CN108220274B - 一种高活性凝血因子xi突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用 - Google Patents
一种高活性凝血因子xi突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用 Download PDFInfo
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Abstract
本发明涉及一种高活性凝血因子XI突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用，核苷酸序列如SEQ ID NO:1‑6，氨基酸序列如SEQ ID NO:7所示。本发明具有很高的凝血活性，可以高效地激活凝血反应，提升机体整体凝血功能，应用于出血性疾病的治疗，具有很好的基因治疗、基因编辑与重组蛋白替代治疗前景。
Description
技术领域
本发明属于出血病治疗领域，特别涉及一种高活性凝血因子XI突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用。
背景技术
凝血因子缺陷或其它人体凝血功能障碍时会导致出血性疾病的发生，其中由于凝血因子 VIII/IX(FVIII/FIX)缺陷所导致的出血性疾病称之为血友病(甲型/乙型)，重型病人的凝血因子VIII/IX活性往往低于正常的1％，经常发生自发的出血导致肌肉血肿或者关节畸形。输注因子VIII/IX制剂(目前通常是体外重组表达的凝血因子VIII/IX蛋白)补充患者体内的凝血因子VIII/IX水平是目前唯一有效的治疗方法，但是需要频繁的给药。基因治疗是目前正在临床试验的治疗方法，将正常的凝血因子VIII/IX基因导入患者体内表达，从而达到提高凝血因子VIII/IX水平，预防出血的目的。虽然应用重组或血浆来源的FVIII/FIX可以有效地治疗血友病A和B，但是约有30％的病人在治疗后产生抗体，使治疗失效。旁路凝血活性药物是治疗有抑制物产生血友病患者的最佳选择。但是目前临床上应用的凝血因子VIIa(FVIIa)，由于半寿期短(～2小时)，所需剂量大(90～100μg/kg体重)，治疗成本高昂，因此如何获得具有更好治疗效果和药物代谢特点的新型旁路凝血途径药物是目前血友病治疗的一个亟待解决的问题。
凝血因子IX是凝血因子XI(FXI)的生理性底物，凝血因子XI的凝血活性主要与其高效裂解活化凝血因子IX的能力有关。但是凝血反应中的其它组分也可能会被凝血因子XI所催化裂解。近年研究表明，凝血因子XI可以催化凝血因子V(FV)和凝血因子X(FX)活化，从而越过凝血因子IX直接激活凝血共同途径；另外一项研究还表明，凝血因子XI可以降解组织因子途径抑制物(TFPI)，从而延长外源途径活化凝血因子FVII(FVIIa)的作用时间，间接地放大外源途径激活的凝血反应。但是野生型凝血因子XI催化裂解包括FX,FV或TFPI 在内的旁路凝血反应底物的效率非常低下，限制了它通过旁路促进凝血反应的能力。
发明内容
本发明所要解决的技术问题是提供一种高活性凝血因子XI突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用，该突变体具有很高的凝血活性，可以通过不依赖于凝血因子IX(FIX)/凝血因子VIII(FVIII)的机制高效的通过旁路途径激活凝血反应，提升机体整体凝血功能，应用于出血性疾病的治疗，具有很好的基因治疗与重组蛋白替代治疗前景。
本发明提供了一种高活性凝血因子XI突变体：
(1)核苷酸序列如SEQ ID NO:1所示，位于1189位的核苷酸为A而非G；
或(2)核苷酸序列如SEQ ID NO:2所示，位于1189位的核苷酸为A而非G，位于1191位的核苷酸为C而非T；
或(3)核苷酸序列如SEQ ID NO:3所示，位于1189位的核苷酸为T而非G，位于1190位的核苷酸为C而非G；
或(4)核苷酸序列如SEQ ID NO:4所示，位于1189位的核苷酸为T而非G，位于1190位的核苷酸为C而非G；位于1191位的核苷酸为C而非T；
或(5)核苷酸序列如SEQ ID NO:5所示，位于1189位的核苷酸为T而非G，位于1190位的核苷酸为C而非G，位于1191位的核苷酸为A而非T；
或(6)核苷酸序列如SEQ ID NO:6所示，位于1189位的核苷酸为T而非G，位于1190位的核苷酸为C而非G，位于1191位的核苷酸为G而非T；
或是任意其他核苷酸在1189位、1190位和1191位突变的组合。
本发明还提供了一种高活性凝血因子XI的突变蛋白，氨基酸序列如SEQ ID NO:7所示，位于397的氨基酸为Ser而非Gly；或是任意其他氨基酸在该位点的改变。
本发明还提供了一种编码高活性凝血因子XI的突变蛋白的核酸，或与所述编码核酸长度相同且与所述编码核酸完全互补的核酸。
本发明还提供了一种表达高活性凝血因子XI的突变蛋白的载体。
本发明还提供了一种高活性凝血因子XI的突变蛋白的制备方法，包括如下步骤：
(1)将人野生型或凝血因子XI Gly397Ser突变的人凝血因子XI基因连入载体中，得到重组载体；
(2)将上述重组载体转化宿主细胞，得到表达重组凝血因子XI Gly397Ser突变细胞克隆；
(3)于无血清培养基中连续灌流培养上述重组细胞克隆，诱导重组凝血因子XIGly397Ser 的突变蛋白的表达；
(4)分离纯化、过滤，最后灌装、冻干，得到所表达的高活性凝血因子XI Gly397Ser突变蛋白。
所述步骤(3)中的无血清培养基为“SAFC Biosciences EX-CELLTM 302”(商品化的试剂)。
所述步骤(4)中的纯化包括初纯和精纯。
本发明还提供了一种表达突变蛋白(FXI Gly397Ser)的质粒载体进行基因转导，其制备和检验包括如下步骤：
(1)将编码FXI Gly397Ser的cDNA连接入含CMV启动子的pcDNA3.1质粒。
(2)将纯化的表达FXI Gly397Ser的质粒载体150μg溶于2mL生理盐水经鼠尾静脉高压注射入4-8周的A型血友病小鼠体内。注射同体积PBS为阴性对照。
(3)注射48小时后，眼眶采血应用凝固法APTT检测凝血因子XI活性，ELISA法检测凝血因子XI抗原。
(4)注射后72小时，直径2mm处断尾，置于温生理盐水中观察10分钟内出血情况。通过检测血红蛋白量估测出血量，以缓冲液注射组出血量为100％，比较不同质粒转导后的血友病小鼠断尾后出血量。
(5)心脏取血进行血栓弹力图检测。
所述高活性凝血因子XI突变蛋白应用于制备基因治疗药物。
所述高活性凝血因子XI突变蛋白应用于制备血友病或其他出血性疾病的重组蛋白治疗药物。
所述高活性凝血因子XI突变蛋白应用于制备凝血因子XI Gly397Ser突变体融合蛋白，并将其施用于血友病或其他出血性疾病的重组蛋白治疗药物。
所述融合蛋白为人白蛋白、免疫球蛋白Fc、转铁蛋白或alpha 1抗胰蛋白酶。
本发明的核酸或氨基酸序列的药物组合物或基因治疗载体，用于诊断，预防和/或治疗疾病，其中所述疾病主要包括出血性疾病或各种原因导致的出血；其中，最可能的出血性疾病是血友病A和B，即由于遗传性凝血因子VIII或IX缺乏导致的出血性疾病，并包括其中有抑制性抗体产生的血友病A和B，或者后天因抑制物产生所导致的获得性凝血因子VIII或IX 缺乏；及其它使用旁路制剂的出血性疾病，例如新生儿凝血障碍；严重的肝脏疾病；高风险手术；创伤性失血；骨髓移植；血小板减少症和血小板功能障碍；口服抗凝的紧急逆转；先天性凝血因子V，VII，X和XI的缺陷；血管性血友病，及血管性血友病因子抑制物导致的获得性血管性血友病，与大量损伤有关的失血，大脑出血，血小板功能障碍。
