新型冠状病毒RBD蛋白原核表达及多克隆抗体的制备

doi:10.19586/j.2095-2341.2022.0114

生物技术进展 ›› 2023, Vol. 13 ›› Issue (1): 102-106.DOI: 10.19586/j.2095-2341.2022.0114

新型冠状病毒RBD蛋白原核表达及多克隆抗体的制备

彭晓燕(), 陆盼盼, 胡祖权()

贵州医科大学生物与工程学院，贵州省免疫细胞与抗体工程研究中心，贵阳 550025

收稿日期:2022-06-29 接受日期:2022-09-28 出版日期:2023-01-25 发布日期:2023-02-07
通讯作者: 胡祖权
作者简介:彭晓燕E-mail：pengxiaoyan@gmc.edu.cn；
基金资助:
国家自然科学基金项目(31660258);贵州省自然科学基金项目（黔科合平台人才〔2021〕5637号;黔科合基础-ZK〔2021〕重点029

Prokaryotic Expression of the RBD Protein of SARS-CoV-2 and its Polyclonal Antibodies Preparation

Xiaoyan PENG(), Panpan LU, Zuquan HU()

Immune Cells and Antibody Engineering Research Center of Guizhou Province，School of Biology and Engineering，Guizhou Medical University，Guiyang 550025，China

Received:2022-06-29 Accepted:2022-09-28 Online:2023-01-25 Published:2023-02-07
Contact: Zuquan HU

摘要/Abstract

摘要：

为原核表达严重急性呼吸综合征冠状病毒2（简称新型冠状病毒，severe acute respiratory syndrome-coronavirus 2，SARS-CoV-2）S蛋白受体结合域（receptor binding domain, RBD）并制备多克隆抗体，利用基因克隆技术将RBD基因连接到原核表达载体pGEX-6p-1和pET-32a(+)上，电转化至大肠杆菌XL1-Blue感受态细胞，利用优化后的表达条件大量表达重组蛋白，经亲和层析纯化后通过SDS-PAGE检测蛋白的表达情况。利用GST-RBD融合蛋白作为免疫抗原免疫小鼠制备多克隆抗体，ELISA和Western blot分析抗血清的效价和特异性。PCR鉴定和序列测定结果显示，成功构建了重组载体pGEX-RBD和pET-RBD，在大肠杆菌中实现了GST-RBD和RBD-His融合蛋白的可溶性高效表达。研究获得的多克隆抗体的滴度达到约1∶3 000，并具有良好的结合特异性。原核表达的可溶性新型冠状病毒RBD重组蛋白具有良好的免疫原性，为后续制备基因工程抗体奠定了实验基础。

关键词: 新型冠状病毒, 受体结合域, 原核表达, 融合蛋白, 多克隆抗体

Abstract:

To obtain the receptor binding domain （RBD） of S protein of severe acute respiratory syndrome-coronavirus 2 （SARS-CoV-2） in prokaryotic cells and further prepare its polyclonal antibodies， the RBD gene was connected into prokaryotic expression vectors of pGEX-6p-1 and pET-32a（+） by gene cloning technology. The recombinant plasmids were then transformed into the competent cells of Escherichia coli strain XL1-blue. The recombinant proteins were largely expressed by the optimized expression conditions. After purification by affinity chromatography， the expression of recombinant proteins was detected by SDS-PAGE. Polyclonal antibodies were prepared by immunizing mice with GST-RBD fusion protein as immune antigen. Their titers and specificity were detected by enzyme-linked immunosorbent assay （ELISA） and Western blot. The PCR identification and sequencing results showed that the recombinant vectors of pGEX-RBD and pET-RBD were successfully constructed. The fusion proteins of GST-RBD and RBD-His were achieved efficient and soluble expression in E. coli. The titers of the obtained polyclonal antibodies reached approximately 1∶3 000 and they had good binding specificities. The soluble recombinant RBD protein of novel coronavirus expressed in prokaryotic cells has good immunogenicity， which layed an experimental foundation for the further preparation of genetic engineering antibodies.

Key words: SARS-CoV-2, receptor binding domain （RBD）, prokaryotic expression, fusion protein, polyclonal antibody

中图分类号:

R392.11

彭晓燕, 陆盼盼, 胡祖权. 新型冠状病毒RBD蛋白原核表达及多克隆抗体的制备[J]. 生物技术进展, 2023, 13(1): 102-106.

Xiaoyan PENG, Panpan LU, Zuquan HU. Prokaryotic Expression of the RBD Protein of SARS-CoV-2 and its Polyclonal Antibodies Preparation[J]. Current Biotechnology, 2023, 13(1): 102-106.

图/表 5

图1 PCR扩增RBD基因片段注：M—DNA分子量标准；1—构建pGEX-RBD重组载体的RBD基因；2—构建pET-RBD重组载体的RBD基因。

Fig. 1 PCR amplification of RBD gene fragment

图2 PCR鉴定阳性转化子A：pGEX-RBD重组载体的转化子；B：pET-RBD重组载体的转化子。M—DNA分子量标准；1~10—重组载体转化子；11：阴性对照。

Fig. 2 PCR identification of the positive transformants

图3 SDS-PAGE检测纯化的融合蛋白A：GST-RBD融合蛋白；B：RBD-His融合蛋白；M—蛋白分子量标准

Fig. 3 SDS-PAGE detection of the purified fusion proteins

图4 ELISA检测多克隆抗体的效价

Fig. 4 ELISA detection of the titers of polyclonal antibodies

图5 Western blot检测多克隆抗体特异性A：1号小鼠；B：2号小鼠；C：3号小鼠；D：对照小鼠；M—蛋白分子量标准

Fig. 5 Western blot detection of the specificities of polyclonal antibodies

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新型冠状病毒RBD蛋白原核表达及多克隆抗体的制备

Prokaryotic Expression of the RBD Protein of SARS-CoV-2 and its Polyclonal Antibodies Preparation

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