CN111138534B - 寨卡病毒囊膜蛋白的鼠源单克隆抗体 - Google Patents

寨卡病毒囊膜蛋白的鼠源单克隆抗体 Download PDF

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CN111138534B
CN111138534B CN201911422479.7A CN201911422479A CN111138534B CN 111138534 B CN111138534 B CN 111138534B CN 201911422479 A CN201911422479 A CN 201911422479A CN 111138534 B CN111138534 B CN 111138534B
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龚睿
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Wuhan Banke Biotechnology Co ltd
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Abstract

本发明公开了一种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

寨卡病毒囊膜蛋白的鼠源单克隆抗体
技术领域
本发明涉及生物技术领域，具体地指一种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B0氨基酸序列的易聚集区。
图1-2为Tango软件预测突变后B8氨基酸序列的易聚集区。
图1-3为B0和B8的重链突变区域比较图。
图2为SDS-PAGE检测B8抗体。
图3为ELISA分析B8抗体与寨卡病毒囊膜蛋白的结合图。
图4为B8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：B8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B0候选克隆的碱基序列，并经氨基酸翻译软件(https://web.expasy.org/translate/)，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B0氨基酸序列进行优化
利用Tango软件(http://tango.crg.es/)对B0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B8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B8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B8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B8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B8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100nM，如图3所示。
实施例5：B8抗体的寨卡病毒中和实验。
将B8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50nM，如图4所示。
SEQUENCE LISTING
<110> 武汉班科生物技术股份有限公司
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Claims (2)

1.一种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SEQ ID No.1所示，其重链序列如SEQ ID No.3所示。
2.根据权利要求1所述寨卡病毒囊膜蛋白的鼠源单克隆抗体，其特征在于，其轻链的编码核苷酸序列如SEQ ID No.4所示，其重链的编码核苷酸序列如SEQ ID No.5所示。
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