有益效果
本发明具有很高的凝血活性，对非生理性底物具有更强的催化能力，从而增强了旁路途径凝血活性，通过不依赖于凝血因子IX(FIX)/凝血因子VIII(FVIII)的机制高效的通过旁路途径激活凝血反应，提升机体整体凝血功能，应用于出血性疾病的治疗，具有很好的基因治疗与重组蛋白替代治疗前景。
附图说明
图1-图7为本发明高活性凝血因子XI突变的核酸与编码蛋白的序列示意图；
图8为本发明载体结构的示意图；
图9为重组凝血因子XI突变体Gly397Ser活性示意图；
图10为凝血因子XI突变体Gly397Ser体外纠正凝血因子VIII缺陷血浆凝血酶生成示意图；
图11为基因转导凝血因子XI突变体Gly397Ser后血友病小鼠的人凝血因子XI抗原水平示意图；
图12为基因转导凝血因子XI突变体Gly397Ser后血友病小鼠的凝血因子XI活性示意图；
图13为基因转导凝血因子XI突变体Gly397Ser纠正血友病小鼠断尾出血量示意图；
图14为血栓弹力图检测基因转导凝血因子XI突变体Gly397Ser纠正血友病小鼠血浆凝血缺陷示意图；其中，a为凝血因子XI突变体Gly397Ser处理组，b为无凝血因子VIII缺陷正常小鼠组，c为缺乏凝血因子VIII的血友病小鼠组。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
高活性凝血因子XI的突变蛋白的氨基酸序列如SEQ ID NO:7所示。
高活性凝血因子XI的突变蛋白的制备方法，包括如下步骤：
(1)将人野生型或凝血因子XI突变体Gly397Ser的人凝血因子XI编码基因连入载体中，得到重组载体；(见图8)
(2)将上述重组载体转化宿主细胞，得到重组表达细胞克隆；
(3)于无血清培养基中培养上述细胞克隆，表达高活性凝血因子XI的突变蛋白的表达；
无血清培养基为“SAFC Biosciences EX-CELLTM 302”(商品化的试剂)。为保证产品安全，防止血液来源制剂传播感染性疾病，故应用无血清培养基用于哺乳动物细胞培养、蛋白表达，细胞经对数期生长达到稳态后，将细胞密度维持在目标区间内，维持凝血因子XI高表达。
(4)分离纯化与冻干，从而得到所表达的凝血因子XI突变蛋白及相关融合蛋白。
在培养基收集后，经深层滤器澄清过滤并进一步分离纯化。纯化步骤分为初纯和精纯两个阶段，初纯：将过滤澄清的培养液在10倍超滤浓缩后通过有机溶剂/洗涤剂法灭活脂包膜病毒即HIV1/2、HCV以及HBV等；精纯：以离子交换(阴离子和阳离子)和分子筛等色谱层析方法进一步去除产品中残留以宿主细胞分泌的其他蛋白为主的杂质。纯化蛋白经过超滤换液、调节配方后再进行即20nm纳米膜除病毒过滤并冻干。冻干过程为速冻、淬火、冷冻、真空、主干燥、后干燥。冻干配方以甘氨酸、甘露醇、氯化钠、氯化钙等惰性糖类以及无机盐为主(组成为甘氨酸、甘露醇、氯化钠、氯化钙等；冻干时间为30小时)。
(5)凝血因子XI突变体活性和抗原检测方法。凝血因子XI的特异性凝血活性由凝血活性与抗原比较推算而得，见图9。由图9可知，凝血因子XI突变体Gly397Ser具有比野生型更高的凝血活性。
凝血因子XI活性和抗原检测方法：
①凝固法检测凝血因子XI活性：
正常混合血浆用OV Buffer分别进行1:10,1:20,1:40,1:80,1:160,1:320稀释，血浆待测样本进行1:10和1:20稀释，细胞上清液不处理。取50微升稀释的正常混合血浆、待测血浆样本或转染凝血因子XI表达载体的细胞上清液，加入50微升凝血因子XI基质血浆，加入APTT 试剂，37℃温育3分钟，再加入50微升氯化钙，ST4半自动血凝仪(Stago公司，法国)上记录凝固时间。以正常混合血浆1:10稀释的凝血因子XI活性为100％，以不同稀释度对应的凝固时间的log值和对应的活性得log值建立标准曲线，若相关系数R2大于0.95，则将待测样本的值带入计算，得到待测样本的凝血因子XI活性。
②双抗夹心法检测凝血因子XI的抗原：
用包被液(1.59g/L碳酸钠和2.94g/L碳酸氢钠，pH 9.6)将包被抗体(F9ELISA试剂盒,Affinity Biologicals,EIA9-0035R1)1:100稀释，加入稀释抗体100ul/孔，室温孵育2小时。重复洗涤3次。将正常混合血浆用样品稀释液(23.8g HEPES(free acid)/L,5.84g/LNaCl,3.72g/ L Na2EDTA,10g/L BSA,0.1％吐温-20，Ph 7.2)分别1:100对倍稀释至1:3200。待测血浆样本以1:200,1:400和1:800稀释，细胞上清液分别为原液、1:10和1:100稀释。每孔加入100ul 稀释好的正混血浆或待测样品，室温放置90min。重复洗涤3次。将检测抗体用样品稀释液 1:100稀释，每孔加入100ul的稀释完的检测抗体，室温放置90min。重复洗涤3次。每孔加入100ul的OPD底物，待出现稳定的黄色之后(约5-10min)，每孔加入100ul的终止液。用酶标仪在450nm的波长下读取吸光度。建立标准曲线，并计算待测样本的抗原值。
(6)凝血因子XI突变体纠正血友病A患者血浆凝血酶生成缺陷
凝血酶生成试验(thrombin generation test,TGT)：是用于监测血浆中凝血酶生成能力的综合性实验。将激活剂(含组织因子及磷脂)加入血浆中启动凝血反应，再加入凝血酶特异性的荧光底物，生成的凝血酶催化底物释放出荧光基团，使用FLUOROSKAN荧光读数仪动态监测生成的荧光信号，使用配套的凝血酶生成实验软件将信号转换成一条凝血酶生成曲线。主要通过曲线的几个参数评价凝血酶生成能力：(1)延迟时间(lag time)，即从反应开始到凝血酶开始生成所需时间；(2)峰值(peak)，即生成的凝血酶最大量；(3)达峰时间(time to peak,ttpeak)，即从反应开始到凝血酶达峰值所需时间；(4)凝血酶生成潜力(endogenous thrombin potential,ETP)，即凝血酶生成曲线下面积，反应凝血酶生成总量。
在血友病A患者的血浆中(乏凝血因子VIII)中加入凝血因子XI或其突变体(浓度为正常生理浓度5ug/mL)，进行凝血酶生成检测，见图10。图10显示凝血因子XI突变体Gly397Ser可以部分纠正由于凝血因子VIII缺乏造成的凝血酶生成障碍。
实施例2
高活性凝血因子XI的基因治疗质粒载体的制备方法，包括如下步骤：
(1)载体质粒的构建将编码FXI Gly397Ser的cDNA连接入含CMV启动子的pcDNA3.1质粒。
(2)将纯化的载体注射A型血友病小鼠。选用4-8周的血友病小鼠，通过尾静脉将质粒载体 150μg溶于2mL生理盐水分别注射6-7只小鼠。PBS注射6只小鼠为负对照。
(3)aPTT检测凝血因子XI活性。注射48小时后，眼眶采血检测凝血因子XI活性和抗原，其中活性为小鼠体内原有小鼠凝血因子XI活性和倒入的人野生型或突变凝血因子XI的活性的总和，而抗原则仅为倒入的人凝血因子XI或其突变体的水平。载体基因转导处理后小鼠血浆凝血因子XI抗原见图11，由图11可知，基因转导的人血浆凝血因子XI突变体Gly397Ser 的表达与野生型凝血因子XI相似。基因转导的人血浆凝血因子XI突变体Gly397Ser小鼠血浆凝血因子XI活性见图12，由图12可知，基因转导表达的人血浆凝血因子XI的突变体 Gly397Ser具有更高的凝血活性。
(4)将小鼠麻醉后，在直径为2mm处断尾，将尾巴浸入PBS中，即刻开始计时10min，再检测血红蛋白，根据血红蛋白量估算出血程度(见图13)。由图13可知，基因转导表达的人血浆凝血因子XI突变体Gly397Ser可在体内纠正凝血因子VIII缺陷所导致的凝血缺陷，减少出血。
