丙型肝炎病毒F蛋白原核表达及初步应用.docVIP

丙型肝炎病毒F蛋白原核表达及初步应用 　 ; 作者：蒋春梅, 刁振宇, 崔国兴, 钱万红, 邓小昭, 许可, 丁伟良, 谈永飞, 张云 毕业论文 【关键词】; 丙型肝炎病毒;,F蛋白;,基因克隆;,蛋白表达;,抗体制备 毕业论文 　　Expression and preliminary study of HCV F protein gene 毕业论文 　　[Abstract]; AIM: To express HCV F protein gene fragment in E.coli and detect if there is its antibody in HCV patients. METHODS: RNA of the virus identified as genetype 1b was choosed as template, the F protein gene was amplified by RTPCR. This gene fragment was inserted into plasmid vector pGEM simple T. F gene was digested by EcoR I and BamH I from pGEM and cloned into plasmid vector pGEX4T2.The ligation mixture was transformed into E.coli. TG1 and F fragment expression was induced by IPTG. The expressed protein was purified from lysates with Glutathione Sepharose 4B. The purified protein was used to identify whether there was antiF antibody in the patients which HCV RNA was positive by ELISA. RESULTS: After IPTG induction, a positive band about 43 kD was detected. 82 of 120 HCV RNA positive?s sera had antiF antibody by ELISA. CONCLUSION: It is possible to efficiently express the HCV F protein in E.coli. The positive rate of antiF antibody in HCV RNA positive patients was 68%. 毕业论文 　　[Keywords]hepatitis C virus; F protein; gene cloning; protein expression; antibody preparation 　　 　　[摘 要]; 目的: 构建丙型肝炎病毒(HCV)F基因的原核表达载体, 并在大肠杆菌TG1中进行表达, 用以检测丙肝患者体内抗体, 并制备抗血清。方法: 提取患者血清中的HCV RNA, 并进行基因分型。取鉴定为HCV 1b型的病毒, 用RTPCR扩增HCV F蛋白基因的序列。将扩增产物插入pGEMT载体中, 测序正确后, 用EcoR I、 BamH I双酶切后, 再插入表达载体 pGEX4T2中, 转化大肠杆菌TG1, 在IPTG诱导下表达GSTF蛋白。用亲和层析法纯化GSTF蛋白免疫BALB/c小鼠制备抗血清。并用纯化的GSTF蛋白进行ELISA法检测丙肝患者血清抗F蛋白抗体。结果: 在细菌裂解液中检测到重组的融合蛋白, 经Glutathione Sepharose 4B 亲和层析柱纯化获得较高纯度的GSTF 融合蛋白。应用Western blot方法检测证实, 用纯化的GSTF蛋白免疫BALB/c小鼠得到了特异性的抗体。用纯化的GSTF蛋白进行ELISA检测120例丙肝患者血清抗F蛋白抗体, 82例呈阳性。结论: 通过上述方法制备的融合蛋白纯度较高, 免疫小鼠获得的抗体具有良好的特异性。用纯化的目的蛋白检测丙肝患者血清抗F蛋白的抗体的阳性率为68%, 与国外的报道接近, 说明在HCV感染的自然病程中有F蛋白的产生, 为研究HCV F蛋白及与HCV相关疾病的诊断和治疗提供了条件。 毕业论文 　　[关键词]丙型肝炎病毒; F蛋白; 基因克隆; 蛋白表达; 抗体制备 ; 　　丙型肝炎病毒（HCV）基因可编码十几种蛋白, 其中核心蛋白的功能最为复杂, 是病毒与宿主免疫相互作用的重要环节。近年发