鸡传染性支气管炎病毒(IBV)ZZ2004株核衣壳蛋白基因的克隆及原核表达

Cloning and Prokaryotic Expression of Nucleoprotein Gene of Infectious Bronchitis Virus (IBV) ZZ2004 Strain

本研究旨在克隆并表达鸡传染性支气管炎病毒(IBV)的核衣壳蛋白(N)。根据IBV ZZ2004株的N基因设计1对引物,提取IBV ZZ2004株的RNA,利用RT-PCR技术扩增大小约1.23kb的片段,将其插入克隆载体pGEM-T构建pGEMT-N。将pGEM-T-N用EcoRⅠ和XhoⅠ进行双酶切,将酶切后的N基因片段克隆到表达载体pGEX-6P-1,转入大肠杆菌BL21(DE3)菌中,经IPTG诱导表达,进行SDS-PAGE电泳,结果显示外源基因获得了表达且为可溶性蛋白;Western blotting检测结果显示N蛋白可与相应的IBV ZZ2004阳性血清发生特异性反应,结果表明N蛋白有一定的生物活性。本研究为IBV诊断试剂盒及新型IBV重组亚单位疫苗研制奠定基础。

This experiment was conducted to clone and express the nucleoprotein （N） gene of infectious bronchitis virus （IBV）. A pair of specific primers was designed and synthesized according to the published sequence of IBV ZZ2004. RNA of IBV ZZ2004 strain was extracted and the fragment of about 1.23 kb was amplified by RT-PCR, which was correct fragment.The fragment was inserted into the cloning vector pGEM-T to construct pGEM T-N, and nucleotide sequence was determined by dideoxy-mediated chain termination method. The pGEM-T-N was excised by EcoR I and Xho I , and inserted into the ex- pression plasmid pGEX 6P-1 to obtain the recombinant expression vector pGEX-6P-1-N, which was used to transform into E. coli BL21 （DE3） competent cells. Then the recombinant pGEX-6P 1-N was induced by IPTG, SD^PAGE and Western blotting results confirmed that the gene had been expressed in recombinant pGEX 6P-l-N, and the protein was soluble. The ex- pression product could specially react to the anti-sera of IBV which showed that N protein had some biological activity. This study had laid a solid foundation for production of diagnostic reagents for testing IB and new type IB gene engineering vaccines.