7型庚型肝炎病毒非结构蛋白NS3在大肠埃希菌中的表达、纯化及其反应原性鉴定  

Expression, Purification of Recombinant GB Virus Type C Gene Type 7 Non-structural Proteins NS3 in E. coli and Its Reactogenicity

作　　者：赵跃[1] 杨晓钰[1] 冯悦[1] 夏雪山[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,昆明市650500

出　　处：《医学分子生物学杂志》2015年第4期207-212,共6页Journal of Medical Molecular Biology

基　　金：资助项目：国家自然科学基金（No.81360247）,武汉大学国家病毒学重点实验室开放基金（No.2014KF009）和云南省科技计划项目（No.2013FB31）

摘　　要：目的研究在大肠埃希菌（Escherichia coli，Ecoli）表达系统中表达和纯化7型庚型肝炎病毒（GBvirus C genotype 7，GBV．CG7）非结构蛋白NS3的149个氨基酸（NS3—149），并对其进行反应原性鉴定。方法将NS3．149基因克隆人原核表达载体pET-28a，并转化人BL21，利用异丙基．B．D．硫代吡喃半乳糖苷（IPTG）诱导其表达。用Ni。’柱对融合蛋白NS3．149进行纯化，最后经Western印迹检测其抗原特异性和反应原性。结果①成功构建了pET-28a-NS3-149重组质粒，该重组质粒转化入BL21后．融合蛋白NS3-149表达的最佳条件为在32℃条件下，A600nm等于0．8时，用1mmol／L的IPTG诱导6h后，融合蛋白表达量达到最高。经过SDS—PAGE分析，融合蛋白以包涵体的形式表达，其大小约27kD，与预期大小基本一致；②用感染GBV．C7型的人阳性血清经Western印迹鉴定，融合蛋白NS3-149具有较好的反应原性．另外，其与GBV-C容易发生共同感染的HIV或HCV阳性血清无非特异性交叉反应，说明了融合蛋白NS3-149具有较好的特异性。结论本实验在大肠埃希菌中表达并纯化并获得具有良好反应原性的GBV-C7型融合蛋白NS3-149，为进一步对GBV-C进行抗体检测的流行病学调查研究奠定了重要的前期工作基础。Objective To express and purify the GB virus C genotype 7 （GBV-C G 7 ） non- structural protein NS3-149 in Escherichia coli （E. coli）, and analyze its reactogenicity. Methods The NS3-149 gene （447 bp） of GBV-C was amplified by RT-PCR, and it was cloned into the pro- karyotic expression vector pET-28a. The recombinant vector pET-28a-NS3-149 was transformed into E. coli cells and induced by Isopropyl-D-1-Thiogalactopyrano-side （IPTG） ; then inclusion body was lysed, and purified via Ni-affinity chromatography; finally, the reactogenicity of recombinant NS3- 149 was detected by western blotting. Results DNA sequencing confirmed that GBV-C-NS3-149 gene had been accurately cloned into pET-28a. The best expression condition for recombinant NS3- 149 gene involved induction with 1mM 1PTG for 6 hours at 32℃ and the A600nm value of 0. 8. SDS- PAGE indicated that the expressed recombinant NS3-149 protein was inclusion bodies, and the size of recombinant NS3-149 protein was approximately 27 kD; ② Western blot analyses showed that the recombinant NS3-149 protein possessed reactogenicity by human positive sera infected with GBV-C 7. In addition, recombinant NS3-149 protein has specificity; it can＇ t cross reaction that human positive sera infected with HIV or HCV. Conclusion GBV-C recombinant NS3-149 protein with reactogenicity was expressed and purified in E. coli, the foundation of assay could be potentially used for epidemiology investigation of the GBV-C infection.

关 键 词：庚型肝炎病毒 NS3蛋白 大肠埃希菌 蛋白表达 反应原性 

分 类 号：Q789[生物学—分子生物学]