摘要
目的表达及纯化新型冠状病毒(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)3C样蛋白酶(3-chymotrypsin-like protease,3CL^(pro))并制备3CL^(pro)多克隆抗体。方法将重组质粒pET28a-Proteinase_3CL-pro转化大肠埃希菌BL21(DE3),利用IPTG诱导重组蛋白表达,超声破碎后用SDS-PAGE凝胶分析蛋白表达情况,利用Ni-NTA进行重组蛋白的纯化。用重组蛋白免疫4~8周龄健康小鼠,制备多克隆抗体,通过Western blot和ELISA对抗体进行定性和定量检测。结果重组蛋白His-3CL^(pro)在重组菌培养上清中高表达,分子质量单位为33.8 ku。制备的多克隆抗体可特异结合重组蛋白3CL^(pro),ELISA检测血清抗体效价可达1∶204800。结论在大肠埃希菌中成功表达3CL^(pro),免疫小鼠并制备高效价的鼠抗3CL^(pro)多克隆抗体,为研究3CL^(pro)功能进而开发新型冠状病毒检测试剂盒及治疗药物奠定了基础。
Objective The expression and purification of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)3-chymotrypsin-like protease(3 CL^(pro))and the preparation of 3 CL^(pro)polyclonal antibodies.Methods Transformation of recombinant plasmid pET28 a-Proteinase_3 CL-pro into Escherichia coli BL21(DE3),IPTG was used to induce the recombinant protein expression,SDS-PAGE was used to analyze the protein expression after ultrasonic crushing.The purification of the recombinant protein was made using Ni-NTA.The purified protein immune 4~8 weeks of healthy mice to prepare polyclonal antibodies.Qualitative and quantitative detection through Western blot and ELISA.Results The recombinant protein His-3 CL^(pro)was highly expressed in the supernatant of recombinant bacteria,the molecular mass unit is 33.8 ku.The preparation of polyclonal antibodies can be a specific combination of recombinant protein 3 CL^(pro);The antibody titer of serum polyclonal antibody was up to 1∶204800.Conclusion 3 CL^(pro)was successfully expressed in E.coli,mice were immunized and polyclonal antibodies against 3 CL^(pro)were prepared,this has laid a foundation for studying the function of 3 CL^(pro)and developing novel Coronavirus detection kits and therapeutic drugs.
作者
郑皖怿
李瑞芳
徐海山
宋桃花
高岩
陈柯
伊正君
付玉荣
ZHENG Wan-yi;LI Rui-fang;XU Hai-shan;SONG Tao-hua;GAO Yan;CHEN Ke;YI Zheng-jun;FU Yu-rong(School of Basic Medicine of Weifang Medical University,Weifang 261053,Shandong,China;School of Medical Laboratory of Weifang Medical University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2022年第1期18-21,共4页
Journal of Pathogen Biology
基金
山东省自然科学基金重点项目(No.ZR2018ZC1054)。
