BVDV E2蛋白的原核表达及间接ELISA方法的建立

Prokaryotic Expression of Bovine Viral Diarrhea Virus E2 Protein and Development of an Indirect ELISA Method

本试验克隆、表达了牛病毒性腹泻病毒(BVDV)的E2蛋白,以表达纯化后的E2蛋白作为包被抗原,通过优化反应条件建立了间接ELISA检测法。确定该方法的阴阳判定临界值为0.33,抗原包被浓度为8μg/mL,一抗的最佳稀释度为1∶100,10%PEG4000为最佳封闭液,酶标二抗最佳稀释度为1∶6000。用建立的ELISA检测方法和IDEXX ELISA抗体检测试剂盒检测180份血清临床样品,总符合率为95%。该方法的建立为进一步研制BVDV ELISA检测试剂盒奠定了基础。

In this experiment,the E2 gene of Bovine viral diarrhea virus(BVDV)was cloned for recombinant protein expression and the resulting E2 protein was purifi ed and used as a coating antigen for development of an indirect ELISA assay.The reaction conditions were optimized and determined to be 0.33 for positive threshold value,8μg/mL for antigen coating concentration,10%PEG4000 for blocking solution,1:100 for the primary antibody dilution,and 1:600 for the enzyme-labeled secondary antibody dilution.Total 180 clinical serum samples were tested using the ELISA assay developed here and the IDEXX ELISA antibody test kit.As a result,the coincidence rate of these two ELIA methods was 95%.These results indicated that the ELISA method described here would be further developed to be a universal BVDV test kit.