(5)小鼠心脏取血，检测血栓弹力图(见图14)
血栓弹力图(thromboelastogram，TEG)：是用于监测全血中全部凝血过程的综合性试验。它不需要血标本处理，用少量全血监测凝血因子、血小板、纤维蛋白原、纤溶系统和其他细胞成分之间的相互作用，准确地提供患者的凝血概况。检测时先将抗凝血加至活化监测试剂瓶中，再吸出一定体积加至特制圆柱形杯中(提前加入CaCl2)。杯子以4°45'的角度和1 周/9s的速度匀速转动，通过一根浸泡在血液中由螺旋丝悬挂的针监测血液的凝固状态，并由计算机绘制凝血速度和强度曲线。主要通过以下几个曲线参数评估凝血过程：(1)反应时间R值，即检测起始至曲线振幅上升至2mm所需时间，是指从标本开始检测至纤维蛋白凝块开始形成所需的时间；(2)凝集时间K值和凝集块形成速率α角，凝集时间K值是记录从凝血时间终点至曲线振幅达20mm所需时间，凝集块形成速率α角是指从血凝块形成点至描记图最大弧度所作切线与水平线的夹角，它们反映纤维蛋白和血小板在血凝块开始形成时的共同作用的结果，主要受纤维蛋白原功能影响；(3)MA值是指描记图上的最大振幅，即最大切变力系数。反映正在形成的纤维蛋白与血小板相互联结的最强动力学特性及血凝块形成的稳定性，其中血小板的作用要比纤维蛋白原大，约占80％；(4)凝血综合指数即CI值，是结合了血栓弹力图曲线的反应时间、凝集时间、凝集块形成速率、最大振幅结合推算得出。反映样本在各种条件下的凝血综合状态，低于-3提示存在低凝，高于3提示存在高凝，介于 -3和3提示为正常。
由图14可知，基因转导表达的人血浆凝血因子XI突变体Gly397Ser可在体内纠正凝血因子VIII缺陷所导致的凝血缺陷。
SEQUENCE LISTING
<110> 上海交通大学医学院附属瑞金医院
王学锋
武文漫
丁秋兰
<120> 一种高活性凝血因子XI突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用
<130> 1
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 1878
<212> DNA
<213> 人工序列
<400> 1
atgattttct tatatcaagt ggtacatttc attttattta cttcagtttc tggtgaatgt 60
gtgactcagt tgttgaagga cacctgcttt gaaggagggg acattactac ggtcttcaca 120
ccaagcgcca agtactgcca ggtagtctgc acttaccacc caagatgttt actcttcact 180
ttcacggcgg aatcaccatc tgaggatccc acccgatggt ttacttgtgt cctgaaagac 240
agtgttacag aaacactgcc aagagtgaat aggacagcag cgatttctgg gtattctttc 300
aagcaatgct cacaccaaat aagcgcttgc aacaaagaca tttatgtgga cctagacatg 360
aagggcataa actataacag ctcagttgcc aagagtgctc aagaatgcca agaaagatgc 420
acggatgacg tccactgcca ctttttcacg tacgccacaa ggcagtttcc cagcctggag 480
catcgtaaca tttgtctact gaagcacacc caaacaggga caccaaccag aataacgaag 540
ctcgataaag tggtgtctgg attttcactg aaatcctgtg cactttctaa tctggcttgt 600
attagggaca ttttccctaa tacggtgttt gcagacagca acatcgacag tgtcatggct 660
cccgatgctt ttgtctgtgg ccgaatctgc actcatcatc ccggttgctt gttttttacc 720
ttcttttccc aggaatggcc caaagaatct caaagaaatc tttgtctcct taaaacatct 780
gagagtggat tgcccagtac acgcattaaa aagagcaaag ctctttctgg tttcagtcta 840
caaagctgca ggcacagcat cccagtgttc tgccattctt cattttacca tgacactgat 900
ttcttgggag aagaactgga tattgttgct gcaaaaagtc acgaggcctg ccagaaactg 960
tgcaccaatg ccgtccgctg ccagtttttt acctataccc cagcccaagc atcctgcaac 1020
gaagggaagg gcaagtgtta cttaaagctt tcttcaaacg gatctccaac taaaatactt 1080
cacgggagag gaggcatctc tggatacaca ttaaggttgt gtaaaatgga taatgagtgt 1140
accaccaaaa tcaagcccag gatcgttgga ggaactgcgt ctgttcgtag tgagtggccg 1200
tggcaggtga ccctgcacac aacctcaccc actcagagac acctgtgtgg aggctccatc 1260
attggaaacc agtggatatt aacagccgct cactgtttct atggggtaga gtcacctaag 1320
attttgcgtg tctacagtgg cattttaaat caatctgaaa taaaagagga cacatctttc 1380
tttggggttc aagaaataat aatccatgat cagtataaaa tggcagaaag cgggtatgat 1440
attgccttgt tgaaactgga aaccacagtg aattacacag attctcaacg acccatatgc 1500
ctgccttcca aaggagatag aaatgtaata tacactgatt gctgggtgac tggatggggg 1560
tacagaaaac taagagacaa aatacaaaat actctccaga aagccaagat acccttagtg 1620
accaacgaag agtgccagaa gagatacaga ggacataaaa taacccataa gatgatctgt 1680
gccggctaca gggaaggagg gaaggacgct tgcaagggag attcgggagg ccctctgtcc 1740
tgcaaacaca atgaggtctg gcatctggta ggcatcacga gctggggcga aggctgtgct 1800
caaagggagc ggccaggtgt ttacaccaac gtggtcgagt acgtggactg gattctggag 1860
aaaactcaag cagtgtga 1878
<210> 2
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<212> DNA
<213> 人工序列
