马乙型脑炎病毒E蛋白的原核表达及间接ELISA的初步应用

Expression of Japanese encephalitis virus envelope protein in prokaryocyte and its application on indirect ELISA diagnosis

目的原核表达马乙型脑炎病毒(Japanese encephalitis virus,JEV)E蛋白,建立JEV间接ELISA诊断方法。方法根据GenBank(AF315119.1)公布的JEVSA14-14-2株E蛋白基因序列设计一对引物,提取病毒RNA经反转录和RT-PCR扩增获得E蛋白编码基因片段,将该片段插入表达载体pET30a(+),转化BL21(DE3)后经IPTG诱导蛋白表达,Western blot检测活性,以表达的E蛋白为包被抗原建立间接ELISA诊断方法。结果获得约1500bp目的片段,与GenBank上的序列同源性100%,其表达产物相对分子质量约为60000,Western blot结果显示该蛋白可与JEV阳性血清结合,具有免疫反应性。建立了间接ELISA检测方法,对不同省份的340份马血清进行检测,其中154份为阳性。结论原核表达JEV的E蛋白具有良好的免疫活性,建立的间接ELISA诊断方法特异性和灵敏性较好,可用于JEV的现地检测。

This project aims to express an E protein of Japanese encephalitis virus（JEV） in prokaryocyte and establish a specific diagnosis assay of JEV.According to the sequence of E protein coding gene in GenBank（AF315119.1）,a pair of specific primes was designed.After reverse transcription and RT-PCR amplification,a 1 500 bp fragment was obtained from the RNA of the virus and cloned into prokaryotic expression vector PET-30a（＋）.The reconstruction plasmid was transformed into E.coli BL21（DE3） and induced by IPTG for expression.A Mr 60 000 protein was expressed and Western blot analysis indicated that the protein had immunological activity after conjugation with positive sera of JEV.In conclusion,a sensitive and specific indirect ELISA with E protein as coating antigen is established and successfully used to test 154 positive sera from 340 horse sera in different provinces.