鸡传染性支气管炎病毒核衣壳(N)蛋白的原核表达及其抗原性分析-预防兽医学专业论文.docxVIP

鸡传染性支气管炎病毒核衣壳（N）蛋白的原核 表达及其抗原性分析 摘 要 鸡传染性支气管炎(Avian Infectious Bronchitis ， IB)是由鸡传染性支气管炎病毒 (Avian Infectious Bronchitis Virus ， IBV)引起的一种急性、高度接触性传染病。由于 IBV 核衣壳（N）蛋白是其主要的结构蛋白，具有很强的免疫原性，是 IBV 诱导体液免疫、 细胞免疫应答的重要免疫原，而且比较保守，因此适宜用作检测 IBV 特异性抗体的抗原。 本研究参照 GenBank 中 IBV N 基因序列设计一对特异性引物，提取 IBV 的 RNA 进行 RT-PCR 扩增，并克隆至 pGEM-T Easy 载体中，转化 E.coli DH5a 感受态细胞，获 得重组质粒 pGEM-T-N，进行序列测定。将目的片段经 EcoR I 和 Xho I 双酶切，克隆入 经同样处理的大肠杆菌原核表达质粒 pET-28a（+），转化 BL21(DE3)感受态细胞并用 IPTG 进行诱导表达，通过镍离子亲和树脂对其进行纯化。SDS结果表明，IBV N 基因在 BL21(DE3)成功表达，相对分子质量约为 50 kDa，表达的目的蛋白主要以可溶形 式表达。经 Western blotting 分析，表明 IBV N 蛋白能被 IBV 阳性血清特异识别，具有 良好的反应原性。 关键词：鸡传染性支气管炎病毒；核衣壳蛋白；原核表达；反应原性 I II II Prokaryotic Expression of the Nucleocapsid Protein of Infectious Bronchitis Virus and the Analysis of Its Antigenicity Abstract Infectious bronchitis (Avian Infectious Bronchitis，IB) is an acute and highly contagious viral disease caused by avian infectious bronchitis virus (Avian Infectious Bronchitis Virus，IBV)． As the characters of the major structural protein of IBV， the highly immunogenic property，the important immunogen of IBV humoral and cellular immune response and the highly conservative property，IBV nucleocapsid (N) protein is considered as a suitable antigen in detecting IBVspecific antibody． A pair of primers were designed and synthesized based on the sequence of the nucleocapsid（N）protein gene of Avian Infectious Bronchitis virus(IBV)，and the N gene was amplified by RT-PCR． The fragment was cloned into pGEM-T vector and transformed into E. coli DH5a to get recombinant plasmid pGEM-T-N. After identification by digesting with EcoRⅠand XhoⅠenzyme， the cloned fragment was subcloned into prokaryotic expression vector pET-28a（+）． Recombined expression plasmid pET-28a-N was transformed into BL21(DE3) and then induced with IPTG． N protein was purified by affinity chromatography taking advantage of the affinity of Ni2+ for His tag protein． It was demonstrated by SDSand Western blotting that the fusion protein which was 50 kDa，was ex