CN113248608A - 一种基孔肯雅病毒e2蛋白兔单克隆抗体及其用途 - Google Patents
一种基孔肯雅病毒e2蛋白兔单克隆抗体及其用途 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域。本发明提供了一株基孔肯雅病毒(CHIKV)E2蛋白兔单克隆抗体的氨基酸序列及其用途。本发明提供的一株CH IKV E2蛋白兔单克隆抗体的氨基酸序列包括抗体重链和轻链的氨基酸序列。用哺乳动物细胞表达的该抗体在体外可中和全球主要流行的CHIKV基因型对细胞的感染，在体内可保护I型干扰素受体基因敲除小鼠抵抗致死性CHIKV的感染。经过改造，如人源化改造或制备成单链抗体，有望用于重症CHIKV感染的治疗。
Description
技术领域
本发明涉及生物医药工程技术领域，具体地，本发明提供了一种CHIKV E2蛋白兔单克隆抗体的氨基酸序列及其在开发抗CHIKV感染治疗性抗体，包括人源化单抗和单链抗体中的应用。
背景技术
基孔肯雅病毒(Chikungunya virus，CHIKV)是引起基孔肯雅热(Chikungunyafever)的病原体，其基因组是一种单股正链RNA，属于披膜病毒科(Togaviridae)，甲病毒属(Alphavirus)成员。CHIKV是一类重要的虫媒病毒，伊蚊是其主要的传播媒介，人群普遍易感，除了引起发热和皮疹等症状以外，常可引起病程长达一年以上的慢性关节炎。近年来有报道CHIKV可引起更严重的疾病，包括脑炎和出血性疾病。目前CHIKV已传播至全球一百多个国家和地区，每年造成数百万人的感染，给人类生命健康带来巨大影响，是全球公共卫生的重大负担。针对CHIKV感染，目前无有效的治疗药物和商业化的疫苗。
CHIKV的基因组编码两个开放阅读框(ORF)。5’端的ORF编码非结构蛋白(nsp1、nsp2、nsp3、nsp4)主要负责基因组RNA的复制；3’端的ORF编码结构蛋白(衣壳蛋白C、包膜蛋白E3、包膜蛋白E2、包膜蛋白E1)，负责病毒颗粒的形成。
结构蛋白的翻译产物为一个多聚蛋白前体，该多聚蛋白氨基端的衣壳蛋白通过自身的蛋白酶水解功能从多聚蛋白中释放出来，羧基端的多肽则在内质网内被宿主信号肽酶切割形成前体pE2蛋白、6K蛋白和E1蛋白，随后pE2由宿主细胞高尔基体中的弗林蛋白酶切割形成E3和E2蛋白。
衣壳蛋白包含两个结构域：N端的RNA结合域和C端的蛋白酶域。RNA结合域富含带正电荷的赖氨酸和精氨酸，与带负电的基因组RNA相互作用。包膜蛋白E2是病毒的受体结合蛋白，主要负责CHIKV与细胞表面受体的结合。E2蛋白在结构上分为膜外区、茎区、跨膜区和膜内区4部分。CHIKV E2蛋白全长包括423个氨基酸残基(aa)，其中胞外区长约260aa，茎区约100aa，跨膜区约30aa，膜内区约33aa。膜外区在病毒吸附宿主细胞以及穿膜感染过程中起关键作用，含有中和抗体的表位；跨膜区进入脂质双分子层，膜内区在病毒在病毒颗粒内部，与核衣壳蛋白相互作用。E1蛋白属于第二类融合蛋白，主要负责膜融合。E1在结构上也分为膜外区、茎区、跨膜区和膜内区4部分。E2蛋白和E2蛋白形成异源二聚体，锚定于病毒颗粒的包膜表面。
CHIKV感染细胞起始于病毒与细胞表面受体的结合，CHIKV的E2蛋白与受体结合后，病毒颗粒通过受体介导的细胞内吞作用形成内体，进入细胞内，随着内体的成熟，内体的酸化引起CHIKV E1-E2异源二聚体构象发生变化，导致E1蛋白中的融合肽暴露并插入到内体膜中诱发病毒包膜与内体膜的融合；膜融合发生后，病毒核衣壳结构被释放到细胞质中，并短时间内迅速解体，释放出病毒基因组RNA，开始病毒基因的复制和蛋白的表达。
抗体治疗是用于治疗严重病毒感染的一种有效方法，在治疗埃博拉病毒、新型冠状病毒、呼吸道合胞病毒等病毒感染上显示了良好的效果。治疗性抗体成为治疗病毒感染的一类新的特效药物，研发抗病毒治疗性抗体是当前抗病毒药物研发领域的热点。虽然小鼠单克隆抗体技术非常成熟，但近年来兴起的兔单克隆抗体技术更具有抗体药物开发的优势。相比小鼠单克隆抗体，兔单克隆抗体具体如下特点：兔B细胞库容量大，抗体谱更广泛；具有高突变与基因转换二重抗体亲和力成熟机制，具有独特的互补决定区结构，因而与抗原的亲和力更高；在重链可变区有额外的二硫键，还有另一个连接可变区和恒定区的二硫键，因此其稳定性更高；兔单抗易于进行人源化改造。
发明内容
CHIKV E2蛋白是介导病毒侵入宿主细胞的关键蛋白，是诱导中和抗体的主要蛋白，本发明以CHIKV E2蛋白为靶抗原，制备兔单克隆抗体，获得了一种可高效、广谱中和全球主要流行基因型CHIKV的中和抗体，可进一步开发为CHIKV治疗性抗体。
具体地，本发明提供了一种CHIKVE2蛋白兔单克隆抗体的氨基酸序列及其用途。该单抗在体外可阻断全球主要流行的基因型CHIKV，包括中亚洲型(Asian)和东/中/南非型(East/Central/South African，ECSA)，以及ECSA基因型的印度洋(India Ocean lineage，IOL)亚型对靶细胞的感染。在体内可保护I型干扰素受体基因敲除小鼠抵抗这些基因型CHIKV的致死性感染。上述抗体重链基因的脱氧核苷酸序列如SEQ ID NO:1所示，抗体重链蛋白的氨基酸序列如SEQ ID NO:2所示。上述抗体轻链基因的脱氧核苷酸序列如SEQ IDNO:3所示，抗体轻链蛋白的氨基酸序列如SEQ ID NO:4所示。
更具体地，本发明提供的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：上述抗体的氨基酸序列，包括抗体重链和抗体轻链；
其中，上述抗体重链的氨基酸序列如SEQ ID NO:2所示；
上述抗体轻链的氨基酸序列如SEQ ID NO:4所示。
进一步地，本发明提供的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
上述抗体重链基因的脱氧核苷酸序列如SEQ ID NO:1所示。
进一步地，本发明提供的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
上述抗体轻链基因的脱氧核苷酸序列如SEQ ID NO:3所示。
进一步地，本发明提供的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
上述抗体作为一种药物、一种治疗用的成分，或用于制备一种药物；
上述药物在体外可阻断CHIKV对靶细胞的感染。
进一步地，本发明提供的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
上述抗体作为一种药物、一种治疗用的成分，或用于制备一种药物；
上述药物在体内可抵抗CHIKV的致死性感染。
进一步地，本发明提供的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
上述抗体作为一种药物、一种治疗用的成分，或用于制备一种药物；
上述药物在体内可保护I型干扰素受体基因敲除小鼠抵抗CHIKV的致死性感染。
即、简单的说：该抗体在体外可阻断全球主要流行的CHIKV基因型，包括亚洲型(Asian)和东/中/南非型(East/Central/South African，ECSA)，以及ECSA型的印度洋系(India Ocean lineage，IOL)对靶细胞的感染。
