中国HTLV-Ⅰ env基因的克隆及Ⅰ+Ⅱ型嵌合基因的原核表达与抗原活性检测  

Cloning of a Chinese Human T-lymphotropic Virus Type Ⅰ env Gene and the Prokaryotic Expression and Immuno-activity Detection of the Type Ⅰ+Ⅱ Chimeric Gene

作　　者：张军[1] 王颖彬[1] 徐颖潇[1] 逄淑强[1] 张国忠 杨海杰[1] 夏宁邵[1] 

机构地区：[1]厦门大学细胞生物学与肿瘤细胞工程教育部重点实验室,厦门361005 [2]福建省莆田市中心血站,福建莆田351100

出　　处：《病毒学报》2001年第1期38-42,共5页Chinese Journal of Virology

基　　金：教育部重点科技项目! (99179)

摘　　要：为尽快研制出国产HTLV抗体诊断试剂 ,首先从福建HTLV流行区 1名HTLV感染者外周血细胞中克隆出HTLV Ⅰ的全长膜基因 (env) ,继而结合文献报道、PSA软件的亲疏水性分析和EPI软件的B细胞表位分析数据 ,选择了gp46中段开始延伸至 gp2 1N端 2 12个氨基酸 (aa185～aa396 )的基因 ,并在 3′端通过 (GlySer) 2 与人工合成的HTLV Ⅱ型的型特异性表位区基因嵌合 ,插入原核表达载体 pRSET ,在 E .coli中得到了高效表达 ,目的蛋白产量约占菌体总蛋白的 30 %。通过Triton X10 0洗涤 ,低浓度尿素逐步变性处理 ,8mol/L尿素溶解后纯度在 75 %左右 ,经电泳洗脱纯化 ,最终纯度可达 95 %左右 ,纯蛋白得率约 40 %。经Westernblotting检测 ,该蛋白对 4份HTLV Ⅰ型和 2份HTLV Ⅱ型血清均有较强反应 ,而对 4份阴性血清无反应 ,从而有可能用于研制HTLV抗体诊断试剂盒。For development of an antibody detection reagent for human T-lymphotropic virus (HTLV), a full-length envelope(env) gene of HTLV type Ⅰ (HTLV-I) was cloned from peripheral blood cells of a HTLV-Ⅰ infected individual in a HTLV endemic region of Fujian, and then a gene fragment of 212 amino acid length from the midpiece of gp46 extending to the N-terminal of gp21 was selected as well as a type specific epitope region of HTLV type Ⅱ env gene to construct a HTLV Ⅰ+Ⅱ chimeric gene. This chimeric fragment was inserted into prokaryotic expression vector pRSET and expressed in E. coli. The yield of the target recombinant protein was about 30%. The final purity is about 95% after electrophoresis elution purification. Detected by Western blotting, this recombinant protein showed strong reaction with four HTLV-Ⅰ positive sera as well as two HTLV-Ⅱ positive sera, and had no response to four HTLV negative sera. Thus, this recombinant antigen can be used to develop a diagnostic reagent for HTLV antibody detection.

关 键 词：人类T淋巴细胞白血病病毒 膜基因 原核表达 抗原 

分 类 号：R373[医药卫生—病原生物学] R733.7[医药卫生—基础医学]