CN108239151B - 重组单链人FVIII-Fc融合蛋白及其应用 - Google Patents
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Abstract
本发明提供了一种重组单链人FVIII‑Fc融合蛋白，其中，FVIII是B结构域缺失的人FVIII；Fc来自包含N297G突变的人IgG1，两个CH2‑CH3结构域通过柔性多肽连接成一条单链，在靠近N端的CH3中引入T366S、L368A和Y407V突变，在靠近C端的CH3中引入T366W突变。本发明提供的重组单链人FVIII‑Fc融合蛋白达到了与市售的重组FVIII相当的凝血活性，而且半衰期延长。另外，由于在Fc链内引入了点突变，本发明的融合蛋白不会形成同源二聚体，融合蛋白的产量大大提高，而且能够通过常规的protein A纯化工艺获得目的蛋白，大大降低了生产成本。
Description
技术领域
本发明涉及重组FVIII-Fc融合蛋白领域，更具体地涉及B结构域缺失的人FVIII与特定的单链Fc连接形成的融合蛋白。
背景技术
A型血友病是由凝血因子VIII(FVIII)活性的缺乏或功能障碍造成的一种遗传性出血障碍。补充有活性的FVIII是治疗A型血友病的有效措施。内源的FVIII的循环半衰期是12-14小时，因而每周需要进行几次预防性治疗，为了提高患者的依从性，减少FVIII每周施用的次数，研究者一直致力于开发半衰期延长的FVIII。
利用活性蛋白与IgG的Fc结构域连接构成融合蛋白来延长活性蛋白在体内的半衰期，是目前本领域技术人员常采用的手段之一。但是早期的Fc融合蛋白均是将两个活性蛋白与一个Fc片段融合形成二价分子。FVIII不仅分子量较大，是目前重组技术表达的分子量最大的活性蛋白，并且其需要与磷脂、FIXa和FX形成复合物才能发挥凝血活性，因此，二价分子形式的FVIII-Fc蛋白会由于空间位阻效应而导致FVIII活性降低或丧失。
专利CN201080062950.7公开了一种嵌合FVIII-Fc蛋白，其中所述嵌合蛋白包含两条链，一条链包括FVIII与IgG1的铰链区-CH2-CH3，另一条链仅包括IgG1的铰链区-CH2-CH3，二条链通过Fc区二聚化而形成杂合蛋白。但是，在该嵌合蛋白生产过程中，由于可同时产生“铰链区-CH2-CH3”同源二聚形式的分子，因此嵌合FVIII-Fc的产量极低，而且不能通过常规的protein A纯化工艺获得目的蛋白，只能采用VIIIselect(GE Healthcare)来纯化，从而极大的增加了生产成本。
发明内容
本发明目的在于提供一种新的重组单链人FVIII-Fc融合蛋白，其中Fc片段中的两个CH2-CH3结构域通过柔性多肽连接成一条单链。并采用真核细胞表达系统进行融合蛋白表达。所表达的重组单链人FVIII-Fc可用常规protein A纯化工艺获得较高纯度蛋白。
本发明的技术方案如下：
本发明首先提供了一种重组单链人FVIII-Fc融合蛋白，其中，FVIII是B结构域缺失的人FVIII；Fc来自包含N297G突变的人IgG1，两个CH2-CH3结构域通过柔性多肽连接成一条单链，在靠近N端的CH3中引入T366S、L368A和Y407V突变，在靠近C端的CH3中引入T366W突变。
优选地，所述FVIII与Fc直接连接或通过柔性多肽连接。
优选地，所述柔性多肽由至少一种选自G、S、A、T的氨基酸组成。
优选地，重组单链人FVIII-Fc融合蛋白，其序列为SEQ ID NO.1。
本发明还提供了编码序列为SEQ ID NO.1的重组单链人FVIII-Fc融合蛋白的核酸，其序列为SEQ ID NO.2。
本发明还提供了包含上述的核酸的表达载体。
本发明还提供了表达前述的重组单链人FVIII-Fc融合蛋白的细胞株。
本发明还提供了一种组合物，其包含前述的重组单链人FVIII-Fc融合蛋白，和药学上可接受的载体。
优选地，上述组合物其还包括FVIII重链、FVIII轻链-Fc。
本发明还提供了上述的重组单链人FVIII-Fc融合蛋白在制备治疗A型血友病的药物中的用途。
本发明提供的重组单链人FVIII-Fc融合蛋白达到了与市售的重组FVIII相当的凝血活性，而且半衰期延长。另外，由于在Fc链内引入了点突变，本发明的融合蛋白不会形成同源二聚体，融合蛋白的产量极高，而且能够通过常规的protein A纯化工艺获得目的蛋白，大大降低了生产成本。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为细胞生长曲线。
图2为细胞培养液残糖和PH变化趋势。
图3为纯化后的重组单链人FVIII-Fc融合蛋白的SEC检测结果。
图4为纯化后的重组单链人FVIII-Fc融合蛋白的SDS-PAGE检测结果。
图5为各组动物的出血量的统计结果。
图6为各组动物的出血时间的统计结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例所用到的试剂均市售可得。
实施例1制备重组单链人FVIII-Fc融合蛋白
1)表达载体的构建
SEQ ID NO.1为重组单链人FVIII-Fc融合蛋白的氨基酸序列，使FVIII的C端通过间隔序列GS与Fc的N端连接，其中，20位到1457位为B结构域缺失的人FVIII序列；1位到19位为人FVIII的天然信号肽，融合蛋白借助于该信号肽分泌到细胞外。Fc结构域优先选择人IgG1，其中N297G突变以去除ADCC效应；两个CH2-CH3结构域通过柔性多肽sggsstasgsgsggsgtagssggagssggsttaggsasgsgstgsgtggassggasgasg连接成一条单链，并在单链Fc片段中靠近N端的CH3中引入T366S(EU编码)、L368A和Y407V突变，在靠近C端的CH3中引入T366W突变。这种设计是为了更好地形成链内Fc结构，而避免形成“FVIII-Fc-Fc”同源二聚体。在后文中也将重组单链人FVIII-Fc融合蛋白称为F8-59L。
