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

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。

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 neu⁃tralization 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 vi⁃rus disease 2019(COVID-19).