流行性乙型脑炎病毒NS1基因克隆及其蛋白表达纯化

Cloning,Expression and Purification of NS1 Gene of Epidemic Encephalitis Type B

为了表达流行性乙型脑炎(epidemic encephalitis type B)非结构蛋白NS1,本研究通过PCR扩增了日本乙脑病毒(Japanese encephalitis virus,JEV)P3株NS1基因,并分别构建了其融合表达His和GST标签的原核表达载体,经PCR、酶切和测序鉴定正确后转化大肠杆菌BL21感受态细胞,用IPTG诱导表达了NS1融合蛋白,通过SDS-PAGE电泳和Western blotting进行鉴定,并用尿素变性复性和镍亲合层析法纯化目的蛋白。结果显示,试验成功构建了NS1的两个原核表达载体pET-42b-NS1和pGEX-KG-NS1,P3株NS1基因全长1 056bp,以包涵体的形式表达约40ku的蛋白,尿素变性复性效果较好,镍亲合层析纯化得到纯度和浓度均较高的NS1蛋白。JEV NS1基因可以通过原核表达系统高效表达蛋白并纯化得到高纯度产物,为后期生物学功能研究奠定基础。

In order to express non-structural protein NS1 of epidemic encephalitis type B,NS1 gene of Japanese encephalitis virus（JEV）P3 strain was amplified by PCR method,and the prokaryotic expressing vectors were constructed by homologous recombination that fusion expressed His or GST tag in this study.The correct plasmids tested by PCR,enzyme digestion and sequencing were transformed into E.coli BL21,and NS1 fusion proteins were induced by IPTG.The proteins were identified using SDS-PAGE electrophoresis and Western blotting,and purified by urea modified and renaturation and affinity chromatography of nickel.The results indicated that two prokaryotic expression vectors of NS1 pET-42 b-NS1 and pGEX-KG-NS1 were constructed successfully,and the length of NS1 gene of P3 strain was 1 056 bp,which expressed about 40 ku protein in the form of inclusion body,the degeneration and renaturation effect of urea was better,and NS1 protein had high purity and concentration by nickel affinity chromatography.JEV NS1 gene could express the protein with high purity through prokaryotic expression system,and which laid a foundation for the later biological function research.