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为了探究7型庚型肝炎病毒E2基因编码蛋白作为ELISA试剂盒研发所需检测抗原的可能性,建立更为可靠的GBV-C检测方法,本研究应用在线软件对GBV-C E2基因序列的编码区进行生物信息学分析,预测了E2基因编码蛋白的抗原表位、空间结构及线性B细胞表位等;通过逆转录PCR从7型GBV-C病毒中克隆出E2基因片段,将其克隆到pET-32a载体上,重组载体pET-32a-E2转化大肠杆菌BL21后诱导表达,用12%SDS-PAGE检测,结果重组蛋白主要以包涵体形式存在,其分子量大小约为55kD,利用His标签抗体对重组蛋白进行Western-blotting验证。结果表明GBV-C E2蛋白有多个抗原表位点,克隆的E2基因序列长度为945bp,重组蛋白以包涵体形式表达,其分子量大小与预期一致,此研究为GBV-C检测试剂盒的研制工作奠定了基础。
In order to explore the possibility of detecting the antigen required for the development of the hepatitis C virus E2 gene as an ELISA kit and to establish a more reliable GBV-C detection method, this study used on-line software to encode the GBV-C E2 gene sequence The bioinformatics analysis was carried out to predict the antigenic epitopes, spatial structure and linear B cell epitopes of E2 protein. The E2 gene fragment was cloned from type 7 GBV-C virus by reverse transcription PCR and cloned into The recombinant protein pET-32a-E2 was transformed into E.coli BL21 and induced by 12% SDS-PAGE. The results showed that the recombinant protein was mainly in the form of inclusion bodies with a molecular weight of about 55 kD. Using the His-tagged antibody The recombinant protein was verified by Western-blotting. The results showed that the GBV-C E2 protein has multiple antigenic epitopes, the length of the cloned E2 gene is 945bp, and the recombinant protein is expressed as a inclusion body. The molecular weight of the GBV-C E2 protein is consistent with the expectation. This study was developed for the GBV-C detection kit Work laid the foundation.