该抗体在体内可保护I型干扰素受体基因敲除小鼠抵抗全球主要流行的CHIKV基因型，包括亚洲型(Asian)、东/中/南非型(East/Central/South African，ECSA)，以及ECSA型的印度系(India Ocean lineage，IOL)的致死性感染。
进一步地，本发明提供的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
在开发CHIKV感染的治疗性抗体中的应用。
进一步地，本发明提供的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
在开发CHIKV感染的治疗性人源化抗体或单链抗体中的应用。
附图说明
图1：ELISA检测WT08G2单抗与CHIKV E2蛋白的结合；
图2A：WT08G2单抗可中和亚洲型37997株CHIKV对靶细胞BHK的感染；
图2B：WT08G2单抗可中和东/中/南非型Ross株CHIKV对靶细胞BHK的感染；
图2CWT08G2单抗可中和东/中/南非型印度洋系LR2006株CHIKV对靶细胞BHK的感染；
图3A：WT08G2单抗可保护A6小鼠抵抗亚洲型CHIKV 37997株CHIKV的感染；
图3B：WT08G2单抗可保护A6小鼠抵抗东/中/南非型Ross株CHIKV的感染；
图3C：WT08G2单抗可保护A6小鼠抵抗东/中/南非型印度洋系LR2006株CHIKV的感染。
具体实施方式
下面结合本发明的实施例和附图对本发明的实施作详细说明，以下实施例是在以本发明技术方案为前提下进行实施，给出了具体的实施方式和操作过程，但本发明的保护范围不限于下述的实施例。本发明中涉及CHIKV病毒的所有操作均在中国人民解放军海军军医大学生物安全三级实验室内进行。
一、主要实验材料
1、CHIKV E2蛋白，用人胚肾293T细胞重组表达的ECSA型CHIKV E2蛋白，由中国人民解放军海军军医大学生物医学防护教研室用无血清培养基悬浮培养生长的freestyle293F细胞重组表达。
2、CHIKV病毒，包括亚洲型37997株、东/中/南非型Ross株、东/中/南非型印度洋系LR2006株，为中国人民解放军海军军医大学生物医学防护教研室用反向遗传学技术合成拯救。
3、I型干扰素受体基因敲除小鼠(A6小鼠)由中国科学院上海巴斯德研究所提供，在中国人民解放军海军军医大学生物安全三级实验室动物实验区繁殖。
4、小鼠抗CHIKV多克隆抗体由中国人民解放军海军军医大学生物医学防护教研室用β-丙内酯灭活的Ross株CHIKV免疫小鼠而制备。
二、实验方法和结果
1、兔单抗的制备与鉴定流程
将重组表达的CHIKV E2蛋白联合完全弗氏佐剂免疫新西兰兔，0.2mgCHIKV E2蛋白溶解于磷酸盐缓冲液(PBS)，终体积0.5ml，与0.5ml完全弗氏佐剂充分乳化混匀，皮下注射免疫新西兰兔，分别在首次免疫的3、6、9周后加强免疫一次，CHIKV E2蛋白用量0.1mg，佐剂用不完全弗氏佐剂混匀，皮下注射免疫。每次免疫3周后均从兔耳缘静脉采血，用ELISA检测兔血清中的IgG抗体(方法如下上述)。第12周后，将兔以异氟烷醚麻醉，心脏穿刺取血，兔死亡以后，取脾脏，剪成碎块，置于尼龙网上研磨、过滤，制备成单细胞悬液，用流式细胞技术分选出表达CHIKV E2 IgG抗体的单个脾细胞，单个脾细胞接种于96孔板，用添加了10％胎牛血清和100U/ml青霉素和100μg/ml链霉素的RPMI 1640培养基培养，将96孔板置于5％CO2、饱和湿度的37℃细胞培养箱内约7-10天，细胞铺满板孔底面时，用ELISA检测细胞培养上清中是否有CHIKV E2 IgG抗体。抽提抗体阳性细胞总RNA，用逆转录聚合酶链反应分别扩增抗体重链和轻链基因。逆转录引物为寡聚脱氧胸腺嘧啶(oligo dT)，重链基因上游引物序列为：5-ATGGAGACTGGGCTGCGCTG-3，重链基因下游引物序列为：5-CTATTTACCCGGAGAGCGGGA-3，轻链基因上游引物序列为：5-ATGGACACGAGGGCCCCCAC-3，轻链基因下游引物序列为：5-CTAACAGTCACCCCTATTGAAG-3。PCR产物经琼脂糖凝胶电泳回收，插入pMD18T载体(Takara公司)，经DNA测序以后，再分别将抗体重链和轻链基因插入到哺乳动物细胞表达载体pcDNA3.1的多克隆位点中，构建成表达质粒。将抗体重链和轻链表达质粒共转染293T细胞(质粒质量比为2：1)，用ELISA检测细胞培养上清中是否有CHIKV E2 IgG抗体。IgG抗体阳性的上清用病毒微量中和实验鉴定其是否具有中和活性。若单抗具有病毒中和活性，则用无血清培养基悬浮培养生长的freestyle 293F细胞进行重组表达，培养freestyle 293F细胞至300ml(1L体积的培养瓶)，细胞密度达到1×106/ml时，用聚乙烯亚胺(polyethylenimine，PEI)试剂将抗体重链和轻链表达质粒共转染freestyle 293F细胞，继续培养细胞72小时，用金黄色葡萄球菌ProteinA亲和层析法从培养上清中纯化兔抗体，进行ELISA、病毒微中和试验和A6小鼠攻毒保护试验。
2、ELISA检测兔抗CHIKV E2 IgG抗体
将CHIKV E2蛋白用50mM的碳酸盐包被液(pH 9.6)稀释，加入酶标板，每孔0.1ml，含CHIKV E2蛋白0.1μg，置于4℃冰箱过夜，次日吸除包被液，每孔用0.2ml PBS缓冲液洗一次，然后每孔加入含3％牛血清白蛋白、5％山羊血清和0.05％Tween 20的PBS缓冲液(封闭液，pH 7.4)0.2ml，室温静置2小时以后，吸除封闭液，用含0.05％Tween 20的PBS缓冲液(洗涤液)洗孔3次，随后加入用封闭液稀释的兔脾细胞培养上清、抗体表达质粒共转染293T细胞的培养上清，或从抗体表达质粒共转染freestyle 293F细胞培养上清中纯化得到的兔单抗，体积0.1ml，室温置于缓慢摇动的水平摇床，反应30分钟，随后弃去孔中的反应液，用洗涤液洗孔5次，再加入用封闭液以1:1000倍稀释的辣根过氧化物酶标记抗兔IgG(Thermo公司产品)，室温置于缓慢摇动的水平摇床，反应30分钟，随后弃去孔中的酶标抗体稀释液，用洗涤液洗孔5次，每孔加入含5,5'-四甲基联苯胺(3,3′,5,5′-Tetramethylbenzidine，TMB)的底物液0.1ml，室温避光放置5分钟后，加入2M硫酸和30％过氧化氢溶液各50μl，混匀，用酶标仪检测450nm波长的光吸收值，参考波长630nm。
图1显示筛选鉴定出的一株兔抗CHIKV E2单克隆抗体(编号为WT08G2)不同浓度得到的ELISA光吸收值，用正常兔IgG(Cell signaling公司)作为阴性对照(Control)。可见单抗WT08G2可特异结合CHIKV E2蛋白。进一步对该单抗进行体外微量中和试验和体内小鼠攻毒保护试验，评价其抗病毒效果。该抗体的重链基因脱氧核苷酸序列如SEQ ID NO:1所示，由基因序列推导出的抗体重链蛋白的氨基酸序列如SEQ ID NO:2。抗体轻链基因脱氧核苷酸序列如SEQ ID NO:3所示，由基因序列推导出的抗体轻链蛋白的氨基酸序列如SEQ IDNO:4所示。
即、SEQ ID NO:1为WT08G2株兔抗CHIKV E2单抗重链基因的脱氧核苷酸序列(1377bp)；
SEQ ID NO:2为WT08G2株兔抗CHIKV E2单抗重链蛋白的氨基酸序列(458aa)；
SEQ ID NO:3为WT08G2株兔抗CHIKV E2单抗轻链基因的脱氧核苷酸序列(711bp)；
SEQ ID NO:4为WT08G2株兔抗CHIKV E2单抗轻链蛋白的氨基酸序列(236aa)；
