传染性支气管炎N基因克隆及在大肠杆菌中的可溶性表达

核衣壳蛋白(N)是鸡传染性支气管炎病毒(IBV)的重要结构蛋白。根据GenBank报道的IBV的M41株的N基因序列设计一对引物,从提取的M41株的RNA中利用RT-PCR技术扩增获得了约1.23kb的片段,将该片段插入克隆载体pMD18-T,经测定核苷酸序列证实N基因扩增正确,表明已成功构建pMD18-T-N。再将pMD18-T-N用BamHⅠ和HindⅢ双酶切,将酶切后的N基因片段克隆到表达载体pET-32a,转化入大肠杆菌Rossetta中,用IPTG诱导,进行SDS-PAGE电泳,电泳结果显示外源基因获得了表达且为可溶性蛋白,Western-blotting检测表明N蛋白可与相应IBV病毒抗体发生特异性的反应,表明N蛋白有一定的生物活性,可为用于生产检测IB的诊断试剂和研制新型IB重组亚单位疫苗奠定基础。

Cloning of Nucleoprotein Gene of Infectious Bronchitis Virus and Its Soluble Expression in E. Coli Rossetta

Nucleocapsid protein(N protein) is one of the major structural protein of infectious bronchitis virus(IBV).A pair of specific primers were designed and synthesized according to the published sequence of N gene of IBV M41 strain in GeneBank,M41 strain from the extracted RNA using RT-PCR,amplified fragment of about 1.23kb,Which is correct fragment.This fragment inserted into the Cloning vector pMD18-T,and its nucleotide sequence was determined by the dideoxy-mediated chain termination method.The results showed that successful construction recombinant vector pMD18-T-N.The NP gene was excised with BamHⅠand HindⅢand inserted into the expression plasmid pET-32a to obtain the recombinant expression vector pET-32a-N,which was used to transform E.coli Rossetta competent cells for gaining recombinant bacteria pET-32a-N(R).Then the recombinant pET-32a-N(R)was induced by IPTG,SDS-PAGE and Western blotting confirmed that the gene had been expressed solubly in recombinant pET-32a-N(R)cells,The expression product could specially react to the anti-sera of IBV that showed N protein has some biological activity.This study has laid a solid foundation for production of diagnostic reagents for testing IB and production of new type IB gene engineering vaccines.