大肠杆菌中融合表达汉滩病毒S,M基因片段的研究  

Study on Prokaryotic Expression of Hantaan Virus S and M Chimeric Gene Segment

作　　者：罗雯[1] 徐志凯[2] 张芳琳[2] 阎岩[2] 吴兴安[2] 刘勇[2] 白文涛[2] 王海涛[2] 

机构地区：[1]西安联合大学化学与生命科学系,陕西西安710065 [2]第四军医大学微生物学教研室,陕西西安710032

出　　处：《西安联合大学学报》2002年第2期11-14,共4页

基　　金：国家自然科学基金项目 ( 3 0 0 70 686)

摘　　要：为在大肠杆菌中融合表达汉滩病毒囊膜糖蛋白G1与NP含主要抗原位点的片段 .将汉滩病毒 76 118株S基因编码区 5′端约 0 .7kb的片段与M基因编码G1的片段连接 ,克隆入 pGEX 4T2 ,构建嵌合基因原核表达载体 pGEX 4T2 S 0 .7G1,在大肠杆菌XL1 Blue中诱导GST S0 .7G1融合蛋白的表达 .表达产物用ELISA和Westernblot进行鉴定 .限制性内切酶酶切鉴定证明 ,成功构建了嵌合原核表达载体 pGEX 4T2 S0 .7G1.经IPTG诱导后 ,ELISA活性测定结果表明 ,该融合蛋白可与抗汉坦病毒NPmAb特异性结合 .Westernblot结果显示 ,诱导出相对分子质量 (Mr)大于 1× 10 5的S0 .7G1与GST的融合蛋白 ,并有一系列大小不等的可与抗汉坦病毒NPmAb特异性结合的蛋白带 .获得具有特异性结合活性的融合蛋白GST S0 .7G1。Aim To obtain fused expression of hantaan virus glycoprotein G1 and nucleoprotein fragment including major antigen sites in E. coli .Methods The prokaryotic fusion expression vector pGEX 4T2 S 0.7G1 was constructed through connection of 0.7 kb segment of 5′terminal in S gene region with G1 gene segment encoded by M gene from hantaan virus 76 118 strain and cloned into pGEX 4T2. The expression of the fusion protein was induced in E.coli XL1 Blue, and then the expression product was analyzed by ELISA and Western blot. Results It was proved that the prokaryotic fusion expression vector pGEX 4T2 S 0.7G1 was constructed successfully by restriction enzyme analysis. After IPTG induction, ELISA results showed that the fusion protein could bind specifically to the hantavirus nucleoprotein specific mAb, and Western blot result exhibited a novel protein band with M r more than 1×10 5 which was encoded by S 0.7G1 and GST gene. Besides, a series of protein bands with different molecular weights which could bind to the hantavirus nucleoprotein specific mAb was detected. Conclusion The fusion protein GST S 0.7G1 with specific binding activity to nucleoprotein is obtained. It lays the foundation for the further research on genetic engeneering vaccine for hantaan virus.

关 键 词：大肠杆菌 融合表达 汉滩病毒 基因片段 

分 类 号：R37[医药卫生—病原生物学] Q786[医药卫生—基础医学]