3、微量中和试验检测兔抗CHIKV E2单抗对CHIKV的中和活性
将培养的幼仓鼠肾(BHK)细胞传代接种于96孔板，培养基为添加了10％胎牛血清和100U/ml青霉素和100μg/ml链霉素的DMEM培养基(以下简称为完全DMEM培养基)，每孔10000个细胞，置于含5％CO2、饱和湿度的37℃细胞培养箱内，培养12小时。
在96孔板内将兔单抗WT08G2进行浓度梯度稀释(稀释液用完全DMEM培养基)，用正常兔IgG(Cell signaling公司)作为阴性对照，各浓度梯度吸出至96孔板，每孔50μl，再每孔加入稀释的CHIKV病毒液50μl，约含300个空斑形成单位(focus forming units,PFUs)CHIKV，稀释液用完全DMEM培养基)，混匀，置于37℃细胞培养箱内，30分钟后，将孔内液体转移至于BHK细胞培养孔内(先吸除原细胞配培养液)，将细胞继续培养18小时，用免疫荧光检测细胞内CHIKV蛋白的表达。具体操作如下：吸除培养板中的培养液，每孔加0.1ml甲醇，将培养板至于-20℃冰箱，20分钟后，取出培养板，吸除甲醇，每孔以磷酸盐缓冲液(PBS)洗孔一次，随后加入100μl含3％牛血清白蛋白(BSA)的PBS(以下简称3％BSA-PBS)，置于水平摇床上，室温缓慢摇1小时，吸除培养板中的3％BSA-PBS，每孔加0.1ml含抗CHIKV多克隆抗体的1％BSA-PBS(抗体500倍稀释)，室温缓慢摇1小时，吸除培养板中的CHIKV多克隆抗体工作液，每孔以PBS洗3次，随后加入0.1ml含荧光素Alexa Fluor 488-标记的抗小鼠IgG的1％BSA-PBS(荧光素抗体1500倍稀释)，室温避光缓慢摇1小时，吸除培养板中的荧光素抗体工作液，每孔加入DAPI细胞核染色液0.1ml，室温避光缓慢摇10分钟，吸除培养板中的DAPI细胞核染色液，每孔以PBS洗3次，用细胞成像及分析系统(BioTek Cytation 5 ImagingReader)对每孔细胞的绿色荧光阳性细胞进行计数，然后计算中和百分率(％)＝WT08G2单抗处理孔或正常兔IgG处理孔阳性细胞数/未加抗体孔的阳性细胞数。
从图2A、2B和2C可见，WT08G2单抗可高效中和亚洲型37997株、东/中/南非型Ross株、东/中/南非型印度洋系LR2006株对靶细胞BHK的感染。
4、用小鼠病毒攻击模型评价单抗在体内的抗病毒活性
8周龄、雌性A6小鼠共32只，随机分为4组，每组8只，分别腹腔注射亚洲型37997株、东/中/南非型Ross株、东/中/南非型印度洋亚型LR2006株各，200μl含有10000PFUs的DMEM培养基，2小时以后，腹腔注射20μg WT08G2单抗或正常兔IgG。在注射病毒以后，每隔24小时称量小鼠质量，当小鼠质量下降超过20％视为死亡，吸入异氟烷醚麻醉，颈椎脱臼处死小鼠。
从图3A、3B和3C可见，WT08G2单抗可有效保护A6小鼠抵抗亚洲型37997株、东/中/南非型Ross株、东/中/南非型印度洋系LR2006株的感染，降低小鼠的病死率。
上述体外和体内试验结果均表明，WT08G2株兔抗CHIKV E2单抗能高效中和CHIKV的感染性和有效治疗CHIKV引起的小鼠发病和死亡，进过改造，如人源化改造或制备成单链抗体，有望用于重症CHIKV感染的治疗。
以上显示和描述了本发明的主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。
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<110> 中国人民解放军海军军医大学
<120> 一种基孔肯雅病毒E2蛋白兔单克隆抗体及其用途
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Ala Thr Gly Gly Ala Gly Ala Cys Thr Gly Gly Gly Cys Thr Gly Cys
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Gly Cys Thr Gly Gly Cys Thr Thr Cys Thr Cys Cys Thr Gly Gly Thr
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Cys Gly Cys Thr Gly Thr Gly Cys Thr Cys Ala Ala Ala Gly Gly Thr
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Gly Thr Cys Cys Ala Gly Thr Gly Thr Cys Gly Gly Thr Cys Gly Gly
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Thr Gly Gly Ala Gly Gly Ala Gly Thr Cys Cys Gly Gly Gly Gly Gly
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Thr Cys Gly Cys Cys Thr Gly Gly Thr Cys Ala Cys Gly Cys Cys Thr
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Gly Gly Gly Ala Cys Ala Cys Cys Cys Cys Thr Gly Ala Cys Ala Cys
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Thr Cys Ala Cys Cys Thr Gly Cys Ala Cys Ala Gly Thr Cys Thr Cys
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Thr Gly Gly Ala Thr Thr Cys Thr Cys Cys Cys Thr Cys Gly Ala Thr
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Gly Thr Cys Thr Ala Thr Gly Cys Ala Gly Thr Gly Ala Gly Thr Thr
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Gly Gly Gly Thr Cys Cys Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys
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Ala Gly Gly Gly Ala Ala Gly Gly Gly Thr Cys Thr Gly Gly Ala Ala
180 185 190
Thr Gly Gly Ala Thr Cys Gly Gly Ala Ala Thr Cys Ala Thr Thr Thr
195 200 205
Ala Thr Gly Gly Gly Ala Cys Thr Cys Cys Thr Ala Ala Cys Gly Cys
210 215 220
