鼠肝炎冠状病毒非结构蛋白1及其突变体的原核表达

目的:构建鼠肝炎冠状病毒(MHV)非结构蛋白1(NSP1)及其突变体(NSP1 mu)的原核重组表达质粒,在大肠杆菌中分别融合表达重组NSP1及NSP1 mu。方法:以现有质粒载体为模板,扩增编码NSP1及NSP1 mu的基因片段,并克隆至pMD18-T克隆载体;菌落PCR鉴定阳性克隆并测序分析;将阳性克隆的目的片段亚克隆至表达载体pET-28a,并转化大肠杆菌TOP10感受态细胞,PCR和双酶切鉴定转化菌落;将阳性质粒转化大肠杆菌BL21(DE3)感受态细胞并加入IPTG诱导表达,SDS-PAGE和免疫印迹分析目的蛋白的表达。结果:PCR扩增得到表达NSP1及NSP1 mu的特异片段,并克隆到pMD18-T载体,测序结果正确无误;构建了NSP1和NSP1 mu的重组表达质粒,并在大肠杆菌BL21(DE3)中分别融合表达了重组NSP1及NSP1 mu,表达的目的蛋白均能与His单克隆抗体特异结合;用Ni-NTA琼脂糖试剂盒纯化重组蛋白,获得可溶性的NSP1及NSP1 mu,相对分子质量分别为27×103和28×103。结论:在大肠杆菌中分别表达并纯化获得了大量可溶性重组NSP1及NSP1 mu。

Prokaryotic Expression of NSP1 and NSP1 Mutant Fusion Proteins of Mouse Hepatitis Virus

Objective: To construct the prokaryotic expression vector for genes encoding the non-structural pro tein 1(NSP1) and NSP1 mutant(NSP1 mu) fusion proteins of mouse hepatitis virus(MHV),express each of the proteins in E.coli BL21(DE3).Methods: nsp1 and nsp1 mu gene were amplified by PCR.The PCR products were cloned into pMD18-T vector and then sequenced.The prokaryotic expression vectors pET-28a-nsp1 and pET-28a-nsp1 mu were constructed respectively by using the purified nsp1 and nsp1 mu gene that were sub cloned into the expression vector pET-28a.These vectors pET-28a-nsp1 and pET-28a-nsp1 mu were transformed into E.coli BL21(DE3),respectively.Expression of proteins was induced by IPTG,and was assayed with SDS-PAGE and Western blot.Results & Conclusion: Expression vectors of fusion proteins of NSP1 and NSP1 mu were constructed.As demonstrated by SDS-PAGE and Western blotting,exogenous proteins with molecular mass of 27 kD and 28 kD approximately were obtained.