【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。更多还原

【Abstract】 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 BamHⅠ and SalⅠ 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.更多还原