagaagcctgcctggcctcattggctgccacagaaaatctgtgtactggcat
gtgattggcatgggcaccacccctgaggtgcacagcatcttcctggagggccacaccttcctggtgagaaaccacagacaggccagcctg
gagatcagccccatcaccttcctgacagcccagactctgctgatggacctgggccagttcctgctgttctgccacatcagcagccaccaacat
gatggcatggaggcctatgtgaaggtggacagctgccctgaggagccccagctgagaatgaaaaacaatgaggaggctgaggactatga
tgatgacctgacagactctgagatggatgtggtgagatttgatgatgacaacagccccagcttcatccagatcagatctgtggccaagaagca
ccccaaaacctgggtgcactacattgctgctgaggaagaggactgggactatgcccccctggtgctggcccctgatgacagaagctataag
tcccagtacctcaacaatggcccccagagaataggcagaaagtacaagaaggtgagattcatggcctacacagatgagaccttcaaaacca
gagaagccatccagcatgagtctggcatcctgggccccctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaaccaagc
cagcagaccttacaacatctacccccatggcatcacagatgtgagacccctgtacagcagaagactgccaaagggggtgaaacacctgaa
ggactttcccatcctgcctggggagatcttcaagtacaagtggacagtgacagtggaggatggccccaccaagtctgaccctagatgcctga
ccagatactacagcagctttgtgaatatggagagagatctggcttctggcctgattggccccctgctgatctgctacaaggagtctgtggacca
aagaggcaaccaaatcatgtctgacaagagaaatgttatcttgttctctgtgtttgatgagaatagaagctggtacctgacagagaacatccag
agattcctgcccaaccctgctggggtgcagctggaggaccctgagttccaggccagcaacatcatgcacagcatcaatggctatgtgtttga
cagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagacagacttcctgtctgtgttcttctctg
gctacaccttcaagcacaagatggtgtatgaggacacactgaccctgttccccttctctggggagacagtgttcatgagcatggagaaccctg
gcctgtggatcctgggctgccacaactctgacttcagaaacagaggcatgacagccctgctgaaggtcagcagctgtgacaagaacacag
gggactactatgaggacagctatgaggacatttctgcctacctgctcagcaagaacaatgccattgagcccagaagctttagccagaacccc
cctgtgctgaaggccgccgctgccgagatcaccagaaccaccttgcaatctgaccaagaggagattgactatgatgacactatctctgtgga
gatgaagaaggaggactttgacatctatgatgaggatgagaaccaaagccccagaagcttccagaaaaagaccagacactacttcattgct
gctgtggagagactgtgggactatggcatgtccagcagcccccatgtgctgagaaacagagcccagtctggctctgtgccccagttcaaga
aggtggtgttccaggagttcacagatggcagcttcacccagcccctgtacagaggggagctgaatgagcacctgggcctgctgggaccct
acatcagagctgaggtggaagacaacattatggtgaccttcagaaatcaggccagcagaccctacagcttctacagcagcctgatcagctat
gaggaagatcagagacagggggctgagcccagaaagaactttgtgaagcctaatgagaccaagacctacttctggaaggtgcagcatcac
atggcccccaccaaggatgagtttgactgcaaggcctgggcctacttttctgatgtggacctggagaaagatgtgcactctggcctgattggc
cccctgctggtgtgccacaccaacaccctgaaccctgcccatggcagacaggtgacagtgcaggagtttgccctgttcttcactatttttgatg
agaccaagtcctggtacttcactgagaatatggaaagaaactgtagagccccctgcaacatccagatggaggaccccaccttcaaggagaa
ctacagattccatgcaataaatggctacatcatggacaccctgcctggcctggtgatggcccaagaccagagaatcagatggtacctgctga
gcatggggtctaatgagaatatccacagcatccacttctctggccatgtgttcacagtgaggaagaaagaggaatacaagatggccctgtac
aacctctaccctggggtgtttgagacagtggagatgctgccctccaaggctggcatctggagagtggagtgtctgattggggagcacctgca
tgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacccccctgggcatggcctctggccacataagagactttcaaattac
agcctctggccagtatggccagtgggcccccaaactggccagactgcactactctggcagcatcaatgcctggagcaccaaggagccctt
cagctggatcaaggtggatctgctggcccccatgatcatccatggcatcaagacccagggggccagacagaagttcagcagcctgtacata
agccagttcatcatcatgtacagcctggatggcaagaagtggcagacctacagaggcaacagcactggcaccctcatggtgttctttggcaa
tgtggacagctctggcattaagcacaacatcttcaacccccctatcattgccagatacatcagactgcatcccacccactacagcatcagaag
cactctgagaatggaactgatgggctgtgacctcaacagctgcagcatgcccctggggatggagagcaaggcaatctctgatgcccagatc
acagccagcagctacttcaccaacatgtttgccacctggagcccctccaaggctagactgcacctgcaaggaagaagcaatgcctggagg
ccccaggtgaacaaccccaaggagtggctgcaggtggacttccagaagaccatgaaggtgacaggggtgaccacccagggggtgaaga
gcctgctgaccagcatgtatgtgaaggagttcctgatcagctctagccaggatggccaccaatggaccctgttcttccagaatggcaaggtca
aggtgttccagggcaaccaggatagcttcacccctgtggtgaacagcctggacccccccctgctgactagatacctgagaatccaccccca
gagctgggtgcaccagattgccctgagaatggaggtgctgggctgtgaggcccaggacttgtactga
核酸hFVIII-L2-6(SEQ ID NO:65)
atgcagattgagctgagcacctgcttctttctgtgcctgctgagattctgcttctctgccaccagaagatactacctgggggctgtggaactgag
ctgggattacatgcagtctgacctgggggagctgcctgtggatgccagattcccccccagagtgcccaagagcttccccttcaacacctctgt
ggtgtacaagaaaaccttgtttgtggagttcacagaccacctgttcaacattgccaagcccagacccccctggatgggcctgctgggcccca
ctatccaggctgaggtgtatgacacagtggtgatcaccctgaagaatatggccagccaccctgtgagcctgcatgctgtgggggtgagctat
tggaaggcctctgagggggctgagtatgatgaccagaccagccagagagagaaggaggatgacaaggtgttccctgggggcagccaca
cctatgtgtggcaggtgctgaaggaaaatggccccatggcctctgaccccctgtgcctgacctacagctacctgagccatgtggacctggtg
aaggacctgaactctgggctgattggggccctgctggtgtgtagagagggcagcctggccaaggagaagacccagaccctgcacaaattc
atattgctgtttgctgtgtttgatgagggcaagagctggcattctgagaccaagaacagcctcatgcaggacagagatgctgcctctgccaga
gcttggcccaaaatgcacacagtgaatggctatgtgaacagaagcctgccaggactgattggctgccacagaaagtctgtgtactggcatgt
gattggcatgggcacaacccctgaggtgcactctatcttcctggagggccataccttcctggtgagaaaccacagacaggccagcctggag
atctcccccatcaccttcctgacagcccagacactgctgatggatctgggccaattcctgctgttctgccacatcagcagccaccagcatgat
ggcatggaggcctatgtgaaggtggacagctgccctgaggagccccagctgagaatgaagaacaatgaggaggctgaggattatgatgat
gacctcacagactctgagatggatgtggtgagatttgatgatgacaactctcccagcttcatccagattagatctgtggccaagaagcacccc
aagacctgggtccactacattgctgctgaggaagaggactgggactatgcccccctggtgctggcacctgatgacagatcctataagagcc
agtacctgaacaatggcccccagagaattggcagaaagtacaagaaggtgagattcatggcctacacagatgagaccttcaagaccagag
aggccatccagcatgagtctggcatcctgggccccctgctgtatggggaggtgggggacaccctgctcatcatcttcaagaaccaggcctc
cagaccctataacatctacccccatggcatcacagatgtgagacccctgtacagcagaagactgcccaagggggtgaagcacctgaagga
cttccccattctgccaggggagatcttcaagtacaagtggacagtgacagtggaggatggccccaccaagtctgaccccagatgcctgacc
agatactacagcagctttgtgaacatggagagagacctggcctctggcctgattggccctctcctgatctgctacaaggagtctgtggaccag
agaggcaaccagatcatgtctgacaagagaaatgtgatcctgttctctgtgtttgatgagaatagaagctggtacctgacagagaacattcaaa
gattcctgcccaaccctgctggggtgcagctggaggaccctgagttccaggccagcaacatcatgcacagcatcaatggctatgtgtttgact
ccctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagacagacttcctgtctgtgtttttctctggct
ataccttcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctctggggagacagtgttcatgagcatggagaatcctggcc
tgtggatcctgggctgccacaactctgacttcagaaacagaggcatgacagccctgctgaaggtgagcagctgtgacaagaacacagggg
actactatgaggacagctatgaggacatctctgcctacttgctgagcaagaacaatgccattgagcccagaagcttcagccagaacccccct
gtgctgaaggccgccgctgccgagatcaccagaaccaccctgcaatctgaccaggaggaaattgactatgatgacaccatctctgtggaaa
tgaagaaggaagactttgacatctatgatgaggatgagaaccagagccccagaagcttccagaagaagaccagacactacttcattgctgct
gtggagagactgtgggactatggcatgagctctagcccccatgtgctgagaaacagagcccagtctggctctgtgccccaattcaagaagg
tggtgttccaggagttcacagatggcagcttcacccagcccctgtacagaggggagctgaatgagcacctgggcctgctgggcccctatat
cagagctgaggtggaggataacatcatggtgacctttagaaatcaggccagcagaccctacagcttttacagcagcctgatcagctatgagg
aggaccagagacagggggctgagcccagaaagaactttgtgaagccaaatgagaccaagacctacttctggaaggtgcagcaccacatg
gcccccacaaaggatgagtttgactgcaaggcctgggcctacttctctgatgtggacctggagaaggatgtgcactctggcctgattggccc
cctgttggtgtgccacaccaacaccctgaaccctgcccatggcagacaggttacagtgcaggagtttgccctgtttttcaccatttttgatgaga
ccaagtcttggtacttcacagagaatatggaaagaaactgtagagccccctgcaacatccagatggaggaccccaccttcaaggagaacta
cagatttcatgccattaatggctacatcatggacaccctgcctggcctggttatggcccaggaccagagaatcagatggtacctgctgagcat
gggctctaatgagaacattcacagcatccacttctctggccatgtgttcacagtgagaaagaaggaagagtacaagatggcactgtacaactt
gtaccctggggtgtttgagacagtggaaatgctgcccagcaaggctggcatctggagagtggaatgtctcattggggagcacctgcatgctg
gcatgagcaccctgttcctggtgtacagcaacaagtgccagacacccctgggcatggcttctggccacatcagagacttccagatcacagc
ctctggccaatatggccagtgggcccccaagctggccagactccactattctggcagcatcaatgcctggagcaccaaggagcccttcagc
tggatcaaggtggacctgctggcccccatgatcatccatggcatcaagacccagggggccagacagaagttcagcagcctgtacatcagc
cagttcatcatcatgtacagcctggatggcaagaaatggcagacctacagaggcaacagcactggcaccctgatggtgttctttggcaatgtg
gacagctctggcatcaagcacaacatcttcaacccccccatcattgccagatatatcagactgcaccccacccactacagcattagaagcac
cctgagaatggagctgatgggctgtgacctgaacagctgcagcatgcccctgggcatggagtccaaggccatctctgatgcccaaatcaca
gccagcagctacttcaccaacatgtttgccacctggagccctagcaaggccagactgcacctgcagggcagaagcaatgcctggagaccc
caggtgaacaaccccaaggagtggctgcaggtggacttccagaagaccatgaaggtgacaggggtgaccacacagggggtgaagagcc
tcctgaccagcatgtatgtgaaggagttcctgattagctctagccaggatggccaccagtggaccctgttcttccagaatggcaaggtgaagg
tcttccagggcaaccaggacagcttcacccctgtggtgaacagcctggacccccctctcctgacaagatacctgagaatccacccccagag
ctgggtgcaccagattgccctgagaatggaggtgctgggctgtgaggcccaggacctctactga
核酸hFVIII-L2-8(SEQ ID NO:66)
atgcagattgagctgagcacctgcttcttcctctgcctgctgaggttctgcttctctgccactagaagatactacctgggggctgtggagctgag
ctgggactacatgcagtctgacctgggggagctgcctgtggatgccagattcccacccagggtgcccaagagcttccccttcaacacctctg
tggtgtacaagaagaccctgtttgtggagtttacagaccacctgttcaatattgccaagcccagacccccctggatggggctgctgggcccca
ccatccaggcagaggtgtatgacacagtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtgggggtgagcta
ctggaaggcctctgagggggctgagtatgatgaccagaccagccagagagagaaggaggatgacaaggtgttccctgggggcagccac
acctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacatacagctacctgagccatgtggacctggt
gaaggatctcaattctggcctgattggggccctgctggtgtgcagagagggcagcctggccaaggagaagacccagaccctgcacaagtt
catcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggacagagatgctgcctctgcca
gagcctggcccaagatgcacacagtgaatggctatgtgaacagaagcctgcctggcctgattggctgccatagaaagtctgtgtactggcat
gtgattggcatgggcaccacccctgaggtgcacagcatcttcctggaaggccacaccttcctggtgagaaaccacagacaggccagcctg
gagatcagccccatcaccttcctgacagcccaaaccctgttgatggacctgggccagttcctgctgttctgtcacatcagctctcaccagcatg
atggcatggaggcctatgtgaaggtggacagctgccctgaggagccccagctgagaatgaagaacaatgaggaggctgaggactatgat
gatgacctgacagactctgagatggatgtggtgagatttgatgatgacaacagccccagcttcatccagatcagatctgtggccaagaagca
ccccaagacctgggtgcattacattgctgctgaggaagaggactgggactatgcccccctggttctggcccctgatgacagaagctacaagt
ctcaatacctgaacaatggcccccagagaattggcagaaagtacaagaaggtgagattcatggcctacacagatgagacctttaagaccag
agaagccatccagcatgagtctggcatcctgggccccctcctgtatggggaggtgggggacaccttgctgatcatcttcaagaaccaggcc
agcagaccctacaacatctacccccatggcatcacagatgtgagacccctgtacagcagaagactccccaagggggtgaagcacctgaag
gacttccccatcctgcctggggagatcttcaagtataagtggacagtgacagtggaggatggccccaccaagtctgaccccagatgcctga
ccagatactacagcagctttgtgaacatggaaagagacctggcatctggcctcattggccctctgctgatttgctacaaggaatctgtggacca
gagaggcaatcagatcatgtctgacaagagaaatgtgatcctgttctctgtgtttgatgagaacagaagctggtacctgacagagaacatcca
aaggttcctgcccaaccctgctggggtgcagctggaggaccctgagttccaggcctctaatatcatgcacagcatcaatggctatgtgtttga
cagcctccagctgtctgtgtgcctgcatgaggtggcctattggtacatcctgagcattggggcccagacagacttcctgtctgtgttcttctctg
gctacaccttcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctctggggagacagtgttcatgagcatggagaaccctg
gcctgtggatcctgggctgccacaactctgacttcagaaacagagggatgactgccctgctgaaggtgagcagctgtgacaagaacacag
gggactactatgaggacagctatgaggacatctctgcttacctgctgagcaagaacaatgccattgagcccagaagcttcagccagaaccc
ccctgtgctgaaggccgccgctgccgagatcaccagaaccaccctgcaatctgaccaggaggagattgactatgatgacaccatctctgtg
gagatgaagaaggaggactttgacatctatgatgaggatgagaaccaatcccccagatccttccagaaaaagaccagacactactttattgct
gctgtggagagactgtgggactatggcatgtccagcagcccccatgtgctgagaaacagagcccagtctggctctgtgccccagttcaaga
aggtggtgttccaggaattcacagatggcagcttcacccagcctctgtacagaggggagctgaatgagcacctgggcctgctgggccccta
catcagagctgaggtggaggacaacatcatggttaccttcagaaaccaagcctctagaccctacagcttctacagcagcctgatcagctatg
aggaagaccagagacagggggctgagcccagaaagaactttgtgaagcccaatgagaccaagacatacttctggaaggtgcagcaccac
atggcccccaccaaggatgagtttgactgcaaggcctgggcctacttctctgatgtggatctggaaaaggatgtgcattctggcctgattggg
cctctgctggtgtgtcacaccaacaccctgaaccctgcccatggcagacaggtgacagtgcaggagtttgccctgttcttcaccatttttgatg
agaccaagagctggtacttcacagagaacatggagagaaactgcagagccccctgcaacatccagatggaggatcccaccttcaaggag
aactacaggttccatgccatcaatggctacatcatggacaccctccctggcctggttatggctcaggaccagagaatcagatggtatctgctg
agcatgggcagcaatgagaatatccacagcatccacttctctggccatgtgttcacagtgagaaagaaggaggagtacaagatggccctgt
acaacctgtaccctggggtgtttgagacagtggagatgctgcccagcaaggctggcatctggagagtggagtgcctgattggggagcacct
gcatgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacacccctgggcatggcctctggccatatcagagacttccaga
ttacagcctctggccagtatggccagtgggcccccaagctggccagattgcactactctggctccataaatgcctggagcaccaaggagcc
cttctcttggatcaaggtggacctgctggcccccatgatcatccatggcatcaagacccagggggccagacagaagttcagctccctgtaca
tcagccagttcatcatcatgtacagcctggatggcaagaagtggcagacctacagagggaactctacaggcaccctgatggtgttctttggca
atgtggacagctctggcatcaagcacaacatcttcaacccccccatcattgccagatacattagactgcaccccacccactacagcatcagaa
gcaccctgagaatggagctgatgggctgtgacctgaacagctgcagcatgcccctgggcatggagagcaaggccatctctgatgcccaga
tcacagcctcctcttacttcaccaacatgtttgccacctggagcccctctaaggccagactgcacctgcagggcagaagcaatgcctggaga
ccccaggtcaacaaccccaaggagtggctgcaggtggacttccagaagactatgaaggtgacaggggtgaccacccaaggggtgaaga
gcctgctgaccagcatgtatgtgaaggagttcctgatcagctctagccaggatggccaccagtggaccctgttcttccagaatggcaaggtg
aaagttttccagggcaaccaggactccttcacccctgtggtgaacagcctggacccccccctgctgactagatacctgagaatccaccccca
gagctgggtgcatcagattgccctcagaatggaggtgctgggctgtgaggcccaggacctgtactga
核酸hFVIII-L2-9(SEQ ID NO:67)
atgcagattgagctgagcacatgcttcttcctgtgcctgctgaggttctgcttttcagctaccagaaggtactacctgggtgctgtggagctgtc
ctgggactacatgcagtctgatctgggagagctgcctgtggatgccaggttcccccctagggtgcctaagagcttccctttcaacacctctgtg
gtgtacaagaagacactgtttgttgagttcacagaccacctgttcaacatagctaagcccagacccccctggatggggctgctgggccccac
cattcaggctgaggtgtatgatactgttgtcatcaccctgaagaacatggcctcccaccctgtgtccctgcatgctgttggtgtgtcctactgga
aggcttcagagggggctgagtatgatgaccagacctcccagagagaaaaggaggatgataaggtgtttccaggtgggtcccacacctatgt
ctggcaggtgctgaaggagaatggtcccatggcttctgatcctctgtgcctgacctactcctacctctcccatgtggacctggttaaggacctg
aactctggcctgataggtgccctgctggtgtgcagagagggctccctggccaaggagaagacccagaccctccacaagttcatcttgctgtt
tgctgtgtttgatgagggcaagtcttggcacagtgagacaaagaactccctgatgcaggacagggatgctgcatcagctagagcttggccca
agatgcacactgtgaatggttatgtgaacaggtccctgcctggcctgattggttgccacagaaagtctgtctactggcatgttataggtatggg
caccaccccagaggtgcactccatctttctggaggggcacacattcctggtgagaaaccacaggcaggcctctctggagatcagccccatc
acctttctgacagctcagaccctgctgatggacctgggccagttcctgctcttctgccacatctcctcccaccagcatgatgggatggaggcct
atgtgaaggtggatagctgccctgaggaaccccagctgaggatgaagaacaatgaggaggcagaggactatgatgatgacctgactgatt
ctgagatggatgtagtgaggtttgatgatgataactccccctctttcatccagatcaggtctgtggccaagaagcaccctaagacctgggtgca
ctacattgctgctgaggaggaggactgggactatgctcccctggtgctggctccagatgacaggagctacaagagccagtacctgaacaat
ggtccccagaggattggtaggaagtacaagaaggtgagattcatggcttacacagatgagaccttcaagaccagggaggccatccagcat
gagagtggtatcctgggtcccctgctgtatggtgaggtgggtgacaccctgctgatcatcttcaagaaccaggcctccagaccctacaacatc
taccctcatggtatcactgatgtgagacctctgtactctagaagactccccaagggggtgaagcacctgaaggactttcccatcctccctggtg
aaatcttcaagtacaagtggactgtcactgttgaggatggccccaccaagagtgatcccaggtgcctgaccaggtactactcctcctttgttaa
catggagagagacctggcctctggtctgattggtcccctgctgatctgctacaaggagtctgttgatcagagagggaaccagatcatgtcaga
taagaggaatgtgatcctgttctctgtgtttgatgagaacaggagctggtacctgactgagaacatccagaggttcctgcccaaccctgctggt
gtgcagctggaggaccctgagttccaggcctccaacatcatgcactctatcaatggttatgtatttgatagcctgcagctgtctgtgtgcctgca
tgaggtggcctactggtacatcctgtccattggtgctcagactgatttcctgtctgtgttcttctctggttacaccttcaagcacaagatggtgtatg
aggacaccctgaccctgttccccttctcaggtgagacagtgttcatgtccatggagaacccaggtctgtggatcctgggctgccacaactcag
atttcaggaacagggggatgacagctctgctgaaggtgtcctcttgtgacaagaacacaggtgactactatgaggactcctatgaggacatct
ctgcctacctgctgagcaagaacaatgccattgagcccaggagcttcagccagaacccacctgtcctgaaggccgccgctgccgagatca
ctagaaccaccctgcagtcagatcaggaggagattgattatgatgacaccatctctgtggagatgaagaaggaggactttgatatttatgatga
ggatgagaaccagagccccagatccttccagaagaagactagacactacttcatagctgctgtggagaggctgtgggactatggtatgtcct
ctagccctcatgtgctgaggaacagggcccagtctggttctgtgccccagttcaagaaggtggtgttccaggagttcactgatggcagcttca
cacagccactctacaggggtgagctcaatgagcacctgggactgctgggcccctacatcagggctgaggtggaggacaacatcatggtca
ccttcagaaaccaggcctccagaccctacagcttctactccagcctgatctcctatgaggaggaccagaggcagggtgctgagcccagaaa
gaactttgttaagcccaatgagaccaagacctacttctggaaggtgcagcaccacatggctcccaccaaggatgagtttgattgcaaggcct
gggcctacttctctgatgtggacctggagaaggatgtgcacagtggtctgattggtcccctgctggtgtgccacaccaacaccctgaaccctg
cccatggtaggcaggtgactgtccaggagtttgctctgttcttcaccatctttgatgagacaaagagctggtatttcacagagaacatggagag
aaactgcagagccccctgcaacatccagatggaggaccccaccttcaaggagaactacagattccatgccatcaatggttacatcatggaca
ccctgcctggcctggtcatggcccaggaccagagaatcaggtggtacctgctgtccatgggctctaatgagaacatccactccatccacttct
ctggtcatgtgttcacagtcagaaagaaggaggagtacaagatggccctgtacaacctgtaccctggtgtgtttgagactgtagagatgctgc
cctccaaggctgggatctggagagtggagtgcctgataggtgagcacctccatgctgggatgtccaccctgttcctggtctactccaataagt
gccagacacccctgggcatggccagtggtcacatcagagacttccagatcacagcttctggtcagtatggtcagtgggccccaaagctggc
caggctccactactctggtagcatcaatgcatggagcacaaaggagcccttctcctggatcaaggtggacctgctggctcctatgatcatcca
tggtatcaagacccagggtgccagacagaagttctcctccctgtatatctctcagttcatcatcatgtactccctggatggtaagaagtggcag
acctacagggggaactccactggtaccctgatggtgttctttggtaatgtagactccagtggtatcaagcacaacatcttcaacccccccatca
ttgctaggtacattaggctgcaccccacccactactccatcaggagcaccctgagaatggagctgatgggctgtgacctgaacagctgctcc
atgcccctgggcatggagtccaaggccatctcagatgcccagatcactgcctcctcctacttcaccaacatgtttgctacctggtccccctcca
aggccaggctgcacctgcagggcagatccaatgcatggaggcctcaggtgaacaaccccaaggagtggctgcaggtggacttccagaag
accatgaaggtgacaggtgtgaccacccagggtgtcaagtcactgctgacctccatgtatgtgaaggagttcctcatctccagctcccaggat
ggtcaccagtggaccctgttcttccagaatggtaaggtgaaggtgttccagggcaaccaggactctttcacccctgtagtgaactccctggac
ccccccctgctgaccagatacctcaggatccatccccagtcttgggtgcaccagattgctctgagaatggaggtgcttggctgtgaagccca
ggacctgtactga
核酸hFVIII-L2-10(SEQ ID NO:68)
atgcagattgagctgtccacctgcttcttcctgtgcctgctgaggttctgtttcagtgctaccaggaggtactacctgggtgctgtggagctgtcc
tgggactatatgcagtctgacctgggtgaactgcctgtggatgctagattccctcccagggtgcccaagtccttccccttcaatacctctgttgt
gtacaagaagaccctgtttgttgagttcactgatcacctgttcaacatagctaagcccagacctccctggatgggcctgctgggccccaccat
ccaggcagaggtgtatgatactgtggtgattaccctgaagaacatggcctcccatcctgtgagcctgcatgctgtgggggtgtcctactggaa
ggcctcagagggtgctgagtatgatgaccagacctcccagagagagaaggaggatgacaaggtgttccctggtggctcccacacctatgtc
