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当前位置: 首页 > 文章 > 基于山羊痘病毒P32蛋白ELISA检测方法的建立 中国兽医学报 2014,34 (12) 1906-1911
Position: Home > Articles > The development of an indirect ELISA method based on the goat pox virus P32 gene Chinese Journal of Veterinary Science 2014,34 (12) 1906-1911

基于山羊痘病毒P32蛋白ELISA检测方法的建立

作  者：
吴锦艳;田宏;陈妍;尚佑军;宋江伟;刘湘涛
单  位：
中国农业科学院兰州兽医研究所
关键词：
山羊痘病毒;p32基因;表达;间接ELISA方法
摘  要：
P32蛋白是羊痘病毒株中特异性很强的免疫原性结构蛋白,本研究利用原核表达系统对山羊痘病毒P32基因进行表达,并应用该蛋白建立了检测山羊痘病毒血清抗体检测方法。将P32基因克隆至pGEX-6P-1表达载体,经抗性筛选,测序鉴定获得重组阳性克隆,成功构建了羊痘病毒重组pGEX-P32表达载体。经IPTG诱导表达,可稳定、高效地表达P32融合蛋白。融合蛋白纯化后SDS-PAGE结果表明,以终浓度为1mmol/L的IPTG进行诱导,8h后表达量最高,表达蛋白为融合蛋白,相对分子质量约56 000,表达产量约占菌体总蛋白的28%。Western-blotting检测表明,表达的融合蛋白能与羊痘阳性血清发生特异性反应,说明表达获得的产物具有良好的反应原性。用表达的融合蛋白建立了检测羊痘血清抗体的ELISA方法,经方阵滴定,确定抗原最佳包被质量浓度为每孔0.4mg/L,最佳血清稀释度为1∶20。符合率试验表明ELISA方法和琼脂扩散试验的阳性符合率为83.44%,并且所建立的方法具有很好的特异性、重复性和敏感性,这为应用该融合蛋白制备山羊痘病毒免疫血清学诊断试剂和疫苗研究奠定了基础。
译  名：
The development of an indirect ELISA method based on the goat pox virus P32 gene
作  者：
WU Jin-yan;TIAN Hong;CHEN Yan;SHANG You-jun;SONG Jiang-wei;LIU Xiang-tao;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences;
关键词：
goat pox virus;;P32gene;;expression;;indirect ELISA method
摘  要：
This experiment was conducted to express the structural protein P32 gene of the goat pox virus by aprokaryotic expression system,and an indirect ELISA method was developed to test goat poxvirus(GPV)serum antibody.The recombinant plasmid pMD-T-P32 was cloned into pGEX-6P-1by molecular biology.The recombinant fusion protein about 56 kDa pGEX-P32 was expressed well in E.coli induced by 1mmol/L IPTG,and expressed production accounts for about 28%of the bacteria protein.Western-blotting test showed that the protein could be recognized by antiserum against goat pox virus.The recombinant fusion protein was detected by ELISA with standard negative and positive serum,The result indicates that the recombinant fusion protein has good immunological characteristics.The indirect ELISA method indicated that the optimal quality concentration of coated recombinant antigen was 0.4mg/L and the serum dilution was 1:20.The rate of agreement rate was 83.44%(121/145)with ELISA and agar diffusion test(ADP),meanwhile that established ELISA diagnosis method has good repeatability,sensitivity and specificity.These results support the potential use of recombinant p32 as antigens for indirect ELISA to detect GPV antibody in goats and laid a foundation in vaccine research.

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