蜱传森林脑炎病毒非结构蛋白1原核表达载体的构建  

Construction of prokaryotic expression vector of nonstructural protein 1 of Tick-borne encephalitis virus

作　　者：宋天一[1] 李菁华[2] 张哲[2] 史红艳[2] 孙延波[2] 

机构地区：[1]吉林大学白求恩医学院 [2]吉林大学白求恩医学院病原生物学系,吉林长春130021

出　　处：《吉林大学学报（医学版）》2013年第3期620-624,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(69301261)

摘　　要：目的:构建蜱传森林脑炎病毒(TBEV)非结构蛋白1(NS1)的原核表达载体,以期获得其高纯度的表达,为制备多克隆抗体及其功能的研究奠定实验基础。方法:根据目的基因序列设计PCR引物,利用基因重组技术构建原核表达载体pEASYTM-E1-NS1。将重组后质粒pEASYTM-E1-NS1转化至BL21细胞,经异丙基硫代半乳糖苷(IPTG)诱导后进行镍离子亲和纯化,利用Bradford法测定蛋白浓度。采用SDS-PAGE和Western blotting法分析融合蛋白的表达水平。结果:含TBEV NS1的原核表达载体pEASYTM-E1-NS1经诱导后,可获得以包涵体形式存在的融合蛋白,通过亲和纯化得到较高纯度的蛋白质。SDS-PAGE和Western blotting,在相对分子质量约40 000处有特异性蛋白条带。结论:成功构建重组pEASYTM-E1-NS1原核表达载体,获得高纯度的重组TBEV NS1的融合蛋白。Objective To construct the prokaryotic expression vector of nonstructural protein 1（NS1） of Tick-borne encephalitis virus（TBEV） and to obtain high purity expression of NS1 and to provide experimental basis for preparation of polyclonal antibody against NS1 and study on its function.Methods The PCR primers were designed according to the target gene,and the prokaryotic expression vector pEASYTM-E1-NS1 was constructed using gene recombination method.The recombinant plasmids pEASYTM-E1-NS1 were transferred into BL21 cells,and the expressed fusion proteins were purified by nickel ion affinity purification and the concentration of protein was analyzed by Bradford method after induction by Isopropy1β-D-1-Thiogalactopyranoside（IPTG）.The expression level of fusion protein was detected by SDS-PAGE and Western blotting methods.Results The prokaryotic expression vector pEASYTM-E1-NS1 carrying TBEV NS1 expressed fusion protein in the form of inclusion body after induction;a kind of highly purified protein was obtained through affinity purification.The SDS-PAGE and Western blotting results showed that a kind of specific protein with a relative molecular mass of about 40 000 was found.Conclusion The prokaryotic expression vector pEASYTM-E1-NS1 is constructed successfully,and the fusion protein of TBEV NS1 with high purity is acquired.

关 键 词：蜱传森林脑炎病毒 非结构蛋白1 原核表达 融合蛋白 

分 类 号：R373.31[医药卫生—病原生物学]