SARS-CoV-2中和性纳米抗体的原核表达及中和活性检测

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。

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

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）.