汉滩病毒M、S基因部分片段在大肠杆菌中的融合表达

Fused expression of hantaan virus M and S partial gene segments in E.coli

目的在大肠杆菌中融合表达含主要抗原位点的汉滩病毒囊膜糖蛋白G1与NP片段。方法将汉滩病毒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,并在大肠杆菌XL1Blue中,诱导GSTG1S0.7融合蛋白的表达。表达产物用ELISA和Westernblot进行鉴定。结果成功地构建了嵌合原核表达载体pGEX4T2G1S0.7。经IPTG诱导后,ELISA活性测定结果表明,该融合蛋白可与抗汉滩病毒NP及糖蛋白的mAb特异性结合。Westernblot结果显示,诱导出相对分子质量Mr大于1×105的G1S0.7与GST的融合蛋白。结论获得具有特异性结合活性的融合蛋白GSTG1S0.7,为汉滩病毒基因工程疫苗的研制奠定了基础。

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 G1S0.7 was constructed through connection of G1 gene segment encoded by M gene with 0.7kb segment of 5′terminal in S gene region from hantann 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 G1S0.7 was constructed successfully by restriction enzyme analysis. After IPTG induction, ELISA results showed that the fusion protein could bind specifically to the hantaan virus nucleoprotein specific mAb and glycoprotein specific mAb, and Western blot result exhibited a novel protein band with Mr more than 1×105 which was encoded by G1S0.7 and GST gene. Conclusion The fusion protein GST G1S0.7 with specific binding activity to nucleoprotein and glycoprotein G1 is obtained. It lays the foundation for the further research on genetic engeneering vaccine for hantann virus.