全长基因序列根据CHO细胞偏爱进行密码子优化，优化的核酸序列为SEQ IDNO.2，其借助于NheI和EcoRI克隆到真核表达载体pcDNA3.1-SD载体上。该载体包含DHFR基因，可通过DHFR和融合蛋白基因的共扩增来实现单链人FVIII-Fc的高水平表达。
2)高表达细胞株的筛选
采用电穿孔的方法转染宿主细胞(CHO DG44)：使用Bio-Rad电转仪，4mm电转杯，设置电压为280V和电击时间为25毫秒。每次电击1×107个细胞，质粒40μg，总体积为0.7ml。电转后细胞转入含有30ml生长培养基的摇瓶中培养。培养24小时后，将培养基换成含200nMMTX的筛选培养基中，并以1000个细胞/孔接种于96孔板中。细胞培养2周左右，直到克隆汇合率达到80％或以上，用抗人FVIII抗体和抗人IgG抗体，采用夹心ELISA方法分析表达量，筛选出表达量相对高的克隆，依次转入24孔板、6孔板、T25细胞培养瓶和细胞培养摇瓶中扩大培养。在转入24孔板时，将培养基中MTX的浓度提高到500nM。
3)融合蛋白的生产
采用Fed-batch的方法生产重组单链人FVIII-Fc融合蛋白。细胞以0.8×106个/mL接种到5L生物反应器(Biobundle 5L，Applikon)，反应条件为：37℃、pH 6.95±0.15、溶氧(DO)40％，培养3到4天后补料流加培养。第10天时，细胞存活率降到88％，终止发酵并收集上清液进行纯化。下罐时，根据发色底物法(见实施例2)测得的FVIII活性估算培养上清中的产量为272.3IU/ml。
图1为细胞生长曲线。细胞生长最高密度达到11.5×106个/mL，细胞倍增时间为37.8h。
图2为细胞培养液残糖和PH变化趋势。在整个发酵过程中，pH全程6.95±0.15；在第3-5天，每天葡萄糖消耗约2.5g，第五天，培养温度降低后，细胞耗糖减少，在第7-9天时，随着蛋白进入高速表达期，耗糖量逐渐增加，最多可达4g/L。
4)融合蛋白的纯化
离心收集细胞培养上清，用0.22μm的硝酸纤维素过滤器过滤。其纯化流程如下：
Protein A亲和层析进行初步捕获及浓缩--G25置换缓冲液--Q-HP阴离子层析去除DNA及HCP等杂质，并去除一定量的聚体成分--Superdex 200分子筛层析去除聚体并将样品置换至制剂缓冲体系中。
从血浆或细胞培养上清中分离纯化得到的活性FVIII由两条链组成--轻链和重链，二者之间通过二价离子(Ca2+)非共价连接。
纯化后的重组单链人FVIII-Fc融合蛋白的SEC检测结果见图3，蛋白纯度为97.80％。SDS-PAGE检测结果见图4，和预测一致，非还原和还原电泳都包括三条带，分别为FVIII重链(约90KDa)，FVIII轻链-Fc(约130kDa)和全长重组人FVIII-Fc(约220kDa)。
实施例2重组融合蛋白的生物活性分析
1.发色底物法测定融合蛋白的生物学活性
采用Chromogenix Coatest SP FVIII试剂盒(Chromogenix，Ref.K824086)测定融合蛋白的FVIII活性，其检测原理如下:当被凝血酶激活后，FVIIIa在磷脂和钙离子存在下，与FIXa结合形成酶复合物，继而可激活因子X转变成其活性形式Xa。活化的Xa继而可使特异性发色底物(S-2765)发生裂解，释放发色基团pNA。在405nm下测定所产生pNA的量，即可知与其量直接成正比关系的FXa的活性大小，其中在体系中因子IXa和因子X的含量是一定且过量的，FXa的活性仅与FVIIIa的含量多少直接相关。用发色法测定纯化的重组单链人FVIII-Fc的生物学活性为899.4IU/ml。根据以下公式计算其比活性为9993.3IU/mg。
比活性(IU/mg)＝生物学活性(IU/ml)/蛋白含量(mg/ml)
2.一期法(凝固法)测定融合蛋白的生物学活性
凝固法测定FVIII生物学活性是通过纠正FVIII因子缺失血浆所导致凝固时间延长的能力而获得的。采用德国Siemens公司生产的试剂盒Coagulation Factor VIIIDeficient Plasma(Cat.No.OTXW17)。检测方法首先是已知效价的FVIII活性标准品WHOInternational Standard 8th International Standard Factor VIII Concentrate(Cat.No.07/350)用5％乏FVIII血浆稀释至1IU/ml，再分别进行稀释10倍、20倍、40倍、80倍，并与乏FVIII基质血浆混合，测定部分凝血活酶时间(APTT)，以FVIII活性标准品溶液效价(IU/ml)的对数对应其相应的凝固时间(s)的对数作直线回归，建立标准曲线。再将待测样本经适度稀释后与乏FVIII基质血浆混合，进行APTT测定。通过代入标准曲线，可知待测样品FVIII的效价为多少，据此可求算出待测样品FVIII的比活性大小，单位为IU/mg。用凝血法直接测定纯化的重组单链人FVIII-Fc的生物学活性为725.6IU/ml。根据以下公式计算其比活性为10321.1IU/mg。
比活性(IU/mg)＝生物学活性(IU/ml)/蛋白含量(mg/ml)
根据报道，重组B结构域缺失的FVIII
的比活性为9110-13700IU/mg。