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atgattttct tatatcaagt ggtacatttc attttattta cttcagtttc tggtgaatgt 60
gtgactcagt tgttgaagga cacctgcttt gaaggagggg acattactac ggtcttcaca 120
ccaagcgcca agtactgcca ggtagtctgc acttaccacc caagatgttt actcttcact 180
ttcacggcgg aatcaccatc tgaggatccc acccgatggt ttacttgtgt cctgaaagac 240
agtgttacag aaacactgcc aagagtgaat aggacagcag cgatttctgg gtattctttc 300
aagcaatgct cacaccaaat aagcgcttgc aacaaagaca tttatgtgga cctagacatg 360
aagggcataa actataacag ctcagttgcc aagagtgctc aagaatgcca agaaagatgc 420
acggatgacg tccactgcca ctttttcacg tacgccacaa ggcagtttcc cagcctggag 480
catcgtaaca tttgtctact gaagcacacc caaacaggga caccaaccag aataacgaag 540
ctcgataaag tggtgtctgg attttcactg aaatcctgtg cactttctaa tctggcttgt 600
attagggaca ttttccctaa tacggtgttt gcagacagca acatcgacag tgtcatggct 660
cccgatgctt ttgtctgtgg ccgaatctgc actcatcatc ccggttgctt gttttttacc 720
ttcttttccc aggaatggcc caaagaatct caaagaaatc tttgtctcct taaaacatct 780
gagagtggat tgcccagtac acgcattaaa aagagcaaag ctctttctgg tttcagtcta 840
caaagctgca ggcacagcat cccagtgttc tgccattctt cattttacca tgacactgat 900
ttcttgggag aagaactgga tattgttgct gcaaaaagtc acgaggcctg ccagaaactg 960
tgcaccaatg ccgtccgctg ccagtttttt acctataccc cagcccaagc atcctgcaac 1020
gaagggaagg gcaagtgtta cttaaagctt tcttcaaacg gatctccaac taaaatactt 1080
cacgggagag gaggcatctc tggatacaca ttaaggttgt gtaaaatgga taatgagtgt 1140
accaccaaaa tcaagcccag gatcgttgga ggaactgcgt ctgttcgtag cgagtggccg 1200
tggcaggtga ccctgcacac aacctcaccc actcagagac acctgtgtgg aggctccatc 1260
attggaaacc agtggatatt aacagccgct cactgtttct atggggtaga gtcacctaag 1320
attttgcgtg tctacagtgg cattttaaat caatctgaaa taaaagagga cacatctttc 1380
tttggggttc aagaaataat aatccatgat cagtataaaa tggcagaaag cgggtatgat 1440
attgccttgt tgaaactgga aaccacagtg aattacacag attctcaacg acccatatgc 1500
ctgccttcca aaggagatag aaatgtaata tacactgatt gctgggtgac tggatggggg 1560
tacagaaaac taagagacaa aatacaaaat actctccaga aagccaagat acccttagtg 1620
accaacgaag agtgccagaa gagatacaga ggacataaaa taacccataa gatgatctgt 1680
gccggctaca gggaaggagg gaaggacgct tgcaagggag attcgggagg ccctctgtcc 1740
tgcaaacaca atgaggtctg gcatctggta ggcatcacga gctggggcga aggctgtgct 1800
caaagggagc ggccaggtgt ttacaccaac gtggtcgagt acgtggactg gattctggag 1860
aaaactcaag cagtgtga 1878
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<212> DNA
<213> 人工序列
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atgattttct tatatcaagt ggtacatttc attttattta cttcagtttc tggtgaatgt 60
gtgactcagt tgttgaagga cacctgcttt gaaggagggg acattactac ggtcttcaca 120
ccaagcgcca agtactgcca ggtagtctgc acttaccacc caagatgttt actcttcact 180
ttcacggcgg aatcaccatc tgaggatccc acccgatggt ttacttgtgt cctgaaagac 240
agtgttacag aaacactgcc aagagtgaat aggacagcag cgatttctgg gtattctttc 300
aagcaatgct cacaccaaat aagcgcttgc aacaaagaca tttatgtgga cctagacatg 360
aagggcataa actataacag ctcagttgcc aagagtgctc aagaatgcca agaaagatgc 420
acggatgacg tccactgcca ctttttcacg tacgccacaa ggcagtttcc cagcctggag 480
catcgtaaca tttgtctact gaagcacacc caaacaggga caccaaccag aataacgaag 540
ctcgataaag tggtgtctgg attttcactg aaatcctgtg cactttctaa tctggcttgt 600
attagggaca ttttccctaa tacggtgttt gcagacagca acatcgacag tgtcatggct 660
cccgatgctt ttgtctgtgg ccgaatctgc actcatcatc ccggttgctt gttttttacc 720
ttcttttccc aggaatggcc caaagaatct caaagaaatc tttgtctcct taaaacatct 780
gagagtggat tgcccagtac acgcattaaa aagagcaaag ctctttctgg tttcagtcta 840
