HSV-TK红色荧光蛋白融合载体在HepG2细胞中的表达  

Construction of TK/RFP Fusion Gene Expression Vector and Its Expression in HepG2 Cells

作　　者：王炜煜[1] 曹利民[2] 司进[3] 王健[1] 易继林[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院普外科,湖北武汉430030 [2]华中科技大学同济医学院免疫学系,湖北武汉430030 [3]江苏省寄生虫病防治研究所,江苏无锡214046

出　　处：《贵阳医学院学报》2005年第4期294-297,301,共5页Journal of Guiyang Medical College

基　　金：卫生部科研基金资助项目(WKJ2004-2-013)

摘　　要：目的:建立红色荧光蛋白(red fluorescent prote in,RFP)标记的单纯疱疹病毒1型TK基因(Herpessimp lex virus thym id ine k inase,HSV-TK)的基因转移系统,并验证其有效性。方法:以pBLuescript-TK为模板,设计引物,将聚合酶链反应(PCR)扩增到的TK基因克隆到真核表达载体pD sRed2-N1中,获得以红色荧光蛋白为报告基因的重组质粒pD sRed2-N1-TK,利用脂质体转染体外培养的HepG2细胞,在活细胞状态下用荧光显微镜直接观察pD sRed2-N1-TK在细胞中的分布和定位,用RT-PCR方法验证其mRNA和蛋白质的表达。结果:空载体pD sRed2-N1转染组中,HepG2细胞内红色荧光呈弥散分布;重组质粒pD sRed2-N1-TK转染组中,红色荧光集中在细胞核中,随着表达量的增加,红色荧光在细胞核中聚集成团块状,2周后稳定表达于细胞浆中;RT-PCR的结果表明,HSV-TK基因在重组质粒转染组中有表达;丙氧鸟苷(GCV)杀伤效应确证了TK基因表达产物胸苷激酶的活性。结论:pD sRed2-N1-TK融合基因真核表达载体在真核细胞HepG2中获得了表达,细胞所表达的融合蛋白具有TK和RFP的双重活性,可用荧光显微镜直接观察其表达情况及亚细胞定位,为HSV-TK/GCV治疗肿瘤研究提供了实用而又方便的工具。Objective： To construct gene transfer system for red fluorescent protein （RFP）marked herpes simplex virus thymidine kinase （HSV-TK） gene into eukaryotic and to investigate its expression in HepG2 ceils. Methods： HSV-TK gene was amplified from pBLuescript-TK by PCR and inserted into plasmid pDsRed2-N1. Using lipofectin method, the recombinant expression plasmid pDsRed2-N1- TK was transfected into HepG2 ceils. The expressed mRNA of TK gene was detect by RT-PCR, and the RFP expression was observed under fluorescence microscope. Results： When detected by fluorescence microscope, the red fluorescence was localized in the nucleus of HepG2 ceils of test group, and in the cytoplasm of control group; TK gene mRNA was detected in the transfected ceils. The expression of recombined plasmid pDsRed2-N1-TK in HepG2 ceils was proved by RT-PCR. Conclusions： The HSV-TK and RFP fusion gene can express in HepG2 ceils. The expressed fusion protein show double activity of HSV-TK and RFP; RFP can be used as a reporting gene for tracing the expression of HSV-TK gcne, which provide practical and feasible tools for the study of gene therapy to cancer using herpes simplex type thymidine kinase gene transfer.

关 键 词：单纯疱疹病毒1型 人 荧光染料 融合蛋白质类 基因扩增 真核细胞 肝肿瘤 聚合酶链反应 

分 类 号：Q813.2[生物学—生物工程] R735.7[医药卫生—肿瘤]