的分子量为170kDa，而重组单链人FVIII-Fc的理论分子量为221kDa，考虑到两个蛋白分子量的差异，说明C末端单链Fc的加入不影响FVIII的活性。
实施例3重组融合蛋白对血友病A(HemA)小鼠急性出血的止血作用
用FVIII基因剔除纯合子HemA小鼠断尾出血模型(tail clip bleeding model)评估实施例1所制备的融合蛋白F8-59L在HemA小鼠体内的止血活性。选取8-12周龄雄性HemA小鼠(购自上海南方模式生物责任有限公司)，适应性饲养一周后将小鼠随机分为6组，分别单次尾静脉注射给予不同活性剂量的融合蛋白F8-59L或对照药物任捷(Xyntha，辉瑞)，另设HemA小鼠+载体组(Vehicle)和正常小鼠C57BL/6+载体组。动物分组情况见下表2：
表2：融合蛋白F8-59L对HemA小鼠止血效果实验动物分组情况
给药前，首先以1.0％戊巴比妥钠(Sigma公司)按照0.1ml/10g剂量腹腔注射麻醉小鼠，然后将小鼠置于37℃的加热垫上以保持其体温。将小鼠尾巴浸入37℃的温水中10分钟，使尾静脉扩张，然后按照上表方式尾静脉注射给药。给药10分钟后，在距小鼠尾末端1.5cm处剪断，迅速将尾端浸入装有约13ml预热生理盐水的离心管中，并开始计时。如果出血在30分钟内停止，记录出血时间和出血量。如果出血时间超过30分钟，则记为30分钟。出血量(ml)＝(采血后离心管重量(g)-采血前离心管重量(g))/1.05。30分钟后，将鼠尾从生盐水管中取出。在24小时内，每隔10分钟观察记录复出血情况，并记录小鼠存活数量。所有数据以均数±标准误
表示，各实验组间比较采用t-test检验分析，分析软件采用Graphpad Prism 5.0，p<0.05认为有统计学意义。
从图5和图6中各组动物的出血量和出血时间的统计结果分析，HemA小鼠给予F8-59L 270IU/kg 10分钟后，其出血时间和出血量与C57+载体组接近；小鼠给药F8-59L 90IU/kg后，在出血时间和出血量上与C57+载体组差异不显著；与HA+载体组比较，F8-59L 30IU/kg给药组的出血量显著减少、出血时间显著缩短(p<0.01；p<0.01)，说明F8-59L促凝血效果明显，可以作为血友病等凝血因子缺乏症发生急性出血情况的有效凝血剂。与F8-59L30IU/kg给药组比较，F8-59L 90IU/kg组出血量显著减少、出血时间显著缩短(p<0.05；p<0.05)，F8-59L270IU/kg组的出血量显著减少、出血时间显著缩短(p<0.01；p<0.01)；另外与F8-59L 90IU/kg组比较，F8-59L 270IU/kg组的出血量减少(p<0.05)，说明融合蛋白F8-59L对HemA小鼠急性出血的止血作用具有一定的剂量-效应关系(详细结果见表3)。
表3：各组HemA小鼠断尾试验的出血时间、出血量和存活率统计
注：a，出血时间超过30分钟，记做1800秒
以上对本发明所提供的重组单链人FVIII-Fc融合蛋白及其应用进行了详细介绍。本文中应用了具体实施例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其中心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护。
序列表
<110> 辅仁药业集团有限公司
<120> 重组单链人FVIII-Fc融合蛋白及其应用
<160> 2
<210> 1
<211> 1976
<212> PRT
<213> 人工序列
<220>
<223> 重组单链人FVIII-Fc融合蛋白序列
<400> 1
MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP PRVPKSFPFN 60
TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY DTVVITLKNM ASHPVSLHAV 120
GVSYWKASEG AEYDDQTSQR EKEDDKVFPG GSHTYVWQVL KENGPMASDP LCLTYSYLSH 180
VDLVKDLNSG LIGALLVCRE GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD 240
AASARAWPKM HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 300
RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE EPQLRMKNNE 360
EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT WVHYIAAEEE DWDYAPLVLA 420
PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY TDETFKTREA IQHESGILGP LLYGEVGDTL 480
LIIFKNQASR PYNIYPHGIT DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP 540
TKSDPRCLTR YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 600
NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL HEVAYWYILS 660
IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS MENPGLWILG CHNSDFRNRG 720
MTALLKVSSC DKNTGDYYED SYEDISAYLL SKNNAIEPRS FSQNPPVLKR HQREITRTTL 780
QSDQEEIDYD DTISVEMKKE DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP 840
HVLRNRAQSG SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 900
RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP TKDEFDCKAW 960
AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE FALFFTIFDE TKSWYFTENM 1020
ERNCRAPCNI QMEDPTFKEN YRFHAINGYI MDTLPGLVMA QDQRIRWYLL SMGSNENIHS 1080
IHFSGHVFTV RKKEEYKMAL YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL 1140
VYSNKCQTPL GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1200
LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV FFGNVDSSGI 1260
KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS MPLGMESKAI SDAQITASSY 1320
FTNMFATWSP SKARLHLQGR SNAWRPQVNN PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM 1380
YVKEFLISSS QDGHQWTLFF QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH 1440
QIALRMEVLG CEAQDLYGSE PKSSDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT 1500
PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY GSTYRVVSVL TVLHQDWLNG 1560
KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLS CAVKGFYPSD 1620
IAVEWESNGQ PENNYKTTPP VLDSDGSFFL VSKLTVDKSR WQQGNVFSCS VMHEALHNHY 1680
TQKSLSLSPG SGGSSTASGS GSGGSGTAGS SGGAGSSGGS TTAGGSASGS GSTGSGTGGA 1740
SSGGASGASG DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 1800
PEVKFNWYVD GVEVHNAKTK PREEQYGSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 1860
PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN 1920
YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 1976
<210> 2
<211> 5934
<212> DNA
<213> 人工序列
<220>
<223> 密码子优化的核酸序列
<400> 2
atgcagatcg aactgtcaac ttgtttcttc ctgtgcctgc tgagattttg cttttccgcc 60
actcgtcgtt actacctagg agccgtggaa ctgagctggg attacatgca gtctgacctg 120
ggagagctgc cagtggacgc tagatttccc cctcgcgtgc ctaagagttt ccccttcaac 180
acctcagtgg tctataagaa aacactgttc gtggagttta ctgatcacct gttcaacatc 240
gccaagccaa gaccaccctg gatgggactg ctgggaccta caatccaggc tgaggtgtac 300
gacactgtgg tcattaccct gaaaaacatg gcaagtcacc cagtgtcact gcatgccgtc 360
ggggtgtcat actggaaggc ttccgaaggt gcagagtatg acgatcagac ctctcagcgc 420
gaaaaagagg acgataaggt gtttcccggc ggaagccata catacgtctg gcaggtgctg 480
aaggagaatg gccccatggc cagcgaccct ctgtgcctga cctactcata tctgtcccac 540