tggcaggtgctgaaggagaatggtcccatggcctctgatcccctgtgtctgacatactcttacctgagccatgtggatctggtcaaggacctg
aactctggtcttataggtgctctgctggtgtgcagggaggggtctctggctaaggagaagactcagaccctccacaaattcatcctgctgtttg
ctgtctttgatgaggggaagtcttggcattctgagaccaagaactccctgatgcaggacagagatgctgcttcagctagagcctggcccaag
atgcacactgttaatggttatgtgaacaggtccctgcctggtctgattggctgccacaggaagtctgtctactggcatgtgattggtatgggcac
cacacctgaggtgcactccatcttcttggagggacacaccttcctggtgaggaaccacaggcaggcatcattggagatcagccccatcacct
tcctgacagctcagaccctgctcatggacctgggacagttcctgctgttctgccacattagctctcaccagcatgatggcatggaggcttatgt
caaggtggactcctgcccagaggagccccagctgaggatgaagaacaatgaggaggcagaggactatgatgatgatctgacagattctga
gatggatgtggtgaggtttgatgatgacaacagccccagcttcatccagatcaggtctgtggccaagaagcaccccaagacctgggtgcact
acatagctgctgaggaggaggactgggactatgccccactggtgctggccccagatgacaggagctacaagtcccagtacctgaacaatg
gccctcagaggataggtaggaagtacaagaaggtgaggttcatggcctacacagatgagaccttcaagaccagggaggccatccagcatg
agtcaggtatcctgggccccctgctgtatggtgaggtgggtgacaccctgctgatcatcttcaagaaccaggcctccagaccctacaacatct
accctcatggtatcacagatgtgaggcccctgtactccaggaggctgcccaagggggtgaagcatctgaaggacttccccatcctgcctggt
gagatcttcaagtacaagtggactgttactgtggaggatgggcctaccaagagtgatcccagatgcctgaccagatactactccagctttgtta
acatggagagggacctggcctcaggtctgattggtcccctcctgatctgctacaaagagtctgtggaccagaggggcaaccagatcatgag
tgataagaggaatgtaatcctgttctctgtctttgatgagaacaggtcctggtacctgacagagaacatccagagattcctgcccaacccagct
ggtgtacagctggaggaccctgagttccaggcctccaacatcatgcactccatcaatggttatgtctttgattccctgcagctttctgtatgcctg
catgaggtggcatactggtacatcctctccattggtgctcagacagatttcctctctgtgttcttcagtggttacaccttcaagcacaagatggtgt
atgaggacaccctgaccctgttccccttctctggtgagactgtcttcatgagtatggagaaccctggtctgtggatcctgggctgccacaactc
agatttcagaaacagggggatgacagctctgctgaaggtgtcttcatgtgacaagaacacaggtgactactatgaggactcctatgaggaca
tctctgcctacctgctgagcaagaacaatgccattgagccaaggagcttcagccagaacccccctgtgctgaaggccgccgctgccgagat
caccagaaccaccctccagtctgatcaggaggagattgattatgatgataccatctctgtggagatgaagaaggaggactttgatatctatgat
gaggatgagaaccagagcccaaggagcttccagaagaagaccaggcactacttcattgctgctgtagagaggctctgggactatggtatgt
cctctagcccccatgtgctgagaaacagggcccagagtggttctgtgccccagttcaagaaggtggtgttccaggagttcactgatgggagc
tttacccagcccctgtacaggggtgaactgaatgagcacctggggctgctgggcccctacatcagagctgaggtggaggacaacatcatgg
tgaccttcagaaaccaggcttccaggccctacagcttctactcctcactgatctcctatgaggaggaccagaggcagggtgcagagcctagg
aagaactttgttaagcctaatgagaccaagacctacttctggaaggtgcagcaccacatggcccctaccaaggatgagtttgattgcaaggcc
tgggcctacttctcagatgtggacctggagaaggatgtgcactcaggcctcattggtcccctgctggtgtgccacaccaacaccctgaaccct
gctcatggtagacaggtgactgtgcaggagtttgctctgttctttacaatctttgatgagaccaagagctggtacttcactgagaacatggagag
gaactgcagggctccatgcaacatccagatggaggaccccaccttcaaggagaactacagattccatgccatcaatggttacatcatggaca
cactgcctggccttgttatggcccaggaccagagaattaggtggtacctgctgtccatgggcagcaatgagaacatccactccatccacttct
ctggtcatgtcttcactgtgaggaagaaggaggagtacaagatggccctgtacaacctgtacccaggtgtgtttgagactgtggagatgctgc
cctccaaggctggtatctggagggtggagtgcctgataggtgagcacctgcatgcaggaatgagcaccctgttcctggtttactccaacaaat
gccagacccccctgggcatggccagtggtcacatcagagacttccagatcactgcttctggtcagtatggtcagtgggcccccaagctggc
caggctgcactactctggtagcatcaatgcttggtccaccaaggagcctttcagctggatcaaggtggacctgctggcccccatgatcatcca
tggtatcaagacccagggggccaggcagaagttctccagcctgtacatctcccagttcatcattatgtactccctggatggcaagaagtggca
gacctacagaggcaacagcactggtaccctgatggtgttctttggtaatgtggactcaagtggtatcaagcacaacatcttcaacccccccatc
atagctagatacattagactgcaccccacccactactcaatcagaagcacactgaggatggagctgatggggtgtgacctgaactcctgctc
catgcccctgggcatggagagcaaggccatcagtgatgcccagatcacagctagctcctacttcaccaacatgtttgctacctggagcccca
gcaaggccaggctgcacctgcagggcaggagcaatgcttggagaccccaggtgaacaaccccaaggagtggctgcaggtggacttcca
gaagaccatgaaggtgacaggtgtgaccacccagggtgttaagagcctgctgaccagtatgtatgtgaaggagtttctgatctcctcctcaca
ggatggtcaccagtggaccctcttcttccagaatggtaaggtgaaggtgttccagggcaaccaggactccttcacccctgtggttaactccct
ggacccccctctgctgaccaggtacctgaggatccacccccagtcctgggtgcaccagattgctctgaggatggaggtgctgggctgtgag
gctcaggacctctactga
上述核酸中，hFVIII-L1-WT、hFVIII-L1-1至hFVIII-L1-10均为编码hFVIII-L1的核酸，其中，hFVIII-L1-1至hFVIII-L1-10在hFVIII-L1-WT基础上进行了密码子优化；hFVIII-L2-WT、hFVIII-L2-1至hFVIII-L2-10均为编码hFVIII-L2的核酸，其中，hFVIII-L2-1至hFVIII-L2-10在hFVIII-L2-WT基础上进行了密码子优化。
优选地，编码凝血因子FVIII蛋白变体的核酸为hFVIII-L1-5、hFVIII-L1-9、hFVIII-L1-10、hFVIII-L2-5、hFVIII-L2-9或hFVIII-L2-10。进一步优选地，编码凝血因子FVIII蛋白变体的核酸为hFVIII-L1-7和hFVIII-L2-7。
相应地，本发明实施例还提供了一种表达载体，其包含本发明实施例所述的核酸。
病毒载体如慢病毒和细小病毒载体，包括AAV血清型和其变体，提供离体、体内、体外递送核酸进细胞的方式，使细胞表达编码的蛋白。AAV是可用于基因治疗载体的病毒，因为其可渗透细胞并将核酸/遗传材料引入，使核酸/遗传材料可稳定保持在细胞中。可用于本发明的病毒载体包括但不限于多种血清型的腺相关病毒(AAV)载体，例如AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10或AAV11血清型。在一个具体实施方案中，表达载体为AAV8。
在一些实施方案中，所述表达载体还包含与所述核酸可操作地连接的启动子、增强子、内含子、一个或多个末端反向重复序列(如5'ITR、3'ITR)、Kozak序列和/或polyA尾。在一些实施方案中，所述增强子和所述启动子依次可操作地连接至所述核酸的序列，或者所述内含子位于所述核酸的序列内部或两侧，或者所述末端反向重复序列位于所述核酸的序列的5'或3'末端，或者所述Kozak序列位于所述核酸的序列的5'末端与所述内含子之间，或者所述polyA尾位于所述核酸的序列的3'末端与所述末端反向重复序列之间。图1示出了本发明一个具体实施方案所涉及的hFVIII基因表达盒的结构，从5'至3'端依次包含5'ITR、增强子、启动子、内含子、Kozak序列、hFVIII目的基因(即，所述核酸序列)、polyA和3'ITR。但本发明提供的hFVIII基因表达盒的结构并不限于此。
如本文所用，术语“可操作地连接”是指表达编码序列必须的调节序列至于相对于编码序列合适的位置，从而产生编码序列的表达。该术语也应用于表达载体中编码序列和转录控制元件(例如启动子、增强子等)的排列。
在一些实施方案中，所述启动子为TTRmut启动子(突变的mini人转甲状腺素启动子)、TTRm启动子(mini人转甲状腺素启动子)或HLP启动子(人α1抗胰蛋白酶核心启动子)，所述启动子的核苷酸序列如SEQ ID NO:16～18中的任一序列所示。
TTRmut启动子(SEQ ID NO:16)
gtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaa
gtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccag
gagaagccgtcacacagatccacaagctcctg
TTRm启动子(SEQ ID NO:17)
gtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatatttgtgtaggttacttattctccttttgttgactaa
gtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccag
gagaagccgtcacacagatccacaagctcctg
HLP启动子(SEQ ID NO:18)
gtttgctgcttgcaatgtttgcccattttagggtggacacaggacgctgtggtttctgagccagggggcgactcagatcccagccagtggactt
agcccctgtttgctcctccgataactggggtgaccttggttaatattcaccagcagcctcccccgttgcccctctggatccactgcttaaatacg
gacgaggacagggccctgtctcctcagcttcaggcaccaccactgacctgggacagtgaatc
在一些实施方案中，所述增强子为TTR增强子(人转甲状腺素增强子)、SerpinA1增强子(人α1抗胰蛋白酶增强子)或CRMSB2增强子(修饰后的人α1抗胰蛋白酶增强子)，所述增强子的核苷酸序列如SEQ ID NO:19～21中的任一序列所示。
TTR增强子(SEQ ID NO:19)
cactgggaggatgttgagtaagatggaaaactactgatgacccttgcagagacagagtattaggacatgtttgaacaggggccgggcgatc
agcaggtag
SerpinA1增强子(SEQ ID NO:20)
gggggaggctgctggtgaatattaaccaaggtcaccccagttatcggaggagcaaacaggggctaagtccac
CRMSB2增强子(SEQ ID NO:21)
gggggaggctgctggtgaatattaaccaagatcaccccagttaccggaggagcaaacagggactaagttcac
在一些实施方案中，所述内含子为EF1a内含子(人源伸长因子1a内含子)、MVM内含子(小鼠微小病毒内含子)或SBR内含子3(修饰后的小鼠微小病毒内含子)，所述内含子的核苷酸序列如SEQ ID NO:22～24中的任一序列所示。
EF1a内含子(SEQ ID NO:22)
caggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattact
MVM内含子(SEQ ID NO:23)
aagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttgg
SBR内含子3(SEQ ID NO:24)
aagaggtaagggtttaagttatcgttagttcgtgcaccattaatgtttaattacctggagcacctgcctgaaatcatttttttttcaggttgg
在一些实施方案中，所述末端反向重复序列为位于5'末端的5'ITR和位于3'末端的3'ITR，所述末端反向重复序列的核苷酸序列如SEQ ID NO:25或SEQ ID NO:26所示.
5'ITR(SEQ ID NO:25)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct
3'ITR(SEQ ID NO:26)
aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgg
gctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
在一些实施方案中，所述Kozak序列为：gccacc
polyA尾是聚腺苷酸化信号。在一些实施方案中，所述polyA尾的核苷酸序列如SEQID NO:27所示。
polyA尾(SEQ ID NO:27)
aataaaagatcagagctctagagatctgtgtgttggttttttgtgt
本发明实施例还进一步对hFVIII基因表达盒中的增强子、启动子、内含子元件组合进行了优化，以获得表达效果更好的元件组合，具体如表1所示。
表1
元件组合0TE(SEQ ID NO:44)
gtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaa
gtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccag
gagaagccgtcacacagatccacaagctcctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggccc
ttgcgtgccttgaattact
元件组合TTM(SEQ ID NO:45)
cactgggaggatgttgagtaagatggaaaactactgatgacccttgcagagacagagtattaggacatgtttgaacaggggccgggcgatc
agcaggtaggtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatatttgtgtaggttacttattctccttt
tgttgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagcc
ccttcaccaggagaagccgtcacacagatccacaagctcctgaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaatt
acctggagcacctgcctgaaatcactttttttcaggttgg
元件组合HLP(SEQ ID NO:46)
gtttgctgcttgcaatgtttgcccattttagggtggacacaggacgctgtggtttctgagccagggggcgactcagatcccagccagtggactt
agcccctgtttgctcctccgataactggggtgaccttggttaatattcaccagcagcctcccccgttgcccctctggatccactgcttaaatacg
gacgaggacagggccctgtctcctcagcttcaggcaccaccactgacctgggacagtgaatc
元件组合STM(SEQ ID NO:47)
gggggaggctgctggtgaatattaaccaaggtcaccccagttatcggaggagcaaacaggggctaagtccacgtctgtctgcacatttcgta
gagcgagtgttccgatactctaatctccctaggcaaggttcatatttgtgtaggttacttattctccttttgttgactaagtcaataatcagaatcagc
aggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacag
atccacaagctcctgaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttt
tttcaggttgg
元件组合CTS(SEQ ID NO:48)
gggggaggctgctggtgaatattaaccaagatcaccccagttaccggaggagcaaacagggactaagttcacgtctgtctgcacatttcgta
gagcgagtgttccgatactctaatctccctaggcaaggttcatatttgtgtaggttacttattctccttttgttgactaagtcaataatcagaatcagc
aggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacag
atccacaagctcctgaagaggtaagggtttaagttatcgttagttcgtgcaccattaatgtttaattacctggagcacctgcctgaaatcatttttttt
tcaggttgg
在一些实施方案中，所述表达载体包含如SEQ ID NO:28～43所示的hFVIII蛋白表达框中的任一个。
蛋白表达框AAV8-0TE-hFVIII-L1-WT-pA(SEQ ID NO:28)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcaaatagagctctccacctgcttctttctgtgccttttgcgattctgctttagtgccacc
agaagatactacctgggtgcagtggaactgtcatgggactatatgcaaagtgatctcggtgagctgcctgtggacgcaagatttcctcctaga
gtgccaaaatcttttccattcaacacctcagtcgtgtacaaaaagactctgtttgtagaattcacggatcaccttttcaacatcgctaagccaagg
ccaccctggatgggtctgctaggtcctaccatccaggctgaggtttatgatacagtggtcattacacttaagaacatggcttcccatcctgtcag
tcttcatgctgttggtgtatcctactggaaagcttctgagggagctgaatatgatgatcagaccagtcaaagggagaaagaagatgataaagtc
ttccctggtggaagccatacatatgtctggcaggtcctgaaagagaatggtccaatggcctctgacccactgtgccttacctactcatatctttct
catgtggacctggtaaaagacttgaattcaggcctcattggagccctactagtatgtagagaagggagtctggccaaggaaaagacacaga
ccttgcacaaatttatactactttttgctgtatttgatgaagggaaaagttggcactcagaaacaaagaactccttgatgcaggatagggatgctg
catctgctcgggcctggcctaaaatgcacacagtcaatggttatgtaaacaggtctctgccaggtctgattggatgccacaggaaatcagtcta
ttggcatgtgattggaatgggcaccactcctgaagtgcactcaatattcctcgaaggtcacacatttcttgtgaggaaccatcgccaggcgtcc
ttggaaatctcgccaataactttccttactgctcaaacactcttgatggaccttggacagtttctactgttttgtcatatctcttcccaccaacatgat
ggcatggaagcttatgtcaaagtagacagctgtccagaggaaccccaactacgaatgaaaaataatgaagaagcggaagactatgatgatg
atcttactgattctgaaatggatgtggtcaggtttgatgatgacaactctccttcctttatccaaattcgctcagttgccaagaagcatcctaaaact
tgggtacattacattgctgctgaagaggaggactgggactatgctcccttagtcctcgcccccgatgacagaagttataaaagtcaatatttga
acaatggccctcagcggattggtaggaagtacaaaaaagtccgatttatggcatacacagatgaaacctttaagactcgtgaagctattcagc
atgaatcaggaatcttgggacctttactttatggggaagttggagacacactgttgattatatttaagaatcaagcaagcagaccatataacatct
accctcacggaatcactgatgtccgtcctttgtattcaaggagattaccaaaaggtgtaaaacatttgaaggattttccaattctgccaggagaa
atattcaaatataaatggacagtgactgtagaagatgggccaactaaatcagatcctcggtgcctgacccgctattactctagtttcgttaatatg
gagagagatctagcttcaggactcattggccctctcctcatctgctacaaagaatctgtagatcaaagaggaaaccagataatgtcagacaag
aggaatgtcatcctgttttctgtatttgatgagaaccgaagctggtacctcacagagaatatacaacgctttctccccaatccagctggagtgca
gcttgaggatccagagttccaagcctccaacatcatgcacagcatcaatggctatgtttttgatagtttgcagttgtcagtttgtttgcatgaggtg
gcatactggtacattctaagcattggagcacagactgacttcctttctgtcttcttctctggatataccttcaaacacaaaatggtctatgaagaca
cactcaccctattcccattctcaggagaaactgtcttcatgtcgatggaaaacccaggtctatggattctggggtgccacaactcagactttcgg
aacagaggcatgaccgccttactgaaggtttctagttgtgacaagaacactggtgattattacgaggacagttatgaagatatttcagcatactt
gctgagtaaaaacaatgccattgaaccaagaagcttctcccaaaacccaccagtcttgaaagggggagggggctctgaaataactcgtacta
ctcttcagtcagatcaagaggaaattgactatgatgataccatatcagttgaaatgaagaaggaagattttgacatttatgatgaggatgaaaat
cagagcccccgcagctttcaaaagaaaacacgacactattttattgctgcagtggagaggctctgggattatgggatgagtagctccccacat
gttctaagaaacagggctcagagtggcagtgtccctcagttcaagaaagttgttttccaggaatttactgatggctcctttactcagcccttatac
cgtggagaactaaatgaacatttgggactcctggggccatatataagagcagaagttgaagataatatcatggtaactttcagaaatcaggcct
ctcgtccctattccttctattctagccttatttcttatgaggaagatcagaggcaaggagcagaacctagaaaaaactttgtcaagcctaatgaaa
ccaaaacttacttttggaaagtgcaacatcatatggcacccactaaagatgagtttgactgcaaagcctgggcttatttctctgatgttgacctgg
aaaaagatgtgcactcaggcctgattggaccccttctggtctgccacactaacacactgaaccctgctcatgggagacaagtgacagtacag
gaatttgctctgtttttcaccatctttgatgagaccaaaagctggtacttcactgaaaatatggaaagaaactgcagggctccctgcaatatccag
atggaagatcccacttttaaagagaattatcgcttccatgcaatcaatggctacataatggatacactacctggcttagtaatggctcaggatca
aaggattcgatggtatctgctcagcatgggcagcaatgaaaacatccattctattcatttcagtggacatgtgttcactgtacgaaaaaaagag
gagtataaaatggcactgtacaatctctatccaggtgtttttgagacagtggaaatgttaccatccaaagctggaatttggcgggtggaatgcct
tattggcgagcatctacatgctgggatgagcacactttttctggtgtacagcaataagtgtcagactcccctgggaatggcttctggacacatta
gagattttcagattacagcttcaggacaatatggacagtgggccccaaagctggccagacttcattattccggatcaatcaatgcctggagca
ccaaggagcccttttcttggatcaaggtggatctgttggcaccaatgattattcacggcatcaagacccagggtgcccgtcagaagttctcca
gcctctacatctctcagtttatcatcatgtatagtcttgatgggaagaagtggcagacttatcgaggaaattccactggaaccttaatggtcttcttt
ggcaatgtggattcatctgggataaaacacaatatttttaaccctccaattattgctcgatacatccgtttgcacccaactcattatagcattcgca
gcactcttcgcatggagttgatgggctgtgatttaaatagttgcagcatgccattgggaatggagagtaaagcaatatcagatgcacagattac
tgcttcatcctactttaccaatatgtttgccacctggtctccttcaaaagctcgacttcacctccaagggaggagtaatgcctggagacctcaggt
gaataatccaaaagagtggctgcaagtggacttccagaagacaatgaaagtcacaggagtaactactcagggagtaaaatctctgcttacca
gcatgtatgtgaaggagttcctcatctccagcagtcaagatggccatcagtggactctcttttttcagaatggcaaagtaaaggtttttcaggga
aatcaagactccttcacacctgtggtgaactctctagacccaccgttactgactcgctaccttcgaattcacccccagagttgggtgcaccaga
ttgccctgaggatggaggttctgggctgcgaggcacaggacctctactgagcggccgcaataaaagatcagagctctagagatctgtgtgtt
ggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgccc
gacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L1-1-pA(SEQ ID NO:29)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgtctacatgcttcttcttgtgtctgctcagattttgcttctctgccacta
ggagatactacttgggagcagtggagctgtcctgggactacatgcagagtgaccttggggaactccctgtagatgcaagatttcctcccaga
gtccccaaaagtttcccctttaacacatcagttgtgtacaagaagaccctgtttgtggagtttacagaccacctgttcaacattgcaaagcccag
acctccatggatgggactcctaggcccaactatccaggcagaggtctatgatacagttgtcataaccttgaagaatatggcatctcatcctgtct
ccctgcatgctgttggagtcagctattggaaggcctctgaaggtgctgagtatgatgaccagactagccaaagagaaaaggaggatgacaa
agtgttccctggagggtctcacacatatgtttggcaggtgctgaaggagaatggacccatggccagtgaccctctgtgcttgacctacagttat
ctgtctcatgtagacctagtgaaggacctcaactcaggactcataggagcattacttgtgtgtagagaggggagcttagctaaggaaaaaacc
cagacacttcataagttcatccttctgtttgcagtttttgatgaggggaaaagttggcactcagagacaaaaaactctctgatgcaggacaggg
atgctgccagtgctagggcctggcctaagatgcacacagtaaatggatatgtgaacagatctctgccaggcctcataggttgccatagaaagt
ctgtttattggcatgtcataggaatggggactaccccagaggtgcactcaatctttctagaaggccacacattcttagtaagaaaccacaggca
agcatctttggagatcagtccaatcacattcctcacagcacaaactctcctgatggacctgggccagttcctcttgttctgccacattagctctca
ccagcatgatggaatggaggcatatgttaaagtagactcatgtccagaagagcctcagttaagaatgaaaaacaatgaagaagcagaagac
tatgatgatgacctgactgactctgagatggatgttgtgaggtttgatgatgataattctccctcctttatacaaataagatctgtagccaaaaagc
atcccaaaacttgggttcactacatagctgctgaggaagaagactgggattatgctcctctggtcctagcccctgatgacagatcctacaaaa
gtcagtacctcaataatggtcctcaaagaattggcaggaagtacaagaaagtcaggttcatggcttacactgatgagactttcaaaaccaggg
aggctatacaacatgagtctggtattctgggaccactgttatatggtgaggtgggagatacactgctgatcatctttaagaatcaggcctcaag
accatacaacatctatcctcatggtatcactgatgtaagacctctgtatagcagaagactgcccaaaggagttaagcaccttaaagattttcctat
ccttcctggagaaatcttcaaatacaaatggactgtcactgttgaggatggtcctactaaatcagaccccaggtgcctaaccagatactacagc
agttttgtgaatatggagagggacttagcttctggactgattgggccattgctgatctgctataaggagtctgtagatcagaggggcaatcagat
catgagtgacaagagaaatgtgattctgttctctgtgtttgatgagaataggagctggtatctgacagagaacatccagagattcctacccaatc
ctgctggggtacaattagaagaccctgagttccaggctagcaatatcatgcactctatcaatgggtatgtgtttgattctcttcagctgtctgtttg
cctacatgaggtagcctactggtacatcctttcaataggggctcagactgacttcctctcagtatttttctctggctacacttttaaacataagatgg
tgtatgaggacactctcacactgttccccttctcaggtgagactgtttttatgagtatggagaacccaggcctctggattttaggctgtcacaaca
gtgatttcaggaacagggggatgactgcactcctgaaggtgtcttcatgtgataagaacactggggactattatgaagattcctatgaggatatt
tctgcatatttgctctctaagaacaatgctattgaacctaggtctttttctcagaacccaccagtattaaagggtgggggaggatctgaaatcact
aggacaaccctgcaatcagaccaagaggaaattgactatgatgacacaatctctgtagagatgaagaaagaagattttgatatctatgatgaa
gatgagaatcagagtcctaggtcttttcagaaaaaaaccaggcactactttatagctgcagtagaaaggctgtgggattatggtatgtctagca
gtccacatgtgctgagaaacagagcccagtcaggcagtgtcccccagtttaagaaggtagttttccaggagttcactgatggcagtttcaccc
aacctctgtacagaggggagctgaatgaacacctgggactgttgggcccttatatcagagctgaggttgaggacaacatcatggtgaccttc
aggaatcaagcatctagaccctattctttctactcttccctcatctcctatgaagaggaccagagacaaggagcagaacctaggaaaaactttg
tcaaacctaatgagaccaagacttacttttggaaagttcagcaccatatggcccctacaaaggatgagtttgattgcaaggcatgggcttacttc
tcagatgtagatttagaaaaagatgtgcatagtggtttaataggtccattgctggtttgccacacaaacacactgaaccctgcccatggcaggc
aggtcacagtgcaagagtttgctctcttctttaccatatttgatgaaactaaatcttggtacttcacagaaaacatggaaagaaactgcagagctc
cctgcaacatacagatggaggatcccactttcaaggaaaactacaggtttcatgccatcaatggttacataatggacacactccctggcctggt
aatggctcaggaccaaaggataaggtggtaccttttaagcatgggatccaatgaaaatatccactcaatccacttttctggtcatgtgttcacag
tcagaaagaaagaagagtataagatggccctctataacctctacccaggggtatttgaaacagtggagatgctcccatctaaggcaggaattt
ggagagtggaatgcttgattggtgaacacctgcatgcaggtatgtcaactctattcctggtgtactctaataagtgccagactccactgggcat
ggcctctggtcacatcagagacttccagatcactgcctcaggccaatatggtcagtgggcccctaagctagccagattgcattattctgggtca
atcaatgcctggagcaccaaagagcctttctcttggatcaaagttgatcttctggctcctatgatcatccatgggatcaagactcagggagcca
ggcaaaagtttagctccctatacatttctcagtttataatcatgtattctctggatggcaaaaaatggcaaacatacaggggtaattctacaggaa