Ala Gly Cys Cys Thr Ala Cys Gly Cys Gly Ala Ala Cys Thr Gly Gly
225 230 235 240
Gly Cys Gly Ala Ala Ala Gly Gly Cys Cys Gly Ala Cys Thr Cys Ala
245 250 255
Cys Cys Ala Thr Cys Thr Cys Cys Ala Ala Ala Ala Cys Cys Thr Cys
260 265 270
Gly Ala Cys Cys Ala Cys Gly Gly Thr Gly Gly Ala Thr Cys Thr Gly
275 280 285
Ala Ala Ala Ala Thr Cys Ala Cys Cys Ala Gly Thr Cys Cys Gly Ala
290 295 300
Cys Ala Ala Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Gly Gly Cys
305 310 315 320
Cys Ala Cys Cys Thr Ala Thr Thr Thr Cys Thr Gly Thr Gly Cys Cys
325 330 335
Ala Gly Ala Thr Cys Cys Cys Thr Cys Thr Ala Thr Cys Cys Thr Ala
340 345 350
Ala Thr Ala Gly Thr Ala Gly Thr Gly Gly Thr Thr Ala Thr Ala Cys
355 360 365
Gly Ala Gly Ala Thr Ala Thr Thr Ala Cys Thr Thr Thr Ala Gly Cys
370 375 380
Thr Thr Gly Thr Gly Gly Gly Gly Cys Cys Ala Ala Gly Gly Cys Ala
385 390 395 400
Cys Cys Cys Gly Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys
405 410 415
Cys Thr Cys Ala Gly Gly Gly Cys Ala Ala Cys Cys Thr Ala Ala Gly
420 425 430
Gly Cys Thr Cys Cys Ala Thr Cys Ala Gly Thr Cys Thr Thr Cys Cys
435 440 445
Cys Ala Cys Thr Gly Gly Cys Cys Cys Cys Cys Thr Gly Cys Thr Gly
450 455 460
Cys Gly Gly Gly Gly Ala Cys Ala Cys Ala Cys Cys Cys Ala Gly Cys
465 470 475 480
Gly Gly Cys Thr Gly Cys Cys Thr Gly Gly Thr Cys Ala Ala Ala Gly
485 490 495
Gly Cys Thr Ala Cys Cys Thr Cys Cys Cys Gly Gly Ala Gly Cys Cys
500 505 510
Ala Gly Thr Gly Ala Cys Cys Gly Thr Gly Ala Cys Cys Thr Gly Gly
515 520 525
Ala Ala Cys Thr Cys Gly Gly Gly Cys Ala Cys Cys Ala Cys Cys Cys
530 535 540
Thr Cys Ala Cys Cys Ala Ala Thr Gly Gly Gly Gly Thr Ala Cys Gly
545 550 555 560
Cys Ala Cys Cys Thr Thr Cys Cys Cys Gly Thr Cys Cys Gly Thr Cys
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Cys Gly Gly Cys Ala Gly Thr Cys Cys Thr Cys Ala Gly Gly Cys Cys
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Thr Cys Thr Ala Cys Thr Cys Gly Ala Gly Cys Ala Gly Cys Gly Thr
595 600 605
Gly Gly Thr Gly Ala Gly Cys Gly Thr Gly Ala Cys Cys Thr Cys Ala
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Ala Gly Cys Ala Gly Cys Cys Ala Gly Cys Cys Cys Gly Thr Cys Ala
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Cys Cys Thr Gly Cys Ala Ala Cys Gly Thr Gly Gly Cys Cys Cys Ala
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Cys Cys Cys Ala Gly Cys Cys Ala Cys Cys Ala Ala Cys Ala Cys Cys
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Ala Ala Ala Gly Thr Gly Gly Ala Cys Ala Ala Gly Ala Cys Cys Gly
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Thr Thr Gly Cys Gly Cys Cys Cys Thr Cys Gly Ala Cys Ala Thr Gly
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Cys Ala Gly Cys Ala Ala Gly Cys Cys Cys Ala Thr Gly Thr Gly Cys
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Ala Gly Cys Ala Ala Gly Cys Cys Cys Ala Thr Gly Thr Gly Cys Cys
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Cys Ala Cys Cys Cys Cys Cys Thr Gly Ala Ala Cys Thr Cys Cys Cys
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Gly Gly Gly Gly Gly Gly Ala Cys Cys Gly Thr Cys Thr Gly Thr Cys
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Thr Thr Cys Ala Thr Cys Thr Thr Cys Cys Cys Cys Cys Cys Ala Ala