gtggacctgg tgaaggatct gaacagcggg ctgatcggtg cactgctggt gtgtagagaa 600
ggctctctgg ccaaggagaa aactcagacc ctgcataagt tcattctgct gttcgccgtg 660
tttgacgaag gaaaaagctg gcactctgag actaagaact ccctgatgca ggacagggat 720
gcagcaagcg cacgagcttg gcccaaaatg cataccgtca acggctacgt gaatcgaagt 780
ctgcctggcc tgatcggatg ccaccgtaag tccgtctatt ggcatgtgat cgggatgggc 840
accacacccg aagtccacag cattttcctg gagggtcata cctttctggt gagaaaccac 900
cgccaggcat ccctggagat cagccctatt actttcctga ccgcccagac actgctgatg 960
gatctgggcc agttcctgct gttttgccac atctccagcc accagcatga tggaatggag 1020
gcatacgtca aagtggactc ttgtcctgag gaaccacaac tgaggatgaa gaacaatgag 1080
gaagccgaag actatgacga tgacctgaca gactccgaga tggatgtggt ccgcttcgat 1140
gacgataact cccctagctt tatccagatt cgaagcgtcg ccaagaaaca cccaaagact 1200
tgggtgcatt acatcgcagc cgaggaagag gactgggatt atgctccact ggtgctggca 1260
cccgatgatc ggagttacaa atcacagtat ctgaacaatg ggcctcagcg aattggtcgt 1320
aagtacaaga aagtgcgatt catggcctat actgatgaaa cctttaagac acgtgaagct 1380
atccagcacg agtctgggat tctgggtcca ctgctgtacg gcgaagtggg agacacactg 1440
ctgatcattt ttaagaacca ggcaagcaga ccttacaata tctatccaca tggaattact 1500
gatgtccggc ctctgtactc taggcggctg ccaaaggggg tgaaacacct gaaggacttc 1560
cccatcctgc ctggtgaaat ttttaagtac aagtggacag tcactgtgga ggatgggcca 1620
acaaagtctg accctcgatg cctgactcgt tactattcta gtttcgtgaa tatggaaaga 1680
gacctggcct ccgggctgat cggtcctctg ctgatttgtt acaaagagtc tgtggatcag 1740
aggggcaacc agatcatgag tgacaagcgg aatgtcattc tgttcagcgt gtttgacgaa 1800
aacaggtctt ggtatctgac cgagaacatc cagcggttcc tgccaaatcc cgcaggcgtg 1860
cagcttgaag atccagagtt tcaggccagc aacatcatgc attctattaa tggatacgtg 1920
ttcgactctc tgcagttgag tgtctgtctg cacgaggtgg cctactggta tatcctgtct 1980
attggcgctc agactgattt cctgtcagtg ttcttttccg gatacacctt taagcataaa 2040
atggtgtatg aggacaccct gacactgttc ccctttagtg gcgaaaccgt gtttatgtca 2100
atggagaatc ctggcctgtg gattctggga tgccacaact ccgatttcag aaatcgcggg 2160
atgaccgctc tgctgaaagt gtcatcctgt gacaagaaca ctggtgacta ctatgaagat 2220
agttacgagg acatctcagc ttatctgctg tccaaaaaca atgcaattga accacgatct 2280
tttagtcaga atcctccagt gctgaagagg caccagcggg agatcacaag gactaccctg 2340
cagagtgatc aggaagagat cgactacgac gatactattt ccgtggaaat gaagaaagag 2400
gacttcgaca tctatgacga agatgagaac cagtccccca ggagcttcca gaagaaaacc 2460
cgtcattact ttattgctgc agtggagcgc ctgtgggatt atggcatgag ctctagtcca 2520
cacgtcctgc gaaatcgtgc ccagtcaggc tccgtgcccc agttcaagaa agtggtcttc 2580
