CN111100201B - 针对寨卡病毒囊膜蛋白的鼠源单克隆抗体 - Google Patents
针对寨卡病毒囊膜蛋白的鼠源单克隆抗体 Download PDFInfo
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Abstract
本发明公开了一种针对寨卡病毒囊膜蛋白的鼠源单克隆抗体，通过筛选和基因改造后，所获抗体的特异性好、中和活性强、抗聚集性增，适用于寨卡病毒的防治。
Description
技术领域
本发明涉及生物技术领域，具体地指一种针对寨卡病毒囊膜蛋白的鼠源单克隆抗体。
背景技术
寨卡病毒(ZIKV)是一种黄病毒科病毒，感染该病毒后可导致新生儿的小头畸形并影响婴幼儿的神经发育，成年人感染后可能发生格林巴利综合征(GBS)导致神经损伤。目前尚无获批的疫苗和特效药物，这里我们利用自己构建的寨卡病毒囊膜蛋白免疫小鼠，获得了具备较强中和活性的鼠源单克隆抗体，将来可以运用于寨卡病毒的防治。
发明内容
本发明的目的在于填补现有技术的空白，提供一种特异性好的针对寨卡病毒囊膜蛋白的鼠源单克隆抗体。
为实现上述目的，本发明提供了一种针对寨卡病毒囊膜蛋白的鼠源单克隆抗体，其轻链序列如SEQ No.1所示，其重链序列如SEQ No.3所示。
上述针对寨卡病毒囊膜蛋白的鼠源单克隆抗体对应的碱基序列，其轻链序列如SEQ No.4所示，其重链序列如SEQ No.5所示。
本申请的发明人在中国专利申请201811613477.1的研究基础上，利用其构建噬菌体文库的方法进行筛选并进一步基因改造，获得了效果较好的鼠源单克隆抗体。
本发明的有益效果：通过筛选和基因改造后，所获抗体的特异性好、中和活性强、抗聚集性增强，适用于寨卡病毒的防治。
附图说明
图1-1为Tango软件预测D10氨基酸序列的易聚集区。
图1-2为Tango软件预测D11氨基酸序列的易聚集区。
图1-3为D10和D11的重链突变区域比较图。
图2为SDS-PAGE检测D11抗体。
图3为ELISA分析D11抗体与寨卡病毒囊膜蛋白的结合图。
图4为D11抗体对寨卡病毒的中和活性图。
具体实施方式
以下结合附图和具体实施例对本发明作进一步的详细描述。以下实施例是在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
实施例1：D10抗体序列的获得
基于已构建的免疫小鼠Fab噬菌体展示文库(该文库的获得方法来自中国专利申请201811613477.1公开的黄病毒科病毒囊膜蛋白的融合蛋白及其制备方法与应用，利用其中的融合蛋白作为免疫原免疫小鼠后获得其cDNA，利用其作为噬菌体展示文库的构建材料)，我们进行了三轮的筛选，并通过单克隆phage ELISA，将其中的阳性克隆送测序，得到了D10候选克隆的碱基序列，并经氨基酸翻译软件，得到了其氨基酸序列，轻链氨基酸序列如SEQ No.1所示，其重链氨基酸序列如SEQ No.2所示。
实施例2：利用Tango软件对获得的D10氨基酸序列进行优化
利用Tango软件(http://tango.crg.es/)对D10候选克隆的氨基酸序列进行易聚集区预测，发现其中存在较强的易聚集区域，如图1-1，因此对该区域进行点突变，得到D11抗体，其轻链氨基酸序列如SEQ No.1所示，其重链氨基酸序列如SEQ No.3所示，对应碱基序列，轻链如SEQ No.4所示，重链如SEQ No.5所示。突变后的氨基酸序列经Tango软件重新预测，发现该易聚集区被明显改善，如图1-2，1-3。
实施例3：293F细胞表达D11抗体并SDS-PAGE检测
将突变后的碱基序列克隆到双启动载体pVitro2-neo-mcs(InvivoGen)上，利用PEI转染293F细胞后进行蛋白的表达，经过5-7天的表达，将293F细胞的培养上清经ProteinA(GE)纯化，将纯化后的蛋白经过浓换液后，得到溶于PBS缓冲液的D11抗体。经SDS-PAGE电泳检测，发现其呈现轻链和重链两条带，如图2所示。
实施例4：ELISA分析D11抗体与寨卡病毒囊膜蛋白的结合
包被寨卡病毒囊膜蛋白在ELISA板上，将D11抗体经梯度稀释后作为一抗，HRP标记的Goat-anti-mouse IgG(Abcam)作为二抗，显色后，通过信号强度变化计算EC50，约在200nM，如图3所示。
实施例5：D11抗体的寨卡病毒中和实验。
将D11抗体进行梯度稀释后分别与100PFU的寨卡病毒在37℃共同孵育1h，然后将该混合物加入到提前一天铺有Vero细胞的24孔板中，37℃孵育1h，弃掉病毒混合液，加入上层覆盖物(DMDM配制的2％羟甲基纤维素，含有2％血清)继续培养2-3天。形成明显病毒噬斑后，弃掉上层覆盖物，并加入3.7％的甲醛溶液进行固定，后加入1％浓度的结晶紫对噬斑进行染色，根据不同抗体浓度下病毒噬斑减少的情况，计算出IC50值，约为100nM，如图4所示。
SEQUENCE LISTING
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<120> 针对寨卡病毒囊膜蛋白的鼠源单克隆抗体
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agaaatagct actcctgttc agtggtccac gagggtctgc acaatcacca cacgactaag 1320
agcttctccc ggactccg 1338
Claims (2)
1.一种针对寨卡病毒囊膜蛋白的鼠源单克隆抗体，其特征在于，其轻链序列如SEQNo.1所示，其重链序列如SEQ No.3所示。
2.根据权利要求1所述针对寨卡病毒囊膜蛋白的鼠源单克隆抗体，其特征在于，所述轻链的编码核苷酸序列如SEQ No.4所示，所述重链的编码核苷酸序列如SEQ No.5所示。
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| WO2018010789A1 (en) * | 2016-07-13 | 2018-01-18 | Humabs Biomed Sa | Novel antibodies specifically binding to zika virus epitopes and uses thereof |
| TW201815821A (zh) * | 2016-07-18 | 2018-05-01 | 美商再生元醫藥公司 | 抗茲卡病毒抗體及使用方法 |
| CN110066333B (zh) * | 2016-08-10 | 2020-09-04 | 中国科学院微生物研究所 | 一种寨卡病毒特异性中和抗体及其应用 |
| CU20210030A7 (es) * | 2016-10-13 | 2021-11-04 | Massachusetts Inst Technology | Anticuerpos que se unen a la proteína de envoltura del virus zika |
| KR102071539B1 (ko) * | 2016-11-18 | 2020-02-03 | 재단법인 바이오나노헬스가드연구단 | 지카 바이러스의 표피단백질과 비구조단백질 1에 특이적인 단클론항체를 이용한 지카 바이러스 항체의 검출방법 및 지카 바이러스 항체 검출을 위한 신속진단키트 |
| CN109081868B (zh) * | 2017-06-14 | 2022-06-24 | 中国科学院上海巴斯德研究所 | 靶向寨卡病毒包膜蛋白保守表位的单克隆抗体及其应用 |
| CN109705222A (zh) * | 2018-12-27 | 2019-05-03 | 中国科学院武汉病毒研究所 | 黄病毒科病毒囊膜蛋白的融合蛋白及其制备方法与应用 |
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