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Ala Ala Cys Cys Cys Ala Ala Gly Gly Ala Cys Ala Cys Cys Cys Thr
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Cys Ala Thr Gly Ala Thr Cys Thr Cys Ala Cys Gly Cys Ala Cys Cys
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Cys Cys Cys Gly Ala Gly Gly Thr Cys Ala Cys Ala Thr Gly Cys Gly
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Thr Gly Gly Thr Gly Gly Thr Gly Gly Ala Cys Cys Cys Cys Gly Ala
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Gly Gly Thr Gly Cys Ala Gly Thr Thr Cys Ala Cys Ala Thr Gly Gly
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Thr Ala Cys Ala Thr Ala Ala Ala Cys Ala Ala Cys Gly Ala Gly Cys
865 870 875 880
Ala Gly Gly Thr Gly Cys Gly Cys Ala Cys Cys Gly Cys Cys Cys Gly
885 890 895
Gly Cys Cys Gly Cys Cys Gly Cys Cys Gly Cys Thr Ala Cys Gly Gly
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Gly Ala Gly Cys Ala Gly Cys Ala Gly Thr Thr Cys Ala Ala Cys Ala
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Gly Cys Ala Cys Gly Ala Thr Cys Cys Gly Cys Gly Thr Gly Gly Thr
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Cys Ala Gly Cys Ala Cys Cys Cys Thr Cys Cys Cys Cys Ala Thr Cys
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Gly Cys Gly Cys Ala Cys Cys Ala Gly Gly Ala Cys Thr Gly Gly Cys
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Thr Gly Ala Gly Gly Gly Gly Cys Ala Ala Gly Gly Ala Gly Thr Thr
980 985 990
Cys Ala Ala Gly Thr Gly Cys Ala Ala Ala Gly Thr Cys Cys Ala Cys
995 1000 1005
Ala Ala Cys Ala Ala Gly Gly Cys Ala Cys Thr Cys Gly Cys Cys Cys
1010 1015 1020
Cys Cys Ala Thr Cys Gly Ala Gly Ala Ala Ala Ala Cys Cys Ala Thr
1025 1030 1035 1040
Cys Thr Cys Cys Ala Ala Ala Gly Cys Cys Ala Gly Ala Gly Gly Gly
1045 1050 1055
Cys Ala Gly Cys Cys Cys Cys Thr Gly Gly Ala Gly Cys Cys Gly Ala
1060 1065 1070
Ala Gly Gly Thr Cys Thr Ala Cys Ala Cys Cys Ala Thr Gly Gly Gly
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Cys Cys Cys Thr Cys Cys Cys Cys Gly Gly Gly Ala Gly Gly Ala Gly
1090 1095 1100
Cys Thr Gly Ala Gly Cys Ala Gly Cys Ala Gly Gly Thr Cys Gly Gly
1105 1110 1115 1120
Thr Cys Ala Gly Cys Cys Thr Gly Ala Cys Cys Thr Gly Cys Ala Thr
1125 1130 1135
Gly Ala Thr Cys Ala Thr Cys Ala Ala Cys Gly Gly Cys Thr Thr Cys
1140 1145 1150
Thr Ala Cys Cys Cys Thr Thr Cys Cys Gly Ala Cys Ala Thr Cys Thr
1155 1160 1165
Cys Gly Gly Thr Gly Gly Ala Gly Thr Gly Gly Gly Ala Gly Ala Ala
1170 1175 1180
Gly Ala Ala Cys Gly Gly Gly Ala Ala Gly Gly Cys Ala Gly Ala Gly
1185 1190 1195 1200
Ala Ala Cys Thr Ala Cys Ala Ala Gly Ala Cys Cys Ala Cys Gly Cys
1205 1210 1215
Cys Gly Ala Cys Cys Gly Thr Gly Cys Thr Gly Gly Ala Cys Ala Gly
1220 1225 1230
Cys Gly Ala Cys Gly Gly Cys Thr Cys Cys Thr Ala Cys Thr Thr Cys
1235 1240 1245
Cys Thr Cys Thr Ala Cys Ala Gly Cys Ala Ala Gly Cys Thr Cys Thr
1250 1255 1260
Cys Ala Gly Thr Gly Cys Cys Cys Ala Cys Gly Ala Gly Thr Gly Ala
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Gly Thr Gly Gly Cys Ala Gly Cys Gly Gly Gly Gly Cys Gly Ala Cys
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Gly Thr Cys Thr Thr Cys Ala Cys Cys Thr Gly Cys Thr Cys Cys Gly
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Thr Gly Ala Thr Gly Cys Ala Cys Gly Ala Gly Gly Cys Cys Thr Thr
