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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
OBJECTIVE: To clone and express the E2 gene of eastern equine encephalitis virus (EEEV) and study the antigenicity of the recombinant protein, so as to lay the foundation for the development of genetic engineering diagnostic reagents for equine equine encephalitis in the east. Methods: Total RNA was prepared from murine brain infected with virus. The full length of EEEVE2 gene was amplified by RT - PCR and sequenced. The E2 gene was cloned into pGEM_Teasy vector and cloned into GST fusion expression vector Expression in pGEX_5x_2. The antigenicity of the expressed recombinant protein was analyzed by Western blotting and ELISA. Results: The RT-PCR amplification and sequencing showed that the sequence of E2 gene was correct and expressed in prokaryotic vector. The target protein accounted for 24.5% of the total bacterial proteins, mainly in the form of inclusion bodies. Western blot analysis showed that the GST_E2 protein has good antigenicity. Inclusion bodies were washed with Tri ton X 100, 1 mol L and 2 mol L urea. After preliminary purification, an indirect ELISA was established by using the expressed E2 fusion protein as the coating antigen. The results showed that Specific reaction with rabbit anti-EEEV serum, but no cross-reaction with rabbit anti-WEEV serum. CONCLUSION: The E2 gene of oriental equine encephalitis virus is stably expressed in E. coli, and the expressed GST_E2 protein has good antigenicity and specificity. In order to study the function of EEEVE2 protein at molecular level and to develop the EEEV genetic engineering diagnostic reagent Foundation