caaagctgca ggcacagcat cccagtgttc tgccattctt cattttacca tgacactgat 900
ttcttgggag aagaactgga tattgttgct gcaaaaagtc acgaggcctg ccagaaactg 960
tgcaccaatg ccgtccgctg ccagtttttt acctataccc cagcccaagc atcctgcaac 1020
gaagggaagg gcaagtgtta cttaaagctt tcttcaaacg gatctccaac taaaatactt 1080
cacgggagag gaggcatctc tggatacaca ttaaggttgt gtaaaatgga taatgagtgt 1140
accaccaaaa tcaagcccag gatcgttgga ggaactgcgt ctgttcgttc tgagtggccg 1200
tggcaggtga ccctgcacac aacctcaccc actcagagac acctgtgtgg aggctccatc 1260
attggaaacc agtggatatt aacagccgct cactgtttct atggggtaga gtcacctaag 1320
attttgcgtg tctacagtgg cattttaaat caatctgaaa taaaagagga cacatctttc 1380
tttggggttc aagaaataat aatccatgat cagtataaaa tggcagaaag cgggtatgat 1440
attgccttgt tgaaactgga aaccacagtg aattacacag attctcaacg acccatatgc 1500
ctgccttcca aaggagatag aaatgtaata tacactgatt gctgggtgac tggatggggg 1560
tacagaaaac taagagacaa aatacaaaat actctccaga aagccaagat acccttagtg 1620
accaacgaag agtgccagaa gagatacaga ggacataaaa taacccataa gatgatctgt 1680
gccggctaca gggaaggagg gaaggacgct tgcaagggag attcgggagg ccctctgtcc 1740
tgcaaacaca atgaggtctg gcatctggta ggcatcacga gctggggcga aggctgtgct 1800
caaagggagc ggccaggtgt ttacaccaac gtggtcgagt acgtggactg gattctggag 1860
aaaactcaag cagtgtga 1878
<210> 4
<211> 1878
<212> DNA
<213> 人工序列
<400> 4
atgattttct tatatcaagt ggtacatttc attttattta cttcagtttc tggtgaatgt 60
gtgactcagt tgttgaagga cacctgcttt gaaggagggg acattactac ggtcttcaca 120
ccaagcgcca agtactgcca ggtagtctgc acttaccacc caagatgttt actcttcact 180
ttcacggcgg aatcaccatc tgaggatccc acccgatggt ttacttgtgt cctgaaagac 240
agtgttacag aaacactgcc aagagtgaat aggacagcag cgatttctgg gtattctttc 300
aagcaatgct cacaccaaat aagcgcttgc aacaaagaca tttatgtgga cctagacatg 360
aagggcataa actataacag ctcagttgcc aagagtgctc aagaatgcca agaaagatgc 420
acggatgacg tccactgcca ctttttcacg tacgccacaa ggcagtttcc cagcctggag 480
catcgtaaca tttgtctact gaagcacacc caaacaggga caccaaccag aataacgaag 540
ctcgataaag tggtgtctgg attttcactg aaatcctgtg cactttctaa tctggcttgt 600
attagggaca ttttccctaa tacggtgttt gcagacagca acatcgacag tgtcatggct 660
cccgatgctt ttgtctgtgg ccgaatctgc actcatcatc ccggttgctt gttttttacc 720
ttcttttccc aggaatggcc caaagaatct caaagaaatc tttgtctcct taaaacatct 780
gagagtggat tgcccagtac acgcattaaa aagagcaaag ctctttctgg tttcagtcta 840
caaagctgca ggcacagcat cccagtgttc tgccattctt cattttacca tgacactgat 900
ttcttgggag aagaactgga tattgttgct gcaaaaagtc acgaggcctg ccagaaactg 960
tgcaccaatg ccgtccgctg ccagtttttt acctataccc cagcccaagc atcctgcaac 1020
gaagggaagg gcaagtgtta cttaaagctt tcttcaaacg gatctccaac taaaatactt 1080
cacgggagag gaggcatctc tggatacaca ttaaggttgt gtaaaatgga taatgagtgt 1140
accaccaaaa tcaagcccag gatcgttgga ggaactgcgt ctgttcgttc cgagtggccg 1200
tggcaggtga ccctgcacac aacctcaccc actcagagac acctgtgtgg aggctccatc 1260
attggaaacc agtggatatt aacagccgct cactgtttct atggggtaga gtcacctaag 1320
attttgcgtg tctacagtgg cattttaaat caatctgaaa taaaagagga cacatctttc 1380
tttggggttc aagaaataat aatccatgat cagtataaaa tggcagaaag cgggtatgat 1440
attgccttgt tgaaactgga aaccacagtg aattacacag attctcaacg acccatatgc 1500
ctgccttcca aaggagatag aaatgtaata tacactgatt gctgggtgac tggatggggg 1560
tacagaaaac taagagacaa aatacaaaat actctccaga aagccaagat acccttagtg 1620
accaacgaag agtgccagaa gagatacaga ggacataaaa taacccataa gatgatctgt 1680
