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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。
OBJECTIVE: To construct NS5 expression system of Dengue virus type 2 (DEN2) and explore suitable conditions of induced expression to overcome the bottleneck of difficult expression of macromolecular protein and to obtain the expression product of DEN2_2NS5 (relative molecular mass of 104 × 103) Activity, inhibitors and other aspects of the study laid the foundation. Methods: The total RNA was extracted from the infected tissue of DEN_2. The NS5 gene fragment (2.7 kb) was amplified by RT-PCR and inserted into plasmid pQE30. The strain XL1 Blue was transformed into E. coli XL1. While optimizing the conditions for the induction of expression and the purification of the protein of interest. Results: After the strain XL1Blue QENS5 was enriched, a new medium was replaced. Under certain temperature range, NS5 protein could be expressed up to 22.8% of the total bacterial proteins, and the target protein could be induced at lower temperature Soluble form of expression, after purification of the purity of NS5 protein up to 90%. CONCLUSION: The expression of DEN_2NS5 protein was first realized in China. The induced expression conditions may have certain reference value for the expression of other macromolecular proteins.