登革2型病毒NS5蛋白在大肠杆菌中的可溶性表达及纯化

目的 :构建登革 2型病毒 (DEN_2 )NS5表达体系 ,摸索适宜的诱导表达条件 ,克服大分子蛋白难以表达的瓶颈 ,获得DEN2_2NS5 (相对分子质量 10 4× 10 3)表达产物 ,为以后对其活性、抑制剂等方面的研究奠定基础。方法 :自DEN_2感染组织提取总RNA ,RT_PCR扩增NS5基因片段 (2 .7kb) ,插入质粒pQE30 ,转化XL1Blue大肠杆菌得表达菌株XL1Blue QENS5。同时优化诱导表达和目的蛋白的纯化条件。结果 :表达菌株XL1Blue QENS5增菌培养后更换新的培养基 ,在一定温度范围条件下诱导可以表达出最高占菌体总蛋白 2 2 .8%的NS5蛋白 ,在较低温度下诱导目的蛋白能以可溶性形式表达 ,经过纯化后的NS5蛋白纯度可达 90 %以上。结论 :在国内首次实现了DEN_2NS5蛋白的表达 ,获得的诱导表达条件可能对于其他大分子蛋白的表达有一定的借鉴意义。

Expression and purification of soluble DEN-2 NS5 protein in E.coli

Objective:To construct a prokaryotic expression system of the NS5 protein of dengue virus-2, and search an appropriate inducing condition to gain soluble expression product.Methods:The NS5 gene was cloned from DEN-2 infected tissues by RT-PCR and inserted into plasmid pQE30 to gain an expression plasmid pQENS5. Then it was transformed into E.coli XL1Blue. The expression and purification condition was optimized. Results:E.coli XL1Blue carrying pQENS5 could express DEN NS5 protein when induced under a range of temperature conditions. Furthermore, it could express NS5 in soluble form at a low temperature. After stepwise purification,a solution was obtained,in which the NS5 accounted for more than 90% of the total protein.Conclusion:It is the first time to successfully express DEN NS5 in China. It may favor the continued work about DEN NS5.The expression condition might be helpful for expressing other large molecule proteins.