gccggctaca gggaaggagg gaaggacgct tgcaagggag attcgggagg ccctctgtcc 1740
tgcaaacaca atgaggtctg gcatctggta ggcatcacga gctggggcga aggctgtgct 1800
caaagggagc ggccaggtgt ttacaccaac gtggtcgagt acgtggactg gattctggag 1860
aaaactcaag cagtgtga 1878
<210> 5
<211> 1878
<212> DNA
<213> 人工序列
<400> 5
atgattttct tatatcaagt ggtacatttc attttattta cttcagtttc tggtgaatgt 60
gtgactcagt tgttgaagga cacctgcttt gaaggagggg acattactac ggtcttcaca 120
ccaagcgcca agtactgcca ggtagtctgc acttaccacc caagatgttt actcttcact 180
ttcacggcgg aatcaccatc tgaggatccc acccgatggt ttacttgtgt cctgaaagac 240
agtgttacag aaacactgcc aagagtgaat aggacagcag cgatttctgg gtattctttc 300
aagcaatgct cacaccaaat aagcgcttgc aacaaagaca tttatgtgga cctagacatg 360
aagggcataa actataacag ctcagttgcc aagagtgctc aagaatgcca agaaagatgc 420
acggatgacg tccactgcca ctttttcacg tacgccacaa ggcagtttcc cagcctggag 480
catcgtaaca tttgtctact gaagcacacc caaacaggga caccaaccag aataacgaag 540
ctcgataaag tggtgtctgg attttcactg aaatcctgtg cactttctaa tctggcttgt 600
attagggaca ttttccctaa tacggtgttt gcagacagca acatcgacag tgtcatggct 660
cccgatgctt ttgtctgtgg ccgaatctgc actcatcatc ccggttgctt gttttttacc 720
ttcttttccc aggaatggcc caaagaatct caaagaaatc tttgtctcct taaaacatct 780
gagagtggat tgcccagtac acgcattaaa aagagcaaag ctctttctgg tttcagtcta 840
caaagctgca ggcacagcat cccagtgttc tgccattctt cattttacca tgacactgat 900
ttcttgggag aagaactgga tattgttgct gcaaaaagtc acgaggcctg ccagaaactg 960
tgcaccaatg ccgtccgctg ccagtttttt acctataccc cagcccaagc atcctgcaac 1020
gaagggaagg gcaagtgtta cttaaagctt tcttcaaacg gatctccaac taaaatactt 1080
cacgggagag gaggcatctc tggatacaca ttaaggttgt gtaaaatgga taatgagtgt 1140
accaccaaaa tcaagcccag gatcgttgga ggaactgcgt ctgttcgttc agagtggccg 1200
tggcaggtga ccctgcacac aacctcaccc actcagagac acctgtgtgg aggctccatc 1260
attggaaacc agtggatatt aacagccgct cactgtttct atggggtaga gtcacctaag 1320
attttgcgtg tctacagtgg cattttaaat caatctgaaa taaaagagga cacatctttc 1380
tttggggttc aagaaataat aatccatgat cagtataaaa tggcagaaag cgggtatgat 1440
attgccttgt tgaaactgga aaccacagtg aattacacag attctcaacg acccatatgc 1500
ctgccttcca aaggagatag aaatgtaata tacactgatt gctgggtgac tggatggggg 1560
tacagaaaac taagagacaa aatacaaaat actctccaga aagccaagat acccttagtg 1620
accaacgaag agtgccagaa gagatacaga ggacataaaa taacccataa gatgatctgt 1680
gccggctaca gggaaggagg gaaggacgct tgcaagggag attcgggagg ccctctgtcc 1740
tgcaaacaca atgaggtctg gcatctggta ggcatcacga gctggggcga aggctgtgct 1800
caaagggagc ggccaggtgt ttacaccaac gtggtcgagt acgtggactg gattctggag 1860
aaaactcaag cagtgtga 1878
<210> 6
<211> 1878
<212> DNA
<213> 人工序列
<400> 6
atgattttct tatatcaagt ggtacatttc attttattta cttcagtttc tggtgaatgt 60
gtgactcagt tgttgaagga cacctgcttt gaaggagggg acattactac ggtcttcaca 120
ccaagcgcca agtactgcca ggtagtctgc acttaccacc caagatgttt actcttcact 180
ttcacggcgg aatcaccatc tgaggatccc acccgatggt ttacttgtgt cctgaaagac 240
agtgttacag aaacactgcc aagagtgaat aggacagcag cgatttctgg gtattctttc 300
aagcaatgct cacaccaaat aagcgcttgc aacaaagaca tttatgtgga cctagacatg 360
aagggcataa actataacag ctcagttgcc aagagtgctc aagaatgcca agaaagatgc 420
acggatgacg tccactgcca ctttttcacg tacgccacaa ggcagtttcc cagcctggag 480
catcgtaaca tttgtctact gaagcacacc caaacaggga caccaaccag aataacgaag 540
ctcgataaag tggtgtctgg attttcactg aaatcctgtg cactttctaa tctggcttgt 600
attagggaca ttttccctaa tacggtgttt gcagacagca acatcgacag tgtcatggct 660
