鼠肝炎冠状病毒非结构蛋白1及其突变体的原核表达  

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

作　　者：柳洪涛[1,2] 林磊[2] 孙走南[3] 罗彦军[2] 何湘[3] 吴晓燕[2] 杨益[3] 苏文莉[3] 祝庆余[2] 刘景梅[3] 常国辉[2,3] 

机构地区：[1]广西医科大学基础医学院,广西南宁530021 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071 [3]军事医学科学院疾病预防控制所,北京100071

出　　处：《生物技术通讯》2012年第4期471-475,共5页Letters in Biotechnology

基　　金：国家青年自然科学基金(31100122);病原微生物生物安全国家重点实验室开放课题(SKLPBS1109)

摘　　要：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。Objective： To construct the prokaryotic expression vector for genes encoding the non-structural protein I（NSP1） and NSP1 mutant（NSP1 mu） fusion proteins of mouse hepatitis virus（MHV）, express each of the proteins in E.coli BL21 （DE3）. Methods： nspl and nspl mu gene were amplified by PCR. The PCR products were cloned into pMD18-T vector and then sequenced. The prokaryotic expression vectors pET-28a-nspl and pET-28a-nspl mu were constructed respectively by using the purified nspl and nspl mu gene that were subcloned into the expression vector pET-28a. These vectors pET-28a-nspl and pET-28a-nspl 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.

关 键 词：鼠肝炎冠状病毒 非结构蛋白1 突变体 表达 纯化 

分 类 号：Q78[生物学—分子生物学]