B亚型禽偏肺病毒重组G蛋白间接ELISA方法的建立及应用  

Development of an indirect ELISA diagnostic method for detecting avian metapneumovirus using recombinant attachment protein and its preliminary application

作　　者：陈琳[1] 刁有祥[1] 邹金峰[1] 唐熠[1] 程彦丽[1] 欧全宾[1] 薛聪[1] 崔京腾[1] 鞠小军[1] 孙晓艳[1] 裴萍萍[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2013年第3期330-335,共6页Chinese Journal of Veterinary Science

基　　金：山东省科技发展计划资助项目(2010GNC10912)

摘　　要：以纯化的B亚型禽偏肺病毒(aMPV)疫苗株(VIR-115B)重组G蛋白作为包被抗原,对各项条件进行优化,确定判定标准,建立检测B亚型aMPV抗体的间接ELISA方法。将构建好的重组质粒转入Rosetta(DE3)感受态细胞进行诱导表达,获得重组G蛋白。利用亲和层析法对表达产物进行纯化,并用Western-blotting对表达产物进行鉴定。以纯化后的重组G蛋白作为诊断抗原,对ELISA反应条件进行优化,初步建立检测B亚型aMPV抗体的间接ELISA诊断方法。结果显示,成功表达并纯化了B亚型aMPV疫苗株(VIR-115B)重组G蛋白,Western-blotting检测结果证明表达产物具有良好的反应原性。以重组G蛋白作为诊断抗原,建立了间接ELISA诊断方法。交叉试验结果表明重组G蛋白与新城疫、禽流感(H9N2)、传染性支气管炎和传染性法氏囊阳性血清均不发生交叉反应。该方法与法国IDEXX公司生产的aMPV抗体检测试剂盒符合率为96.0%。采用本试验建立的间接ELISA方法对山东省7个地区鸡场的1 139份鸡血清进行了检测,结果表明,山东省部分地区aMPV的抗体阳性率为37.05%。本试验建立的间接ELISA方法具有较好的特异性、敏感性和重复性,利用该方法初步证实山东省部分地区存在aMPV感染现象。这一研究为aMPV诊断试剂盒的研制奠定了基础,并为该病的诊断与流行病学调查提供有效的技术手段。The ELISA plate was coated with the strain （VIR-115 B） recombinant attachment（G） purified avian metapneumovirus （aMPV） vaccine antigen and the optimal reaction conditions of in- direct ELISA were determined by experiments. The indirect ELISA method for detecting aMPV antibody was established by determining the cut off value. The recombinant E. coli strain Rosetta containing the recombinant plasmid pET-32a-G was induced by IPTG. The recombinant G protein was expressed at high level, and then it was purified by affinity chromatography method. The re- combinant G protein was identified by Western-blotting. Taking the purified recombinant G pro- tein as diagnostic antigen, the indirect ELISA method for detecting aMPV antibody was estab- lished. The recombinant G protein was successfully expressed and purified,it was proved that the recombinant protein has good reactogenicity by Western-blotting. Taking the purified recombinant G protein as diagnostic antigen,the indirect ELISA method for detecting aMPV antibody was es- tablished. The results showed that there was no cross reaction between the recombinant G protein and the positive serum of NDV, H9N2, IBV and IBDV. Compared with the aMPV antibody detec- tion kit of IDEXX in French,the results indicated that the specificity, sensitivity and coincidenceof the developed indirect ELISA was 98.3% ,92.5% and 96.0% ,respectively. A total of 1 139 se- ra of chicken, which obtained from seven cities in Shandong province,were tested by the developed indirect ELISA method and the positive rate was 37.05%. The developed indirect ELISA has good specificity, sensitivity and reproducibility, it proved that aMPV existed in some areas in Shandong province.

关 键 词：禽偏肺病毒 B亚型 重组G蛋白 间接ELISA 

分 类 号：S852.657[农业科学—基础兽医学]