1315 1320 1325
Gly Cys Ala Cys Ala Ala Cys Cys Ala Cys Thr Ala Cys Ala Cys Gly
1330 1335 1340
Cys Ala Gly Ala Ala Gly Thr Cys Cys Ala Thr Cys Thr Cys Cys Cys
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Gly Cys Thr Cys Thr Cys Cys Gly Gly Gly Thr Ala Ala Ala Thr Ala
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Gly
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Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
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Val Gln Cys Arg Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
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Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Asp
35 40 45
Val Tyr Ala Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60
Trp Ile Gly Ile Ile Tyr Gly Thr Pro Asn Ala Ala Tyr Ala Asn Trp
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Ala Lys Gly Arg Leu Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
85 90 95
Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala
100 105 110
Arg Ser Leu Tyr Pro Asn Ser Ser Gly Tyr Thr Arg Tyr Tyr Phe Ser
115 120 125
Leu Trp Gly Gln Gly Thr Arg Val Thr Val Ser Ser Gly Gln Pro Lys
130 135 140
Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser
145 150 155 160
Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp
165 170 175
Asn Ser Gly Thr Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val
180 185 190
Arg Gln Ser Ser Gly Leu Tyr Ser Ser Ser Val Val Ser Val Thr Ser
195 200 205
Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr
210 215 220
Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Met Cys
225 230 235 240
Ser Lys Pro Met Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser Val
245 250 255
Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
260 265 270
Pro Glu Val Thr Cys Val Val Val Asp Pro Glu Val Gln Phe Thr Trp
275 280 285
Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Pro Leu Arg
290 295 300
Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile
305 310 315 320
Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His
325 330 335
Asn Lys Ala Leu Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly
340 345 350
Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu
355 360 365
Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Ile Asn Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu
385 390 395 400
Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe
405 410 415
Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp
420 425 430
Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
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Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
450 455
<210> 3
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Ala Thr Gly Gly Ala Cys Ala Cys Gly Ala Gly Gly Gly Cys Cys Cys
1 5 10 15
Cys Cys Ala Cys Thr Cys Ala Gly Cys Thr Gly Cys Thr Gly Gly Gly
20 25 30
Gly Cys Thr Cys Cys Thr Gly Cys Thr Gly Cys Thr Cys Thr Gly Gly
35 40 45
Cys Thr Cys Cys Cys Ala Gly Gly Thr Gly Cys Cys Ala Gly Ala Thr
50 55 60
Gly Thr Gly Cys Cys Thr Ala Thr Gly Ala Thr Ala Thr Gly Ala Cys
65 70 75 80
Cys Cys Ala Gly Ala Cys Thr Cys Cys Ala Gly Cys Cys Thr Cys Thr
85 90 95
Gly Thr Gly Gly Ala Gly Gly Thr Ala Gly Cys Thr Gly Thr Gly Gly
