14型蓝舌病病毒VP7蛋白原核表达及间接ELISA抗体检测方法的建立

Prokaryotic expression of VP7 protein of serotype 14 bluetongue virus and establishment of an indirect ELISA antibody detection method

为建立蓝舌病(bluetongue,BT)的血清学诊断方法,本研究通过得到14型蓝舌病病毒(bluetongue virus,BTV)的s7基因,并在大肠杆菌表达系统中进行表达,原核表达得到VP7蛋白,并对表达的包涵体VP7蛋白进行纯化,Western blot分析表明,纯化的VP7蛋白具有良好的抗原性。以纯化的VP7蛋白作为包被抗原,建立间接ELISA检测方法。该方法与羊痘阳性血清,羊口蹄疫阳性血清和羊的小反刍兽疫阳性血清均无交叉反应,与中和试验比较,两者符合率为90%,ELISA批内和批间重复性试验显示,D值的变异系数小于10%。说明本方法具有良好的特异性和重复性。本研究在国内首次建立了14型BTV抗体间接ELISA方法,可用于BTV感染的流行病学调查以及疫苗接种动物的抗体水平监测,为以后相关BTV ELISA检测试剂盒研发提供技术平台。

A pair of PCR primers were designed for amplication of BTV14 s7 gene.Then,the VP7 protein of BTV14 was expressed in E.coli and the recombinant VP7 protein in the inclusion body pellets was purified.Western blot results showed that His-tag fusion protein of VP7 shows a solid antigenicity.The purified VP7 protein was used as a coating antigen to establish the indirect ELISA method,the test results showed that the VP7 antigen had no cross reaction to antibodies of CPV,FMDV and PPRV.The coincidence rate of the results of VN was 90%.The CV of intro-batch duplication tests and inter-batch duplication tests were both less than 10%.It means that the ELISA has good specificity and sensitivity.Therefore,this method can be used for clinical detection of antibody and epidemiological investigation of BTV,which makes a good foundation for further development of a rapid diagnostic kit of BTV.