CN114409793A - 抗河豚毒素抗体k548、制备方法及用途 - Google Patents
抗河豚毒素抗体k548、制备方法及用途 Download PDFInfo
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- CN114409793A CN114409793A CN202111600231.2A CN202111600231A CN114409793A CN 114409793 A CN114409793 A CN 114409793A CN 202111600231 A CN202111600231 A CN 202111600231A CN 114409793 A CN114409793 A CN 114409793A
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- antibody
- tetrodotoxin
- humanized antibody
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Abstract
本发明涉及生物医药技术领域，提供了一种针对河豚毒素的人源化抗体K548及应用，该人源化抗体重链可变区和轻链可变区分别具有SEQ ID NO.1‑2所示的氨基酸序列，通过亲和力分析本发明的抗体具有良好的亲和力，通过小动物实验证明，预先注射本发明抗体的抗体保护组小鼠在注射河豚毒素后，无任何小鼠出现中毒症状，连续观察一个月无毒素致死情况，说明本发明的抗体具有优良的抗河豚毒素的作用，对河豚相关生物伤具有优良的预防或治疗作用，具备广阔的临床应用前景。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种用于抗河豚毒中毒的人源化抗体K548、其制备方法及在制备河豚毒素制剂方面的用途。
背景技术
河豚的鱼肉鮮嫩味美，带有肉刺的鱼皮胶质浓厚，食之黏口，味觉美感远胜于鱼翅、海参，因此被誉为“长江三鲜”之首。但河豚大都含有河豚毒素(tetrodotoxin,TTX)，毒素集中于卵巢、肝脏、肾脏、血液、眼睛、鱼鳃及皮肤中。河豚毒素比较稳定，用盐腌、日晒、一般加热烧煮等方法都不易消除。
河豚毒素的化学研究始于1909年，1964年以后由Woodward测定了TTX的结构，1972年Kishi等采用化学方法成功合成了河豚毒素。河豚毒素分子式为C11H17N3O8，分子量为319.27；分子主要由3个氮原子组成，它们与氢氧原子形成特殊的结构；其结构特征含有1个碳环，1个胍基，6个羟基，在C—5和C—10位有一个半醛糖内酯连接着分开的环。有专家将其称为“自然界最奇特的分子之一”，也是世界上最致命的毒药之一。1g河豚毒素的毒性是1g氰化物的1万倍。
为了解除河豚毒素，科学家试验了多种方法，抗体属于其中有效方式之一。由于抗体能够高效、特异地与体内和体外的各种抗原蛋白结合，使得抗体不仅能应用于调节免疫系统功能，还可以应用于各种高灵敏的检测方法。目前，抗体药物是生物技术药物最重要的组成部分，抗体试剂也是医学诊断和生物学研究中最常用到的试剂之一。因此，抗体相关的生物产品具有极高的应用前景和商业价值。抗体可以通过多种途径获得，对于毒素蛋白而言，抗体具有高度的中和性，是一种潜在的抗毒药物。
发明内容
本发明的目的在于，依托上述研究背景，研究抗河豚毒素人源化抗体K548、制备方法和用途，即提供了一种全新的人源化抗体、其制备方法和用途。
本发明的第一方面，提供了人源化抗河豚毒素抗体K548，该人源化抗体重链可变区由FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4区组成，轻链可变区由FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4区组成。该抗体重链可变区和轻链可变区，具有SEQ ID NO.1-2所示的氨基酸序列所示。
人源化抗体重链可变区氨基酸序列(SEQ ID NO.1)如下：
EVQLVESGGGLYKHLIYDDPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAIFWTISHNWGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVKFSKFVMLDYWGQGTLVTVSS
人源化抗体轻链可变区氨基酸序列(SEQ ID NO.2)如下：
DIQMTQSPSSLSASVGDRVTITCRANTNGLCDPQDNWYQQKPGKAPKLLIYAYRCWNWSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQWPYGMYNKRPFGQGTKVEIK
关于人源化抗体的获得，先构建人源化抗体噬菌体展示文库，然后对人源化抗体进行筛选，并采用噬菌体的酶联免疫方法(ELISA)筛选特异性阳性克隆，经序列分析后得到人源化抗体，将人源化抗体在293系统中的表达及纯化后，获得高纯度的人源化抗体。
本发明的第二方面，提供了所述人源化抗体的制备方法，包括以下步骤：
(A)根据抗体可变区基因全基因合成人源化抗体全长；
(B)采用PCR技术对步骤(A)中获得的全长人源化抗体克隆到表达载体，测序验证后确认获得正确的克隆；
(C)将上述表达载体引入宿主细胞内进行融合蛋白的表达。
本发明中，任何合适的载体都适用，优选为pGEM-T、Pet32a，pcDNA3.1、pEE6.4、pEE12.4、pDHFR或pDR1，所述表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列。