cccgatgctt ttgtctgtgg ccgaatctgc actcatcatc ccggttgctt gttttttacc 720
ttcttttccc aggaatggcc caaagaatct caaagaaatc tttgtctcct taaaacatct 780
gagagtggat tgcccagtac acgcattaaa aagagcaaag ctctttctgg tttcagtcta 840
caaagctgca ggcacagcat cccagtgttc tgccattctt cattttacca tgacactgat 900
ttcttgggag aagaactgga tattgttgct gcaaaaagtc acgaggcctg ccagaaactg 960
tgcaccaatg ccgtccgctg ccagtttttt acctataccc cagcccaagc atcctgcaac 1020
gaagggaagg gcaagtgtta cttaaagctt tcttcaaacg gatctccaac taaaatactt 1080
cacgggagag gaggcatctc tggatacaca ttaaggttgt gtaaaatgga taatgagtgt 1140
accaccaaaa tcaagcccag gatcgttgga ggaactgcgt ctgttcgttc ggagtggccg 1200
tggcaggtga ccctgcacac aacctcaccc actcagagac acctgtgtgg aggctccatc 1260
attggaaacc agtggatatt aacagccgct cactgtttct atggggtaga gtcacctaag 1320
attttgcgtg tctacagtgg cattttaaat caatctgaaa taaaagagga cacatctttc 1380
tttggggttc aagaaataat aatccatgat cagtataaaa tggcagaaag cgggtatgat 1440
attgccttgt tgaaactgga aaccacagtg aattacacag attctcaacg acccatatgc 1500
ctgccttcca aaggagatag aaatgtaata tacactgatt gctgggtgac tggatggggg 1560
tacagaaaac taagagacaa aatacaaaat actctccaga aagccaagat acccttagtg 1620
accaacgaag agtgccagaa gagatacaga ggacataaaa taacccataa gatgatctgt 1680
gccggctaca gggaaggagg gaaggacgct tgcaagggag attcgggagg ccctctgtcc 1740
tgcaaacaca atgaggtctg gcatctggta ggcatcacga gctggggcga aggctgtgct 1800
caaagggagc ggccaggtgt ttacaccaac gtggtcgagt acgtggactg gattctggag 1860
aaaactcaag cagtgtga 1878
<210> 7
<211> 625
<212> PRT
<213> 人工序列
<400> 7
Met Ile Phe Leu Tyr Gln Val Val His Phe Ile Leu Phe Thr Ser Val
1 5 10 15
Ser Gly Glu Cys Val Thr Gln Leu Leu Lys Asp Thr Cys Phe Glu Gly
20 25 30
Gly Asp Ile Thr Thr Val Phe Thr Pro Ser Ala Lys Tyr Cys Gln Val
35 40 45
Val Cys Thr Tyr His Pro Arg Cys Leu Leu Phe Thr Phe Thr Ala Glu
50 55 60
Ser Pro Ser Glu Asp Pro Thr Arg Trp Phe Thr Cys Val Leu Lys Asp
65 70 75 80
Ser Val Thr Glu Thr Leu Pro Arg Val Asn Arg Thr Ala Ala Ile Ser
85 90 95
Gly Tyr Ser Phe Lys Gln Cys Ser His Gln Ile Ser Ala Cys Asn Lys
100 105 110
Asp Ile Tyr Val Asp Leu Asp Met Lys Gly Ile Asn Tyr Asn Ser Ser
115 120 125
Val Ala Lys Ser Ala Gln Glu Cys Gln Glu Arg Cys Thr Asp Asp Val
130 135 140
His Cys His Phe Phe Thr Tyr Ala Thr Arg Gln Phe Pro Ser Leu Glu
145 150 155 160
His Arg Asn Ile Cys Leu Leu Lys His Thr Gln Thr Gly Thr Pro Thr
165 170 175
Arg Ile Thr Lys Leu Asp Lys Val Val Ser Gly Phe Ser Leu Lys Ser
180 185 190
Cys Ala Leu Ser Asn Leu Ala Cys Ile Arg Asp Ile Phe Pro Asn Thr
195 200 205
Val Phe Ala Asp Ser Asn Ile Asp Ser Val Met Ala Pro Asp Ala Phe
210 215 220
Val Cys Gly Arg Ile Cys Thr His His Pro Gly Cys Leu Phe Phe Thr
225 230 235 240
Phe Phe Ser Gln Glu Trp Pro Lys Glu Ser Gln Arg Asn Leu Cys Leu
245 250 255
Leu Lys Thr Ser Glu Ser Gly Leu Pro Ser Thr Arg Ile Lys Lys Ser
260 265 270
Lys Ala Leu Ser Gly Phe Ser Leu Gln Ser Cys Arg His Ser Ile Pro
275 280 285
Val Phe Cys His Ser Ser Phe Tyr His Asp Thr Asp Phe Leu Gly Glu
290 295 300
Glu Leu Asp Ile Val Ala Ala Lys Ser His Glu Ala Cys Gln Lys Leu
305 310 315 320
Cys Thr Asn Ala Val Arg Cys Gln Phe Phe Thr Tyr Thr Pro Ala Gln
325 330 335
Ala Ser Cys Asn Glu Gly Lys Gly Lys Cys Tyr Leu Lys Leu Ser Ser
340 345 350
Asn Gly Ser Pro Thr Lys Ile Leu His Gly Arg Gly Gly Ile Ser Gly
355 360 365
Tyr Thr Leu Arg Leu Cys Lys Met Asp Asn Glu Cys Thr Thr Lys Ile