ccctcatggtgttctttgggaatgtagactcctcaggcataaagcataatatattcaaccctccaattatagcaaggtacatcagactgcatccaa
cacattatagcattagatctaccctgagaatggaactaatggggtgtgacctgaactcctgttctatgcctctgggtatggagtctaaggctatct
ctgatgctcaaatcactgcctcttcctatttcactaatatgtttgctacctggagcccaagcaaagccaggctgcacctgcagggcaggtctaat
gcctggagaccccaagttaacaaccccaaggaatggctgcaagttgacttccaaaagaccatgaaagtgacaggtgtcactacccaggga
gtaaaatccttgttgactagcatgtatgtaaaggagttcctcatctcttcttctcaagatggtcaccaatggactctgtttttccagaatggtaaggt
caaggtatttcaaggtaaccaagacagcttcaccccagtggtaaattccttagaccctcctctgttgactaggtatctcagaattcacccacaga
gctgggtccaccagattgctttgagaatggaagttctgggttgtgaggcccaagacctgtactgagcggccgcaataaaagatcagagctct
agagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcg
accaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L1-2-pA(SEQ ID NO:30)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctttccacctgttttttcctctgcctattgagattctgttttagtgccacca
gaagatactacctaggagctgtagaactgagctgggactatatgcagagtgacctgggagaactgcctgtagatgctagatttcctcctagag
tacctaaaagcttcccatttaacactagtgtggtttacaaaaaaactctctttgtagagtttactgaccacctcttcaacattgctaagcctaggcc
cccttggatgggactgttgggacccacaatccaggctgaagtctatgacactgtagtaatcacactaaagaacatggcctctcatccagtttct
cttcatgctgttggtgtgagttattggaaggctagtgaaggggctgagtatgatgaccaaacctcacagagagaaaaggaggatgacaaggt
gtttcctggggggtctcacacatatgtttggcaggtcttaaaagagaatggcccaatggcttctgatcccctgtgcttgacctacagctacttatc
acatgttgatttggtcaaggatctgaactctggcctcataggtgccctactggtatgtagggagggctccctggccaaagagaagactcagac
cttgcacaagttcatactactgtttgctgtgtttgatgagggtaaaagctggcattctgagaccaaaaactctctgatgcaagatagagatgcag
cctctgccagagcctggcctaagatgcacacagttaatgggtatgtaaacagatccttaccaggattgataggctgccataggaaaagtgtgt
actggcatgtcattgggatgggcaccaccccagaggtacactctattttccttgagggccacacattcttagtcagaaaccatagacaagcttc
attggagatttctcctattacttttctaacagcacagaccttactcatggacctagggcaatttttactgttctgccacatcagttcccaccagcatg
atggaatggaggcttatgtgaaggttgactcttgccctgaagaacctcagctgagaatgaagaacaatgaagaggctgaggattatgatgat
gacctgactgattcagagatggatgtagtcaggtttgatgatgataacagcccctccttcatacaaatcagatcagtagcaaagaagcatccaa
agacatgggtacactatatagcagctgaggaggaagattgggactatgctcctttagtgctggcccctgatgatagatcctacaagagccaat
acctgaacaatggcccccagaggattggtagaaaatacaaaaaggttaggttcatggcctatacagatgagacatttaagacaagggaggct
atccagcatgagtcaggaatcctaggtcctttattatatggggaagtgggggatactcttctcatcattttcaagaaccaggcttcaagaccttac
aacatctacccacatggcatcactgatgttaggcccctgtattctaggagactgccaaagggagtgaagcatctaaaggactttccaatcttac
caggtgagatattcaagtataaatggactgtgacagtagaggatggcccaactaaatcagaccctagatgtttgactagatactacagttccttt
gtcaacatggaaagagacttagcctctggtctcataggaccactcctcatctgctataaagagtctgtggatcagagaggcaatcaaattatga
gtgataagagaaatgtgatcctgttttctgtctttgatgagaataggtcttggtacttgacagaaaacatacagaggttccttcctaaccctgctgg
ggtacagctagaagacccagagtttcaggcctccaatattatgcatagtattaatggctatgtgtttgactccctgcagctatcagtgtgccttca
tgaagtggcctattggtatatactttctattggtgcccagacagacttcttatctgtcttcttttctggctatactttcaaacacaaaatggtttatgag
gacactctgacactttttcccttctctggtgagacagtttttatgtctatggaaaaccctggcttatggatcctgggctgccacaacagtgattttag
aaacagaggcatgactgcactgctaaaggtttcttcttgtgacaaaaatactggggactattatgaggattcctatgaagatatctctgcttacct
gctgagcaaaaataatgccattgaacctagaagcttctctcagaatcccccagtactcaaaggtggggggggctctgagatcactaggacaa
ctctccagagtgaccaagaggagattgattatgatgataccattagtgttgagatgaaaaaggaggactttgatatttatgatgaggatgaaaa
ccagtctcccagatctttccagaaaaaaacaagacattactttattgcagcagtagaaagattatgggactatggcatgtctagtagccctcatg
ttctgaggaacagggcccagagtggttcagtaccccaattcaagaaagtagtctttcaggaattcacagatggctcattcacacagcctctgta
taggggtgagcttaatgaacacctgggccttctaggcccctacatcagagctgaggtagaggacaatataatggtgactttcaggaatcagg
cttctaggccttactctttctactcatctctgatcagctatgaggaggaccaaagacaaggagcagagcctagaaagaactttgtgaagcccaa
tgaaactaaaacctacttttggaaggttcagcatcacatggctcccacaaaggatgaatttgattgtaaggcctgggcttatttttcagatgtgga
tttggaaaaggatgttcactctgggttaataggtcctctgctggtgtgccacacaaatactcttaatcctgcacatggtagacaagtgactgtgc
aggaatttgcactatttttcacaatttttgatgaaactaagagttggtattttacagaaaacatggagagaaattgcagggctccatgcaatatcca
aatggaagatcctactttcaaggaaaattataggttccatgctattaatggttatataatggatacactgccaggactggtgatggcccaagatc
aaagaattagatggtacctcttgtccatgggttctaatgaaaacattcactccatccacttcagtggacatgtatttacagtaagaaagaaagag
gaatacaaaatggcactctacaatctatacccaggagtgtttgagacagttgaaatgttgcccagtaaggcaggaatctggagggtagagtg
cctcattggtgaacacctgcatgctggcatgagtacccttttcttagtttacagtaacaagtgtcagacccccctggggatggccagtggacac
ataagagatttccagataacagcatctggtcagtatggtcagtgggctccaaaactggccagattgcactactcaggttcaatcaatgcatggt
ctactaaggaacctttcagttggataaaggtggacttgttagctcctatgatcattcatggcatcaaaacccagggagcaaggcagaagtttag
tagtctgtacatcagccaattcatcataatgtactctctggatggcaaaaagtggcagacatacagggggaattctactgggactctgatggtgt
tctttggcaatgttgactctagtggtatcaagcacaacatcttcaaccctcccattatagcaagatacatcagacttcatccaacccactactctat
cagatcaacattaaggatggaattgatgggctgtgaccttaattcttgctctatgcccctgggaatggaatccaaggcaatctcagatgcccag
atcacagctagcagctacttcaccaatatgtttgcaacttggtccccatctaaggccagactgcacctgcaaggcagatctaatgcctggagg
ccacaagtgaataatcctaaagaatggctgcaagtagactttcaaaaaaccatgaaggttactggtgttacaacacaaggtgtcaagtcactct
taacctctatgtatgtgaaagaatttctgattagttccagccaggatggtcaccagtggacccttttctttcagaatggcaaggtcaaggtctttca
aggaaaccaggatagttttacaccagtggttaattctctggatcctcctctccttacaagatacctaagaatccaccctcagagctgggtgcacc
agattgccctcagaatggaagtacttgggtgtgaagctcaagatctatactgagcggccgcaataaaagatcagagctctagagatctgtgtg
ttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc
ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L1-3-pA(SEQ ID NO:31)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcaaattgagctgagtacctgctttttcctatgcttgctaagattctgcttctctgcaaca
aggagatactatttaggagcagttgaactctcttgggattatatgcagtctgaccttggagaattacctgtagatgcaaggttccctccaagagt
gcccaagagcttcccttttaacacttctgttgtttacaagaagacactctttgtagagtttactgaccatctgttcaacattgctaagcctaggccc
ccatggatggggctcctgggcccaaccattcaagcagaggtctatgacacagtagtgattactttaaaaaacatggcttcccaccctgtcagc
ctgcatgcagtaggtgtgtcttattggaaggcctctgagggtgctgaatatgatgatcagactagtcaaagagagaaagaagatgacaaagtc
tttccaggtggatcacacacatatgtgtggcaggtgctgaaggagaatggtcctatggcttcagatcctttgtgtctgacttacagctacttatct
catgtggaccttgtcaaggacttaaacagtggactgataggggctctgctggtgtgcagagaggggagcctggcaaaggagaagactcaa
actttgcataaatttattctactgtttgctgtttttgatgaggggaagagttggcactctgaaactaagaacagccttatgcaggacagagatgct
gcttctgcaagagcctggcctaaaatgcacacagtaaatggatatgtgaacagatcccttccagggctcataggctgtcacagaaagagtgt
gtactggcatgtaatagggatgggcacaactcctgaggtgcatagcatatttctagaaggccacacattcctagttagaaatcatagacaagc
atctctagagatcagtcctatcacattcttaactgcacagaccctattgatggacttaggccagtttcttcttttttgccacattagctcccaccagc
atgatggcatggaggcctatgtcaaagtggactcatgccctgaagaaccccaactcaggatgaaaaacaatgaggaggctgaggactatga
tgatgacctgacagacagtgagatggatgttgtgaggtttgatgatgataacagcccctctttcatccagatcagatcagttgcaaagaaacac
ccaaagacttgggtgcactacattgctgctgaggaagaggactgggactatgctcctttagtgttggcaccagatgacaggagttacaaatcc
caatacctgaataatggtcctcagaggattggcagaaagtataagaaggtcaggttcatggcttacacagatgagacattcaagaccagaga
agctattcagcatgaatctggcattctggggcctttactctatggggaagtgggtgacacactactcattattttcaaaaatcaagcctccagac
cttacaacatctacccccatggaatcactgatgttaggcccctttacagcagaagactacccaagggggtgaaacaccttaaagattttccaat
cctgcctggagagattttcaaatacaaatggacagtcacagtggaagatggacctaccaagagtgatcctaggtgtttgactaggtactatagt
tcatttgtcaatatggagagagacttggcctcaggcctaatagggccacttctcatctgctataaggaatcagtagaccagagaggaaaccag
atcatgtcagacaaaaggaatgttatcctcttttcagtgtttgatgagaacaggtcttggtacctcacagaaaacatccagagatttttaccaaac
ccagcaggtgttcaattggaagaccctgaattccaagcctccaacatcatgcactcaataaatgggtatgtatttgactctctccagctcagtgt
gtgccttcatgaggtagcttattggtacatcttatctattggagctcagactgactttttatctgtgttttttagtggttatacattcaaacacaagatg
gtgtatgaagataccctgactttattccctttctctggagaaactgtgttcatgtctatggagaacccaggcctgtggattctggggtgtcacaat
agtgacttcagaaacagaggaatgacagcactgcttaaggtgagcagttgtgataagaatacaggagactactatgaggactcttatgagga
tatcagtgcatatctgctaagcaagaataatgcaattgaaccaagatctttcagccagaaccctcctgttctcaaagggggaggaggatcaga
aataaccaggacaactctccagtctgatcaggaagaaattgactatgatgacacaatctcagtggagatgaaaaaagaggactttgacatcta
tgatgaggatgaaaatcagtctcctaggtcatttcagaagaagaccaggcactactttatagcagctgtggaaagactatgggactatggtatg
agctcctccccccatgtgctgaggaatagagctcaaagtggctcagtgcctcagttcaagaaggtggtcttccaagaatttactgatgggagc
ttcacacagcccctgtatagaggggagttaaatgaacatctgggactactgggaccatacattagagcagaggtggaggacaacatcatggt
tactttcagaaaccaggccagtagaccatacagtttctacagctctctaatatcttatgaagaagatcaaagacaaggagcagaacccagaaa
gaactttgtaaaacctaatgagacaaagacctatttctggaaggtccagcaccacatggccccaaccaaggatgaatttgactgtaaggcatg
ggcttacttttctgatgtagatctggagaaggatgtgcacagtggcctaattggaccattactagtctgccataccaacacactcaatcctgcac
atggtagacaggtcacagtgcaagaatttgctctgtttttcaccatttttgatgaaactaaaagttggtactttactgagaacatggagaggaact
gcagagccccatgcaatatccagatggaagatccaactttcaaagagaattatagattccatgccataaatggttacattatggacacactgcc
tggcctggtcatggctcaggaccagaggatcagatggtatctcttgagtatgggtagcaatgagaacatacactccatccacttttcaggacat
gtctttacagtcagaaaaaaagaggagtataaaatggccttatacaacttatatccaggagtgtttgagacagtggagatgctccctagcaagg
caggtatctggagagtagaatgtctcataggggaacacctccatgctggtatgtcaacactgttcctggtgtatagtaacaagtgccaaacacc
actgggcatggccagtggccatattagagactttcaaataactgcctctgggcaatatggtcagtgggcccctaaactggctaggctgcacta
ctctgggtcaatcaatgcttggtcaaccaaagaaccattcagctggattaaggttgacctgctggctcctatgataattcatggcatcaaaaccc
agggagccagacaaaagttttcttccctgtacatctctcaatttataataatgtactctttggatggaaaaaaatggcagacttacagagggaact
caacagggaccctgatggtgttctttgggaatgtagactcttcaggtatcaaacataatattttcaaccctcccatcatagctagatatataagact
tcaccccacccactattctataaggtctactctcaggatggagttaatggggtgtgacttaaatagctgcagtatgcctctggggatggaaagc
aaggctatctctgatgcacagattacagctagcagctacttcactaatatgtttgctacatggtctccatctaaggcaagactgcatctccaggg
aagatctaatgcatggaggcctcaagtgaacaacccaaaagagtggctgcaagtggacttccaaaaaacaatgaaggttacaggagtcaca
acacagggggtgaaatctttactgacatcaatgtatgtaaaggagtttctcatttcaagtagtcaggatggacaccagtggactttattctttcag
aatggtaaggtcaaggtcttccaaggaaatcaggacagctttacaccagtggtcaacagcctggatcctcccttactcaccagatacctgagg
atccacccacagtcttgggttcaccaaattgctctgagaatggaagtgctgggatgtgaggcacaggacctctactgagcggccgcaataaa
agatcagagctctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcact
gaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggc
caa
蛋白表达框AAV8-0TE-hFVIII-L1-4-pA(SEQ ID NO:32)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgagcacctgcttcttcctgtgcctgctgagattctgcttctctgcca
ccagaagatactacctgggggctgtggagctgagctgggactacatgcagtctgacctgggggagctgcctgtggatgccagattcccccc
cagagtgcccaagagctttcccttcaacacctctgtggtgtacaagaagaccctgtttgtggaattcacagaccatctgttcaacattgcaaag
cccagacccccctggatgggcttgctgggccccaccatccaggctgaggtctatgacacagtggtgatcaccctgaagaacatggccagc
caccctgtgagcctgcatgctgtgggggtgagctactggaaagcttctgagggggctgagtatgatgaccagacctcccagagagagaag
gaggatgacaaggtgttccctgggggcagccatacctatgtgtggcaggtgctgaaagagaatggccccatggcctctgaccccctgtgcc
tgacctacagctacctgagccatgtggacctggtgaaagacctcaattctggcctgattggggccttgctggtgtgcagagagggcagcctg
gctaaggagaagacccagaccctgcacaagttcatattgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacag
cctgatgcaggacagagatgctgcctctgccagagcctggcctaaaatgcatacagtgaatggctatgtgaacagaagcctgcctggcctg
attggctgccacagaaagtctgtttactggcatgtgattggcatgggcaccacccctgaggtgcacagcatcttcctggagggccacaccttc
ctggtgagaaaccacaggcaggccagcctggagatcagccccatcaccttcctgacagcccagaccctgctgatggatctgggacagttc
ctgctgttctgtcacatctctagccaccagcatgatggcatggaggcctatgtgaaggtggactcctgccctgaggagcctcagctgagaatg
aagaacaatgaggaggctgaggactatgatgatgacctgacagactctgagatggatgtggtgagatttgatgatgacaacagccctagctt
catccagatcagatctgtggccaagaagcacccaaagacctgggtgcactacattgctgctgaggaagaggactgggactatgcccccctg
gtgctggcccctgatgacagaagctacaaaagccagtacctgaataatggcccccagagaattggcagaaagtacaagaaggtgagattc
atggcttacacagatgagaccttcaagaccagagaggccatccagcatgagtctggcatcttgggccctctgctgtatggggaggtggggg
acaccctgctgatcatcttcaagaatcaagccagcagaccttacaacatctacccacatggcatcacagatgtgagaccactgtacagcaga
agactgcccaaaggggtgaagcacctgaaggacttcccaatcctgcctggggagatcttcaagtataagtggacagtgacagtggaggatg
gccccactaagtctgaccccagatgcctgaccagatactacagcagctttgtgaacatggaaagagatctggcctctggcctgattggcccc
ctcctgatctgctacaaggagtctgtggaccagagaggcaatcagatcatgtctgacaagagaaatgtgatactgttctctgtgtttgatgaga
acagaagctggtacctcacagagaacattcagagattcctgcccaaccctgctggggtgcaactggaagaccctgagttccaggccagcaa
catcatgcacagcatcaatggctatgtctttgactccctgcagctgtctgtgtgtctgcatgaggtggcctactggtacatcttgagcattgggg
cccagacagacttcctgtctgtgttcttctctggctacacattcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctctggg
gagacagtgttcatgagcatggagaacccaggcctgtggatcctgggctgccacaactctgacttcaggaacagaggcatgacagccctct
tgaaggtgtccagctgtgacaagaacacaggggactactatgaggacagctatgaggacatatctgcctacctgctgagcaagaacaatgc
cattgagcccagatcttttagccagaacccacctgtgctgaaggggggggggggctctgagatcaccagaaccaccctccagtctgaccag
gaggagattgactatgatgacaccatctctgtggagatgaaaaaggaggactttgacatctatgatgaggatgagaaccagagcccaagatc
tttccagaagaagaccagacactacttcattgctgctgtggagagactgtgggactatggcatgagctctagcccccatgtgctgagaaacag
agcacagtctggctctgtgccccagttcaagaaggtggtgttccaggagttcacagatggcagcttcacccagcccctgtacagaggggag
ctgaatgagcacctgggcctcctgggcccctatatcagggctgaggtggaggacaacatcatggtgaccttcagaaaccaggccagcaga
ccctacagcttctatagcagcctgataagctatgaggaggatcagagacagggggctgagcctagaaagaactttgtgaagcccaatgaga
ccaaaacctacttctggaaggtgcagcaccacatggcccccaccaaggatgagtttgactgtaaggcctgggcctacttctctgatgtggac
ctggagaaagatgtgcactctggcctgattggccctctgctggtgtgccacaccaacaccttgaaccctgcccatggcagacaggtgacagt
gcaggagtttgccctgttcttcaccatctttgatgagaccaagagctggtacttcacagagaatatggagagaaattgtagagccccctgcaat
atccagatggaggaccccaccttcaaggagaactacagattccatgccatcaatggctacatcatggacaccctccctggcctggtcatggc
ccaggaccagagaatcagatggtatctgctgagcatgggcagcaatgagaatatccactccatccacttctctggccatgtgttcacagtgag
aaagaaggaggagtacaagatggctctgtacaacctgtaccctggggtgtttgagacagtggagatgctccccagcaaggctggcatctgg
agagtggagtgcctgattggggaacacctgcatgctggcatgagcaccctgttcctggtgtactccaacaagtgccaaacccccctgggcat
ggcctctggccacataagagacttccagatcacagcctctggccagtatggccagtgggcccccaagctggccagactgcactactctggc
agcatcaatgcctggagcactaaggagcccttcagctggatcaaggtggatctgctggcccccatgatcatccatggcatcaagacccagg
gggccagacagaagttcagcagcctgtacataagccagttcatcatcatgtacagcctggatggcaaaaagtggcagacctacagaggca
acagcacaggcaccctgatggtgttctttggcaatgtggacagctctggcatcaagcacaacatcttcaacccccccatcattgccagataca
tcagactgcaccccacccactacagcatcagaagcaccctgagaatggagctcatgggatgtgacctgaactcctgcagcatgcctctggg
catggaatctaaggccatctctgatgcccagattacagccagctcttacttcaccaacatgtttgccacctggagccctagcaaggccagact
ccacctgcagggcagaagcaatgcctggagaccccaggtgaacaaccccaaggagtggctgcaggtggacttccagaagaccatgaag
gtgacaggggtgaccacccagggggtgaagagcctgctgaccagcatgtatgtgaaggagttcctgatcagctctagccaggatggccac
cagtggacactgttcttccagaatggcaaggtgaaggtgttccaaggcaaccaggacagcttcacccctgtggtgaacagcttggaccccc
ccctgctgactaggtacctgagaatccacccccagagctgggtgcaccagattgccctgagaatggaagtcctgggctgtgaggcacagg
acctgtactgagcggccgcaataaaagatcagagctctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactc
cctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgag
cgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L1-5-pA(SEQ ID NO:33)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgagcacctgcttcttcctgtgcctgctgagattctgcttctctgcca
ccagaagatactacctgggagctgtggagctgagctgggattacatgcagtctgacctgggggagctgcctgtggatgccagattcccccc
cagagtgcccaagagcttccccttcaacacctctgtggtctacaagaagaccctgtttgtggaattcacagaccacttgtttaacatagccaag
cccagacctccctggatgggcctcctgggccccaccatccaggctgaggtctatgacacagtggtgatcacactgaagaacatggccagc
caccctgtgagcctccatgctgtgggggtgagctactggaaggcctctgagggggctgagtatgatgaccagacctctcagagagagaag
gaagatgacaaggtgttccctgggggcagccacacctatgtgtggcaggtgctgaaggagaatggccccatggcttctgaccccctgtgcc
tcacctacagctacctgagccatgtggacctggtgaaggacctgaactctggcctgataggggccctgctggtgtgcagagaaggcagcct
ggccaaggagaaaacccagaccctgcacaagttcatcctgctgtttgctgtgtttgatgaggggaagagctggcactctgagaccaagaac
agcctgatgcaggacagagatgctgcctctgccagagcctggcccaagatgcacacagtgaatggctatgtgaacagaagcctgcctggc
ctcattggctgccacagaaaatctgtgtactggcatgtgattggcatgggcaccacccctgaggtgcacagcatcttcctggagggccacac
cttcctggtgagaaaccacagacaggccagcctggagatcagccccatcaccttcctgacagcccagactctgctgatggacctgggccag
ttcctgctgttctgccacatcagcagccaccaacatgatggcatggaggcctatgtgaaggtggacagctgccctgaggagccccagctga
gaatgaaaaacaatgaggaggctgaggactatgatgatgacctgacagactctgagatggatgtggtgagatttgatgatgacaacagccc
cagcttcatccagatcagatctgtggccaagaagcaccccaaaacctgggtgcactacattgctgctgaggaagaggactgggactatgcc
cccctggtgctggcccctgatgacagaagctataagtcccagtacctcaacaatggcccccagagaataggcagaaagtacaagaaggtg
agattcatggcctacacagatgagaccttcaaaaccagagaagccatccagcatgagtctggcatcctgggccccctgctgtatggggagg
tgggggacaccctgctgatcatcttcaagaaccaagccagcagaccttacaacatctacccccatggcatcacagatgtgagacccctgtac
agcagaagactgccaaagggggtgaaacacctgaaggactttcccatcctgcctggggagatcttcaagtacaagtggacagtgacagtg
gaggatggccccaccaagtctgaccctagatgcctgaccagatactacagcagctttgtgaatatggagagagatctggcttctggcctgatt
ggccccctgctgatctgctacaaggagtctgtggaccaaagaggcaaccaaatcatgtctgacaagagaaatgttatcttgttctctgtgtttga
tgagaatagaagctggtacctgacagagaacatccagagattcctgcccaaccctgctggggtgcagctggaggaccctgagttccaggc
cagcaacatcatgcacagcatcaatggctatgtgtttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc
attggggcccagacagacttcctgtctgtgttcttctctggctacaccttcaagcacaagatggtgtatgaggacacactgaccctgttcccctt
ctctggggagacagtgttcatgagcatggagaaccctggcctgtggatcctgggctgccacaactctgacttcagaaacagaggcatgaca
gccctgctgaaggtcagcagctgtgacaagaacacaggggactactatgaggacagctatgaggacatttctgcctacctgctcagcaaga
acaatgccattgagcccagaagctttagccagaacccccctgtgctgaaggggggggggggctctgagatcaccagaaccaccttgcaat
ctgaccaagaggagattgactatgatgacactatctctgtggagatgaagaaggaggactttgacatctatgatgaggatgagaaccaaagc
cccagaagcttccagaaaaagaccagacactacttcattgctgctgtggagagactgtgggactatggcatgtccagcagcccccatgtgct