本发明中，哺乳动物或昆虫宿主细胞或原核细胞培养系统均可用于本发明的融合蛋白的表达。可用的宿主细胞为含有上述载体的原核细胞，可以为DH5a、Top10、BL21(DE3)、TG1之一。
本发明的融合蛋白可在以下细胞中容易地产生：哺乳动物细胞，诸如CHO、NSO、HEK293、BHK或者COS细胞；细菌细胞，诸如大肠杆菌、枯草芽自杆菌或荧光假单胞菌；昆虫细胞，或真菌或酵母细胞，所述细胞使用本领域中己知的技术培养。
本发明中公开的融合蛋白的制备方法为在表达条件下，培养上述的宿主细胞，从而表达、分离、纯化所述融合蛋白。利用上述方法，可以将抗体纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。
可以利用亲和层析的方法对本发明公开的融合蛋白进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的融合蛋白多肽。
可采用各种蛋白纯化方法，并且此类方法是本领域中己知的并且描述于例如(Wilchek andBayer,1990,Methods in enzymology)(Scopes,2013,Proteinpurification:principles and practice)。
通过Biacore分析，本发明的人源化抗体具有良好的亲和力，通过小动物实验证明，预先注射本发明人源化抗体的保护组小鼠在注射河豚毒素后，无任何小鼠出现神经中毒症状，连续观察一个月无毒素致死情况。说明本发明的人源化抗体具有优良的抗河豚毒素的作用。
因此，本发明的第三方面，提供了一种含有所述人源化抗体的药物组合物。该药物组合物除了包括所述人源化抗体，还包括药学上可接受的药物载体。
本发明的所述人源化抗体和药学上可以接受的辅料一起组成药物制剂组合物，从而更稳定地发挥疗效，这些制剂可以保证本发明公开的所述人源化抗体氨基酸核心序列的构像完整性，同时还要保护蛋白质的多官能团，防止其降解(包括但不限于凝聚、脱氨或氧化)。
通常情况下，液体制剂可以在2℃-8℃条件下保存至少稳定一年，冻干制剂在30℃至少六个月保持稳定。制剂可为制药领域常用的混悬、水针、冻干等制剂，优选水针或冻干制剂。
对于本发明公开的上述所述人源化抗体的水针或冻干制剂，药学上可以接受的辅料包括表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合。其中，表面活性剂包括非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)；poloxamer(如poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸；Pluronics；MONAQUATTM等，其加入量应使双功能双特异性抗体蛋白的颗粒化趋势最小；溶液稳定剂可以为糖类，包括还原性糖和非还原性糖，氨基酸类包括谷氨酸单钠或组氨酸，醇类包括三元醇、高级糖醇、丙二醇、聚乙二醇之一或其组合，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态；等渗调节剂可以为氯化钠、甘露醇之一；缓冲液可以为TRIS、组氨酸缓冲液、磷酸盐缓冲液之一。
上述制剂为包含所述人源化抗体的组合物，在对包括人在内的动物给药后，抗河豚毒素效果明显。具体来讲，对河豚毒中毒的预防和/或治疗有效，可以作为抗河豚毒素药物使用。
本发明中所述人源化抗体及其组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。具体讲静脉注射的剂量是1～1800mg/天。
本发明的第四方面，提供了一种所述人源化抗体的用途，具体是在制备抗河豚毒素制剂药物中的用途，用于预防或治疗河豚毒中毒。
具体实施方式
以下实施例、实验例对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质拉的方法，将编码蛋白的基因插入到这样的载体和质拉的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.andManiais，T.(1989)Molecular Cloning：A Laboratory Manual，2ndedition，Cold spring Harbor Laboratory Press。
实施例1.人源化抗体的制备及表达
所述人源化抗体来源于噬菌体展示抗体库。抗体本身的构建和表达的方法为领域内的常规实验技术，简单描述如下：
(1)全基因合成抗体的重链和轻链，其中重链可变区和轻链可变区氨基酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
(2)抗体的表达纯化
按照文献[Hu S,Fu W,Li T,et al.Antagonism of EGFR and Notch limitsresistance to EGFR inhibitors and radiation by decreasing tumor-initiatingcell frequency[J].Science translational medicine,2017,9(380)]方法进行抗体的表达和纯化，抗体纯度利用SDS-PAGE鉴定达95％以上。
实施例2Biacore分析
将抗河豚毒素包被在CM5M5芯片(GE公司)上，捕获被检测抗体后，用Biacore T100(GE Healthcare)检测各融合蛋白的亲和力，具体检测亲和力数值见表1。
表1.Biacore分析结果