370 375 380
Lys Pro Arg Ile Val Gly Gly Thr Ala Ser Val Arg Ser Glu Trp Pro
385 390 395 400
Trp Gln Val Thr Leu His Thr Thr Ser Pro Thr Gln Arg His Leu Cys
405 410 415
Gly Gly Ser Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala Ala His Cys
420 425 430
Phe Tyr Gly Val Glu Ser Pro Lys Ile Leu Arg Val Tyr Ser Gly Ile
435 440 445
Leu Asn Gln Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe Gly Val Gln
450 455 460
Glu Ile Ile Ile His Asp Gln Tyr Lys Met Ala Glu Ser Gly Tyr Asp
465 470 475 480
Ile Ala Leu Leu Lys Leu Glu Thr Thr Val Asn Tyr Thr Asp Ser Gln
485 490 495
Arg Pro Ile Cys Leu Pro Ser Lys Gly Asp Arg Asn Val Ile Tyr Thr
500 505 510
Asp Cys Trp Val Thr Gly Trp Gly Tyr Arg Lys Leu Arg Asp Lys Ile
515 520 525
Gln Asn Thr Leu Gln Lys Ala Lys Ile Pro Leu Val Thr Asn Glu Glu
530 535 540
Cys Gln Lys Arg Tyr Arg Gly His Lys Ile Thr His Lys Met Ile Cys
545 550 555 560
Ala Gly Tyr Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly
565 570 575
Gly Pro Leu Ser Cys Lys His Asn Glu Val Trp His Leu Val Gly Ile
580 585 590
Thr Ser Trp Gly Glu Gly Cys Ala Gln Arg Glu Arg Pro Gly Val Tyr
595 600 605
Thr Asn Val Val Glu Tyr Val Asp Trp Ile Leu Glu Lys Thr Gln Ala
610 615 620
Val
625
Claims (10)
1.一种凝血因子XI突变体，其特征在于：
(1)核苷酸序列如SEQ ID NO:1所示；
或(2)核苷酸序列如SEQ ID NO:2所示；
或(3)核苷酸序列如SEQ ID NO:3所示；
或(4)核苷酸序列如SEQ ID NO:4所示；
或(5)核苷酸序列如SEQ ID NO:5所示；
或(6)核苷酸序列如SEQ ID NO:6所示。
2.一种如权利要求1所述的凝血因子XI的突变蛋白，其特征在于：氨基酸序列如SEQ IDNO:7所示。
3.编码如权利要求2中所述的凝血因子XI的突变蛋白的核酸。
4.一种表达如权利要求2中所述的凝血因子XI的突变蛋白的载体。
5.一种凝血因子XI的突变蛋白的制备方法，包括如下步骤：
(1)将如权利要求1所述的凝血因子XI突变体的编码基因连入载体中，得到重组载体；
(2)将上述重组载体转化宿主细胞，得到表达重组凝血因子XI Gly397Ser突变细胞克隆；
(3)于无血清培养基中连续灌流培养上述重组细胞克隆，诱导重组凝血因子XIGly397Ser的突变蛋白的表达；
(4)分离纯化、过滤，最后灌装、冻干，得到所表达的凝血因子XI Gly397Ser突变蛋白。
6.一种凝血因子XI突变蛋白及其编码核酸的应用，其特征在于：应用权利要求1或3所述的核苷酸于制备基因治疗药物，包括将其与启动子和/或终止子连接，构建表达质粒，基因治疗病毒或非病毒载体，表达权利要求2中的凝血因子XI蛋白。
7.一种如权利要求2所述的凝血因子XI的突变蛋白的应用，其特征在于：用于基因编辑的病毒或非病毒载体或模板。
8.一种如权利要求2所述的凝血因子XI的突变蛋白的应用，其特征在于：应用于制备血友病或其他出血性疾病的重组蛋白治疗药物。
9.一种如权利要求2所述的凝血因子XI的突变蛋白的应用，其特征在于：应用于制备凝血因子XI Gly397Ser突变体融合蛋白，并将其用于制备血友病或其他出血性疾病的重组蛋白治疗药物。
10.根据权利要求9所述的一种凝血因子XI的突变蛋白的应用，其特征在于：所述融合蛋白为人白蛋白、免疫球蛋白Fc、转铁蛋白或alpha 1抗胰蛋白酶。
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| PCT/CN2018/103201 WO2019114324A1 (zh) | 2017-12-11 | 2018-08-30 | 一种高活性凝血因子xi突变体及其基因治疗/编辑载体、重组/融合蛋白的制备与应用 |
| US16/771,700 US11913042B2 (en) | 2017-12-11 | 2018-08-30 | Methods for preparing and using highly active blood coagulation factor XI mutant and gene therapy/editing vector and recombinant/fusion protein thereof |
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| CN109260462B (zh) * | 2018-08-24 | 2021-05-11 | 上海交通大学医学院附属瑞金医院 | 一种凝血酶原突变体蛋白及其编码核酸的应用 |
| CN110423776B (zh) * | 2019-06-20 | 2023-04-11 | 广州辉园苑医药科技有限公司 | 表达重组凝血因子fix的载体及其构建方法和应用 |
| CN112126636B (zh) * | 2020-09-09 | 2022-01-18 | 上海交通大学医学院附属瑞金医院 | 一种高活性凝血因子XI突变体Ala570Thr |
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| CN102559720A (zh) * | 2010-12-16 | 2012-07-11 | 中国科学院福建物质结构研究所 | 人源凝血因子ⅺ催化结构域的表达、纯化及结晶 |
| WO2016207858A1 (en) * | 2015-06-26 | 2016-12-29 | Novartis Ag | Factor xi antibodies and methods of use |
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| WO2016207858A1 (en) * | 2015-06-26 | 2016-12-29 | Novartis Ag | Factor xi antibodies and methods of use |
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