汉滩病毒M,S基因不同拼接方式原核表达效果比较研究  

Comparison of prokaryotic expression results of different connection manners of Hantaan virus M and S gene segments

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

机构地区：[1]第四军医大学微生物学教研室,陕西西安710032

出　　处：《西北大学学报（自然科学版）》2003年第3期359-362,367,共5页Journal of Northwest University（Natural Science Edition）

基　　金：国家自然科学基金资助项目(30070686);国家教育部骨干教师资助计划项目

摘　　要：为了比较汉滩病毒囊膜糖蛋白G1与核蛋白(NP)主要抗原位点片段不同拼接方式的原核表达效果,将汉滩病毒76-118株M基因编码G1的片段与S基因编码区5′端约0.7kb的片段连接,克隆入pGEX-4T2,构建嵌合基因原核表达载体pGEX-4T2-G1S0.7,pGEX-4T2-S0.7G1,在大肠杆菌XL1-Blue中诱导表达GST-G1S0.7或GST-S0.7G1融合蛋白。经IPTG诱导后,ELISA活性测定结果表明,两种融合蛋白均可与抗汉坦病毒NP的mAb特异性结合,融合蛋白GST-G1S0.7还可与抗汉滩病毒糖蛋白的mAb特异性结合。Westernblot结果显示,诱导出G1S0.7或S0.7G1与GST的融合蛋白,其中G1S0.7嵌合基因的表达产物降解较少。研究证明:两种拼接方式的嵌合基因均可在大肠杆菌中表达出有生物学活性的融合蛋白,但表达效果不同,为汉滩病毒基因工程疫苗的研究奠定了基础。To compare the prokaryotic expression results of different connection manners of Hantaan virus glycoprotein G1 and nucleoprotein fragment including major antigen sites,G1 gene segment encoded by M gene and 0.7 kb segment of 5′terminal in S gene region from Hantann virus 76118 strain were connected and cloned into pGEX4T2 to construct the prokaryotic fusion expression vectors pGEX4T2G1S0.7 and pGEX4T2S0.7G1. The expression of the fusion protein GSTG1S0.7 or GST-S0.7G1 was induced in E.coli XL1Blue.After IPTG induction,ELISA results showed that both fusion proteins could bind specifically to the hantavirus nucleoprotein specific mAb, and the fusion protein GSTG1S0.7 could bind Hantaan virus glycoprotein specific mAb.Western blot results showed that the fusion protein GSTG1S0.7 or GSTS0.7G1 was expressed after induction,and the expression product of chimeric gene G1S0.7 degradated relatively less than the one of S0.7G1.The results suggest that two different chimeric genes both can express biologically active fusion proteins in E.coli,but with different expression efficiency.It has laid the foundation for developing Hantaan virus genetic engineering vaccine.

关 键 词：汉滩病毒 原核表达 融合蛋白 

分 类 号：Q786[生物学—分子生物学]