【作者】 陈少渠；

【导师】 王泽霖；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 禽流感病毒(Avian Influenza Virus，AIV)的非结构蛋白(NS1)可作为一种区分野毒感染和灭活疫苗免疫鸡群的鉴别诊断标记，但不能区分AIV亚型，具有A型流感特异性。本文利用本河南农业大学禽病研究所实验室已构建的含有H9N2亚型AIV NS1蛋白基因的重组表达载体阳性菌，在37℃条件下，用终浓度1mmol／L的IPTG诱导表达5h，经15％的SDS-PAGE分析，表明NS1基因在大肠杆菌中得到高效表达。用HisTrap HP Kit进行复性和纯化，获得了纯度较高、生物活性较好的NS1蛋白。以此NS1蛋白作为抗原免疫Balb／c小鼠，应用杂交瘤技术，通过ELISA筛选，建立了2株抗H9N2亚型AIV NS1单克隆抗体(Monoclonal Antibody，McAb，简称单抗)杂交瘤细胞株689和6C2，其分泌单抗抗能力稳定。将2株杂交瘤细胞株分别注射小鼠收获的腹水单抗效价分别为100×212和100×210。制备的单抗有良好的热稳定性和较高的特异性，只与AIVNS1蛋白反应，而与H5N2亚型AIV、H9N2亚型AIV、NDV、IBV、IBDV、ILTV、EDS76V均不发生反应。NS1蛋白单抗的成功研制为利用该单抗开发一种快速、特异、敏感的鉴别野毒感染禽群和疫苗免疫禽群的方法及进一步研制诊断试纸条或组装成试剂盒奠定了基础，也为深入研究NS1蛋白的结构和功能奠定了良好的基础，对AI的早期诊断、适时监控和净化具有重要现实意义。 目前H9亚型AI仍是我国的流行主型，由于AIV抗原性和致病力的易变异性及不同血清型毒株间缺乏交叉保护性，加大了AI防制的难度。本试验将本实验室已建立的4株抗H9N2亚型AIV血凝素(HA)单克隆抗体与保存的1998-2005年间的25株H9N2亚型AIV毒株进行交叉ELISA试验，发现不同H9N2亚型AIV毒株血凝素(HA)抗原表位及毒力有明显差异，可将25株H9N2型AIV毒株分为4类，进一步证实了不同H9N2型AIV毒株HA抗原表位和毒力的变异性和多态性。其中2株毒株(A3和A18)比较特殊，与4株H9N2亚型AIV HA单抗均不发生反应，缺少4株单抗所识别的HA抗原表位，但其毒力很强。对25株H9N2亚型AIV毒株进行深入研究将对H9N2型AIV的防制在理论和实践上均具有一定的指导意义。更多还原

【Abstract】 Nonstructural genel (NS1) of AIV (H9N2) was cloned into the pET-28(a) vector to construct a recombinant expression plamid.After the recombinant expression plasmid was transform into E.coli ,the recombinant protein was induced by 1mmol/L IPTG at 37°C for 5 hours and analyzed by SDS-PAGE. The result showed that NS1 protein was highly expressed in a way of inclusion body. Its molecular weight was 28KD as expected. The expression NS1 protein was denatured and refolded and purified by HisTrap HP Kit. The result indicated that the high pure and active NS1 protein was obtained.Eight-week Balb/c mice were immunized by NS1 protein according to Kazuhiro’s produre.Two hybridoma were generated by fusion of SP2/0 myelomas and spleen cells from immunized mice and screened by ELISA and named 6B9、 6C2 respectively. The monoclonal antibodies(McAbs) were produced and proved to be IgG1、IgG2b by AGP.Mouse ascetic fluid was purified (protein concentration: 19mg/ml、14.9mg/ml) and proved to react with NS1 protein of AIV (H9N2)by HI and ELISA. The ELISA or rapid diagnosis strip which was based on the McAbs will provide a kind of sensitive, specific differentiating diagnostic approach and an effective method for early diagnosis, real-time detection and eradication of AIV.In order to study the variation of antigenicity in HA of AIV (H9N2), twenty-five AIV (H9N2) strains of China during 1998-2005 were detected by ELISA based on four McAbs specific to hemagglutinin (HA) of AIV (H9N2). The results of ELISA demonstrated that antigenicity and virulence had variated in the virus strains during 1998-2005, the antigenic site and virulence of A3 and A18 strain among them had become stronger. The results of our study would provide scientific reference for prevention against AIV and selection of vaccine strains.更多还原