100 105 110
Gly Ala Gly Gly Cys Ala Cys Ala Ala Cys Ala Gly Thr Cys Ala Cys
115 120 125
Cys Ala Thr Cys Ala Ala Gly Thr Gly Cys Cys Ala Gly Gly Cys Cys
130 135 140
Ala Gly Thr Cys Ala Gly Ala Gly Cys Ala Thr Thr Gly Gly Thr Ala
145 150 155 160
Gly Thr Thr Ala Cys Thr Thr Gly Thr Cys Cys Thr Gly Gly Thr Ala
165 170 175
Thr Cys Gly Cys Cys Ala Gly Ala Ala Ala Cys Cys Ala Gly Gly Gly
180 185 190
Cys Ala Gly Cys Cys Thr Cys Cys Cys Ala Ala Gly Cys Thr Cys Cys
195 200 205
Thr Gly Ala Thr Cys Thr Ala Cys Cys Ala Gly Gly Cys Ala Thr Cys
210 215 220
Cys Ala Ala Ala Cys Thr Gly Gly Cys Ala Thr Cys Thr Gly Gly Gly
225 230 235 240
Cys Cys Ala Thr Cys Gly Cys Gly Ala Thr Thr Cys Ala Ala Ala Gly
245 250 255
Gly Cys Ala Gly Thr Gly Gly Ala Thr Cys Thr Gly Gly Gly Ala Cys
260 265 270
Ala Gly Ala Gly Thr Ala Cys Ala Cys Thr Cys Thr Cys Ala Cys Cys
275 280 285
Ala Thr Thr Ala Gly Cys Gly Gly Cys Gly Thr Gly Cys Ala Gly Thr
290 295 300
Gly Thr Gly Ala Cys Gly Ala Thr Gly Cys Thr Gly Cys Cys Ala Cys
305 310 315 320
Thr Thr Ala Cys Thr Ala Cys Thr Gly Thr Cys Ala Ala Cys Ala Gly
325 330 335
Gly Gly Thr Thr Ala Thr Ala Gly Thr Ala Gly Thr Gly Ala Thr Ala
340 345 350
Ala Thr Ala Thr Ala Gly Ala Thr Ala Thr Thr Ala Cys Thr Thr Thr
355 360 365
Cys Gly Gly Cys Gly Gly Ala Gly Gly Gly Ala Cys Cys Gly Ala Gly
370 375 380
Gly Thr Gly Gly Thr Gly Gly Thr Cys Ala Ala Ala Gly Gly Thr Gly
385 390 395 400
Ala Thr Cys Cys Ala Gly Thr Thr Gly Cys Ala Cys Cys Thr Ala Cys
405 410 415
Thr Gly Thr Cys Cys Thr Cys Ala Thr Cys Thr Thr Cys Cys Cys Ala
420 425 430
Cys Cys Ala Gly Cys Thr Gly Cys Thr Gly Ala Thr Cys Ala Gly Gly
435 440 445
Thr Gly Gly Cys Ala Ala Cys Thr Gly Gly Ala Ala Cys Ala Gly Thr
450 455 460
Cys Ala Cys Cys Ala Thr Cys Gly Thr Gly Thr Gly Thr Gly Thr Gly
465 470 475 480
Gly Thr Gly Thr Gly Thr Gly Thr Gly Gly Cys Gly Ala Ala Thr Ala
485 490 495
Ala Ala Thr Ala Cys Thr Thr Thr Cys Cys Cys Gly Ala Thr Gly Thr
500 505 510
Cys Ala Cys Cys Gly Thr Cys Ala Cys Cys Thr Gly Gly Gly Ala Gly
515 520 525
Gly Thr Gly Gly Ala Thr Gly Gly Cys Ala Cys Cys Ala Cys Thr Gly
530 535 540
Gly Cys Ala Thr Cys Gly Ala Gly Ala Ala Cys Ala Gly Thr Ala Ala
545 550 555 560
Ala Ala Cys Ala Cys Cys Gly Cys Ala Gly Ala Ala Thr Thr Cys Thr
565 570 575
Gly Cys Ala Gly Ala Thr Thr Gly Thr Ala Cys Cys Thr Ala Cys Ala
580 585 590
Ala Cys Cys Thr Cys Ala Gly Cys Ala Gly Cys Ala Cys Thr Cys Thr
595 600 605
Gly Ala Cys Ala Cys Thr Gly Ala Cys Cys Ala Gly Cys Ala Cys Ala
610 615 620
Cys Ala Gly Thr Ala Cys Ala Ala Cys Ala Gly Cys Cys Ala Cys Ala
625 630 635 640
Ala Ala Gly Ala Gly Thr Ala Cys Ala Cys Cys Thr Gly Cys Ala Ala
645 650 655
Gly Gly Thr Gly Ala Cys Cys Cys Ala Gly Gly Gly Cys Ala Cys Gly
660 665 670
Ala Cys Cys Thr Cys Ala Gly Thr Cys Gly Thr Cys Cys Ala Gly Ala
675 680 685
Gly Cys Thr Thr Cys Ala Ala Thr Ala Gly Gly Gly Gly Thr Gly Ala
690 695 700
Cys Thr Gly Thr Thr Ala Gly
705 710
<210> 4
<211> 236
<212> PRT
<213> Artificial
<400> 4
Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser
20 25 30
Val Glu Val Ala Val Gly Gly Thr Thr Val Thr Ile Lys Cys Gln Ala
35 40 45
Ser Gln Ser Ile Gly Ser Tyr Leu Ser Trp Tyr Arg Gln Lys Pro Gly
50 55 60
Gln Pro Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly
