【作者】 祖立闯；

【导师】 薛飞；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 牛传染性鼻气管炎(Infectious Bovine Rhinotracheitis,IBR),是由牛传染性鼻气管炎病毒(Infectious bovine rhinotracheitis virus,IBRV)引起的牛的一种急性、热性、接触性传染病。IBR被世界动物卫生组织(OIE)列为B类传染病,也是我国进境动物和国际动物贸易中规定的重点检疫对象。因此,加强IBR诊断技术的研究工作对预防和控制本病具有重要的现实意义。目前,国内还没有商品化的检测IBR的诊断试剂,口岸动物IBR的检测处于严重依赖国外试剂盒的状况。为此,本研究用大肠杆菌表达了牛传染性鼻气管炎病毒gD基因,将重组蛋白纯化后作为包被抗原建立了检测牛传染性鼻气管炎病毒抗体的间接ELISA诊断方法。gD糖蛋白位于病毒囊膜及感染细胞的表面,是刺激机体产生中和抗体的主要结构蛋白,是IBR诊断试剂中特异性抗原的首选蛋白。本实验将IBRV gD基因的重组质粒pET30a-gD转化BL21(DE3)表达菌,经IPTG诱导得到了大量可溶性表达的重组蛋白,采用Ni柱亲和层析法在非变性的条件下成功地纯化了重组蛋白,纯化的重组蛋白浓度为0.128 mg/ml,纯度为96 %。Western blot和间接ELISA检测证明纯化后的重组蛋白可以被牛传染性鼻气管炎病毒抗血清所识别,具有良好的反应原性。将大肠杆菌表达的牛传染性鼻气管炎病毒重组gD蛋白,纯化后作为包被抗原建立了检测牛传染性鼻气管炎病毒抗体的间接ELISA诊断方法,将其命名为gD-ELISA。通过一系列试验,确定了底物液中TMB最佳使用浓度(0.4mg/ml);DMSO最佳使用浓度(15%);0.75 %过氧化氢尿素最佳使用浓度(25.6μl/ml);重组抗原最佳包被浓度(6.4μg/ml);最佳封闭液(含10%马血清的PBST)和最佳封闭时间(1h);血清样本最佳稀释度(1:80)和最佳作用时间(1.5 h);酶标二抗最佳工作浓度(1:5000)和最佳作用时间(1h);底物最佳显色时间(15min)和终止液最佳作用时间(5min);判定临界值(PR≥38.0为阳性,PR<30.8为阴性)。交叉反应试验表明,该重组抗原与其它常见5种牛病的阳性血清不发生交叉反应。阻断试验表明,IBRV细胞培养液能在很大程度上阻断重组抗原与阳性血清的反应,而对阴性血清没有明显的影响。重复性试验表明,批内重复的变异系数小于5%,批间重复的变异系数小于15%。与病毒中和试验相比,符合率、敏感性和特异性分别为84.1 %、85.0 %和83.4 %。与本实验室已建立的IBRV全病毒ELISA诊断方法相比,符合率、敏感性和特异性分别为91.9 %、94.2 %和90.2 %。应用IBRV gD-ELISA检测了国内11个省份的2012份血清样本,得出国内平均血清抗体阳性率为46.0 %(926/2012)。上述结果表明,IBRV gD-ELISA诊断方法具有良好的敏感性和特异性,显示出了良好的应用前景。本研究成功诱导表达了gD基因,获得了以可溶性表达的目的蛋白,为研究gD基因的功能奠定了良好的基础。利用纯化的重组蛋白成功建立了快速简便的IBRV gD-ELISA诊断方法,为IBR的检疫、诊断、抗体监测、预防及控制措施提供了坚强的技术支持,为开发IBR商品化诊断试剂盒奠定了基础。更多还原

【Abstract】 Infectious bovine rhinotracheitis(IBR) is an acute, feverish and contagious infectious disease of cattle, caused by infectious bovine rhinotracheitis virus (IBRV). IBR is B species contagious disease lined up by World Organisation for Animal Health(OIE), and also focal point quarantine object for country to enter border animals and international animal trades had rated. Accordingly, strengthen IBR diagnostic technique of research work has significant practical importance to prevent and control primary disease. At present domestic don’t have commercial detect diagnostic reagent for IBR, seaport detect IBR depend on abroad kit seriously. So this research induce and express IBRV gD gene, using the recombinant and purified gD protein as coating antigen, developed an indirect ELISA to detect antibody of IBRV.Glycoprotein D located at peplos and infect cell surface, it’s major structural protein to stimulate organism generate neutralizing antibody, it’s specific antigen and leader protein for IBR diagnostic reagent. The recombinant plasmid pET30a-gD was transformed into E. coli BL21 (DE3), the recombinant protein were expressed in partially soluble form after induction with IPTG, and was purified by immobilized Ni ion affinity chromatography under native conditions. The concentration of purified protein was 0.128 mg/ml with high purity (96%). Western blot and indirect enzyme-linked immunosorbent assay analysis suggested that the recombinant protein was recognized by IBRV anti-sera, having a good responsiveness.Using the recombinant and purified gD protein as antigen expressed in Escherichia coli BL21, an indirect ELISA was successfully developed to detect antibody to IBRV, the indirect ELISA was named gD-ELISA, and through a series of test determinate the optimal working parameters, including TMB at 0.4 mg/ml, DMSO at 15 %, 0.75 % urea hydrogen peroxide at 25.6μl/ml, coating antigen at 6.4μg/ml, testing sera dilution at 1:80, serum reaction time at 1.5 h, blocking solution at 10 % horse serum, blocking solution reaction time at 1 h, anti-bovine IgG peroxidase conjugate dilution at 1:5000, anti-bovine IgG peroxidase conjugate reaction time at 1h, substrate reaction time at 15 min, stop buffer reaction time at 5 min, the standard of determining as positive sample PR≥38.0 and negative sample PR<30.8. The recombinant gD protein antigen showed no cross-reaction with the positive sera of the five kinds of bovine diseases. IBRV strain can block recombinant antigen react with positive serum in a deep degree, however, it has no clear effect with negative serum. Coefficient of variability percent(C.V %) of intro-batch and inter-batch duplicativity test was less 5 % and 15 % respectively. Comparing with the Virus Neutralization testing(VN), the agreement rate was 84.1 %, the sensitivity was 85.0 %, the specificity was 83.4 %. The agreement rate, sensitivity and specificity of IBRV gD-ELISA relative to IBRV whole virus ELISA was 91.9%, 94.2 % and 90.2 %, respectively. In the field test, IBRV gD-ELISA was used to detect 2012 serum samples collected from eleven provinces and autonomous regions in China, the result of gD-ELISA demonstrated that the average seroprevalence was 46.0 %(926/2012). Therefore, this IBRV gD-ELISA based on recombinant gD protein antigen has good specificity and sensitivity, suggesting an application prospection.In conclusion, IBRV gD gene was successfully expressed in soluble forms in E.coli and purified, they lay satisfactory foundation for further studies of the protein structure and function of glycoprotein D. The simple and fast diagnosis method using recombinant gD protein expressed as diagnosis antigen can be a technique support for further quarantine, diagnosis, antibody monitoring, prevention and control of IBR in China, and also settle foundation for developing the commercial kit of diagnosis.更多还原