重组人HMGN2在大肠杆菌中表达及其体外抗乙型肝炎病毒活性研究  

Inhibitory Effect of Recombinant HMGN2 Protein on Human Hepatitis B Viral

作　　者：何兴俐[1] 张永红[1] 金淑慧[1] 冯云[1] 

机构地区：[1]四川大学口腔疾病研究国家重点实验室,成都610041

出　　处：《四川大学学报（医学版）》2011年第4期461-465,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金项目(批准号:30972764)资助

摘　　要：目的制备重组人高迁移率非组蛋白N2蛋白（HMGN2）,研究重组HMGN2的抗乙型肝炎病毒（HBV）活性。方法通过RT-PCR分离纯化人HMGN2基因,并将其构建到原核表达质粒pGEX-4T-1中,经IPTG诱导表达。产物经GST蛋白纯化系统进行纯化,用SDS-PAGE及Western blot分析鉴定。采用MTT法检测重组HMGN2蛋白对稳定转染复制型HBV重组质粒的肝癌细胞株HepG2.2.15细胞的细胞毒性;不同浓度HMGN2蛋白作用于HepG2.2.15细胞,分别在第3 d和6 d收集细胞培养上清液,ELISA检测上清中HBV表面抗原（HBsAg）及e抗原（HBeAg）,采用实时荧光定量PCR法检测上清液HBV DNA的含量。结果成功构建了HMGN2蛋白原核表达载体pGEX-HMGN2,并分离提纯了原核表达的HMGN2蛋白。Western blot结果显示,该蛋白能被抗HMGN2抗体识别。在检测的1~100μg/mL范围内,HMGN2蛋白对HepG2.2.15细胞的无细胞毒性;HMGN2蛋白在1~5μg/mL水平即可抑制HBeAg和HBsAg的表达,可降低HBV DNA拷贝数。结论成功构建HMGN2原核表达载体,并分离提纯了纯度较高的HMGN2蛋白;重组的HMGN2蛋白在体外具有较强的抗HBV活性。Objective To construct a prokaryotic expression recombinant for the expression of HMGN2 and to evaluate its antiviral activity against human hepatitis virus.Methods The extracellular region cDNA of HMGN2 was isolated and amplified by RT-PCR,and introduced to the prokaryotic expression vector pGEX-4T-1.HMGN2 protein was expressed under IPTG induction and purified by GST protein purification system,then identified by SDS-PAGE and Western blot.The cytotoxicity of fusion HMGN2 to HBV-transfected HepG2.2.15 cell was evaluated with MTT assay.Different concentration of fusion HMGN2 was applied on the HepG2.2.15 cell and the cell culture supernatants were harvested after 3 and 6 days treatment.The HBsAg and HBeAg in the supernatants were detected by ELISA and the HBV DNA was detected by RT-PCR.Results In the range of tested 1-100 μg/mL of HMGN2,no cytotoxicity to HepG2.2.15 cells was detected by MTT assay.When incubated with HMGN2 at 1-5 μg/mL for 72 h or 144 h,there was a significant reduction in HBeAg and HBsAg expression as well as the HBV DNA copies.Conclusion pGEX-4T-1/HMGN2 vector was success constructed,and the recombinant HMGN2 protein could inhibit HBV expression and replication in vitro remarkably.

关 键 词：高迁移率非组蛋白N2 原核表达 抗HBV活性 

分 类 号：R512.62[医药卫生—内科学]