gagaaacagagcccagtctggctctgtgccccagttcaagaaggtggtgttccaggagttcacagatggcagcttcacccagcccctgtac
agaggggagctgaatgagcacctgggcctgctgggaccctacatcagagctgaggtggaagacaacattatggtgaccttcagaaatcag
gccagcagaccctacagcttctacagcagcctgatcagctatgaggaagatcagagacagggggctgagcccagaaagaactttgtgaag
cctaatgagaccaagacctacttctggaaggtgcagcatcacatggcccccaccaaggatgagtttgactgcaaggcctgggcctacttttct
gatgtggacctggagaaagatgtgcactctggcctgattggccccctgctggtgtgccacaccaacaccctgaaccctgcccatggcagac
aggtgacagtgcaggagtttgccctgttcttcactatttttgatgagaccaagtcctggtacttcactgagaatatggaaagaaactgtagagcc
ccctgcaacatccagatggaggaccccaccttcaaggagaactacagattccatgcaataaatggctacatcatggacaccctgcctggcct
ggtgatggcccaagaccagagaatcagatggtacctgctgagcatggggtctaatgagaatatccacagcatccacttctctggccatgtgtt
cacagtgaggaagaaagaggaatacaagatggccctgtacaacctctaccctggggtgtttgagacagtggagatgctgccctccaaggct
ggcatctggagagtggagtgtctgattggggagcacctgcatgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagaccc
ccctgggcatggcctctggccacataagagactttcaaattacagcctctggccagtatggccagtgggcccccaaactggccagactgca
ctactctggcagcatcaatgcctggagcaccaaggagcccttcagctggatcaaggtggatctgctggcccccatgatcatccatggcatca
agacccagggggccagacagaagttcagcagcctgtacataagccagttcatcatcatgtacagcctggatggcaagaagtggcagacct
acagaggcaacagcactggcaccctcatggtgttctttggcaatgtggacagctctggcattaagcacaacatcttcaacccccctatcattgc
cagatacatcagactgcatcccacccactacagcatcagaagcactctgagaatggaactgatgggctgtgacctcaacagctgcagcatg
cccctggggatggagagcaaggcaatctctgatgcccagatcacagccagcagctacttcaccaacatgtttgccacctggagcccctcca
aggctagactgcacctgcaaggaagaagcaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccagaa
gaccatgaaggtgacaggggtgaccacccagggggtgaagagcctgctgaccagcatgtatgtgaaggagttcctgatcagctctagcca
ggatggccaccaatggaccctgttcttccagaatggcaaggtcaaggtgttccagggcaaccaggatagcttcacccctgtggtgaacagc
ctggacccccccctgctgactagatacctgagaatccacccccagagctgggtgcaccagattgccctgagaatggaggtgctgggctgtg
aggcccaggacttgtactgagcggccgcaataaaagatcagagctctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggag
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L1-6-pA(SEQ ID NO:34)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgagcacctgcttctttctgtgcctgctgagattctgcttctctgccac
cagaagatactacctgggggctgtggaactgagctgggattacatgcagtctgacctgggggagctgcctgtggatgccagattccccccc
agagtgcccaagagcttccccttcaacacctctgtggtgtacaagaaaaccttgtttgtggagttcacagaccacctgttcaacattgccaagc
ccagacccccctggatgggcctgctgggccccactatccaggctgaggtgtatgacacagtggtgatcaccctgaagaatatggccagcca
ccctgtgagcctgcatgctgtgggggtgagctattggaaggcctctgagggggctgagtatgatgaccagaccagccagagagagaagg
aggatgacaaggtgttccctgggggcagccacacctatgtgtggcaggtgctgaaggaaaatggccccatggcctctgaccccctgtgcct
gacctacagctacctgagccatgtggacctggtgaaggacctgaactctgggctgattggggccctgctggtgtgtagagagggcagcctg
gccaaggagaagacccagaccctgcacaaattcatattgctgtttgctgtgtttgatgagggcaagagctggcattctgagaccaagaacag
cctcatgcaggacagagatgctgcctctgccagagcttggcccaaaatgcacacagtgaatggctatgtgaacagaagcctgccaggactg
attggctgccacagaaagtctgtgtactggcatgtgattggcatgggcacaacccctgaggtgcactctatcttcctggagggccataccttcc
tggtgagaaaccacagacaggccagcctggagatctcccccatcaccttcctgacagcccagacactgctgatggatctgggccaattcct
gctgttctgccacatcagcagccaccagcatgatggcatggaggcctatgtgaaggtggacagctgccctgaggagccccagctgagaat
gaagaacaatgaggaggctgaggattatgatgatgacctcacagactctgagatggatgtggtgagatttgatgatgacaactctcccagctt
catccagattagatctgtggccaagaagcaccccaagacctgggtccactacattgctgctgaggaagaggactgggactatgcccccctg
gtgctggcacctgatgacagatcctataagagccagtacctgaacaatggcccccagagaattggcagaaagtacaagaaggtgagattca
tggcctacacagatgagaccttcaagaccagagaggccatccagcatgagtctggcatcctgggccccctgctgtatggggaggtggggg
acaccctgctcatcatcttcaagaaccaggcctccagaccctataacatctacccccatggcatcacagatgtgagacccctgtacagcaga
agactgcccaagggggtgaagcacctgaaggacttccccattctgccaggggagatcttcaagtacaagtggacagtgacagtggaggat
ggccccaccaagtctgaccccagatgcctgaccagatactacagcagctttgtgaacatggagagagacctggcctctggcctgattggcc
ctctcctgatctgctacaaggagtctgtggaccagagaggcaaccagatcatgtctgacaagagaaatgtgatcctgttctctgtgtttgatga
gaatagaagctggtacctgacagagaacattcaaagattcctgcccaaccctgctggggtgcagctggaggaccctgagttccaggccagc
aacatcatgcacagcatcaatggctatgtgtttgactccctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattgg
ggcccagacagacttcctgtctgtgtttttctctggctataccttcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctctgg
ggagacagtgttcatgagcatggagaatcctggcctgtggatcctgggctgccacaactctgacttcagaaacagaggcatgacagccctg
ctgaaggtgagcagctgtgacaagaacacaggggactactatgaggacagctatgaggacatctctgcctacttgctgagcaagaacaatg
ccattgagcccagaagcttcagccagaacccccctgtgctgaaggggggggggggctctgagatcaccagaaccaccctgcaatctgac
caggaggaaattgactatgatgacaccatctctgtggaaatgaagaaggaagactttgacatctatgatgaggatgagaaccagagcccca
gaagcttccagaagaagaccagacactacttcattgctgctgtggagagactgtgggactatggcatgagctctagcccccatgtgctgaga
aacagagcccagtctggctctgtgccccaattcaagaaggtggtgttccaggagttcacagatggcagcttcacccagcccctgtacagag
gggagctgaatgagcacctgggcctgctgggcccctatatcagagctgaggtggaggataacatcatggtgacctttagaaatcaggccag
cagaccctacagcttttacagcagcctgatcagctatgaggaggaccagagacagggggctgagcccagaaagaactttgtgaagccaaa
tgagaccaagacctacttctggaaggtgcagcaccacatggcccccacaaaggatgagtttgactgcaaggcctgggcctacttctctgatg
tggacctggagaaggatgtgcactctggcctgattggccccctgttggtgtgccacaccaacaccctgaaccctgcccatggcagacaggtt
acagtgcaggagtttgccctgtttttcaccatttttgatgagaccaagtcttggtacttcacagagaatatggaaagaaactgtagagccccctg
caacatccagatggaggaccccaccttcaaggagaactacagatttcatgccattaatggctacatcatggacaccctgcctggcctggttat
ggcccaggaccagagaatcagatggtacctgctgagcatgggctctaatgagaacattcacagcatccacttctctggccatgtgttcacagt
gagaaagaaggaagagtacaagatggcactgtacaacttgtaccctggggtgtttgagacagtggaaatgctgcccagcaaggctggcatc
tggagagtggaatgtctcattggggagcacctgcatgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacacccctgg
gcatggcttctggccacatcagagacttccagatcacagcctctggccaatatggccagtgggcccccaagctggccagactccactattct
ggcagcatcaatgcctggagcaccaaggagcccttcagctggatcaaggtggacctgctggcccccatgatcatccatggcatcaagacc
cagggggccagacagaagttcagcagcctgtacatcagccagttcatcatcatgtacagcctggatggcaagaaatggcagacctacaga
ggcaacagcactggcaccctgatggtgttctttggcaatgtggacagctctggcatcaagcacaacatcttcaacccccccatcattgccaga
tatatcagactgcaccccacccactacagcattagaagcaccctgagaatggagctgatgggctgtgacctgaacagctgcagcatgcccct
gggcatggagtccaaggccatctctgatgcccaaatcacagccagcagctacttcaccaacatgtttgccacctggagccctagcaaggcc
agactgcacctgcagggcagaagcaatgcctggagaccccaggtgaacaaccccaaggagtggctgcaggtggacttccagaagaccat
gaaggtgacaggggtgaccacacagggggtgaagagcctcctgaccagcatgtatgtgaaggagttcctgattagctctagccaggatgg
ccaccagtggaccctgttcttccagaatggcaaggtgaaggtcttccagggcaaccaggacagcttcacccctgtggtgaacagcctggac
ccccctctcctgacaagatacctgagaatccacccccagagctgggtgcaccagattgccctgagaatggaggtgctgggctgtgaggccc
aggacctctactgagcggccgcaataaaagatcagagctctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttggcc
actccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag
cgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L1-pA(SEQ ID NO:35)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgccac
caggaggtattacctgggggctgtggagctgagctgggactatatgcagtctgacctgggggagctgcctgtggatgctaggttccccccc
agggtgcccaagagcttcccctttaacacttctgtggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacattgccaagcc
caggcccccctggatggggctgctggggcccaccatccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggccagcca
ccctgtgagcctgcatgctgtgggggtgagctactggaaggcttctgagggggctgagtatgatgaccagactagccagagggagaagga
ggatgacaaggtgtttcctgggggcagccatacctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctg
acctacagctacctgtctcatgtggacctggtgaaggacctgaactctggcctgattggggctctgctggtgtgtagggagggcagcctggc
taaggaaaagacccagaccctgcataagtttatcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcct
gatgcaggatagggatgctgcctctgccagggcttggcctaagatgcacactgtgaatgggtatgtgaataggagcctgcctggcctgattg
gctgccacaggaagtctgtgtactggcatgtgattgggatgggcaccacccctgaggtccatagcatcttcctggagggccacactttcctgg
tgaggaaccacagacaggcctctctggagatctctcccatcaccttcctgactgctcagactctgctgatggacctgggccagttcctgctgtt
ttgccatattagcagccaccagcatgatgggatggaggcctatgtgaaggtggatagctgccctgaggagcctcagctgaggatgaagaac
aatgaggaggctgaagactatgatgatgacctgactgattctgagatggatgtggtgaggtttgatgatgacaatagccccagcttcattcaga
tcaggtctgtggccaagaaacaccccaagacctgggtgcactacattgctgctgaggaagaggactgggactatgctcccctggtgctggc
ccctgatgataggtcttataagagccagtacctgaacaatgggccccagaggattggcaggaagtacaagaaggtgaggttcatggcctac
actgatgaaaccttcaaaaccagggaggccattcagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctgc
tgatcatcttcaagaaccaggccagcaggccctacaacatctatcctcatggcatcactgatgtgaggcccctgtacagcaggaggctgccc
aagggggtgaagcacctgaaagacttccccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatggccctaccaa
gtctgaccccaggtgtctgaccaggtactattctagctttgtgaacatggagagggacctggcctctggcctgattgggcccctgctgatctgc
tacaaggagtctgtggaccagaggggcaaccagatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgagaataggagctgg
tacctgactgagaacatccagaggtttctgcccaatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcatgcatagc
atcaatggctatgtgtttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagactgact
ttctgtctgtgttcttttctggctataccttcaagcacaagatggtgtatgaggataccctgaccctgttccccttctctggggagactgtgttcatg
agcatggagaatcctgggctgtggatcctggggtgccacaactctgattttaggaacagggggatgactgccctgctgaaggtgtctagctg
tgataagaacactggggactactatgaggacagctatgaggacatttctgcttatctgctgtctaagaataatgccattgagcccagaagcttc
agccagaatccccctgtgctgaaggggggagggggctctgagatcaccagaactaccctgcagtctgatcaggaggagattgactatgat
gacactatctctgtggagatgaagaaggaggactttgacatctatgatgaggatgagaatcagtctcccaggagctttcagaagaagaccag
acattacttcattgctgctgtggagaggctgtgggactatggcatgagctctagccctcatgtgctgaggaacagggcccagtctggctctgt
gccccagttcaagaaggtggtgttccaggaattcactgatggcagcttcacccagcccctgtacaggggggagctgaatgagcacctgggc
ctgctggggccttatatcagggctgaggtggaggataatattatggtgactttcaggaaccaggccagcaggccctactctttctatagcagc
ctgatctcttatgaggaggatcagaggcagggggctgagcctaggaagaactttgtgaagcccaatgagactaagacctacttctggaaggt
ccagcaccacatggcccctaccaaggatgagtttgactgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtccattctgg
gctgattggccccctgctggtgtgccacactaacactctgaatcctgcccatggcaggcaggtgactgtccaggagtttgccctgttcttcact
atctttgatgagaccaagagctggtactttactgagaacatggagaggaactgcagagctccttgcaatattcagatggaggaccccaccttc
aaggagaattacaggttccatgccattaatgggtacatcatggacaccctgcctggcctggtgatggctcaggaccagaggatcaggtggta
cctgctgagcatgggctctaatgagaatatccacagcatccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatgg
ctctgtataatctgtaccctggggtgtttgaaactgtggagatgctgccctctaaggctggcatctggagggtggagtgcctgattggggagc
acctgcatgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacccccctgggcatggcctctggccacatcagggacttc
cagatcactgcctctggccagtatggccagtgggcccccaagctggccaggctgcactattctggcagcatcaatgcctggagcaccaagg
agcccttcagctggatcaaggtggacctgctggcccccatgatcattcatggcatcaagacccagggggccaggcagaagttcagctctct
gtacatctctcagttcatcatcatgtactctctggatgggaagaagtggcagacctacaggggcaacagcactggcaccctgatggtgttcttt
gggaatgtggactcttctggcatcaagcacaacatcttcaatccccccatcattgctaggtatattaggctgcatcccacccactacagcatca
ggtctaccctgaggatggagctgatgggctgtgacctgaactcttgcagcatgcccctgggcatggagtctaaggccatctctgatgcccag
attactgccagcagctacttcaccaacatgtttgccacctggagcccctctaaggccaggctgcatctgcaggggaggagcaatgcctgga
ggcctcaggtgaacaaccccaaggagtggctgcaggtggatttccagaagaccatgaaggtgactggggtgaccacccagggggtcaag
agcctgctgaccagcatgtatgtgaaggagttcctgatcagcagcagccaggatggccaccagtggactctgttctttcagaatgggaaggt
gaaggtgtttcagggcaatcaggactctttcacccctgtggtgaacagcctggacccccccctgctgaccagatacctgaggatccaccccc
agtcttgggtgcatcagattgccctgaggatggaggtgctgggctgtgaggctcaggatctgtactgagcggccgcaataaaagatcagag
ctctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgg
gcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L1-8-pA(SEQ ID NO:36)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgagcacctgcttcttcctctgcctgctgaggttctgcttctctgcca
ctagaagatactacctgggggctgtggagctgagctgggactacatgcagtctgacctgggggagctgcctgtggatgccagattcccacc
cagggtgcccaagagcttccccttcaacacctctgtggtgtacaagaagaccctgtttgtggagtttacagaccacctgttcaatattgccaag
cccagacccccctggatggggctgctgggccccaccatccaggcagaggtgtatgacacagtggtgatcaccctgaagaacatggccag
ccaccctgtgagcctgcatgctgtgggggtgagctactggaaggcctctgagggggctgagtatgatgaccagaccagccagagagaga
aggaggatgacaaggtgttccctgggggcagccacacctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgt
gcctgacatacagctacctgagccatgtggacctggtgaaggatctcaattctggcctgattggggccctgctggtgtgcagagagggcag
cctggccaaggagaagacccagaccctgcacaagttcatcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaaga
acagcctgatgcaggacagagatgctgcctctgccagagcctggcccaagatgcacacagtgaatggctatgtgaacagaagcctgcctg
gcctgattggctgccatagaaagtctgtgtactggcatgtgattggcatgggcaccacccctgaggtgcacagcatcttcctggaaggccac
accttcctggtgagaaaccacagacaggccagcctggagatcagccccatcaccttcctgacagcccaaaccctgttgatggacctgggcc
agttcctgctgttctgtcacatcagctctcaccagcatgatggcatggaggcctatgtgaaggtggacagctgccctgaggagccccagctg
agaatgaagaacaatgaggaggctgaggactatgatgatgacctgacagactctgagatggatgtggtgagatttgatgatgacaacagcc
ccagcttcatccagatcagatctgtggccaagaagcaccccaagacctgggtgcattacattgctgctgaggaagaggactgggactatgc
ccccctggttctggcccctgatgacagaagctacaagtctcaatacctgaacaatggcccccagagaattggcagaaagtacaagaaggtg
agattcatggcctacacagatgagacctttaagaccagagaagccatccagcatgagtctggcatcctgggccccctcctgtatggggaggt
gggggacaccttgctgatcatcttcaagaaccaggccagcagaccctacaacatctacccccatggcatcacagatgtgagacccctgtac
agcagaagactccccaagggggtgaagcacctgaaggacttccccatcctgcctggggagatcttcaagtataagtggacagtgacagtg
gaggatggccccaccaagtctgaccccagatgcctgaccagatactacagcagctttgtgaacatggaaagagacctggcatctggcctca
ttggccctctgctgatttgctacaaggaatctgtggaccagagaggcaatcagatcatgtctgacaagagaaatgtgatcctgttctctgtgtttg
atgagaacagaagctggtacctgacagagaacatccaaaggttcctgcccaaccctgctggggtgcagctggaggaccctgagttccagg
cctctaatatcatgcacagcatcaatggctatgtgtttgacagcctccagctgtctgtgtgcctgcatgaggtggcctattggtacatcctgagc
attggggcccagacagacttcctgtctgtgttcttctctggctacaccttcaagcacaagatggtgtatgaggacaccctgaccctgttcccctt
ctctggggagacagtgttcatgagcatggagaaccctggcctgtggatcctgggctgccacaactctgacttcagaaacagagggatgact
gccctgctgaaggtgagcagctgtgacaagaacacaggggactactatgaggacagctatgaggacatctctgcttacctgctgagcaaga
acaatgccattgagcccagaagcttcagccagaacccccctgtgctgaaggggggggggggctctgagatcaccagaaccaccctgcaa
tctgaccaggaggagattgactatgatgacaccatctctgtggagatgaagaaggaggactttgacatctatgatgaggatgagaaccaatc
ccccagatccttccagaaaaagaccagacactactttattgctgctgtggagagactgtgggactatggcatgtccagcagcccccatgtgct
gagaaacagagcccagtctggctctgtgccccagttcaagaaggtggtgttccaggaattcacagatggcagcttcacccagcctctgtaca
gaggggagctgaatgagcacctgggcctgctgggcccctacatcagagctgaggtggaggacaacatcatggttaccttcagaaaccaag
cctctagaccctacagcttctacagcagcctgatcagctatgaggaagaccagagacagggggctgagcccagaaagaactttgtgaagc
ccaatgagaccaagacatacttctggaaggtgcagcaccacatggcccccaccaaggatgagtttgactgcaaggcctgggcctacttctct
gatgtggatctggaaaaggatgtgcattctggcctgattgggcctctgctggtgtgtcacaccaacaccctgaaccctgcccatggcagaca
ggtgacagtgcaggagtttgccctgttcttcaccatttttgatgagaccaagagctggtacttcacagagaacatggagagaaactgcagagc
cccctgcaacatccagatggaggatcccaccttcaaggagaactacaggttccatgccatcaatggctacatcatggacaccctccctggcc
tggttatggctcaggaccagagaatcagatggtatctgctgagcatgggcagcaatgagaatatccacagcatccacttctctggccatgtgtt
cacagtgagaaagaaggaggagtacaagatggccctgtacaacctgtaccctggggtgtttgagacagtggagatgctgcccagcaaggc
tggcatctggagagtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagaca
cccctgggcatggcctctggccatatcagagacttccagattacagcctctggccagtatggccagtgggcccccaagctggccagattgc
actactctggctccataaatgcctggagcaccaaggagcccttctcttggatcaaggtggacctgctggcccccatgatcatccatggcatca
agacccagggggccagacagaagttcagctccctgtacatcagccagttcatcatcatgtacagcctggatggcaagaagtggcagaccta
cagagggaactctacaggcaccctgatggtgttctttggcaatgtggacagctctggcatcaagcacaacatcttcaacccccccatcattgc
cagatacattagactgcaccccacccactacagcatcagaagcaccctgagaatggagctgatgggctgtgacctgaacagctgcagcatg
cccctgggcatggagagcaaggccatctctgatgcccagatcacagcctcctcttacttcaccaacatgtttgccacctggagcccctctaag
gccagactgcacctgcagggcagaagcaatgcctggagaccccaggtcaacaaccccaaggagtggctgcaggtggacttccagaaga
ctatgaaggtgacaggggtgaccacccaaggggtgaagagcctgctgaccagcatgtatgtgaaggagttcctgatcagctctagccagg
atggccaccagtggaccctgttcttccagaatggcaaggtgaaagttttccagggcaaccaggactccttcacccctgtggtgaacagcctg
gacccccccctgctgactagatacctgagaatccacccccagagctgggtgcatcagattgccctcagaatggaggtgctgggctgtgagg
cccaggacctgtactgagcggccgcaataaaagatcagagctctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttg
gccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagt
gagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L1-9-pA(SEQ ID NO:37)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgagcacatgcttcttcctgtgcctgctgaggttctgcttttcagctac
cagaaggtactacctgggtgctgtggagctgtcctgggactacatgcagtctgatctgggagagctgcctgtggatgccaggttccccccta
gggtgcctaagagcttccctttcaacacctctgtggtgtacaagaagacactgtttgttgagttcacagaccacctgttcaacatagctaagccc
agacccccctggatggggctgctgggccccaccattcaggctgaggtgtatgatactgttgtcatcaccctgaagaacatggcctcccaccc
tgtgtccctgcatgctgttggtgtgtcctactggaaggcttcagagggggctgagtatgatgaccagacctcccagagagaaaaggaggatg
ataaggtgtttccaggtgggtcccacacctatgtctggcaggtgctgaaggagaatggtcccatggcttctgatcctctgtgcctgacctactc
ctacctctcccatgtggacctggttaaggacctgaactctggcctgataggtgccctgctggtgtgcagagagggctccctggccaaggaga
agacccagaccctccacaagttcatcttgctgtttgctgtgtttgatgagggcaagtcttggcacagtgagacaaagaactccctgatgcagg
acagggatgctgcatcagctagagcttggcccaagatgcacactgtgaatggttatgtgaacaggtccctgcctggcctgattggttgccaca
gaaagtctgtctactggcatgttataggtatgggcaccaccccagaggtgcactccatctttctggaggggcacacattcctggtgagaaacc
acaggcaggcctctctggagatcagccccatcacctttctgacagctcagaccctgctgatggacctgggccagttcctgctcttctgccaca
tctcctcccaccagcatgatgggatggaggcctatgtgaaggtggatagctgccctgaggaaccccagctgaggatgaagaacaatgagg
aggcagaggactatgatgatgacctgactgattctgagatggatgtagtgaggtttgatgatgataactccccctctttcatccagatcaggtct
gtggccaagaagcaccctaagacctgggtgcactacattgctgctgaggaggaggactgggactatgctcccctggtgctggctccagatg
acaggagctacaagagccagtacctgaacaatggtccccagaggattggtaggaagtacaagaaggtgagattcatggcttacacagatga
