丙型肝炎病毒F蛋白的原核表达及初步应用  

Expression and preliminary study of HCV F protein gene

作　　者：蒋春梅[1] 刁振宇[2] 崔国兴 钱万红[2] 邓小昭[1,4] 许可[1] 丁伟良 谈永飞 张云[1,4] 

机构地区：[1]南京医科大学公共卫生学院流行病与卫生统计系 [2]南京军区军事医学研究所 [3]宜兴市第一人民医院 [4]南京军区军事医学研究所,江苏南京210002

出　　处：《细胞与分子免疫学杂志》2007年第2期134-137,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：江苏省高校自然科学研究指导性计划资助项目基金资助(03KJD330144);江苏省自然科学基金资助(BK2004011)

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HCVRNA,并进行基因分型。取鉴定为HCV1b型的病毒,用RT-PCR扩增HCVF蛋白基因的序列。将扩增产物插入pGEM-T载体中,测序正确后,用EcoRI、BamHI双酶切后,再插入表达载体pGEX-4T-2中,转化大肠杆菌TG1,在IPTG诱导下表达GST-F蛋白。用亲和层析法纯化GST-F蛋白免疫BALB/c小鼠制备抗血清。并用纯化的GST-F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GlutathioneSepharose4B亲和层析柱纯化获得较高纯度的GST-F融合蛋白。应用Westernblot方法检测证实,用纯化的GST-F蛋白免疫BALB/c小鼠得到了特异性的抗体。用纯化的GST-F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HCVF蛋白及与HCV相关疾病的诊断和治疗提供了条件。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 RT-PCR. 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 pGEX-4T-2. The ligation mixture was transformed into E. coli. TGI 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 anti-F 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 anti-F antibody by ELISA. CONCLUSION： It is possible to efficiently express the HCV F protein in E, coil The positive rate of anti-F antibody in HCV RNA positive patients was 68%.

关 键 词：丙型肝炎病毒 F蛋白 基因克隆 蛋白表达 抗体制备 

分 类 号：R373.2[医药卫生—病原生物学]