猪传染性胃肠炎病毒N基因的克隆及其在大肠杆菌中的表达  

Cloning and expression of nucleoprotein gene of transmissible gastroenteritis virus and purification of recombinant nucleoprotein

作　　者：黄小波[1] 曹三杰[1] 文心田[1] 肖国生[1] 肖驰[1] 

机构地区：[1]四川农业大学动物科技学院,四川雅安625014

出　　处：《中国兽医学报》2008年第5期489-492,共4页Chinese Journal of Veterinary Science

基　　金：四川省重点科技攻关资助项目(01NG018-01);教育部长江学者和创新团队发展计划资助项目(IRT0555-9)

摘　　要：应用RT-PCR扩增猪传染性胃肠炎病毒SC-H株N基因,将扩增的N基因克隆到pMD18-T载体中,进行BamHⅠ和SalⅠ双酶切鉴定,将获得的N基因片段以pET32a(+)载体进行亚克隆,构建了pET32-N重组表达质粒并进行鉴定。将pET32-N重组质粒转化BL21(DE3)大肠杆菌中进行表达,并对表达条件进行了选择,对表达产物进行了定位和纯化。结果表明,N基因全长1149 bp,编码382个氨基酸,克隆的N基因包含完整的阅读框且能在大肠杆菌中正确表达,表达的最佳IPTG诱导浓度为0.8 mmol/L,最佳诱导时间为3 h,表达的N蛋白约为47 000,主要以包涵体形式存在于细胞质中。The nucleoprotein gene of transmissible gastroenteritis virus stain SC-H was amplified by RT-PCR and the fragment was cloned into pMD18-T vector. The recombinant vector was identified by restriction endonuclease analysis, then the recombinant vector was cleavaged by BamH Ⅰ and Sal Ⅰ and the fragment was subcloned site specially into pET-32a（＋ ） expression vector. The recombinant pET32-N expression plasmid was confirmed to be constructed successfully by restriction endonuclease analysis and nucleotide sequencing. Then recombinant plasmid was transformed into BL21（DE） to express and the optimal expression conditions was determined, the cellular localization of the recombinant nucleoprotein was done and the recombinant protein was purified by specific kit with histidine tag. The results showed that cloned N gene covered the whole open reading frame and it has been correctly inserted into expression vector, the full-length was 1 149 bp and encoded 382 amino acids. The expression product was identified by SDS-PAGE with the molecular weight of about 47 000. The optimal induction concentration of IPTG was 0.8 mmol/L and the optimal induction time was three hours. The recombinant nucleoprotein was localized in the cell cytoplasm with soluble protein and inclusion body. A simple protein strap could be found after purification.

关 键 词：猪传染性胃肠炎病毒 N基因 克隆 表达 纯化 

分 类 号：S852.65[农业科学—基础兽医学]