目的: 优化新型冠状病毒木瓜样蛋白酶(papain-like protease, PLpro)在大肠杆菌中的表达条件,并制备高特异性大鼠抗PLpro多克隆抗体。方法: 通过优化诱导时间、诱导温度和IPTG诱导浓度,确定PLpro在大肠杆菌中的最佳表达条件,以HisTrapTM亲和层析柱分离纯化PLpro后,将其作为抗原免疫大鼠,制备抗PLpro多克隆抗体,以酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)和蛋白免疫印迹实验(Western blot)测定多克隆抗体的效价、抗原特异性和灵敏性。结果: PLpro最佳诱导温度为20 ℃、诱导时间为10 h、IPTG浓度为0.2 mmol/L;抗PLpro多克隆抗体的效价可达1∶256 000,且具有良好的抗原特异性;抗PLpro多克隆抗体对抗原的检测限可达12.5 ng,具有良好的灵敏性。结论: 本研究成功地进行了PLpro在大肠杆菌中的表达条件优化及高特异性大鼠抗PLpro多克隆抗体的制备,为研究PLpro在新冠肺炎中的免疫学功能奠定了基础。
Objective: To optimize the expression conditions of SARS-CoV-2 papain-like protease (papain-like protease, PLpro) in Escherichia coli (E.coli) and prepare highly specific polyclonal antibody against rat PLpro. Methods: The optimal expression conditions of PLpro in E.coli were determined by optimizing induction time, induction temperature and IPTG concentration. After PLpro being isolated and purified by HisTrapTM affinity chromatography column, the rat was immunized with PLpro as antigen to prepare anti-PLpro polyclonal antibody. The titer, antigen specificity and sensitivity of polyclonal antibody were determined by enzyme-linked immunosorbent assay (ELISA) and Western Blot experiments. Results: The optimum induction temperature of PLpro was 20 ℃, the induction time was 10 h, and the concentration of IPTG was 0.2 mmol/L. The titer of anti-PLpro polyclonal antibody reached to 1∶256 000 with good antigen specificity, and the detection limit of anti-PLpro polyclonal antibody was 12.5 ng with good sensitivity. Conclusion: The expression conditions of PLpro in E.coli was successfully optimized and highly specific polyclonal antibody against rat PLpro successfully prepared, which laid a foundation for the study of the immunological function of PLpro in COVID-19.
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