caggagttta cagacggctc ctttactcag ccactgtaca gaggagaact gaacgagcat 2640
ctgggcctgc tgggacccta tatccgcgcc gaagtcgagg ataacattat ggtgaccttc 2700
agaaatcagg ccagccgccc ctactctttt tattcatccc tgatcagcta cgaagaggac 2760
cagagacagg gcgctgaacc ccgcaaaaac ttcgtgaagc ctaatgagac taaaacctac 2820
ttttggaagg tgcagcacca catggcacct acaaaagacg agttcgattg caaggcatgg 2880
gcctattttt cagacgtcga tctggagaag gacgtgcatt ctgggctgat cggtcccctg 3940
ctggtgtgtc atacaaacac tctgaatcct gctcacggca ggcaggtcac cgtgcaggaa 3000
tttgcactgt tctttaccat ctttgatgag acaaagtctt ggtactttac agaaaacatg 3060
gagagaaatt gccgcgctcc ttgtaatatt cagatggaag acccaacttt caaggagaac 3120
tatcggtttc atgcaatcaa tggctatatt atggataccc tgcctggact ggtcatggcc 3180
caggaccaga ggattcggtg gtatctgctg tctatgggga gtaacgagaa tatccacagt 3240
attcatttct caggtcacgt ctttaccgtg aggaagaaag aagagtataa aatggccctg 3300
tacaacctgt atccaggcgt cttcgaaaca gtggagatgc tgccctccaa ggctggaatc 3360
tggcgggtgg aatgcctgat tggggagcac ctgcatgcag gcatgtccac actgtttctg 3420
gtgtacagca ataagtgtca gactccactg gggatggcca gcggtcatat ccgggatttc 3480
cagattaccg cttctggcca gtacggacag tgggctccca agctggctag actgcactat 3540
agcggctcta tcaacgcctg gtccactaaa gagcccttct cctggattaa ggtggacctg 3600
ctggctccca tgatcattca tgggatcaaa acccagggtg cacgccagaa gttcagctct 3660
ctgtacatct ctcagtttat catcatgtac agtctggatg gaaagaaatg gcagacctac 3720
cgaggcaatt ccaccggaac actgatggtc ttctttggca acgtggacag ttcaggaatc 3780
aagcacaaca ttttcaatcc ccctatcatt gctcgataca tccgtctgca ccctacccat 3840
tattcaatta ggtccacact gcggatggaa ctgatggggt gcgatctgaa cagttgttca 3900
atgccactgg gtatggagtc caaggcaatc agcgacgccc agattaccgc ttccagctac 3960
ttcactaata tgtttgccac ctggtccccc agcaaagcta ggctgcatct gcagggccga 4020
agcaacgcct ggcgtccaca ggtcaacaat cccaaggagt ggctgcaggt ggattttcag 4080
aaaacaatga aggtcactgg cgtgacaact cagggagtca aatctctgct gacaagtatg 4140
tacgtgaagg agttcctgat ctctagttca caggacggac accagtggac tctgttcttt 4200
cagaacggga aggtcaaagt gttccagggt aatcaggatt ccttcacccc tgtggtcaac 4260
tctctagacc cacccctgct gaccaggtat ctgcgaatcc acccacagag ctgggtccat 4320
cagattgctc tgagaatgga agtgctgggg tgcgaggcac aggatctgta tggatctgaa 4380
cctaaaagct ccgacaaaac acatacctgt ccaccatgcc ctgcacctga gctcctgggc 4440
ggtccaagcg tttttctttt tcctcctaag cctaaggaca ccctgatgat ctccagaact 4500
cctgaggtta cttgcgtggt agtggatgtt tcccacgaag acccagaggt gaaattcaat 4560
tggtatgtgg acggagtcga agtgcacaac gcaaagacca agccaagaga agaacaatac 4620