gaccttcaagaccagggaggccatccagcatgagagtggtatcctgggtcccctgctgtatggtgaggtgggtgacaccctgctgatcatct
tcaagaaccaggcctccagaccctacaacatctaccctcatggtatcactgatgtgagacctctgtactctagaagactccccaagggggtga
agcacctgaaggactttcccatcctccctggtgaaatcttcaagtacaagtggactgtcactgttgaggatggccccaccaagagtgatccca
ggtgcctgaccaggtactactcctcctttgttaacatggagagagacctggcctctggtctgattggtcccctgctgatctgctacaaggagtct
gttgatcagagagggaaccagatcatgtcagataagaggaatgtgatcctgttctctgtgtttgatgagaacaggagctggtacctgactgag
aacatccagaggttcctgcccaaccctgctggtgtgcagctggaggaccctgagttccaggcctccaacatcatgcactctatcaatggttat
gtatttgatagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgtccattggtgctcagactgatttcctgtctgtgttctt
ctctggttacaccttcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctcaggtgagacagtgttcatgtccatggagaac
ccaggtctgtggatcctgggctgccacaactcagatttcaggaacagggggatgacagctctgctgaaggtgtcctcttgtgacaagaacac
aggtgactactatgaggactcctatgaggacatctctgcctacctgctgagcaagaacaatgccattgagcccaggagcttcagccagaacc
cacctgtcctgaagggggggggtggcagtgagatcactagaaccaccctgcagtcagatcaggaggagattgattatgatgacaccatctc
tgtggagatgaagaaggaggactttgatatttatgatgaggatgagaaccagagccccagatccttccagaagaagactagacactacttcat
agctgctgtggagaggctgtgggactatggtatgtcctctagccctcatgtgctgaggaacagggcccagtctggttctgtgccccagttcaa
gaaggtggtgttccaggagttcactgatggcagcttcacacagccactctacaggggtgagctcaatgagcacctgggactgctgggcccc
tacatcagggctgaggtggaggacaacatcatggtcaccttcagaaaccaggcctccagaccctacagcttctactccagcctgatctcctat
gaggaggaccagaggcagggtgctgagcccagaaagaactttgttaagcccaatgagaccaagacctacttctggaaggtgcagcacca
catggctcccaccaaggatgagtttgattgcaaggcctgggcctacttctctgatgtggacctggagaaggatgtgcacagtggtctgattgg
tcccctgctggtgtgccacaccaacaccctgaaccctgcccatggtaggcaggtgactgtccaggagtttgctctgttcttcaccatctttgatg
agacaaagagctggtatttcacagagaacatggagagaaactgcagagccccctgcaacatccagatggaggaccccaccttcaaggag
aactacagattccatgccatcaatggttacatcatggacaccctgcctggcctggtcatggcccaggaccagagaatcaggtggtacctgct
gtccatgggctctaatgagaacatccactccatccacttctctggtcatgtgttcacagtcagaaagaaggaggagtacaagatggccctgta
caacctgtaccctggtgtgtttgagactgtagagatgctgccctccaaggctgggatctggagagtggagtgcctgataggtgagcacctcc
atgctgggatgtccaccctgttcctggtctactccaataagtgccagacacccctgggcatggccagtggtcacatcagagacttccagatca
cagcttctggtcagtatggtcagtgggccccaaagctggccaggctccactactctggtagcatcaatgcatggagcacaaaggagcccttc
tcctggatcaaggtggacctgctggctcctatgatcatccatggtatcaagacccagggtgccagacagaagttctcctccctgtatatctctca
gttcatcatcatgtactccctggatggtaagaagtggcagacctacagggggaactccactggtaccctgatggtgttctttggtaatgtagact
ccagtggtatcaagcacaacatcttcaacccccccatcattgctaggtacattaggctgcaccccacccactactccatcaggagcaccctga
gaatggagctgatgggctgtgacctgaacagctgctccatgcccctgggcatggagtccaaggccatctcagatgcccagatcactgcctc
ctcctacttcaccaacatgtttgctacctggtccccctccaaggccaggctgcacctgcagggcagatccaatgcatggaggcctcaggtga
acaaccccaaggagtggctgcaggtggacttccagaagaccatgaaggtgacaggtgtgaccacccagggtgtcaagtcactgctgacct
ccatgtatgtgaaggagttcctcatctccagctcccaggatggtcaccagtggaccctgttcttccagaatggtaaggtgaaggtgttccagg
gcaaccaggactctttcacccctgtagtgaactccctggacccccccctgctgaccagatacctcaggatccatccccagtcttgggtgcacc
agattgctctgagaatggaggtgcttggctgtgaagcccaggacctgtactgagcggccgcaataaaagatcagagctctagagatctgtgt
gttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcg
cccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L1-10-pA(SEQ ID NO:38)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgtccacctgcttcttcctgtgcctgctgaggttctgtttcagtgctac
caggaggtactacctgggtgctgtggagctgtcctgggactatatgcagtctgacctgggtgaactgcctgtggatgctagattccctcccag
ggtgcccaagtccttccccttcaatacctctgttgtgtacaagaagaccctgtttgttgagttcactgatcacctgttcaacatagctaagcccag
acctccctggatgggcctgctgggccccaccatccaggcagaggtgtatgatactgtggtgattaccctgaagaacatggcctcccatcctgt
gagcctgcatgctgtgggggtgtcctactggaaggcctcagagggtgctgagtatgatgaccagacctcccagagagagaaggaggatga
caaggtgttccctggtggctcccacacctatgtctggcaggtgctgaaggagaatggtcccatggcctctgatcccctgtgtctgacatactct
tacctgagccatgtggatctggtcaaggacctgaactctggtcttataggtgctctgctggtgtgcagggaggggtctctggctaaggagaag
actcagaccctccacaaattcatcctgctgtttgctgtctttgatgaggggaagtcttggcattctgagaccaagaactccctgatgcaggaca
gagatgctgcttcagctagagcctggcccaagatgcacactgttaatggttatgtgaacaggtccctgcctggtctgattggctgccacagga
agtctgtctactggcatgtgattggtatgggcaccacacctgaggtgcactccatcttcttggagggacacaccttcctggtgaggaaccaca
ggcaggcatcattggagatcagccccatcaccttcctgacagctcagaccctgctcatggacctgggacagttcctgctgttctgccacatta
gctctcaccagcatgatggcatggaggcttatgtcaaggtggactcctgcccagaggagccccagctgaggatgaagaacaatgaggagg
cagaggactatgatgatgatctgacagattctgagatggatgtggtgaggtttgatgatgacaacagccccagcttcatccagatcaggtctgt
ggccaagaagcaccccaagacctgggtgcactacatagctgctgaggaggaggactgggactatgccccactggtgctggccccagatg
acaggagctacaagtcccagtacctgaacaatggccctcagaggataggtaggaagtacaagaaggtgaggttcatggcctacacagatg
agaccttcaagaccagggaggccatccagcatgagtcaggtatcctgggccccctgctgtatggtgaggtgggtgacaccctgctgatcat
cttcaagaaccaggcctccagaccctacaacatctaccctcatggtatcacagatgtgaggcccctgtactccaggaggctgcccaagggg
gtgaagcatctgaaggacttccccatcctgcctggtgagatcttcaagtacaagtggactgttactgtggaggatgggcctaccaagagtgat
cccagatgcctgaccagatactactccagctttgttaacatggagagggacctggcctcaggtctgattggtcccctcctgatctgctacaaag
agtctgtggaccagaggggcaaccagatcatgagtgataagaggaatgtaatcctgttctctgtctttgatgagaacaggtcctggtacctga
cagagaacatccagagattcctgcccaacccagctggtgtacagctggaggaccctgagttccaggcctccaacatcatgcactccatcaat
ggttatgtctttgattccctgcagctttctgtatgcctgcatgaggtggcatactggtacatcctctccattggtgctcagacagatttcctctctgt
gttcttcagtggttacaccttcaagcacaagatggtgtatgaggacaccctgaccctgttccccttctctggtgagactgtcttcatgagtatgga
gaaccctggtctgtggatcctgggctgccacaactcagatttcagaaacagggggatgacagctctgctgaaggtgtcttcatgtgacaaga
acacaggtgactactatgaggactcctatgaggacatctctgcctacctgctgagcaagaacaatgccattgagccaaggagcttcagccag
aacccccctgtgctgaagggggggggtggctctgagatcaccagaaccaccctccagtctgatcaggaggagattgattatgatgataccat
ctctgtggagatgaagaaggaggactttgatatctatgatgaggatgagaaccagagcccaaggagcttccagaagaagaccaggcacta
cttcattgctgctgtagagaggctctgggactatggtatgtcctctagcccccatgtgctgagaaacagggcccagagtggttctgtgcccca
gttcaagaaggtggtgttccaggagttcactgatgggagctttacccagcccctgtacaggggtgaactgaatgagcacctggggctgctgg
gcccctacatcagagctgaggtggaggacaacatcatggtgaccttcagaaaccaggcttccaggccctacagcttctactcctcactgatct
cctatgaggaggaccagaggcagggtgcagagcctaggaagaactttgttaagcctaatgagaccaagacctacttctggaaggtgcagc
accacatggcccctaccaaggatgagtttgattgcaaggcctgggcctacttctcagatgtggacctggagaaggatgtgcactcaggcctc
attggtcccctgctggtgtgccacaccaacaccctgaaccctgctcatggtagacaggtgactgtgcaggagtttgctctgttctttacaatcttt
gatgagaccaagagctggtacttcactgagaacatggagaggaactgcagggctccatgcaacatccagatggaggaccccaccttcaag
gagaactacagattccatgccatcaatggttacatcatggacacactgcctggccttgttatggcccaggaccagagaattaggtggtacctg
ctgtccatgggcagcaatgagaacatccactccatccacttctctggtcatgtcttcactgtgaggaagaaggaggagtacaagatggccctg
tacaacctgtacccaggtgtgtttgagactgtggagatgctgccctccaaggctggtatctggagggtggagtgcctgataggtgagcacct
gcatgcaggaatgagcaccctgttcctggtttactccaacaaatgccagacccccctgggcatggccagtggtcacatcagagacttccaga
tcactgcttctggtcagtatggtcagtgggcccccaagctggccaggctgcactactctggtagcatcaatgcttggtccaccaaggagccttt
cagctggatcaaggtggacctgctggcccccatgatcatccatggtatcaagacccagggggccaggcagaagttctccagcctgtacatc
tcccagttcatcattatgtactccctggatggcaagaagtggcagacctacagaggcaacagcactggtaccctgatggtgttctttggtaatgt
ggactcaagtggtatcaagcacaacatcttcaacccccccatcatagctagatacattagactgcaccccacccactactcaatcagaagcac
actgaggatggagctgatggggtgtgacctgaactcctgctccatgcccctgggcatggagagcaaggccatcagtgatgcccagatcaca
gctagctcctacttcaccaacatgtttgctacctggagccccagcaaggccaggctgcacctgcagggcaggagcaatgcttggagacccc
aggtgaacaaccccaaggagtggctgcaggtggacttccagaagaccatgaaggtgacaggtgtgaccacccagggtgttaagagcctg
ctgaccagtatgtatgtgaaggagtttctgatctcctcctcacaggatggtcaccagtggaccctcttcttccagaatggtaaggtgaaggtgtt
ccagggcaaccaggactccttcacccctgtggttaactccctggacccccctctgctgaccaggtacctgaggatccacccccagtcctggg
tgcaccagattgctctgaggatggaggtgctgggctgtgaggctcaggacctctactgagcggccgcaataaaagatcagagctctagaga
tctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaa
aggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-0TE-hFVIII-L2-pA(SEQ ID NO:39)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgccac
caggaggtattacctgggggctgtggagctgagctgggactatatgcagtctgacctgggggagctgcctgtggatgctaggttccccccc
agggtgcccaagagcttcccctttaacacttctgtggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacattgccaagcc
caggcccccctggatggggctgctggggcccaccatccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggccagcca
ccctgtgagcctgcatgctgtgggggtgagctactggaaggcttctgagggggctgagtatgatgaccagactagccagagggagaagga
ggatgacaaggtgtttcctgggggcagccatacctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctg
acctacagctacctgtctcatgtggacctggtgaaggacctgaactctggcctgattggggctctgctggtgtgtagggagggcagcctggc
taaggaaaagacccagaccctgcataagtttatcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcct
gatgcaggatagggatgctgcctctgccagggcttggcctaagatgcacactgtgaatgggtatgtgaataggagcctgcctggcctgattg
gctgccacaggaagtctgtgtactggcatgtgattgggatgggcaccacccctgaggtccatagcatcttcctggagggccacactttcctgg
tgaggaaccacagacaggcctctctggagatctctcccatcaccttcctgactgctcagactctgctgatggacctgggccagttcctgctgtt
ttgccatattagcagccaccagcatgatgggatggaggcctatgtgaaggtggatagctgccctgaggagcctcagctgaggatgaagaac
aatgaggaggctgaagactatgatgatgacctgactgattctgagatggatgtggtgaggtttgatgatgacaatagccccagcttcattcaga
tcaggtctgtggccaagaaacaccccaagacctgggtgcactacattgctgctgaggaagaggactgggactatgctcccctggtgctggc
ccctgatgataggtcttataagagccagtacctgaacaatgggccccagaggattggcaggaagtacaagaaggtgaggttcatggcctac
actgatgaaaccttcaaaaccagggaggccattcagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctgc
tgatcatcttcaagaaccaggccagcaggccctacaacatctatcctcatggcatcactgatgtgaggcccctgtacagcaggaggctgccc
aagggggtgaagcacctgaaagacttccccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatggccctaccaa
gtctgaccccaggtgtctgaccaggtactattctagctttgtgaacatggagagggacctggcctctggcctgattgggcccctgctgatctgc
tacaaggagtctgtggaccagaggggcaaccagatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgagaataggagctgg
tacctgactgagaacatccagaggtttctgcccaatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcatgcatagc
atcaatggctatgtgtttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagactgact
ttctgtctgtgttcttttctggctataccttcaagcacaagatggtgtatgaggataccctgaccctgttccccttctctggggagactgtgttcatg
agcatggagaatcctgggctgtggatcctggggtgccacaactctgattttaggaacagggggatgactgccctgctgaaggtgtctagctg
tgataagaacactggggactactatgaggacagctatgaggacatttctgcttatctgctgtctaagaataatgccattgagcccagaagcttc
agccagaatccccctgtgctgaaggccgccgctgccgagatcaccagaactaccctgcagtctgatcaggaggagattgactatgatgaca
ctatctctgtggagatgaagaaggaggactttgacatctatgatgaggatgagaatcagtctcccaggagctttcagaagaagaccagacatt
acttcattgctgctgtggagaggctgtgggactatggcatgagctctagccctcatgtgctgaggaacagggcccagtctggctctgtgcccc
agttcaagaaggtggtgttccaggaattcactgatggcagcttcacccagcccctgtacaggggggagctgaatgagcacctgggcctgct
ggggccttatatcagggctgaggtggaggataatattatggtgactttcaggaaccaggccagcaggccctactctttctatagcagcctgat
ctcttatgaggaggatcagaggcagggggctgagcctaggaagaactttgtgaagcccaatgagactaagacctacttctggaaggtccag
caccacatggcccctaccaaggatgagtttgactgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtccattctgggctg
attggccccctgctggtgtgccacactaacactctgaatcctgcccatggcaggcaggtgactgtccaggagtttgccctgttcttcactatcttt
gatgagaccaagagctggtactttactgagaacatggagaggaactgcagagctccttgcaatattcagatggaggaccccaccttcaagg
agaattacaggttccatgccattaatgggtacatcatggacaccctgcctggcctggtgatggctcaggaccagaggatcaggtggtacctg
ctgagcatgggctctaatgagaatatccacagcatccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggctctg
tataatctgtaccctggggtgtttgaaactgtggagatgctgccctctaaggctggcatctggagggtggagtgcctgattggggagcacctg
catgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacccccctgggcatggcctctggccacatcagggacttccagat
cactgcctctggccagtatggccagtgggcccccaagctggccaggctgcactattctggcagcatcaatgcctggagcaccaaggagcc
cttcagctggatcaaggtggacctgctggcccccatgatcattcatggcatcaagacccagggggccaggcagaagttcagctctctgtaca
tctctcagttcatcatcatgtactctctggatgggaagaagtggcagacctacaggggcaacagcactggcaccctgatggtgttctttgggaa
tgtggactcttctggcatcaagcacaacatcttcaatccccccatcattgctaggtatattaggctgcatcccacccactacagcatcaggtcta
ccctgaggatggagctgatgggctgtgacctgaactcttgcagcatgcccctgggcatggagtctaaggccatctctgatgcccagattactg
ccagcagctacttcaccaacatgtttgccacctggagcccctctaaggccaggctgcatctgcaggggaggagcaatgcctggaggcctca
ggtgaacaaccccaaggagtggctgcaggtggatttccagaagaccatgaaggtgactggggtgaccacccagggggtcaagagcctgc
tgaccagcatgtatgtgaaggagttcctgatcagcagcagccaggatggccaccagtggactctgttctttcagaatgggaaggtgaaggtg
tttcagggcaatcaggactctttcacccctgtggtgaacagcctggacccccccctgctgaccagatacctgaggatccacccccagtcttgg
gtgcatcagattgccctgaggatggaggtgctgggctgtgaggctcaggatctgtactgagcggccgcaataaaagatcagagctctagag
atctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgacca
aaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-TTM-hFVIII-L1-pA(SEQ ID NO:40)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtcactgggaggatgttgagtaagatggaa
aactactgatgacccttgcagagacagagtattaggacatgtttgaacaggggccgggcgatcagcaggtaggtctgtctgcacatttcgtag
agcgagtgttccgatactctaatctccctaggcaaggttcatatttgtgtaggttacttattctccttttgttgactaagtcaataatcagaatcagca
ggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagat
ccacaagctcctgaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttt
tcaggttgggccaccatgcagattgagctgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgccaccaggaggtattacctggg
ggctgtggagctgagctgggactatatgcagtctgacctgggggagctgcctgtggatgctaggttcccccccagggtgcccaagagcttc
ccctttaacacttctgtggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacattgccaagcccaggcccccctggatgg
ggctgctggggcccaccatccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgc
tgtgggggtgagctactggaaggcttctgagggggctgagtatgatgaccagactagccagagggagaaggaggatgacaaggtgtttcc
tgggggcagccatacctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacctacagctacctgtctc
atgtggacctggtgaaggacctgaactctggcctgattggggctctgctggtgtgtagggagggcagcctggctaaggaaaagacccaga
ccctgcataagtttatcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggatagggatg
ctgcctctgccagggcttggcctaagatgcacactgtgaatgggtatgtgaataggagcctgcctggcctgattggctgccacaggaagtct
gtgtactggcatgtgattgggatgggcaccacccctgaggtccatagcatcttcctggagggccacactttcctggtgaggaaccacagaca
ggcctctctggagatctctcccatcaccttcctgactgctcagactctgctgatggacctgggccagttcctgctgttttgccatattagcagcca
ccagcatgatgggatggaggcctatgtgaaggtggatagctgccctgaggagcctcagctgaggatgaagaacaatgaggaggctgaag
actatgatgatgacctgactgattctgagatggatgtggtgaggtttgatgatgacaatagccccagcttcattcagatcaggtctgtggccaag
aaacaccccaagacctgggtgcactacattgctgctgaggaagaggactgggactatgctcccctggtgctggcccctgatgataggtctta
taagagccagtacctgaacaatgggccccagaggattggcaggaagtacaagaaggtgaggttcatggcctacactgatgaaaccttcaaa
accagggaggccattcagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaacc
aggccagcaggccctacaacatctatcctcatggcatcactgatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacc
tgaaagacttccccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatggccctaccaagtctgaccccaggtgtct
gaccaggtactattctagctttgtgaacatggagagggacctggcctctggcctgattgggcccctgctgatctgctacaaggagtctgtgga
ccagaggggcaaccagatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgagaataggagctggtacctgactgagaacat
ccagaggtttctgcccaatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcatgcatagcatcaatggctatgtgttt
gacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagactgactttctgtctgtgttcttttct
ggctataccttcaagcacaagatggtgtatgaggataccctgaccctgttccccttctctggggagactgtgttcatgagcatggagaatcctg
ggctgtggatcctggggtgccacaactctgattttaggaacagggggatgactgccctgctgaaggtgtctagctgtgataagaacactggg
gactactatgaggacagctatgaggacatttctgcttatctgctgtctaagaataatgccattgagcccagaagcttcagccagaatccccctgt
gctgaaggggggagggggctctgagatcaccagaactaccctgcagtctgatcaggaggagattgactatgatgacactatctctgtggag
atgaagaaggaggactttgacatctatgatgaggatgagaatcagtctcccaggagctttcagaagaagaccagacattacttcattgctgct
gtggagaggctgtgggactatggcatgagctctagccctcatgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaagg
tggtgttccaggaattcactgatggcagcttcacccagcccctgtacaggggggagctgaatgagcacctgggcctgctggggccttatatc
agggctgaggtggaggataatattatggtgactttcaggaaccaggccagcaggccctactctttctatagcagcctgatctcttatgaggag
gatcagaggcagggggctgagcctaggaagaactttgtgaagcccaatgagactaagacctacttctggaaggtccagcaccacatggcc
cctaccaaggatgagtttgactgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtccattctgggctgattggccccctgc
tggtgtgccacactaacactctgaatcctgcccatggcaggcaggtgactgtccaggagtttgccctgttcttcactatctttgatgagaccaag
agctggtactttactgagaacatggagaggaactgcagagctccttgcaatattcagatggaggaccccaccttcaaggagaattacaggttc
catgccattaatgggtacatcatggacaccctgcctggcctggtgatggctcaggaccagaggatcaggtggtacctgctgagcatgggctc
taatgagaatatccacagcatccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggctctgtataatctgtaccct
ggggtgtttgaaactgtggagatgctgccctctaaggctggcatctggagggtggagtgcctgattggggagcacctgcatgctggcatga
gcaccctgttcctggtgtacagcaacaagtgccagacccccctgggcatggcctctggccacatcagggacttccagatcactgcctctggc
cagtatggccagtgggcccccaagctggccaggctgcactattctggcagcatcaatgcctggagcaccaaggagcccttcagctggatca
aggtggacctgctggcccccatgatcattcatggcatcaagacccagggggccaggcagaagttcagctctctgtacatctctcagttcatca
tcatgtactctctggatgggaagaagtggcagacctacaggggcaacagcactggcaccctgatggtgttctttgggaatgtggactcttctg
gcatcaagcacaacatcttcaatccccccatcattgctaggtatattaggctgcatcccacccactacagcatcaggtctaccctgaggatgga
gctgatgggctgtgacctgaactcttgcagcatgcccctgggcatggagtctaaggccatctctgatgcccagattactgccagcagctactt
caccaacatgtttgccacctggagcccctctaaggccaggctgcatctgcaggggaggagcaatgcctggaggcctcaggtgaacaaccc
caaggagtggctgcaggtggatttccagaagaccatgaaggtgactggggtgaccacccagggggtcaagagcctgctgaccagcatgt
atgtgaaggagttcctgatcagcagcagccaggatggccaccagtggactctgttctttcagaatgggaaggtgaaggtgtttcagggcaat
caggactctttcacccctgtggtgaacagcctggacccccccctgctgaccagatacctgaggatccacccccagtcttgggtgcatcagatt
gccctgaggatggaggtgctgggctgtgaggctcaggatctgtactgagcggccgcaataaaagatcagagctctagagatctgtgtgttg
gttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccg
acgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-HLP-hFVIII-L1-pA(SEQ ID NO:41)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtttgctgcttgcaatgtttgcccattttagg
gtggacacaggacgctgtggtttctgagccagggggcgactcagatcccagccagtggacttagcccctgtttgctcctccgataactgggg
tgaccttggttaatattcaccagcagcctcccccgttgcccctctggatccactgcttaaatacggacgaggacagggccctgtctcctcagct
tcaggcaccaccactgacctgggacagtgaatcgccaccatgcagattgagctgagcacctgcttcttcctgtgtctgctgaggttctgcttct
ctgccaccaggaggtattacctgggggctgtggagctgagctgggactatatgcagtctgacctgggggagctgcctgtggatgctaggttc
ccccccagggtgcccaagagcttcccctttaacacttctgtggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacattgc
caagcccaggcccccctggatggggctgctggggcccaccatccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggc
cagccaccctgtgagcctgcatgctgtgggggtgagctactggaaggcttctgagggggctgagtatgatgaccagactagccagaggga
gaaggaggatgacaaggtgtttcctgggggcagccatacctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctg
tgcctgacctacagctacctgtctcatgtggacctggtgaaggacctgaactctggcctgattggggctctgctggtgtgtagggagggcag
cctggctaaggaaaagacccagaccctgcataagtttatcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaa
cagcctgatgcaggatagggatgctgcctctgccagggcttggcctaagatgcacactgtgaatgggtatgtgaataggagcctgcctggc
ctgattggctgccacaggaagtctgtgtactggcatgtgattgggatgggcaccacccctgaggtccatagcatcttcctggagggccacact
ttcctggtgaggaaccacagacaggcctctctggagatctctcccatcaccttcctgactgctcagactctgctgatggacctgggccagttcc
tgctgttttgccatattagcagccaccagcatgatgggatggaggcctatgtgaaggtggatagctgccctgaggagcctcagctgaggatg
aagaacaatgaggaggctgaagactatgatgatgacctgactgattctgagatggatgtggtgaggtttgatgatgacaatagccccagcttc
attcagatcaggtctgtggccaagaaacaccccaagacctgggtgcactacattgctgctgaggaagaggactgggactatgctcccctggt
gctggcccctgatgataggtcttataagagccagtacctgaacaatgggccccagaggattggcaggaagtacaagaaggtgaggttcatg
gcctacactgatgaaaccttcaaaaccagggaggccattcagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggaca
ccctgctgatcatcttcaagaaccaggccagcaggccctacaacatctatcctcatggcatcactgatgtgaggcccctgtacagcaggagg
ctgcccaagggggtgaagcacctgaaagacttccccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatggccc
taccaagtctgaccccaggtgtctgaccaggtactattctagctttgtgaacatggagagggacctggcctctggcctgattgggcccctgct
gatctgctacaaggagtctgtggaccagaggggcaaccagatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgagaatagg
agctggtacctgactgagaacatccagaggtttctgcccaatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcat
gcatagcatcaatggctatgtgtttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccag
actgactttctgtctgtgttcttttctggctataccttcaagcacaagatggtgtatgaggataccctgaccctgttccccttctctggggagactgt
gttcatgagcatggagaatcctgggctgtggatcctggggtgccacaactctgattttaggaacagggggatgactgccctgctgaaggtgt
ctagctgtgataagaacactggggactactatgaggacagctatgaggacatttctgcttatctgctgtctaagaataatgccattgagcccag
aagcttcagccagaatccccctgtgctgaaggggggagggggctctgagatcaccagaactaccctgcagtctgatcaggaggagattga
ctatgatgacactatctctgtggagatgaagaaggaggactttgacatctatgatgaggatgagaatcagtctcccaggagctttcagaagaa
gaccagacattacttcattgctgctgtggagaggctgtgggactatggcatgagctctagccctcatgtgctgaggaacagggcccagtctg
gctctgtgccccagttcaagaaggtggtgttccaggaattcactgatggcagcttcacccagcccctgtacaggggggagctgaatgagca
cctgggcctgctggggccttatatcagggctgaggtggaggataatattatggtgactttcaggaaccaggccagcaggccctactctttctat
agcagcctgatctcttatgaggaggatcagaggcagggggctgagcctaggaagaactttgtgaagcccaatgagactaagacctacttct
ggaaggtccagcaccacatggcccctaccaaggatgagtttgactgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtc
cattctgggctgattggccccctgctggtgtgccacactaacactctgaatcctgcccatggcaggcaggtgactgtccaggagtttgccctgt
tcttcactatctttgatgagaccaagagctggtactttactgagaacatggagaggaactgcagagctccttgcaatattcagatggaggaccc
caccttcaaggagaattacaggttccatgccattaatgggtacatcatggacaccctgcctggcctggtgatggctcaggaccagaggatca
ggtggtacctgctgagcatgggctctaatgagaatatccacagcatccacttctctgggcatgtgttcactgtgaggaagaaggaggagtaca
agatggctctgtataatctgtaccctggggtgtttgaaactgtggagatgctgccctctaaggctggcatctggagggtggagtgcctgattgg
ggagcacctgcatgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacccccctgggcatggcctctggccacatcagg
gacttccagatcactgcctctggccagtatggccagtgggcccccaagctggccaggctgcactattctggcagcatcaatgcctggagca
ccaaggagcccttcagctggatcaaggtggacctgctggcccccatgatcattcatggcatcaagacccagggggccaggcagaagttca
gctctctgtacatctctcagttcatcatcatgtactctctggatgggaagaagtggcagacctacaggggcaacagcactggcaccctgatgg
tgttctttgggaatgtggactcttctggcatcaagcacaacatcttcaatccccccatcattgctaggtatattaggctgcatcccacccactaca
gcatcaggtctaccctgaggatggagctgatgggctgtgacctgaactcttgcagcatgcccctgggcatggagtctaaggccatctctgat
gcccagattactgccagcagctacttcaccaacatgtttgccacctggagcccctctaaggccaggctgcatctgcaggggaggagcaatg
cctggaggcctcaggtgaacaaccccaaggagtggctgcaggtggatttccagaagaccatgaaggtgactggggtgaccacccagggg
gtcaagagcctgctgaccagcatgtatgtgaaggagttcctgatcagcagcagccaggatggccaccagtggactctgttctttcagaatgg
gaaggtgaaggtgtttcagggcaatcaggactctttcacccctgtggtgaacagcctggacccccccctgctgaccagatacctgaggatcc
acccccagtcttgggtgcatcagattgccctgaggatggaggtgctgggctgtgaggctcaggatctgtactgagcggccgcaataaaaga
tcagagctctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgag
gccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa蛋白表达框AAV8-STM-hFVIII-L1-pA(SEQ ID NO:42)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgggggaggctgctggtgaatattaacca
aggtcaccccagttatcggaggagcaaacaggggctaagtccacgtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccct
aggcaaggttcatatttgtgtaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatca
gcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagctcctgaagaggtaagggt
ttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttgggccaccatgcagattgag
ctgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgccaccaggaggtattacctgggggctgtggagctgagctgggactatat
gcagtctgacctgggggagctgcctgtggatgctaggttcccccccagggtgcccaagagcttcccctttaacacttctgtggtgtacaagaa
gaccctgtttgtggagttcactgaccacctgttcaacattgccaagcccaggcccccctggatggggctgctggggcccaccatccaggctg
aggtgtatgacactgtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtgggggtgagctactggaaggcttct
gagggggctgagtatgatgaccagactagccagagggagaaggaggatgacaaggtgtttcctgggggcagccatacctatgtgtggca
ggtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacctacagctacctgtctcatgtggacctggtgaaggacctgaact
ctggcctgattggggctctgctggtgtgtagggagggcagcctggctaaggaaaagacccagaccctgcataagtttatcctgctgtttgctg
tgtttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggatagggatgctgcctctgccagggcttggcctaagat
gcacactgtgaatgggtatgtgaataggagcctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattgggatgggca
ccacccctgaggtccatagcatcttcctggagggccacactttcctggtgaggaaccacagacaggcctctctggagatctctcccatcacct
tcctgactgctcagactctgctgatggacctgggccagttcctgctgttttgccatattagcagccaccagcatgatgggatggaggcctatgt
gaaggtggatagctgccctgaggagcctcagctgaggatgaagaacaatgaggaggctgaagactatgatgatgacctgactgattctgag
atggatgtggtgaggtttgatgatgacaatagccccagcttcattcagatcaggtctgtggccaagaaacaccccaagacctgggtgcactac
attgctgctgaggaagaggactgggactatgctcccctggtgctggcccctgatgataggtcttataagagccagtacctgaacaatgggcc
ccagaggattggcaggaagtacaagaaggtgaggttcatggcctacactgatgaaaccttcaaaaccagggaggccattcagcatgagtct
ggcatcctgggccctctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaaccaggccagcaggccctacaacatctatc
ctcatggcatcactgatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacctgaaagacttccccatcctgcctgggg
agatctttaagtataagtggactgtgactgtggaggatggccctaccaagtctgaccccaggtgtctgaccaggtactattctagctttgtgaac
atggagagggacctggcctctggcctgattgggcccctgctgatctgctacaaggagtctgtggaccagaggggcaaccagatcatgtctg
acaagaggaatgtgatcctgttttctgtgtttgatgagaataggagctggtacctgactgagaacatccagaggtttctgcccaatcctgctggg
gtgcagctggaggatcctgagttccaggccagcaatatcatgcatagcatcaatggctatgtgtttgacagcctgcagctgtctgtgtgcctgc
atgaggtggcctactggtacatcctgagcattggggcccagactgactttctgtctgtgttcttttctggctataccttcaagcacaagatggtgt
atgaggataccctgaccctgttccccttctctggggagactgtgttcatgagcatggagaatcctgggctgtggatcctggggtgccacaact
ctgattttaggaacagggggatgactgccctgctgaaggtgtctagctgtgataagaacactggggactactatgaggacagctatgaggac
atttctgcttatctgctgtctaagaataatgccattgagcccagaagcttcagccagaatccccctgtgctgaaggggggagggggctctgag
atcaccagaactaccctgcagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggaggactttgacatctat
gatgaggatgagaatcagtctcccaggagctttcagaagaagaccagacattacttcattgctgctgtggagaggctgtgggactatggcat
gagctctagccctcatgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttccaggaattcactgatggca
gcttcacccagcccctgtacaggggggagctgaatgagcacctgggcctgctggggccttatatcagggctgaggtggaggataatattat
ggtgactttcaggaaccaggccagcaggccctactctttctatagcagcctgatctcttatgaggaggatcagaggcagggggctgagccta
ggaagaactttgtgaagcccaatgagactaagacctacttctggaaggtccagcaccacatggcccctaccaaggatgagtttgactgcaag
gcctgggcctatttctctgatgtggatctggagaaggatgtccattctgggctgattggccccctgctggtgtgccacactaacactctgaatcc
tgcccatggcaggcaggtgactgtccaggagtttgccctgttcttcactatctttgatgagaccaagagctggtactttactgagaacatggag
aggaactgcagagctccttgcaatattcagatggaggaccccaccttcaaggagaattacaggttccatgccattaatgggtacatcatggac
accctgcctggcctggtgatggctcaggaccagaggatcaggtggtacctgctgagcatgggctctaatgagaatatccacagcatccactt
ctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggctctgtataatctgtaccctggggtgtttgaaactgtggagatgctg
ccctctaaggctggcatctggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctggtgtacagcaacaa
gtgccagacccccctgggcatggcctctggccacatcagggacttccagatcactgcctctggccagtatggccagtgggcccccaagctg
gccaggctgcactattctggcagcatcaatgcctggagcaccaaggagcccttcagctggatcaaggtggacctgctggcccccatgatca
ttcatggcatcaagacccagggggccaggcagaagttcagctctctgtacatctctcagttcatcatcatgtactctctggatgggaagaagtg
gcagacctacaggggcaacagcactggcaccctgatggtgttctttgggaatgtggactcttctggcatcaagcacaacatcttcaatccccc
catcattgctaggtatattaggctgcatcccacccactacagcatcaggtctaccctgaggatggagctgatgggctgtgacctgaactcttgc
agcatgcccctgggcatggagtctaaggccatctctgatgcccagattactgccagcagctacttcaccaacatgtttgccacctggagcccc
tctaaggccaggctgcatctgcaggggaggagcaatgcctggaggcctcaggtgaacaaccccaaggagtggctgcaggtggatttcca
gaagaccatgaaggtgactggggtgaccacccagggggtcaagagcctgctgaccagcatgtatgtgaaggagttcctgatcagcagcag
ccaggatggccaccagtggactctgttctttcagaatgggaaggtgaaggtgtttcagggcaatcaggactctttcacccctgtggtgaacag
cctggacccccccctgctgaccagatacctgaggatccacccccagtcttgggtgcatcagattgccctgaggatggaggtgctgggctgt
gaggctcaggatctgtactgagcggccgcaataaaagatcagagctctagagatctgtgtgttggttttttgtgtaggaacccctagtgatgga
gttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctc
agtgagcgagcgagcgcgcagagagggagtggccaa
蛋白表达框AAV8-CTS-hFVIII-L1-pA(SEQ ID NO:43)
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgggggaggctgctggtgaatattaacca
agatcaccccagttaccggaggagcaaacagggactaagttcacgtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccct
aggcaaggttcatatttgtgtaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatca
gcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagctcctgaagaggtaagggt
ttaagttatcgttagttcgtgcaccattaatgtttaattacctggagcacctgcctgaaatcatttttttttcaggttgggccaccatgcagattgagc
tgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgccaccaggaggtattacctgggggctgtggagctgagctgggactatatg
cagtctgacctgggggagctgcctgtggatgctaggttcccccccagggtgcccaagagcttcccctttaacacttctgtggtgtacaagaag
accctgtttgtggagttcactgaccacctgttcaacattgccaagcccaggcccccctggatggggctgctggggcccaccatccaggctga
ggtgtatgacactgtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtgggggtgagctactggaaggcttctg
agggggctgagtatgatgaccagactagccagagggagaaggaggatgacaaggtgtttcctgggggcagccatacctatgtgtggcag
gtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacctacagctacctgtctcatgtggacctggtgaaggacctgaactct
ggcctgattggggctctgctggtgtgtagggagggcagcctggctaaggaaaagacccagaccctgcataagtttatcctgctgtttgctgtg
tttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggatagggatgctgcctctgccagggcttggcctaagatgc
acactgtgaatgggtatgtgaataggagcctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattgggatgggcacc
acccctgaggtccatagcatcttcctggagggccacactttcctggtgaggaaccacagacaggcctctctggagatctctcccatcaccttc
ctgactgctcagactctgctgatggacctgggccagttcctgctgttttgccatattagcagccaccagcatgatgggatggaggcctatgtga
aggtggatagctgccctgaggagcctcagctgaggatgaagaacaatgaggaggctgaagactatgatgatgacctgactgattctgagat
ggatgtggtgaggtttgatgatgacaatagccccagcttcattcagatcaggtctgtggccaagaaacaccccaagacctgggtgcactacat
tgctgctgaggaagaggactgggactatgctcccctggtgctggcccctgatgataggtcttataagagccagtacctgaacaatgggcccc
agaggattggcaggaagtacaagaaggtgaggttcatggcctacactgatgaaaccttcaaaaccagggaggccattcagcatgagtctgg
catcctgggccctctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaaccaggccagcaggccctacaacatctatcctc
atggcatcactgatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacctgaaagacttccccatcctgcctggggaga
tctttaagtataagtggactgtgactgtggaggatggccctaccaagtctgaccccaggtgtctgaccaggtactattctagctttgtgaacatg
gagagggacctggcctctggcctgattgggcccctgctgatctgctacaaggagtctgtggaccagaggggcaaccagatcatgtctgaca
agaggaatgtgatcctgttttctgtgtttgatgagaataggagctggtacctgactgagaacatccagaggtttctgcccaatcctgctggggtg
cagctggaggatcctgagttccaggccagcaatatcatgcatagcatcaatggctatgtgtttgacagcctgcagctgtctgtgtgcctgcatg
aggtggcctactggtacatcctgagcattggggcccagactgactttctgtctgtgttcttttctggctataccttcaagcacaagatggtgtatg
aggataccctgaccctgttccccttctctggggagactgtgttcatgagcatggagaatcctgggctgtggatcctggggtgccacaactctg
attttaggaacagggggatgactgccctgctgaaggtgtctagctgtgataagaacactggggactactatgaggacagctatgaggacattt
ctgcttatctgctgtctaagaataatgccattgagcccagaagcttcagccagaatccccctgtgctgaaggggggagggggctctgagatc
accagaactaccctgcagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggaggactttgacatctatgat
gaggatgagaatcagtctcccaggagctttcagaagaagaccagacattacttcattgctgctgtggagaggctgtgggactatggcatgag
ctctagccctcatgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttccaggaattcactgatggcagctt
cacccagcccctgtacaggggggagctgaatgagcacctgggcctgctggggccttatatcagggctgaggtggaggataatattatggtg
actttcaggaaccaggccagcaggccctactctttctatagcagcctgatctcttatgaggaggatcagaggcagggggctgagcctaggaa
gaactttgtgaagcccaatgagactaagacctacttctggaaggtccagcaccacatggcccctaccaaggatgagtttgactgcaaggcct
gggcctatttctctgatgtggatctggagaaggatgtccattctgggctgattggccccctgctggtgtgccacactaacactctgaatcctgcc
catggcaggcaggtgactgtccaggagtttgccctgttcttcactatctttgatgagaccaagagctggtactttactgagaacatggagagga
actgcagagctccttgcaatattcagatggaggaccccaccttcaaggagaattacaggttccatgccattaatgggtacatcatggacaccct
gcctggcctggtgatggctcaggaccagaggatcaggtggtacctgctgagcatgggctctaatgagaatatccacagcatccacttctctg
ggcatgtgttcactgtgaggaagaaggaggagtacaagatggctctgtataatctgtaccctggggtgtttgaaactgtggagatgctgccct
ctaaggctggcatctggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctggtgtacagcaacaagtg
ccagacccccctgggcatggcctctggccacatcagggacttccagatcactgcctctggccagtatggccagtgggcccccaagctggc
caggctgcactattctggcagcatcaatgcctggagcaccaaggagcccttcagctggatcaaggtggacctgctggcccccatgatcattc
atggcatcaagacccagggggccaggcagaagttcagctctctgtacatctctcagttcatcatcatgtactctctggatgggaagaagtggc
agacctacaggggcaacagcactggcaccctgatggtgttctttgggaatgtggactcttctggcatcaagcacaacatcttcaatcccccca
tcattgctaggtatattaggctgcatcccacccactacagcatcaggtctaccctgaggatggagctgatgggctgtgacctgaactcttgcag
catgcccctgggcatggagtctaaggccatctctgatgcccagattactgccagcagctacttcaccaacatgtttgccacctggagcccctct
aaggccaggctgcatctgcaggggaggagcaatgcctggaggcctcaggtgaacaaccccaaggagtggctgcaggtggatttccagaa
gaccatgaaggtgactggggtgaccacccagggggtcaagagcctgctgaccagcatgtatgtgaaggagttcctgatcagcagcagcca
ggatggccaccagtggactctgttctttcagaatgggaaggtgaaggtgtttcagggcaatcaggactctttcacccctgtggtgaacagcct
ggacccccccctgctgaccagatacctgaggatccacccccagtcttgggtgcatcagattgccctgaggatggaggtgctgggctgtgag
gctcaggatctgtactgagcggccgcaataaaagatcagagctctagagatctgtgtgttggttttttgtgtaggaacccctagtgatggagttg
gccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagt
gagcgagcgagcgcgcagagagggagtggccaa
本发明实施例还提供了一种宿主细胞，其包含本发明提供的所述表达载体。在一些实施方案中，所述宿主细胞生产由所述核酸编码的FVIII蛋白，并对生产的FVIII蛋白进行回收。这些由细胞生产的FVIII蛋白，可选地通过分离和/或纯化，用于制备相应的药物或用于给予有需要的受试者。在一个具体实施方案中，所述宿主细胞为HepG2细胞。
本发明实施例还提供了一种药物组合物，其包含本发明提供的所述凝血因子FVIII蛋白变体、所述核酸和/或所述表达载体，以及药学上可接受的辅料。所述药物组合物具有以下一种或多种特性：1)表现出细胞或动物中的表达增强；2)表现出增强的细胞分泌；3)表现出增强的活性，例如凝血增加。
本发明所述核酸、所述表达载体可结合进所述药物组合物中，用于体内或体外给药，或者递送至有需要的受试者。
本发明实施例提供的所述凝血因子FVIII蛋白变体、所述核酸和/或所述表达载体可用于表达凝血因子FVIII蛋白。本发明提供的所述凝血因子FVIII蛋白变体、所述核酸和/或所述表达载体还可以用于制备治疗需要凝血因子FVIII蛋白的疾病的药物。在一些实施方案中，所述需要凝血因子FVIII蛋白的疾病为：A型血友病、血管性血友病和相关创伤出血、受伤、血栓症、血小板减少症、中风、凝血病、弥散性血管内凝血(DIC)等。
本发明实施例提供的用途包括使其他化合物、试剂、药物、治疗方案、治疗方案、过程或补救减少需要或使用的用途。例如，对于凝血疾病，在有需要的受试者中，更少频率或更少剂量或停止给予凝血因子FVIII蛋白以补充受试者内生凝血因子的不足或缺陷中方面，本发明实施例提供的用途具有益处。
在一些实施方案中，所述有需要的受试者包括动物，如哺乳动物，例如人、非人的灵长类、家养动物(如狗、猫等)、家畜(如鸡、鸭、马、牛、山羊、绵羊、猪等)、实验动物(如小鼠、大鼠、兔子、豚鼠)等。
在另一个方面，本发明提供了一种生产凝血因子FVIII蛋白的方法，其包括：
S1、在细胞中表达本发明提供的所述核酸和/或所述表达载体；
S2、回收所述细胞产生的凝血因子FVIII蛋白。
具体地，S1中，所述细胞可以是上述的宿主细胞。使所述细胞表达所述核酸和/或所述表达载体的方法可以是本领域已知的方法。S2中，所述回收方法也可以是本领域已知的方法，此处不再赘述。
以下结合具体实施例来对本发明进行进一步描述。然而，下述具体实施例仅用于解释但不以任何方式限制本发明的范围。
实验材料和方法
hFVIII表达盒和AAV载体
在本发明实施例中使用的FVIII基因野生型和密码子优化的DNA序列由金斯瑞或金唯智合成。AAV载体介导的hFVIII表达盒如图1所示。表达盒序列克隆至穿梭质粒载体获得含AAV载体介导的表达hFVIII目的基因的穿梭质粒，本发明实施例中涉及的hFVIII穿梭质粒如表2所示。
表2
其中，hFVIII-SQ的密码子优化序列(hFVIII-SQ-7)为如SEQ ID NO:49所示的序列，AAV8-0TE-hFVIII-SQ-pA的序列如SEQ ID NO:50所示。
hFVIII-SQ-7(SEQ ID NO:49)：
atgcagattgagctgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgccaccaggaggtattacctgggggctgtggagctgag
ctgggactatatgcagtctgacctgggggagctgcctgtggatgctaggttcccccccagggtgcccaagagcttcccctttaacacttctgt
ggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacattgccaagcccaggcccccctggatggggctgctggggccca
ccatccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtgggggtgagcta
ctggaaggcttctgagggggctgagtatgatgaccagactagccagagggagaaggaggatgacaaggtgtttcctgggggcagccata
cctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacctacagctacctgtctcatgtggacctggtga
aggacctgaactctggcctgattggggctctgctggtgtgtagggagggcagcctggctaaggaaaagacccagaccctgcataagtttatc
ctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggatagggatgctgcctctgccagggc
ttggcctaagatgcacactgtgaatgggtatgtgaataggagcctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgatt
gggatgggcaccacccctgaggtccatagcatcttcctggagggccacactttcctggtgaggaaccacagacaggcctctctggagatct
