猴B病毒被膜糖蛋白gC的原核表达及诊断价值评价

目的:原核表达猴B病毒糖蛋白gC胞外区片段,评价其在猴B病毒血清学检测中的特异性和敏感性,为猴B病毒检测奠定基础。方法:利用PCR方法扩增gC胞外区基因片段,同时合成该片段的优化密码子序列,将其插入表达载体pET-28b(+)形成重组质粒,转化至大肠杆菌BL21(DE3)并进行诱导表达;纯化蛋白先以Western印迹进行验证,再以ELISA方法和标准阳性、阴性血清评价其诊断价值。结果:直接从病毒DNA模板上获得的基因片段未能表达,而优化后的基因片段表达水平较高,表达蛋白的相对分子质量约为48×103,为包涵体形式,占菌体总蛋白的30%左右;Western印迹显示,重组蛋白能与猴B病毒阳性血清和His单克隆抗体发生免疫反应;34份标准阳性血清和25份阴性血清的ELISA检测结果显示,重组蛋白的敏感性和特异性分别为94.1%(32/34)和100%(25/25)。结论:利用原核表达系统制备的gC胞外区重组蛋白,可作为猴B病毒血清学检测抗原,其特异性和敏感性都较好。

Production of Recombinant Monkey B Virus Glycoprotein C and Evaluation of its Diagnostic Potential

Objective： To express monkey B virus glycoprotein C extracellular domain gene in prokaryotic expression system and evaluate its specificity and sensitivity in serological detection,and to establish the foundation for the detection of monkey B virus.Methods： The extracellular domain gene of glycoprotein C was amplified by PCR and the optimizing codon sequence of extracelluar domain gene was obtained by chemical synthesis.Two gene fragments were cloned into the prokaryotic expressive vector pET-28b（＋） respectively,and the recombinant plasmids were transformed into E.coli BL21（DE3） and protein expression was induced by IPTG.After confirming by Western blot,the diagnostic value of purified protein was evaluated by ELISA with B virus standard positive serum and negative serum.Results： The gene fragment amplified from the DNA template didn＇t express,whereas optimizing fragments expressed the protein with a higher level.The recombinant protein had a Mr of 48 kD and accounted for 30% total proteins,and expressed in form of inclusion body.The Western blot showed that the protein could react specifically to the monkey B virus positive sera and anti-histidine monoclonal antibody.The indirect ELISA of 34 standard positive sera and 25 negative sera showed the sensitivity and specificity were 94.1%（32/34） and 100%（25/25）.Conclusion： The recombinant glycoprotein C from prokaryotic expression system has perfect specificity and sensitivity,which would suite for B virus serodiagnosis in monkey.