东部马脑炎病毒E2基因的克隆与表达

目的 :克隆表达东部马脑炎病毒 (easternequineencephalomyelitisvirus ,EEEV)E2基因 ,研究重组蛋白的抗原性 ,为研制东部马脑炎病毒基因工程诊断试剂奠定基础。方法 :由病毒感染的鼠脑制备总RNA ,利用RT_PCR技术扩增EEEVE2基因全长序列 ,并将其克隆至pGEM_Teasy载体测序 ,再将E2基因克隆至谷胱甘肽转移酶 (GST)融合表达载体pGEX_5x_2中表达。采用免疫印迹试验 (Westernblotting)和ELISA法分析表达的重组蛋白的抗原性。结果 :经过RT_PCR扩增和测序 ,证明得到的E2基因片段的序列是正确的 ,并且在原核载体中得到表达 ,目的蛋白占总菌体蛋白的 2 4 .5 % ,主要以包涵体形式存在。Western印迹分析表明GST_E2蛋白有良好的抗原性 ;包涵体经Tri tonX_10 0 ,1mol L和 2mol L尿素洗涤 ,初步纯化后 ,用表达的E2融合蛋白作为包被抗原初步建立了间接ELISA法 ,结果表明与兔抗EEEV血清起特异反应 ,但与兔抗WEEV血清无交叉反应。结论 :东方马脑炎病毒的E2基因在大肠杆菌中得到稳定表达 ,表达的GST_E2蛋白具有良好的抗原性和特异性 ,为从分子水平上对EEEVE2蛋白的功能性研究和发展EEEV基因工程诊断试剂奠定了基础

Cloning and expression of E2 gene of eastern equine encephalomyelitis virus

Objective:To clone and express the E2 gene of eastern equine encephalomyelitis virus(EEEV) in order to study the antigenicity of recombinant protein and to lay a foundation for development of genetic engineering diagnostic reagents.Methods:The E2 gene of EEEV was amplified from total RNA of infected mouse brain by RT_PCR and then cloned into pGEM_T easy vector. After DNA sequencing, the E2 gene fragment was cloned into GST fusion protein expression vector pGEX_5x_2 and expressed in E.coli . The antigenicity of recombinant protein was identified by Western_blotting and ELISA. Results:After insertion of E2 gene into the prokaryotic fusion protein expression vector pGEX_5x_2, the fusion protein GST_E2 was expressed in E.coli DH5a and its amount was about 24.5% of total bacterial protein. GST_E2 protein was obtained from inclusion body separation and washed with Triton X_100, 1*!mol/L and 2*!mol/L urea. GST_E2 was demonstrated to have good antigenicity by Western_blotting and indirect ELISA.Conclusions: GST_E2 was expressed stably in E.coli and this recombinant protein has strong antigenicity which can be used to develop the genetic engineering diagnostic reagent.