新型冠状病毒Nsp16蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression of SARS-CoV-2 Nsp16 protein and preparation of polyclonal antibody

目的原核表达新型冠状病毒Nsp16重组蛋白并制备兔抗Nsp16多克隆抗体。方法通过琼脂糖凝胶电泳对含有Nsp16基因的重组质粒进行双酶切鉴定,并将鉴定后的重组质粒转化大肠埃希菌BL21(DE3)感受态细胞;通过IPTG诱导重组蛋白的表达,利用镍柱亲和层析法纯化该重组蛋白并进行Western blot鉴定;将鉴定后的目的蛋白与弗氏佐剂以1:1的体积比混匀,乳化后免疫新西兰大白兔,利用ELISA和Western blot分别进行多克隆抗体效价的测定及其特异性鉴定。结果Nsp16基因重组质粒经双酶切后得到912bp的目的片段,重组质粒转化大肠埃希菌表达出分子质量单位为33.3ku的重组蛋白,且主要存在于上清中。用纯化的重组蛋白免疫大白兔,获得兔抗Nsp16特异性多克隆抗体,且其效价达1:409600以上。结论重组Nsp16蛋白可以在大肠埃希菌中正确表达,免疫大白兔后获得高效价抗Nsp16多克隆抗体,即具有免疫反应性。为SARS-CoV-2Nsp16蛋白在新型冠状病毒肺炎中的进一步研究奠定了基础。

Objective Novel coronavirus Nsp16 recombinant protein was expressed in prokaryotes and rabbit polyclonal antibody against Nsp16 was prepared.Methods The recombinant plasmid containing Nsp16 gene was identified by agarose gel electrophoresis and transformated into EscherichiacoliBL21(DE3)competent cells.The recombinant protein was induced by IPTG,purified by affinity chromatography with nickel column and identified by Western blot.The identified target protein was mixed with Freund’s adjuvant at a volume ratio of 1:1,and then New Zealand white rabbits were immunized after emulsification.ELISA and Western blot were used to determine the titer of polyclonal antibody and its specificity.Results The Nsp16 recombinant plasmid was digested with double enzymes to obtain a 912 bp target fragment.The recombinant plasmid was transformated into E.coli and expressed 33.3 ku recombinant protein,which mainly existed in supernatant.Rabbit polyclonal antibody against Nsp16 was obtained by immunizing white rabbits with purified recombinant protein,and its titer was more than 1:409600.Conclusion The recombinant Nsp16 protein can be expressed correctly in E.coli,and rabbit polyclonal antibody against Nsp16 was characterized by high potency,indicating its immunoreactivity.It laid a foundation for further study of SARS-COV-2 Nsp16 protein in COVID-19.