65 70 75 80
Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr
85 90 95
Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
Gly Tyr Ser Ser Asp Asn Ile Asp Ile Thr Phe Gly Gly Gly Thr Glu
115 120 125
Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro
130 135 140
Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val
145 150 155 160
Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu
165 170 175
Val Asp Gly Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser
180 185 190
Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr
195 200 205
Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr
210 215 220
Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235
<210> 5
<211> 20
<212> PRT
<213> 引物(Artificial)
<400> 5
Ala Thr Gly Gly Ala Gly Ala Cys Thr Gly Gly Gly Cys Thr Gly Cys
1 5 10 15
Gly Cys Thr Gly
20
<210> 6
<211> 21
<212> PRT
<213> 引物(Artificial)
<400> 6
Cys Thr Ala Thr Thr Thr Ala Cys Cys Cys Gly Gly Ala Gly Ala Gly
1 5 10 15
Cys Gly Gly Gly Ala
20
<210> 7
<211> 20
<212> PRT
<213> 引物(Artificial)
<400> 7
Ala Thr Gly Gly Ala Cys Ala Cys Gly Ala Gly Gly Gly Cys Cys Cys
1 5 10 15
Cys Cys Ala Cys
20
<210> 8
<211> 22
<212> PRT
<213> 引物(Artificial)
<400> 8
Cys Thr Ala Ala Cys Ala Gly Thr Cys Ala Cys Cys Cys Cys Thr Ala
1 5 10 15
Thr Thr Gly Ala Ala Gly
20
Claims (8)
1.一种CHIKV E2蛋白兔单克隆抗体，其特征在于：所述抗体的氨基酸序列，包括抗体重链和抗体轻链；
其中，所述抗体重链的氨基酸序列如SEQ ID NO:2所示；
所述抗体轻链的氨基酸序列如SEQ ID NO:4所示。
2.如权利要求1所述的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
所述抗体重链基因的脱氧核苷酸序列如SEQ ID NO:1所示。
3.如权利要求1所述的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
所述抗体轻链基因的脱氧核苷酸序列如SEQ ID NO:3所示。
4.如权利要求1所述的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
所述抗体作为一种治疗性成分、一种药物、或用于制备一种药物；
所述治疗性成分/药物在体外可阻断CHIKV对靶细胞的感染。
5.如权利要求1所述的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
所述抗体作为一种治疗性成分、一种药物、或用于制备一种药物；
所述治疗性成分/药物在体内可抵抗CHIKV的致死性感染。
6.如权利要求1所述的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
所述抗体作为一种治疗性成分、一种药物、或用于制备一种药物；
所述治疗性成分/药物在体内可保护I型干扰素受体基因敲除小鼠抵抗CHIKV的致死性感染。
7.如权利要求1所述的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
在开发CHIKV感染的治疗性抗体中的应用。
8.如权利要求1所述的一种CHIKV E2蛋白兔单克隆抗体，其特征在于：
在开发CHIKV感染的治疗性人源化抗体或单链抗体中的应用。
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| CN114656554A (zh) * | 2022-03-07 | 2022-06-24 | 中山大学 | 基孔肯雅热病毒e2蛋白的纳米抗体及其应用 |
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| CN102282171A (zh) * | 2007-09-07 | 2011-12-14 | 巴斯德研究所 | 抗基孔肯雅病毒单克隆抗体及其用途 |
| CN107973850A (zh) * | 2017-12-13 | 2018-05-01 | 中国农业科学院哈尔滨兽医研究所 | 重组猪瘟病毒e2蛋白猪源化单克隆抗体及其制备方法和应用 |
| CN110903386A (zh) * | 2019-12-07 | 2020-03-24 | 中国人民解放军军事科学院军事医学研究院 | 一种高中和活性抗基孔肯雅热的全人源单克隆抗体及应用 |
| CN110922478A (zh) * | 2019-12-07 | 2020-03-27 | 中国人民解放军军事科学院军事医学研究院 | 针对特异表位的抗基孔肯雅热的全人源单克隆抗体及应用 |
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| CN102282171A (zh) * | 2007-09-07 | 2011-12-14 | 巴斯德研究所 | 抗基孔肯雅病毒单克隆抗体及其用途 |
| CN107973850A (zh) * | 2017-12-13 | 2018-05-01 | 中国农业科学院哈尔滨兽医研究所 | 重组猪瘟病毒e2蛋白猪源化单克隆抗体及其制备方法和应用 |
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| CN114656554A (zh) * | 2022-03-07 | 2022-06-24 | 中山大学 | 基孔肯雅热病毒e2蛋白的纳米抗体及其应用 |
| CN114656554B (zh) * | 2022-03-07 | 2023-05-23 | 中山大学 | 基孔肯雅热病毒e2蛋白的纳米抗体及其应用 |
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