ggttcaactt accgggttgt gtccgtgctg acagtgcttc atcaggactg gctcaatggc 4680
aaagaatata agtgtaaagt ttctaacaag gccctccccg cccccattga gaagaccata 4740
agcaaagcaa aggggcagcc acgagagccc caggtgtata ctctcccccc ttctcgggag 4800
gagatgacaa agaatcaagt cagtctgtcc tgcgcagtca agggcttcta tccctccgat 4860
atcgcagtgg agtgggagag caatggtcag cctgaaaaca attacaagac cacccctccc 4920
gtgttggatt ccgatgggag ctttttcctg gtgtccaagc tgacagtgga caagagccga 5980
tggcaacagg gcaacgtttt ctcctgtagc gttatgcacg aggcactcca caaccactac 5040
acacaaaagt cattgtccct gagtcccgga tccggtggaa gttctacagc aagtggtagt 5100
ggatcaggtg gatcaggcac agctggtagt tctggaggtg caggaagcag tggaggtagt 5160
acaaccgccg gaggtagtgc atctggttct ggtagcactg gaagtggcac aggtggagca 5220
tctagtggag gtgctagtgg tgcatcaggt gacaaaacac atacctgtcc accatgccct 5280
gcacctgagc tcctgggcgg tccaagcgtt tttctttttc ctcctaagcc taaggacacc 5340
ctgatgatct ccagaactcc tgaggttact tgcgtggtag tggatgtttc ccacgaagac 5400
ccagaggtga aattcaattg gtatgtggac ggagtcgaag tgcacaacgc aaagaccaag 5460
ccaagagaag aacaatacgg ttcaacttac cgggttgtgt ccgtgctgac agtgcttcat 5520
caggactggc tcaatggcaa agaatataag tgtaaagttt ctaacaaggc cctccccgcc 5580
cccattgaga agaccataag caaagcaaag gggcagccac gagagcccca ggtgtatact 5640
ctcccccctt ctcgggagga gatgacaaag aatcaagtca gtctgtggtg cctcgtcaag 5700
ggcttctatc cctccgatat cgcagtggag tgggagagca atggtcagcc tgaaaacaat 5760
tacaagacca cccctcccgt gttggattcc gatgggagct ttttcctgta ctccaagctg 5820
acagtggaca agagccgatg gcaacagggc aacgttttct cctgtagcgt tatgcacgag 5880
gcactccaca accactacac acaaaagtca ttgtccctga gtcccggatg ataa 5934
Claims (7)
1.一种重组单链人FVIII-Fc融合蛋白，其中，FVIII是B结构域缺失的人FVIII；Fc来自包含N297G突变的人IgG1，两个CH2-CH3结构域通过柔性多肽连接成一条单链，在靠近N端的CH3中引入T366S、L368A和Y407V突变，在靠近C端的CH3中引入T366W突变；所述重组单链人FVIII-Fc融合蛋白的序列为SEQ ID NO.1。
2.编码权利要求1所述的重组单链人FVIII-Fc融合蛋白的核酸，其序列为SEQ IDNO.2。
3.包含权利要求2所述的核酸的表达载体。
4.表达权利要求1所述的重组单链人FVIII-Fc融合蛋白的细胞株。
5.一种组合物，其包含权利要求1所述的重组单链人FVIII-Fc融合蛋白，和药学上可接受的载体。
6.根据权利要求5所述的组合物，其还包含FVIII重链、FVIII轻链-Fc，其中Fc来自包含N297G突变的人IgG1，两个CH2-CH3结构域通过柔性多肽连接成一条单链，在靠近N端的CH3中引入T366S、L368A和Y407V突变，在靠近C端的CH3中引入T366W突变。
7.权利要求1所述的重组单链人FVIII-Fc融合蛋白在制备治疗A型血友病的药物中的用途。
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| CN105143252A (zh) * | 2013-03-15 | 2015-12-09 | 豪夫迈·罗氏有限公司 | IL-22多肽和IL-22Fc融合蛋白及其使用方法 |
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| CN105143252A (zh) * | 2013-03-15 | 2015-12-09 | 豪夫迈·罗氏有限公司 | IL-22多肽和IL-22Fc融合蛋白及其使用方法 |
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