ctcccatcaccttcctgactgctcagactctgctgatggacctgggccagttcctgctgttttgccatattagcagccaccagcatgatgggatg
gaggcctatgtgaaggtggatagctgccctgaggagcctcagctgaggatgaagaacaatgaggaggctgaagactatgatgatgacctg
actgattctgagatggatgtggtgaggtttgatgatgacaatagccccagcttcattcagatcaggtctgtggccaagaaacaccccaagacc
tgggtgcactacattgctgctgaggaagaggactgggactatgctcccctggtgctggcccctgatgataggtcttataagagccagtacctg
aacaatgggccccagaggattggcaggaagtacaagaaggtgaggttcatggcctacactgatgaaaccttcaaaaccagggaggccatt
cagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctgctgatcatcttcaagaaccaggccagcaggccct
acaacatctatcctcatggcatcactgatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacctgaaagacttccccat
cctgcctggggagatctttaagtataagtggactgtgactgtggaggatggccctaccaagtctgaccccaggtgtctgaccaggtactattct
agctttgtgaacatggagagggacctggcctctggcctgattgggcccctgctgatctgctacaaggagtctgtggaccagaggggcaacc
agatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgagaataggagctggtacctgactgagaacatccagaggtttctgccc
aatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcatgcatagcatcaatggctatgtgtttgacagcctgcagctg
tctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagactgactttctgtctgtgttcttttctggctataccttcaagc
acaagatggtgtatgaggataccctgaccctgttccccttctctggggagactgtgttcatgagcatggagaatcctgggctgtggatcctgg
ggtgccacaactctgattttaggaacagggggatgactgccctgctgaaggtgtctagctgtgataagaacactggggactactatgaggac
agctatgaggacatttctgcttatctgctgtctaagaataatgccattgagcccagaagcttcagccagaatccccctgtgctgaagagacatc
agagggagatcaccagaactaccctgcagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggaggacttt
gacatctatgatgaggatgagaatcagtctcccaggagctttcagaagaagaccagacattacttcattgctgctgtggagaggctgtgggac
tatggcatgagctctagccctcatgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttccaggaattcact
gatggcagcttcacccagcccctgtacaggggggagctgaatgagcacctgggcctgctggggccttatatcagggctgaggtggaggat
aatattatggtgactttcaggaaccaggccagcaggccctactctttctatagcagcctgatctcttatgaggaggatcagaggcagggggct
gagcctaggaagaactttgtgaagcccaatgagactaagacctacttctggaaggtccagcaccacatggcccctaccaaggatgagtttga
ctgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtccattctgggctgattggccccctgctggtgtgccacactaacact
ctgaatcctgcccatggcaggcaggtgactgtccaggagtttgccctgttcttcactatctttgatgagaccaagagctggtactttactgagaa
catggagaggaactgcagagctccttgcaatattcagatggaggaccccaccttcaaggagaattacaggttccatgccattaatgggtacat
catggacaccctgcctggcctggtgatggctcaggaccagaggatcaggtggtacctgctgagcatgggctctaatgagaatatccacagc
atccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggctctgtataatctgtaccctggggtgtttgaaactgtgg
agatgctgccctctaaggctggcatctggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctggtgtac
agcaacaagtgccagacccccctgggcatggcctctggccacatcagggacttccagatcactgcctctggccagtatggccagtgggcc
cccaagctggccaggctgcactattctggcagcatcaatgcctggagcaccaaggagcccttcagctggatcaaggtggacctgctggccc
ccatgatcattcatggcatcaagacccagggggccaggcagaagttcagctctctgtacatctctcagttcatcatcatgtactctctggatgg
gaagaagtggcagacctacaggggcaacagcactggcaccctgatggtgttctttgggaatgtggactcttctggcatcaagcacaacatct
tcaatccccccatcattgctaggtatattaggctgcatcccacccactacagcatcaggtctaccctgaggatggagctgatgggctgtgacct
gaactcttgcagcatgcccctgggcatggagtctaaggccatctctgatgcccagattactgccagcagctacttcaccaacatgtttgccacc
tggagcccctctaaggccaggctgcatctgcaggggaggagcaatgcctggaggcctcaggtgaacaaccccaaggagtggctgcaggt
ggatttccagaagaccatgaaggtgactggggtgaccacccagggggtcaagagcctgctgaccagcatgtatgtgaaggagttcctgatc
agcagcagccaggatggccaccagtggactctgttctttcagaatgggaaggtgaaggtgtttcagggcaatcaggactctttcacccctgtg
gtgaacagcctggacccccccctgctgaccagatacctgaggatccacccccagtcttgggtgcatcagattgccctgaggatggaggtgc
tgggctgtgaggctcaggatctgtactga
AAV8-0TE-hFVIII-SQ-pA(SEQ ID NO:50)：
ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca
gtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgt
tccgatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgttgactaagtcaataatcagaatcagcaggtttggag
tcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagct
cctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacat
ccactttttctttttctccacaggtttaaacgccaccatgcagattgagctgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgccac
caggaggtattacctgggggctgtggagctgagctgggactatatgcagtctgacctgggggagctgcctgtggatgctaggttccccccc
agggtgcccaagagcttcccctttaacacttctgtggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacattgccaagcc
caggcccccctggatggggctgctggggcccaccatccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggccagcca
ccctgtgagcctgcatgctgtgggggtgagctactggaaggcttctgagggggctgagtatgatgaccagactagccagagggagaagga
ggatgacaaggtgtttcctgggggcagccatacctatgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctg
acctacagctacctgtctcatgtggacctggtgaaggacctgaactctggcctgattggggctctgctggtgtgtagggagggcagcctggc
taaggaaaagacccagaccctgcataagtttatcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcct
gatgcaggatagggatgctgcctctgccagggcttggcctaagatgcacactgtgaatgggtatgtgaataggagcctgcctggcctgattg
gctgccacaggaagtctgtgtactggcatgtgattgggatgggcaccacccctgaggtccatagcatcttcctggagggccacactttcctgg
tgaggaaccacagacaggcctctctggagatctctcccatcaccttcctgactgctcagactctgctgatggacctgggccagttcctgctgtt
ttgccatattagcagccaccagcatgatgggatggaggcctatgtgaaggtggatagctgccctgaggagcctcagctgaggatgaagaac
aatgaggaggctgaagactatgatgatgacctgactgattctgagatggatgtggtgaggtttgatgatgacaatagccccagcttcattcaga
tcaggtctgtggccaagaaacaccccaagacctgggtgcactacattgctgctgaggaagaggactgggactatgctcccctggtgctggc
ccctgatgataggtcttataagagccagtacctgaacaatgggccccagaggattggcaggaagtacaagaaggtgaggttcatggcctac
actgatgaaaccttcaaaaccagggaggccattcagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctgc
tgatcatcttcaagaaccaggccagcaggccctacaacatctatcctcatggcatcactgatgtgaggcccctgtacagcaggaggctgccc
aagggggtgaagcacctgaaagacttccccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatggccctaccaa
gtctgaccccaggtgtctgaccaggtactattctagctttgtgaacatggagagggacctggcctctggcctgattgggcccctgctgatctgc
tacaaggagtctgtggaccagaggggcaaccagatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgagaataggagctgg
tacctgactgagaacatccagaggtttctgcccaatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcatgcatagc
atcaatggctatgtgtttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagcattggggcccagactgact
ttctgtctgtgttcttttctggctataccttcaagcacaagatggtgtatgaggataccctgaccctgttccccttctctggggagactgtgttcatg
agcatggagaatcctgggctgtggatcctggggtgccacaactctgattttaggaacagggggatgactgccctgctgaaggtgtctagctg
tgataagaacactggggactactatgaggacagctatgaggacatttctgcttatctgctgtctaagaataatgccattgagcccagaagcttc
agccagaatccccctgtgctgaagagacatcagagggagatcaccagaactaccctgcagtctgatcaggaggagattgactatgatgaca
ctatctctgtggagatgaagaaggaggactttgacatctatgatgaggatgagaatcagtctcccaggagctttcagaagaagaccagacatt
acttcattgctgctgtggagaggctgtgggactatggcatgagctctagccctcatgtgctgaggaacagggcccagtctggctctgtgcccc
agttcaagaaggtggtgttccaggaattcactgatggcagcttcacccagcccctgtacaggggggagctgaatgagcacctgggcctgct
ggggccttatatcagggctgaggtggaggataatattatggtgactttcaggaaccaggccagcaggccctactctttctatagcagcctgat
ctcttatgaggaggatcagaggcagggggctgagcctaggaagaactttgtgaagcccaatgagactaagacctacttctggaaggtccag
caccacatggcccctaccaaggatgagtttgactgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtccattctgggctg
attggccccctgctggtgtgccacactaacactctgaatcctgcccatggcaggcaggtgactgtccaggagtttgccctgttcttcactatcttt
gatgagaccaagagctggtactttactgagaacatggagaggaactgcagagctccttgcaatattcagatggaggaccccaccttcaagg
agaattacaggttccatgccattaatgggtacatcatggacaccctgcctggcctggtgatggctcaggaccagaggatcaggtggtacctg
ctgagcatgggctctaatgagaatatccacagcatccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggctctg
tataatctgtaccctggggtgtttgaaactgtggagatgctgccctctaaggctggcatctggagggtggagtgcctgattggggagcacctg
catgctggcatgagcaccctgttcctggtgtacagcaacaagtgccagacccccctgggcatggcctctggccacatcagggacttccagat
cactgcctctggccagtatggccagtgggcccccaagctggccaggctgcactattctggcagcatcaatgcctggagcaccaaggagcc
cttcagctggatcaaggtggacctgctggcccccatgatcattcatggcatcaagacccagggggccaggcagaagttcagctctctgtaca
tctctcagttcatcatcatgtactctctggatgggaagaagtggcagacctacaggggcaacagcactggcaccctgatggtgttctttgggaa
tgtggactcttctggcatcaagcacaacatcttcaatccccccatcattgctaggtatattaggctgcatcccacccactacagcatcaggtcta
ccctgaggatggagctgatgggctgtgacctgaactcttgcagcatgcccctgggcatggagtctaaggccatctctgatgcccagattactg
ccagcagctacttcaccaacatgtttgccacctggagcccctctaaggccaggctgcatctgcaggggaggagcaatgcctggaggcctca
ggtgaacaaccccaaggagtggctgcaggtggatttccagaagaccatgaaggtgactggggtgaccacccagggggtcaagagcctgc
tgaccagcatgtatgtgaaggagttcctgatcagcagcagccaggatggccaccagtggactctgttctttcagaatgggaaggtgaaggtg
tttcagggcaatcaggactctttcacccctgtggtgaacagcctggacccccccctgctgaccagatacctgaggatccacccccagtcttgg
gtgcatcagattgccctgaggatggaggtgctgggctgtgaggctcaggatctgtactgagcggccgcaataaaagatcagagctctagag
atctgtgtgttggttttttgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgacca
aaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa
AAV8载体生产及纯化方法
AAV8载体的生产采用三质粒系统，即用含hFVIII目的基因的穿梭质粒、含AAV载体rep2，cap8基因的pAAV-R2C8-Kan质粒(SEQ ID NO:51)和辅助质粒pHelper-Kan(SEQ IDNO:52)，以PEI为转染试剂，共转染HEK293细胞，重组包装出AAV8病毒载体。转染后48-72小时进行收获，收获液经纯化后获得一定纯度的重组AAV8病毒载体。纯化方法如下：
首先对收获液进行预处理：充分裂解HEK293细胞，释放出细胞内的AAV8病毒载体，同时加入核酸酶对游离的核酸进行消化，消化结束后，采用深层过滤除去大分子杂质和细胞碎片，深层过滤后滤液用二次过滤得到澄清液用于亲和上样。
亲和层析利用配基与蛋白的特异性吸附捕获收获液中的AAV8病毒载体，并除去大部分工艺相关杂质，达到浓缩与除杂的效果。将收集的洗脱液混匀，并用中和缓冲液中和，保存在无菌储液瓶中作为阴离子层析上样液。
阴离子层析利用不同组分的等电点差异分离实心与空壳AAV8病毒，同时继续去除残留杂质，洗脱液收集在新的无菌储液瓶中，再经超滤浓缩的方法将缓冲液置换为制剂稳定的缓冲液，最后除菌过滤分装后备用。
pAAV-R2C8-Kan质粒(SEQ ID NO:51)：
atgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgacagctttgtgaactgggtggccg
agaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccgtggccgagaagctgcagcgcg
actttctgacggaatggcgccgtgtgagtaaggccccggaggcccttttctttgtgcaatttgagaagggagagagctacttccacatgcacg
tgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaactgattcagagaatttaccgcggga
tcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatcc
ccaattacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgtttgaatctcacggagc
gtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccgg
tgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggatccagg
aggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagc
ctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgg
gtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaa
ctaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaac
gactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaag
caaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgt
gattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactt
tgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaag
ggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgt
cagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagac
aatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtt
tctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatg
tggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggctgccgatggttatcttccagattggctcgaggacaacctctctg
agggcattcgcgagtggtgggcgctgaaacctggagccccgaagcccaaagccaaccagcaaaagcaggacgacggccggggtctgg
tgcttcctggctacaagtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggacgcagcggccctcgagcac
gacaaggcctacgaccagcagctgcaggcgggtgacaatccgtacctgcggtataaccacgccgacgccgagtttcaggagcgtctgca
agaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaagaagcgggttctcgaacctctcggtctggttgaggaaggc
gctaagacggctcctggaaagaagagaccggtagagccatcaccccagcgttctccagactcctctacgggcatcggcaagaaaggcca
acagcccgccagaaaaagactcaattttggtcagactggcgactcagagtcagttccagaccctcaacctctcggagaacctccagcagcg
ccctctggtgtgggacctaatacaatggctgcaggcggtggcgcaccaatggcagacaataacgaaggcgccgacggagtgggtagttcc
tcgggaaattggcattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgaacctgggccctgcccacctacaacaac
cacctctacaagcaaatctccaacgggacatcgggaggagccaccaacgacaacacctacttcggctacagcaccccctgggggtattttg
actttaacagattccactgccacttttcaccacgtgactggcagcgactcatcaacaacaactggggattccggcccaagagactcagcttca
agctcttcaacatccaggtcaaggaggtcacgcagaatgaaggcaccaagaccatcgccaataacctcaccagcaccatccaggtgtttac
ggactcggagtaccagctgccgtacgttctcggctctgcccaccagggctgcctgcctccgttcccggcggacgtgttcatgattccccagt
acggctacctaacactcaacaacggtagtcaggccgtgggacgctcctccttctactgcctggaatactttccttcgcagatgctgagaaccg
gcaacaacttccagtttacttacaccttcgaggacgtgcctttccacagcagctacgcccacagccagagcttggaccggctgatgaatcctc
tgattgaccagtacctgtactacttgtctcggactcaaacaacaggaggcacggcaaatacgcagactctgggcttcagccaaggtgggcct
aatacaatggccaatcaggcaaagaactggctgccaggaccctgttaccgccaacaacgcgtctcaacgacaaccgggcaaaacaacaat
agcaactttgcctggactgctgggaccaaataccatctgaatggaagaaattcattggctaatcctggcatcgctatggcaacacacaaagac
gacgaggagcgtttttttcccagtaacgggatcctgatttttggcaaacaaaatgctgccagagacaatgcggattacagcgatgtcatgctca
ccagcgaggaagaaatcaaaaccactaaccctgtggctacagaggaatacggtatcgtggcagataacttgcagcagcaaaacacggctc
ctcaaattggaactgtcaacagccagggggccttacccggtatggtctggcagaaccgggacgtgtacctgcagggtcccatctgggccaa
gattcctcacacggacggcaacttccacccgtctccgctgatgggcggctttggcctgaaacatcctccgcctcagatcctgatcaagaaca
cgcctgtacctgcggatcctccgaccaccttcaaccagtcaaagctgaactctttcatcacgcaatacagcaccggacaggtcagcgtggaa
attgaatgggagctgcagaaggaaaacagcaagcgctggaaccccgagatccagtacacctccaactactacaaatctacaagtgtggact
ttgctgttaatacagaaggcgtgtactctgaaccccgccccattggcacccgttacctcacccgtaatctgtaattgcttgttaatcaataaaccg
tttaattcgtttcagttgaactttggtctctgcgaagggcgaattcgtttaaacctgcaggactagaggtcctgtattagaggtcacgtgagtgtttt
gcgacattttgcgacaccatgtggtcacgctgggtatttaagcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcg
cagccgccaagccgaattctgcagatatccatcacactggcggccgctcgactagagcggccgccaccgcggtggagctccagcttttgtt
ccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacg
agccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcggg
aaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactga
ctcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgca
ggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccct
gacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctcc
ctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgt
aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaac
tatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcg
gtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcgg
aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaa
aggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaa
aaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttagaaaaactca
tcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccg
aggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaat
aaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggc
cagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgt
taaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattctt
ctaatacctggaatgctgttttcccagggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagag
gcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcat
cgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaa
tttaatcgcggcctagagcaagacgtttcccgttgaatatggctcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatga
gcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctaaattgtaagcgttaata
ttttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccg
agatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagg
gcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccc
cgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggc
aagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcg
caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggta
acgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggc
cccccctcgatcgaggtcgacggtatcgggggagctcgcagggtctccattttgaagcgggaggtttgaacgcgcagccgcc
辅助质粒pHelper-Kan(SEQ ID NO:52)：
ggtacccaactccatgcttaacagtccccaggtacagcccaccctgcgtcgcaaccaggaacagctctacagcttcctggagcgccactcg
ccctacttccgcagccacagtgcgcagattaggagcgccacttctttttgtcacttgaaaaacatgtaaaaataatgtactaggagacactttca
ataaaggcaaatgtttttatttgtacactctcgggtgattatttaccccccacccttgccgtctgcgccgtttaaaaatcaaaggggttctgccgcg
catcgctatgcgccactggcagggacacgttgcgatactggtgtttagtgctccacttaaactcaggcacaaccatccgcggcagctcggtg
aagttttcactccacaggctgcgcaccatcaccaacgcgtttagcaggtcgggcgccgatatcttgaagtcgcagttggggcctccgccctg
cgcgcgcgagttgcgatacacagggttgcagcactggaacactatcagcgccgggtggtgcacgctggccagcacgctcttgtcggagat
cagatccgcgtccaggtcctccgcgttgctcagggcgaacggagtcaactttggtagctgccttcccaaaaagggtgcatgcccaggctttg
agttgcactcgcaccgtagtggcatcagaaggtgaccgtgcccggtctgggcgttaggatacagcgcctgcatgaaagccttgatctgctta
aaagccacctgagcctttgcgccttcagagaagaacatgccgcaagacttgccggaaaactgattggccggacaggccgcgtcatgcacg
cagcaccttgcgtcggtgttggagatctgcaccacatttcggccccaccggttcttcacgatcttggccttgctagactgctccttcagcgcgc
gctgcccgttttcgctcgtcacatccatttcaatcacgtgctccttatttatcataatgctcccgtgtagacacttaagctcgccttcgatctcagcg
cagcggtgcagccacaacgcgcagcccgtgggctcgtggtgcttgtaggttacctctgcaaacgactgcaggtacgcctgcaggaatcgc
cccatcatcgtcacaaaggtcttgttgctggtgaaggtcagctgcaacccgcggtgctcctcgtttagccaggtcttgcatacggccgccaga
gcttccacttggtcaggcagtagcttgaagtttgcctttagatcgttatccacgtggtacttgtccatcaacgcgcgcgcagcctccatgcccttc
tcccacgcagacacgatcggcaggctcagcgggtttatcaccgtgctttcactttccgcttcactggactcttccttttcctcttgcgtccgcata
ccccgcgccactgggtcgtcttcattcagccgccgcaccgtgcgcttacctcccttgccgtgcttgattagcaccggtgggttgctgaaaccc
accatttgtagcgccacatcttctctttcttcctcgctgtccacgatcacctctggggatggcgggcgctcgggcttgggagaggggcgcttct
ttttctttttggacgcaatggccaaatccgccgtcgaggtcgatggccgcgggctgggtgtgcgcggcaccagcgcatcttgtgacgagtctt
cttcgtcctcggactcgagacgccgcctcagccgcttttttgggggcgcgcggggaggcggcggcgacggcgacggggacgacacgtc
ctccatggttggtggacgtcgcgccgcaccgcgtccgcgctcgggggtggtttcgcgctgctcctcttcccgactggccatttccttctcctat
aggcagaaaaagatcatggagtcagtcgagaaggaggacagcctaaccgccccctttgagttcgccaccaccgcctccaccgatgccgc
caacgcgcctaccaccttccccgtcgaggcacccccgcttgaggaggaggaagtgattatcgagcaggacccaggttttgtaagcgaaga
cgacgaggatcgctcagtaccaacagaggataaaaagcaagaccaggacgacgcagaggcaaacgaggaacaagtcgggcgggggg
accaaaggcatggcgactacctagatgtgggagacgacgtgctgttgaagcatctgcagcgccagtgcgccattatctgcgacgcgttgca
agagcgcagcgatgtgcccctcgccatagcggatgtcagccttgcctacgaacgccacctgttctcaccgcgcgtaccccccaaacgcca
agaaaacggcacatgcgagcccaacccgcgcctcaacttctaccccgtatttgccgtgccagaggtgcttgccacctatcacatctttttccaa
