IBV HH06株N基因原核表达及间接ELISA方法的建立

Prokaryotic expression of N gene of IBV HH06 strain and development of indirect IBV N protein-mediated ELISA

鸡传染性支气管炎(Avian infectious bronchitis,IB)是由传染性支气管炎病毒(Infectious bronchitis virus,IBV)引起的一种急性、高度接触性传染病。试验根据NCBI发表的IBV基因序列设计引物,利用RT-PCR方法扩增病毒的N基因,并将其导入pET-30a(+)载体中,构建原核表达质粒。SDS-PAGE及Western-blot检测结果表明,重组蛋白得到表达并能与IBV阳性血清有很好的反应性。以纯化后的重组蛋白作为包被抗原,在包被量为2.5μg·mL-1、血清1??40倍稀释条件下,建立并优化间接ELISA检测方法。临床样品检测表明,该方法可高效检测出IBV抗体,并与IBV病毒作为包被抗原ELISA检测结果一致,为IB的快速诊断及流行病学研究奠定基础。

Avian infectious bronchitis（IB） is an acute and highly contagious disease which caused by infectious bronchitis virus（IBV）. In the study, according to IBV gene sequences published in GenBank, specific primers were designed to clone N gene by RT-PCR, and then this gene was inserted into pET- 30a（＋） vector resulting in a prokaryotic expression plasma pET- 30a- N. The results of SDSPAGE and Western-blot analysis showed that the recombinant protein was expressed successfully and had good reactivity with IBV positive serum. Using purified recombinant N protein as coating antigen, we established and optimized the indirect ELISA protocol, in which N protein was 2.5 μg · mL- 1of concentration, sample serum of 1：40 dilution. For clinical specimen, the IBV antibodies could be detected by this method efficiently, and got nearly the same results as those of IBV-mediated ELISA. It will provide a good tool for rapid diagnosis and epidemiological study of avian infectious bronchitis.