SARS-CoV-2中和性纳米抗体的原核表达及中和活性检测

doi:10.19586/j.2095-2341.2022.0042

生物技术进展 ›› 2022, Vol. 12 ›› Issue (5): 754-759.DOI: 10.19586/j.2095-2341.2022.0042

SARS-CoV-2中和性纳米抗体的原核表达及中和活性检测

王海宁¹^,²^,³(), 刘兴健³, 高新桃³, 李轶女³, 沈兴家¹^,², 张志芳³, 易咏竹¹^,²()

^1.江苏科技大学生物技术学院，江苏省蚕桑生物学与生物技术重点实验室，江苏镇江 212100
^2.中国农业科学院蚕业研究所，农业农村部蚕桑遗传改良重点实验室，江苏镇江 212100
^3.中国农业科学院生物技术研究所，北京 100081

收稿日期:2022-03-21 接受日期:2022-07-07 出版日期:2022-09-25 发布日期:2022-09-30
通讯作者: 易咏竹
作者简介:王海宁 E-mail: 1585676756@qq.com；
基金资助:
宁夏回族自治区重点研发计划(2020BFG02017);国家自然科学基金项目(32002236)

Prokaryotic Expression and Neutralization Activity Detection of SARS-CoV-2 Neutralizing Nanobody

Haining WANG¹^,²^,³(), Xingjian LIU³, Xintao GAO³, Yinv LI³, Xingjia SHEN¹^,², Zhifang ZHANG³, Yongzhu YI¹^,²()

^1.Jiangsu Key Laboratory of Sericulture Biology and Biotechnology，College of Biotechnology，Jiangsu University of Science and Technology，Jiangsu Zhenjiang 212100，China
^2.Key Laboratory of Sericulture Genetic Improvement，Ministry of Agriculture and Rural Affairs，Institute of Sericulture，Chinese Academy of Agricultural Sciences，Jiangsu Zhenjiang 212100，China
^3.Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2022-03-21 Accepted:2022-07-07 Online:2022-09-25 Published:2022-09-30
Contact: Yongzhu YI

摘要/Abstract

摘要：

纳米抗体（nanobody，Nb）作为目前已知的能与目标抗原结合的最小单位抗体，在生物医药、临床研究等方面具有良好的应用前景。根据大肠杆菌密码子偏好性优化合成严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome-coronavirus 2，SARS-CoV-2）中和性纳米抗体H11-D4基因，将其克隆到pET28a表达载体上后，转化至大肠杆菌感受态细胞Rosetta（DE3）进行诱导表达，通过镍柱纯化、质谱分析、Western Blot鉴定H11-D4的表达情况并使用中和试验验证其中和活性。研究结果显示，纳米抗体H11-D4可成功在大肠杆菌中表达，最佳诱导条件为IPTG终浓度1.0 mmol·L^-1，37 ℃诱导5 h。H11-D4抗体的分子量大小约为17.9 kD，与预测值相符。经镍柱纯化后，产量为25.16 mg·L^-1。透析复性后利用TritonX-114快速有效地去除了内毒素，中和试验成功验证了H11-D4的中和活性（IC₅₀）为171.1 nmol·L^-1，研究结果可为纳米抗体的原核表达和新型冠状病毒肺炎（corona virus disease 2019，COVID-19）的预防及治疗提供基础数据支撑。

关键词: 纳米抗体, 大肠杆菌, 新冠病毒, 中和试验

Abstract:

Nanobody， as the smallest unit known to bind to target antigen， has a good application prospect in biomedicine and clinical research. According to the optimization of Escherichia coli codon preference， the gene of nanobody H11-D4 neutralizing severe acute respiratory syndrome-coronavirus 2（SARS-CoV-2） was synthesized and cloned into pET28a expression vector and transformed into E. coli competent cell Rosetta（DE3）for induced expression. The expression of H11-D4 was identified by nickel column purification， mass spectrometry and Western blot， its activity was verified by neutralization test. The experimental results showed that the nanobody H11-D4 had been successfully expressed in E. coli. The optimal induction conditions were determined to be an induction temperature of 37 ℃，an induction time of 5 h，and IPTG final concentration of 1.0 mmol·L^-1. The molecular weight was about 17.9 kD， which was consistent with the predicted value. After nickel column purification， the yield of H11-D4 protein was 25.16 mg·L^-1. After dialysis renaturation， endotoxin was removed quickly and effectively by Triton X-114. The neutralization test successfully verified that the neutralization activity（IC₅₀）of H11-D4 was 171.1 nmol·L^-1. The results obtained in this study could provide basic data support for the prokaryotic expression of nanobody and prevention and treatment of corona virus disease 2019（COVID-19）.

Key words: nanobody, Escherichia coli, SARS-CoV-2, neutralization test

中图分类号:

R392-33

王海宁, 刘兴健, 高新桃, 李轶女, 沈兴家, 张志芳, 易咏竹. SARS-CoV-2中和性纳米抗体的原核表达及中和活性检测[J]. 生物技术进展, 2022, 12(5): 754-759.

Haining WANG, Xingjian LIU, Xintao GAO, Yinv LI, Xingjia SHEN, Zhifang ZHANG, Yongzhu YI. Prokaryotic Expression and Neutralization Activity Detection of SARS-CoV-2 Neutralizing Nanobody[J]. Current Biotechnology, 2022, 12(5): 754-759.

图/表 7

图1 重组质粒pET28a-H11-D4双酶切鉴定注：M-DNA Marker；1-pET28a空载双酶切产物；2-pET28a-H11-D4双酶切产物。

Fig. 1 Identification of recombinant plasmid pET28a-H11-D4 by double enzyme digestion

图2 重组蛋白H11-D4的SDS-PAGE分析注：M-标准蛋白分子量；1-pET28a；2-pET28a-H11-D4（未诱导）；3~7-pET28a-H11-D4（分别用1.0、0.8、0.5、0.3、0.1 mmol·L-1IPTG诱导）。

Fig. 2 SDS-PAGE analysis of recombinant protein H11-D4

图3 H11-D4纯化后的SDS-PAGE验证与浓度测定A：纯化后的SDS-PAGE验证。M—标准蛋白分子量；1—未纯化蛋白；2—纯化后蛋白。B：BCA法测纯化后H11-D4蛋白浓度

Fig. 3 SDS-PAGE analysis and concentration determination after H11-D4 purification

图4 H11-D4 Western Blot验证图注：M-标准蛋白分子量；1-pET28a；2-pET28a-H11-D4。

Fig. 4 Western Blot analysis of H11-D4

图5 绿色荧光蛋白ACE2融合蛋白在293T细胞中的表达

Fig. 5 Expression of ACE2-GFP in 293T cells

图6 假病毒感染力测定

Fig. 6 Determination of pseudovirus infectivity

图7 H11-D4对假病毒的中和活性曲线

Fig. 7 Neutralization activity curve of H11-D4 to pseudovirus

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SARS-CoV-2中和性纳米抗体的原核表达及中和活性检测

Prokaryotic Expression and Neutralization Activity Detection of SARS-CoV-2 Neutralizing Nanobody

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