实施例3.小动物实验
取体重(20±2)g的C57小鼠32只，实验前禁食12h(不禁水)。将小鼠随机分组，雌雄各半，将小鼠分为应用半数致死剂量毒素组12只、预先注射人源化抗体10mg/kg药物保护组10只、以及空白对照组(生理盐水)10只。小鼠腹腔注射给药，空白对照组小鼠给药后1h内，全部出现典型的神经中毒症状，而抗体保护组无小鼠出现神经中毒症状，存活连续观察一个月无毒素致死情况，具体结果见表2。
表2抗体抗毒素作用检测结果
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
序列表
<110> 中国人民解放军海军军医大学
<120> 抗河豚毒素抗体K548、制备方法及用途
<130> 权利要求书、说明书
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 120
<212> PRT
<213> 人工序列(Artificial sequence )
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Lys Gly Leu Glu Trp Val Ser Ala Ile Phe Trp Thr Ile Ser His Asn
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Trp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Lys Val Lys Phe Ser Lys Phe Val Met Leu Asp Tyr Trp Gly Gln
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Gly Thr Leu Val Thr Val Ser Ser
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<213> 人工序列(Artificial sequence )
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Pro Gln Asp Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
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Leu Ile Tyr Ala Tyr Arg Cys Trp Asn Trp Ser Gly Val Pro Ser Arg
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Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Trp Pro Tyr Gly
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Met Tyr Asn Lys Arg Pro Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
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Claims (9)
1.一种抗河豚毒素抗体K548，其特征在于，其人源化抗体重链可变区氨基酸序列如SEQID NO.1所示，人源化抗体轻链可变区氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的抗河豚毒素抗体K548，其特征在于，该人源化抗体重链可变区由FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4区组成；轻链可变区由FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4区组成。
3.编码权利要求1所述的抗河豚毒素抗体K548的核苷酸。
4.权利要求1所述的抗河豚毒素抗体K548的制备方法，其特征在于，包括以下步骤：
(A)根据抗体可变区基因全基因合成人源化抗体全长；
(B)采用PCR技术对步骤(A)中获得的全长人源化抗体克隆到表达载体，测序验证后确认获得正确的克隆；
(C)将上述表达载体引入宿主细胞内进行融合蛋白的表达。
5.根据权利要求4所述的抗河豚毒素抗体K548的制备方法，其特征在于：
其中，所述表达载体为pGEM-T、Pet32a，pcDNA3.1、pEE6.4、pEE12.4、pDHFR或pDR1，所述表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列，
所述宿主细胞为原核细胞、哺乳动物细胞、细菌细胞、昆虫细胞或真菌细胞。
6.含有权利要求1～2任一项所述的抗河豚毒素抗体K548的药物组合物，其特征在于，还包括药学上可接受的药物载体。
7.根据权利要求6所述的药物组合物，其特征在于：
其中，所述药物组合物为水针或冻干制剂，
所述药学上可接受的药物载体包括表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或组合。
8.根据权利要求6所述的药物组合物，其特征在于：
其中，所述水针或冻干制剂静脉注射的剂量是1～1800mg/天。
9.权利要求1～2任一项所述的抗河豚毒素抗体K548在制备抗河豚毒素药物中的应用。
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