aactgcaagatacccctatcctgccgtgccaaccgcagccgagcggacaagcagctggccttgcggcagggcgctgtcatacctgatatc
gcctcgctcgacgaagtgccaaaaatctttgagggtcttggacgcgacgagaaacgcgcggcaaacgctctgcaacaagaaaacagcga
aaatgaaagtcactgtggagtgctggtggaacttgagggtgacaacgcgcgcctagccgtgctgaaacgcagcatcgaggtcacccacttt
gcctacccggcacttaacctaccccccaaggttatgagcacagtcatgagcgagctgatcgtgcgccgtgcacgacccctggagagggat
gcaaacttgcaagaacaaaccgaggagggcctacccgcagttggcgatgagcagctggcgcgctggcttgagacgcgcgagcctgccg
acttggaggagcgacgcaagctaatgatggccgcagtgcttgttaccgtggagcttgagtgcatgcagcggttctttgctgacccggagatg
cagcgcaagctagaggaaacgttgcactacacctttcgccagggctacgtgcgccaggcctgcaaaatttccaacgtggagctctgcaacc
tggtctcctaccttggaattttgcacgaaaaccgcctcgggcaaaacgtgcttcattccacgctcaagggcgaggcgcgccgcgactacgtc
cgcgactgcgtttacttatttctgtgctacacctggcaaacggccatgggcgtgtggcagcaatgcctggaggagcgcaacctaaaggagct
gcagaagctgctaaagcaaaacttgaaggacctatggacggccttcaacgagcgctccgtggccgcgcacctggcggacattatcttcccc
gaacgcctgcttaaaaccctgcaacagggtctgccagacttcaccagtcaaagcatgttgcaaaactttaggaactttatcctagagcgttcag
gaattctgcccgccacctgctgtgcgcttcctagcgactttgtgcccattaagtaccgtgaatgccctccgccgctttggggtcactgctacctt
ctgcagctagccaactaccttgcctaccactccgacatcatggaagacgtgagcggtgacggcctactggagtgtcactgtcgctgcaacct
atgcaccccgcaccgctccctggtctgcaattcgcaactgcttagcgaaagtcaaattatcggtacctttgagctgcagggtccctcgcctga
cgaaaagtccgcggctccggggttgaaactcactccggggctgtggacgtcggcttaccttcgcaaatttgtacctgaggactaccacgccc
acgagattaggttctacgaagaccaatcccgcccgccaaatgcggagcttaccgcctgcgtcattacccagggccacatccttggccaattg
caagccatcaacaaagcccgccaagagtttctgctacgaaagggacggggggtttacctggacccccagtccggcgaggagctcaaccc
aatccccccgccgccgcagccctatcagcagccgcgggcccttgcttcccaggatggcacccaaaaagaagctgcagctgccgccgccg
ccacccacggacgaggaggaatactgggacagtcaggcagaggaggttttggacgaggaggaggagatgatggaagactgggacagc
ctagacgaagcttccgaggccgaagaggtgtcagacgaaacaccgtcaccctcggtcgcattcccctcgccggcgccccagaaattggca
accgttcccagcatcgctacaacctccgctcctcaggcgccgccggcactgcctgttcgccgacccaaccgtagatgggacaccactgga
accagggccggtaagtctaagcagccgccgccgttagcccaagagcaacaacagcgccaaggctaccgctcgtggcgcgggcacaag
aacgccatagttgcttgcttgcaagactgtgggggcaacatctccttcgcccgccgctttcttctctaccatcacggcgtggccttcccccgtaa
catcctgcattactaccgtcatctctacagcccctactgcaccggcggcagcggcagcggcagcaacagcagcggtcacacagaagcaaa
ggcgaccggatagcaagactctgacaaagcccaagaaatccacagcggcggcagcagcaggaggaggagcgctgcgtctggcgccca
acgaacccgtatcgacccgcgagcttagaaataggatttttcccactctgtatgctatatttcaacaaagcaggggccaagaacaagagctga
aaataaaaaacaggtctctgcgctccctcacccgcagctgcctgtatcacaaaagcgaagatcagcttcggcgcacgctggaagacgcgg
aggctctcttcagcaaatactgcgcgctgactcttaaggactagtttcgcgccctttctcaaatttaagcgcgaaaactacgtcatctccagcgg
ccacacccggcgccagcacctgtcgtcagcgccattatgagcaaggaaattcccacgccctacatgtggagttaccagccacaaatggga
cttgcggctggagctgcccaagactactcaacccgaataaactacatgagcgcgggaccccacatgatatcccgggtcaacggaatccgc
gcccaccgaaaccgaattctcctcgaacaggcggctattaccaccacacctcgtaataaccttaatccccgtagttggcccgctgccctggtg
taccaggaaagtcccgctcccaccactgtggtacttcccagagacgcccaggccgaagttcagatgactaactcaggggcgcagcttgcg
ggcggctttcgtcacagggtgcggtcgcccgggcgttttagggcggagtaacttgcatgtattgggaattgtagtttttttaaaatgggaagtg
acgtatcgtgggaaaacggaagtgaagatttgaggaagttgtgggttttttggctttcgtttctgggcgtaggttcgcgtgcggttttctgggtgt
tttttgtggactttaaccgttacgtcattttttagtcctatatatactcgctctgtacttggccctttttacactgtgactgattgagctggtgccgtgtc
gagtggtgttttttaataggtttttttactggtaaggctgactgttatggctgccgctgtggaagcgctgtatgttgttctggagcgggagggtgct
attttgcctaggcaggagggtttttcaggtgtttatgtgtttttctctcctattaattttgttatacctcctatgggggctgtaatgttgtctctacgcctg
cgggtatgtattcccccgggctatttcggtcgctttttagcactgaccgatgttaaccaacctgatgtgtttaccgagtcttacattatgactccgg
acatgaccgaggaactgtcggtggtgctttttaatcacggtgaccagtttttttacggtcacgccggcatggccgtagtccgtcttatgcttataa
gggttgtttttcctgttgtaagacaggcttctaatgtttaaatgtttttttttttgttattttattttgtgtttaatgcaggaacccgcagacatgtttgaga
gaaaaatggtgtctttttctgtggtggttccggaacttacctgcctttatctgcatgagcatgactacgatgtgcttgcttttttgcgcgaggctttg
cctgattttttgagcagcaccttgcattttatatcgccgcccatgcaacaagcttacataggggctacgctggttagcatagctccgagtatgcgt
gtcataatcagtgtgggttcttttgtcatggttcctggcggggaagtggccgcgctggtccgtgcagacctgcacgattatgttcagctggccc
tgcgaagggacctacgggatcgcggtatttttgttaatgttccgcttttgaatcttatacaggtctgtgaggaacctgaatttttgcaatcatgattc
gctgcttgaggctgaaggtggagggcgctctggagcagatttttacaatggccggacttaatattcgggatttgcttagagacatattgataag
gtggcgagatgaaaattatttgggcatggttgaaggtgctggaatgtttatagaggagattcaccctgaagggtttagcctttacgtccacttgg
acgtgagggcagtttgccttttggaagccattgtgcaacatcttacaaatgccattatctgttctttggctgtagagtttgaccacgccaccggag
gggagcgcgttcacttaatagatcttcattttgaggttttggataatcttttggaataaaaaaaaaaaaacatggttcttccagctcttcccgctcct
cccgtgtgtgactcgcagaacgaatgtgtaggttggctgggtgtggcttattctgcggtggtggatgttatcagggcagcggcgcatgaagg
agtttacatagaacccgaagccagggggcgcctggatgctttgagagagtggatatactacaactactacacagagcgagctaagcgacga
gaccggagacgcagatctgtttgtcacgcccgcacctggttttgcttcaggaaatatgactacgtccggcgttccatttggcatgacactacga
ccaacacgatctcggttgtctcggcgcactccgtacagtagggatcgcctacctccttttgagacagagacccgcgctaccatactggagga
tcatccgctgctgcccgaatgtaacactttgacaatgcacaacgtgagttacgtgcgaggtcttccctgcagtgtgggatttacgctgattcag
gaatgggttgttccctgggatatggttctgacgcgggaggagcttgtaatcctgaggaagtgtatgcacgtgtgcctgtgttgtgccaacattg
atatcatgacgagcatgatgatccatggttacgagtcctgggctctccactgtcattgttccagtcccggttccctgcagtgcatagccggcgg
gcaggttttggccagctggtttaggatggtggtggatggcgccatgtttaatcagaggtttatatggtaccgggaggtggtgaattacaacatg
ccaaaagaggtaatgtttatgtccagcgtgtttatgaggggtcgccacttaatctacctgcgcttgtggtatgatggccacgtgggttctgtggt
ccccgccatgagctttggatacagcgccttgcactgtgggattttgaacaatattgtggtgctgtgctgcagttactgtgctgatttaagtgagat
cagggtgcgctgctgtgcccggaggacaaggcgtctcatgctgcgggcggtgcgaatcatcgctgaggagaccactgccatgttgtattcc
tgcaggacggagcggcggcggcagcagtttattcgcgcgctgctgcagcaccaccgccctatcctgatgcacgattatgactctaccccca
tgtaggcgtggacttccccttcgccgcccgttgagcaaccgcaagttggacagcagcctgtggctcagcagctggacagcgacatgaactt
aagcgagctgcccggggagtttattaatatcactgatgagcgtttggctcgacaggaaaccgtgtggaatataacacctaagaatatgtctgtt
acccatgatatgatgctttttaaggccagccggggagaaaggactgtgtactctgtgtgttgggagggaggtggcaggttgaatactagggtt
ctgtgagtttgattaaggtacggtgatcaatataagctatgtggtggtggggctatactactgaatgaaaaatgacttgaaattttctgcaattgaa
aaataaacacgttgaaacataacatgcaacaggttcacgattctttattcctgggcaatgtaggagaaggtgtaagagttggtagcaaaagttt
cagtggtgtattttccactttcccaggaccatgtaaaagacatagagtaagtgcttacctcgctagtttctgtggattcactagaatcgatgtagg
atgttgcccctcctgacgcggtaggagaaggggagggtgccctgcatgtctgccgctgctcttgctcttgccgctgctgaggaggggggcg
catctgccgcagcaccggatgcatctgggaaaagcaaaaaaggggctcgtccctgtttccggaggaatttgcaagcggggtcttgcatgac
ggggaggcaaacccccgttcgccgcagtccggccggcccgagactcgaaccgggggtcctgcgactcaacccttggaaaataaccctcc
ggctacagggagcgagccacttaatgctttcgctttccagcctaaccgcttacgccgcgcgcggccagtggccaaaaaagctagcgcagc
agccgccgcgcctggaaggaagccaaaaggagcgctcccccgttgtctgacgtcgcacacctgggttcgacacgcgggcggtaaccgc
atggatcacggcggacggccggatccggggttcgaaccccggtcgtccgccatgatacccttgcgaatttatccaccagaccacggaaga
gtgcccgcttacaggctctccttttgcacggtctagagcgtcaacgactgcgcacgcctcaccggccagagcgtcccgaccatggagcactt
tttgccgctgcgcaacatctggaaccgcgtccgcgactttccgcgcgcctccaccaccgccgccggcatcacctggatgtccaggtacatct
acggattacgtcgacgtttaaaccatatgatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaa
catgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagca
tcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgc
tctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctc
agttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttg
agtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacag
agttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagtt
ggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaa
gaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttc
acctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttagaaaaactcatcgagcatca
aatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttcc
ataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaa
gtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacg
ctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggaca
attacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctgg
aatgctgttttcccagggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattcc
gtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttccca
tacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggc
ctagagcaagacgtttcccgttgaatatggctcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacata
tttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaatt
cgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggtt
gagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcc
cactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagag
cttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcg
gtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgatggatcc
AAV8载体的滴度定量
在AAV8病毒纯化完成之后，需要对病毒的含量进行测定，基因组滴度是表征AAV物理滴度最经典的检项。通过针对rAAV的基因组序列设计引物探针，然后进行Q-PCR检测是基因组滴度检测最通用的方法。
考虑到本发明中针对hFVIII ORF编码框进行了密码子优化，涉及到多个不同载体结构的筛选，本发明中共设计两套引物和探针用于AAV8病毒的滴度检测。第一套引物和探针针对0TE元件组合(SEQ ID NO:44)区域设计，用于AAV8-0TE-hFVIII-L1-WT-pA(SEQ IDNO:28)，AAV8-0TE-hFVIII-L1-1～6-pA(SEQ ID NO:29～34)，AAV8-0TE-hFVIII-L1-8～10-pA(SEQ ID NO:36～38)，AAV8-0TE-hFVIII-L1-pA(SEQ ID NO:35)病毒的滴度检测；其中F1引物序列为：5'-gccgtcacacagatccacaag-3'(SEQ ID NO:53)，R1引物序列为：5'-agtggatgtcagtgtcagtaattca-3'(SEQ ID NO:54)，P1探针序列为：5'-Fam-ttcccgcgggcctggcctcttt-BHQ1-3'(SEQ ID NO:55)。第二套因引物和探针针对hFVIII-L1-7(SEQ ID NO:10)区域设计，用于AAV8-0TE-hFVIII-L1\L2\SQ-pA(SEQ ID NO:35，39，50)，和AAV8-TTM，HLP，STM，CTS-hFVIII-L1-pA(SEQ ID NO:40～43)病毒的滴度检测，其中F2引物序列为：5'-gtctcatgtggacctggtga-3'(SEQ ID NO:56)，R2引物序列为：5'-ggtcttttccttagccaggc-3'(SEQ ID NO:57)，P2探针序列为：：5'-Fam-ctccctacacaccagcagagccccaatc-BHQ1-3'(SEQ ID NO:58)。
在基因组滴度检测过程中，首先要进行标准曲线的建立，阳性标准品使用样品稀释液稀释至2×107、2×106、2×105、2×104、2×103、2×102拷贝/μl，作为标准曲线模板，标准曲线需要控制其线性和扩增效率，一般要求R2>0.99，扩增效率在90％-110％之间。之后采用预处理完成的rAAV样品稀释后进行QPCR检测，确保样品检测Ct值在标准曲线范围内，根据样品的Ct值代入到标准曲线中进行rAAV样品基因组滴度的计算，对产品的含量进行标识。
HepG2-AAVR细胞病毒侵染
消化处理HepG2-AAVR细胞，按1.5e5细胞/孔接种细胞至24孔细胞培养皿中；24小时后，取1个孔的细胞消化后细胞计数，根据病毒侵染的MOI计算每孔需加入的病毒量，将病毒原液用DMEM+10％FBS培养基稀释至合适的浓度；去除24孔细胞培养皿中的培养基，每孔加入500μl病毒液侵染，置于CO2恒温培养箱中培养5天；5天后，收集细胞培养液上清，加入合适体积的DMEM+10％FBS培养基定量至500μl。
小鼠尾静脉注射病毒样品
将小鼠放入合适的容器中，放置红外灯下，打开开关，照射若干分钟，取出小鼠，用酒精棉球擦拭尾部，使尾部静脉充分扩张。将小鼠放入小鼠固定器中，露出尾部。使用合适的注射器及针头抽取待注射样本，注射使用1mL注射器的针头，匹配1mL注射器的针筒。抽取对应体积的待注射样本，通过尾静脉进行注射，在大于10秒时间推注完毕，注射需缓慢且速度均一。注射完毕后，用干棉球按压注射部位进行止血。
小鼠血浆采集
使用异氟烷将小鼠麻醉，用毛细管从小鼠眼眶中采集180μl全血，加入20μl 3.8％柠檬酸钠抗凝剂，上下颠倒混匀后5000rpm离心10min，吸取上清液即为小鼠血浆。
Elisa方法测量小鼠血浆中hFVIII蛋白表达量
使用Matched-PairAntibody Set for ELISA ofhuman Factor VIII antigen(FVIII:C)试剂盒(F8C-E1A，Affinity Biologicals)进行小鼠血浆中hFVIII蛋白表达量检测，实验过程如下：用50mM碳酸盐缓冲液1:100稀释Capture antibody，按100μl/孔加至96孔Elisa板，室温孵育2小时；去除孔内液体，加入300μl PBST溶液清洗5次，加入100μl标准品(0.59ng/ml～37.5ng/ml)或待测样品(标准品或待测样品用试剂盒中样品稀释液稀释)，室温孵育2小时；去除孔内液体，加入300μl PBST溶液清洗5次，加入100μl Detectingantibody，室温孵育1h；去除孔内液体，加入300μl PBST溶液清洗5次，加入100μl TMB底物室温孵育15min，加入100μl终止液，用酶标仪测量OD450nm；根据标准曲线计算小鼠血浆样品中hFVIII蛋白表达量。
CSA方法测量小鼠血浆中hFVIII蛋白活性
使用Chromogenix SP4Factor VIII试剂盒(K824094，Diapharma)进行小鼠血浆中hFVIII蛋白活性检测，实验过程如下：取1份体积样品稀释液存储液(10×)和9份体积无菌水，混匀后配制成样品稀释液；按试剂盒中方法配制2.5％～80％标曲稀释液(标准品原液浓度为1IU/ml)；取5μl待测样品，加入400μl稀释液进行样品稀释；取1份体积磷脂试剂和5份体积凝血因子试剂，混匀后配制成RP试剂；在96孔板中加入40μl RP溶液和20μl标准品或待测样品，混匀后37℃孵育5min；加入20μl CaCl2溶液(37℃预热)，混匀37℃孵育10min；加入40μl S-2765+I-2581溶液(37℃预热)，混匀后37℃孵育10min；加入20μl20％乙酸溶液，混匀后用酶标仪测量A405nm吸光度；根据标准曲线计算小鼠血浆样品中hFVIII蛋白活性。
Elisa方法测量小鼠血浆中hFVIII结合抗体检测
将hFVIII标准品(Xyntha，Pfizer)用PBS稀释至1μg/mL，按100μl/孔加至96孔Elisa板，4℃过夜孵育；去除孔内液体，加入300μl PBST溶液清洗5次，加入300μl封闭液(5％脱脂奶粉)封闭，室温孵育2小时；去除孔内液体，加入300μl PBST溶液清洗5次，加入100μl Murine Anti-Factor VIII标准品(GMA-8024，Green Mountain Antibodies)(1.56ng/mL～100ng/ml)或待测样品(标准品或待测样品用酪蛋白Buffer稀释)，室温300rpm孵育2小时；去除孔内液体，加入300μl PBST溶液清洗5次，加入100μl、1：100000稀释的山羊抗小鼠IgG(H+L)二抗(Goat anti-Mouse IgG(H+L)SecondaryAntibody)、HRP抗体，室温300rpm孵育2小时；去除孔内液体，加入300μl PBST溶液清洗5次，加入100μlTMB底物室温孵育15min，加入100μl终止液，用酶标仪测量OD450nm。
小鼠断尾实验
使用异氟烷将小鼠麻醉，并在整个实验过程中用气麻机保持麻醉状态；将小鼠置于37℃保温垫上保持体温，小鼠尾巴置于37℃恒温水浴锅中保温5分钟；取出小鼠尾巴，用一次性手术刀片从尖端横切6mm小鼠尾巴，切除后将小鼠尾部立即置于已预热至37℃的15mL生理盐水中，记录30min内小鼠流血时间。
qPCR方法测量小鼠肝脏组织中hFVIII DNA拷贝数
使用基于TaqMan探针的qPCR方法进行hFVIII DNA拷贝数分析。使用DNeasyBlood&Tissue Kit试剂盒(69506，Qiagen)从小鼠肝脏组织中提取基因组DNA，按如下反应体系建立qPCR反应：10μl 2xTaqMan Gene Expression Master Mix(4369016，ABI)，6μlddH2O，0.5μl 10μM F2引物，0.5μl 10μM R2引物，0.5μl 10μM P2探针，2.5μl基因组DNA(500ng)或2.5μl阳性标准品(2e1～2e7拷贝/μl)，总反应体系为20μl，每份标准品或待检样品设置2个复孔。使用Quant studio 5(ABI)qPCR仪进行qPCR反应，反应条件如下：50℃2分钟，95℃10分钟，95℃15秒和60℃30秒进行40个循环反应，采用483-533nm荧光检测hFVIII载体基因组对应的扩增子。根据标准曲线线性回归方程和样本Ct值计算出每个二倍体基因组中hFVIII DNA拷贝数。
qPCR方法测量小鼠肝脏组织中hFVIII mRNA拷贝数
使用RNeasy Mini Kit试剂盒(74106，Qiagen)从小鼠肝脏组织中提取总RNA，参考PrimeScript RTreagent kit with gDNAEraser(RR047A，TaKaRa)试剂盒所明书进行基因组DNA去除和逆转录反应。按如下反应体系建立qPCR反应：5μl 2x TB Green Premix EXTaq II(RR820A，TaKaRa)，0.4μl 10μM F2引物或mTbp-F内参引物(5’-ccgtgaatcttggctgtaaact-3’，SEQ ID NO:69)，0.4μl 10μM R2引物或mTbp-R内参引物(5’-tgtccgtggctctcttattc-3’，SEQ ID NO:70)，0.2μl Rox II，4μl逆转录产物(7x稀释)，总反应体系为10μl，每份标准品或待检样品设置2个复孔。使用Quant studio 5(ABI)qPCR仪进行qPCR反应，反应条件如下：95℃30秒，95℃10秒和60℃30秒进行40个循环反应，采用483-533nm荧光检测hFVIII和mTbp内参信号。根据样本hFVIII Ct值和mTbp内参Ct值差异计算hFVIII mRNA相对于mTbp mRNA表达的倍数。
实施例1：在HepG2-AAVR细胞中比较不同hFVIII结构表达效果
本实施例评价了hFVIII-L1，hFVIII-L2，hFVIII-SQ在HepG2-AAVR细胞中的表达效果。
HepG2-AAVR细胞提前一天接种至24孔细胞培养皿中，每孔1.5e5细胞，培养基为DMEM+10％胎牛血清。24小时后，将AAV8-0TE-hFVIII-L1-pA，AAV8-0TE-hFVIII-L2-pA，AAV8-0TE-hFVIII-SQ-pA病毒侵染至HepG2-AAVR细胞，侵染MOI设置2e4，1e5，4e5三个梯度，每组设置3个复孔。病毒侵染5天后，收集培养上清液，分别使用Elisa方法测量hFVIII蛋白表达量和CSA方法测量hFVIII蛋白活性。
不同hFVIII结构病毒侵染HepG2-AAVR细胞后，hFVIII蛋白表达量和活性检测结果如图2和图3所示。结果显示，hFVIII蛋白表达量和活性与病毒侵染的MOI均呈剂量依赖的增加；在相同的MOI，AAV8-0TE-hFVIII-L1-pA和AAV8-0TE-hFVIII-L2-pA病毒hFVIII蛋白表达量和活性均显著高于AAV8-0TE-hFVIII-SQ-pA病毒。AAV8-0TE-hFVIII-L1-pA病毒hFVIII蛋白表达量和活性最高，hFVIII蛋白表达量分别为AAV8-0TE-hFVIII-SQ-pA病毒的8倍(MOI：2e4)，16倍(MOI：1e5)，34倍(MOI：4e5)，hFVIII蛋白活性分别为AAV8-0TE-hFVIII-SQ-pA病毒的5倍(MOI：2e4)，17倍(MOI：1e5)，24倍(MOI：4e5)。
实施例2：在野生型C57BL/6小鼠体内比较不同hFVIII优化基因表达效果
本实施例评价了不同hFVIII优化基因在野生型C57BL/6小鼠体内的表达效果。
选用8～10周周龄的雄性野生型C57BL/6小鼠(n＝4只/组)，按4e12 vg/kg的给药剂量，尾静脉给药AAV8-0TE-hFVIII-L1-1～6-pA，AAV8-0TE-hFVIII-L1-8～10-pA，AAV8-0TE-hFVIII-L1-pA和AAV8-0TE-hFVIII-L1-WT-pA病毒，给药后4周时间点，从小鼠眼眶采集血浆，使用Elisa方法测量hFVIII蛋白表达量。
第一批小鼠给药AAV8-0TE-hFVIII-L1-WT\1\2\3\9\10-pA和AAV8-0TE-hFVIII-L1-pA病毒，hFVIII蛋白表达量检测结果如图4所示，结果显示给药4周后，AAV8-0TE-hFVIII-L1-pA、AAV8-0TE-hFVIII-L1-9-pA和AAV8-0TE-hFVIII-L1-10-pA表达量均大幅高于AAV8-0TE-hFVIII-L1-WT-pA，分别为431±186ng/ml、324±78ng/ml和317±79ng/ml，为AAV8-0TE-hFVIII-L1-WT-pA的17倍、13倍和13倍。第二批小鼠给药AAV8-0TE-hFVIII-L1-WT-pA\4\5\6\8-pA和AAV8-0TE-hFVIII-L1-pA病毒，hFVIII蛋白表达量检测结果如图5所示，结果显示给药4周后，AAV8-0TE-hFVIII-L1-pA和AAV8-0TE-hFVIII-L1-5-pA表达量均大幅高于AAV8-0TE-hFVIII-L1-WT-pA，分别为674±463ng/ml和379±172ng/ml，为AAV8-0TE-hFVIII-L1-WT-pA的19倍和11倍。
实施例3：在野生型C57BL/6小鼠体内比较不同元件组合表达hFVIII效果
本实施例评价了0TE，TTM，HLP，STM，CTS元件组合在野生型C57BL/6小鼠体内表达hFVIII的效果。
选用8～10周周龄的雄性野生型C57BL/6小鼠(n＝4只/组)，按4e12 vg/kg的给药剂量，尾静脉给药AAV8-0TE-hFVIII-L1-pA，AAV8-TTM-hFVIII-L1-pA，AAV8-HLP-hFVIII-L1-pA，AAV8-STM-hFVIII-L1-pA，AAV8-CTS-hFVIII-L1-pA病毒，给药4周后，从小鼠眼眶采集血浆，使用Elisa方法测量hFVIII蛋白表达量。
小鼠给药4周后，hFVIII蛋白表达量检测结果如图6所示，结果显示AAV8-0TE-hFVIII-L1-pA，AAV8-TTM-hFVIII-L1-pA，AAV8-CTS-hFVIII-L1-pA病毒具有相似的hFVIII表达水平，稍好于AAV8-HLP-hFVIII-L1-pA和AAV8-STM-hFVIII-L1-pA病毒。
实施例4：不同hFVIII结构病毒在FVIII KO小鼠体内药效评价
本实施例评价了hFVIII-L1，hFVIII-L2，hFVIII-SQ结构病毒在FVIII KO小鼠体内的表达活性和对小鼠凝血异常表型的改善。
选用8～10周周龄的雄性FVIII KO小鼠(n＝5只/组)，按4e12 vg/kg的给药剂量，尾静脉给药AAV8-0TE-hFVIII-L1-pA，AAV8-0TE-hFVIII-L2-pA，AAV8-0TE-hFVIII-SQ-pA病毒。给药后1，2，3，4，6周时间点，从小鼠眼眶采集血浆，使用CSA方法测量hFVIII蛋白活性。在给药后8周的实验终点，进行小鼠断尾实验，测量小鼠流血时间来评价对小鼠凝血异常表型的改善。
小鼠给药后1，2，3，4，6周时间点，hFVIII蛋白活性检测结果如图7所示，结果显示给药AAV8-0TE-hFVIII-L1-pA和AAV8-0TE-hFVIII-L2-pA病毒的小鼠hFVIII蛋白活性均高于给药AAV8-0TE-hFVIII-SQ-pA病毒的小鼠，在hFVIII稳定表达的6周时间点，hFVIII蛋白活性分别为AAV8-0TE-hFVIII-SQ-pA病毒的7.1倍(hFVIII-L1)和5.9倍(hFVIII-L2)。
在给药后8周时间点，测量了小鼠断尾后的流血时间，检测结果如图8所示，结果显示给药AAV8-0TE-hFVIII-L1-pA病毒的小鼠，对小鼠凝血异常表型的改善最明显。
实施例5：hFVIII病毒不同给药剂量在FVIII KO小鼠体内药效评价
本实施例评价了AAV8-0TE-hFVIII-L1-pA病毒在不同给药剂量下，在FVIII KO小鼠体内的表达活性和对小鼠凝血异常表型的改善。
选用8～10周周龄的雄性FVIII KO小鼠(n＝6只/组)，按4e12，8e11 vg/kg的给药剂量，尾静脉给药AAV8-0TE-hFVIII-L1-pA病毒，同时设置FVIII KO小鼠给药溶媒组和野生型C57BL/6小鼠给药溶媒组对照。给药后2，4，6周时间点，从小鼠眼眶采集血浆，分别使用Elisa方法测量hFVIII蛋白表达量和CSA方法测量hFVIII蛋白活性。在给药后6周的实验终点，进行小鼠断尾实验，测量小鼠流血时间来评价对小鼠凝血异常表型的改善；同时采集小鼠肝脏组织，qPCR方法测量hFVIII DNA拷贝数。
小鼠给药后2，4，6周时间点，hFVIII蛋白表达量和hFVIII蛋白活性检测结果如图9和图10所示。结果显示在4e12 vg/kg和8e11 vg/kg的给药剂量下，检测到hFVIII蛋白的有效表达，给药后6周时间点，hFVIII蛋白表达量分别为513±222ng/ml和53±34ng/ml，hFVIII蛋白活性分别为279％±144％和39％±21％。表3列出了同类产品在FVIII KO小鼠体内表达数据，结果显示本发明提供的编码人凝血因子FVIII蛋白的核酸可以在更低的给药剂量下实现更高的hFVIII蛋白表达效果。
小鼠给药后6周实验终点，对小鼠进行断尾实验测量小鼠流血时间，检测结果如图11所示。结果显示在4e12 vg/kg和8e11 vg/kg的给药剂量下，小鼠断尾后流血时间分别为3.7±5.1和7.2±7.3分钟，相对于溶媒组给药的FVIII KO小鼠断尾流血时间分别缩短85.5％和71.8％，具有明显的小鼠凝血异常表型改善效果。
表3
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种凝血因子FVIII蛋白变体，其氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
2.一种编码凝血因子FVIII蛋白变体的核酸，其核苷酸序列如SEQ ID NO:3～15、SEQID NO:60～68中的任一序列所示。
3.一种表达载体，其包含权利要求2所述的核酸。
4.根据权利要求3所述的表达载体，其特征在于，所述表达载体选自由腺相关病毒载体、反转录病毒载体、腺病毒载体、质粒或慢病毒载体组成的组中的任一种；
优选地，所述腺相关病毒载体包含AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10或AAV11血清型。
5.根据权利要求3或4所述的表达载体，其特征在于，所述表达载体还包含与所述核酸可操作地连接的启动子、增强子、内含子、一个或多个末端反向重复序列、Kozak序列和/或polyA尾；
可选地，所述增强子和所述启动子依次可操作地连接至所述核酸的序列，或者所述内含子位于所述核酸的序列内部或两侧，或者所述末端反向重复序列位于所述核酸的序列的5'或3'末端，或者所述Kozak序列位于所述核酸的序列的5'末端与所述内含子之间，或者所述polyA尾位于所述核酸的序列的3'末端与所述末端反向重复序列之间。
6.根据权利要求5所述的表达载体，其特征在于，所述启动子的核苷酸序列如SEQ IDNO:16～18中的任一序列所示；所述增强子的核苷酸序列如SEQ ID NO:19～21中的任一序列所示；所述内含子的核苷酸序列如SEQ ID NO:22～24中的任一序列所示；所述末端反向重复序列的核苷酸序列如SEQ ID NO:25或SEQ ID NO:26所示；所述Kozak序列为gccacc；所述polyA尾的核苷酸序列如SEQ ID NO:27所示；
优选地，所述表达载体包含如SEQ ID NO:28～43所示的任一序列。
7.一种宿主细胞，其包含权利要求2所述的核酸或权利要求3-6任一项所述的表达载体。
8.一种药物组合物，其包含权利要求1所述的凝血因子FVIII蛋白变体、权利要求2所述的核酸和/或权利要求3-6任一项所述的表达载体，以及药学上可接受的辅料。
9.权利要求1所述的凝血因子FVIII蛋白变体、权利要求2所述的核酸和/或权利要求3-6任一项所述的表达载体在表达凝血因子FVIII蛋白中的用途，或在制备治疗需要凝血因子FVIII蛋白的疾病的药物中的用途。
10.一种生产凝血因子FVIII蛋白的方法，其特征在于，包括：
在细胞中表达权利要求2所述的核酸和/或权利要求3-6任一项所述的表达载体；
回收所述细胞产生的凝血因子FVIII蛋白。
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