人T细胞白血病病霉Ⅰ型env基因的克隆与表达  

CLONING AND EXPRESSION OF THE ENVELOPE GENEOF HUMAN T CELL LEUKEMIA TYPE

作　　者：段震峰[1] 滕智平[1] 曾毅[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《病毒学报》1995年第3期228-233,共6页Chinese Journal of Virology

摘　　要：本文通过聚合酶链式反应（ＰＣＲ），从整合有人Ｔ细胞白血病病毒Ⅰ型（ＨＴＬＶ－Ｉ）的ＭＴ－２细胞株中，扩增出ＨＴＬＶ－Ｉ外膜蛋白（ｅｎｖ）的全基因（１．４５ｋｂ），并成功地克隆入ｐＵＣ１９载体，构建成ｅｎｖ基因克隆ｅｎｖ／ｐＵＣ１９．利用原核高效表达载体ｐＧＥＸ－２Ｔ，在大肠杆菌中有效地表达了与谷胱甘肽Ｓ－转移酶（ＧＳＴ）融合的ｅｎｖ羧基末端抗原的重组蛋白，融合基因的转录由ｔａｃ启动子调控。ＳＤＳ－ＰＡＧＥ结果表明，有一约５２ｋＤ的目的蛋白带，表达量占菌体总蛋白的１０％；ＷｅｓｔｅｒＮｂｌｏｔｔ及ＥＬＩＳＡ结果显示，表达产物能与ＨＴＬＶ－Ｉ多抗血清结合。本研究为研制ＨＴＬＶ－Ｉ的诊断试剂及进一步了解ｅｎｖ的免疫学和生物学性质打下了基础。sing the polymerase chain(PCR)techniques ,we have amplified the envelope(env)gene(1.46kb)of human T cell leukenia virus type I (HTLV-I)from the MT-2 cells,in which the HTLV-I provirus is integrated. The env fragment was then inserted into pUC19vector,producing the construction env/pUC19 .The gene fragment containing the COOH-terminalof env gene was introduced into the lacterial expression vector,pGEX-2T, by means of fusion to the glutathione S-transferase(GST)gene of the vector. Transcription of this hybrid gene was controlled by the well -regulated tac promoter.SDS-PAGEanalysis indicated that the 52kD chimeric protein was stably induced when the Escherichia coli strain DH5α,that carried the GST-env gene fusion plasmid,was induced by IPTG;this represented 10%of total cellular protein.The resrlt of ELISA and Western blot showed that the fusion protein could react with serum from HTLV-I patients. This work may be important in strdying the structure and function of env gene of HTLV-I,and in developing ananti-HTLV-Iassat .

关 键 词：白血病 人T细胞白血病 病毒 ENV基因 基因克隆 

分 类 号：R733.703[医药卫生—肿瘤]