四环素调控HCVNS3/4A丝氨酸蛋白酶表达细胞系的建立  

Establishment of a tetracycline-regulated cell line expressing high HCV NS3/4A serine protease

作　　者：雷迎峰[1] 杨敬[1] 尹文[1] 吕欣[1] 安群星[2] 黄小军[1] 姚敏[1] 徐志凯[1] 

机构地区：[1]第四军医大学基础部微生物学教研室,陕西西安710032 [2]第四军医大学西京医院输血科,陕西西安710033

出　　处：《中国病毒病杂志》2012年第1期30-33,共4页Chinese Journal of Viral Diseases

基　　金：国家自然科学基金(30600564;30700710;3070030)

摘　　要：目的建立双稳定表达HCV NS3/4A蛋白酶的HepG2细胞系。方法将pTET-ON质粒转染HepG2细胞后用G418压力选择,利用Tet反应性质粒pTER2luc筛选高表达、低背景的TET-ON稳定转染的细胞。HindⅢ单切pMD18T-NS3/4A,平端化后再用BamHⅠ单切,然后与用BamHⅠ与EcoRⅤ双切的pTER2hyg质粒连接,转化菌株JM109感受态细胞,获得重组质粒pTER2hyg-NS3/4A,经PCR鉴定及序列测定。将pTER2hyg-NS3/4A用脂质体法转染TET-ON稳定转染HepG2细胞,经持续潮霉素压力选择和克隆化获稳定转染的细胞系,用实时荧光RT-PCR筛选出高水平表达NS3/4A的细胞株,并用Western blot验证。结果成功建立了双稳定的可诱导表达NS3/4A蛋白酶的HepG2细胞株。结论本研究构建的细胞株为建立以细胞为基础的抗HCV药物筛选系统提供了有效的筛选细胞系。Objective To establish a double stable HepG2 cell lines expressing HCV NS3/4A protease.Methods The plasmid pTET-ON was transfected into HepG2 cells and the stable transformants with low background and high inducibility were selected using Tetrocycline（Tet）-responsive plasmid pTER2luc for the detection of luciferase activity.Recombinant plasmid pMD18T-NS3/4A was digested with HindⅢ,and then blunted and digested with BamHⅠ.The fragment NS3/4A was purified and ligated to BamHⅠand EcoRⅤ-digested pTER2hyg.After being verified by PCR and sequencing,the new construct pTER2hyg-NS3/4A was transfected into TET-ON HepG2 cells and thereafter,double stable transformants under hymogrocin were selected using real-time PCR.To further identify the most highly inducible transformants,Western blot was applied to measure the expression of NS3/4A.Results A double stably transfected HepG2 cell line which can be induced to express high NS3/4A protease was cloned.Conclusions The high expression of NS3/4A of the established HepG2 cell clone could possibly be used for cell-based drug-evaluation with HCV serine protease as the target.

关 键 词：丙型肝炎病毒 NS3/4A蛋白酶 质粒转染 细胞系 

分 类 号：R512.63[医药卫生—内科学]