| 山羊痘病毒p32基因原核表达及间接ELISA抗体检测方法的建立 |
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| 引用本文： | 王芳,雷震,于力.山羊痘病毒p32基因原核表达及间接ELISA抗体检测方法的建立[J].中国预防兽医学报,2009,31(12). |
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| 作者姓名： | 王芳 雷震 于力 |
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| 作者单位： | 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/大动物传染病研究室,黑龙江,哈尔滨,150001 |
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| 摘 要： | 为了建立山羊痘病毒(GPV)抗体快速检测方法,本研究通过人工合成密码子优化的GPV p32基因,并在大肠杆菌中进行截短表达(p32-opti).表达的重组p32-opti蛋白主要以包涵体形式存在,Western blot分析表明,p32-opti与GPV标准阳性血清具有良好的抗原性.以纯化的重组p32-opti蛋白作为ELISA包被抗原,建立间接ELISA抗体检测方法.该方法检测小反刍兽疫、蓝舌病和口蹄疫阳性血清均无交叉反应;与中和试验(VNT)比较,两者的符合率为95.7%;ELISA批内和批间重复性试验显示,OD值的变异系数小于10%.上述结果表明.该ELISA检测方法具有良好的特异性、敏感性和重复性.对来自黑龙江、内蒙古和新疆自治区的390份山羊和绵羊血清进行检测,抗体阳性率为80.2%,表明这些地区的山羊和绵羊群普遍存在羊痘病毒抗体.本研究建立的间接ELISA方法可用于GPV感染的流行病学调查以及疫苗接种动物的抗体水平监测.
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| 关 键 词： | p32基因 大肠杆菌表达 |
Development of an indirect ELISA for detection of antibodies against Goat pox virus using recombinant p32 protein as antigen |
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| WANG Fang,LEI Zhen,YU Li.Development of an indirect ELISA for detection of antibodies against Goat pox virus using recombinant p32 protein as antigen[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(12). |
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| Authors: | WANG Fang LEI Zhen YU Li |
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| Abstract: | In order to develop an indirect ELISA for detection of antibodies against Goat pox virus (GPV), GPV p32 gene was codon-optimized and synthesized on basis of E.coli. codon usage, and the truncated p32 protein (p32-opti) with deletion of trans-membrane domain was expressed in E.coli. BL21 (DE3) cells. An indirect ELISA for detection of antibodies against Goat pox virus was developed using p32-opti protein as coat antigen. Concordance of the indirect ELISA relative to VN was 95.7 %. The p32-opti antigen showed no cross-reaction with the positive sera of other goat diseases. The coefficient of variability percent(C. V) of intro-batch and inter-batch duplicativity was less than 10 %. These results demonstrated that the indirect ELISA method was specific, sensitive and reproducible. A total of 390 goat and sheep serum samples collected from Heilongjiang province, Inner Mongolian autonomous region and Xinjiang autonomous region were detected by the indirect ELISA and 80.2 % samples were detected positive, indicating that GPV antibody was prevalent in goats and sheep of these regions. The indirect ELISA would be useful tool for GPV epidemiological surveys as well as detection of GPV antibodies in the vaccinated animals. |
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| Keywords: | GPV ELISA GPV p32 gene E coli expression ELISA |
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