B、C基因型乙型肝炎病毒X蛋白对MDR1基因的反式激活能力不同

发布时间：2017-12-30 21:07

本文关键词：B、C基因型乙型肝炎病毒X蛋白对MDR1基因的反式激活能力不同　出处：《福建医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:研究B、C基因型乙型肝炎病毒(HBV)X蛋白对多药耐药基因1(Multidrug resisitance gene 1,MDR1)反式激活能力的差别。方法: PCR扩增B、C基因型HBV X基因并克隆于pcDNA3.1/HisC载体(重组载体分别为pcDNA3.1/HisC-XB及pcDNA3.1/HisC-XC)。以FuGENE6将重组表达载体转染HepG2细胞,采用融合表达的X-press多肽表位抗体,通过western blot检测目的蛋白在不同时间段的表达。PCR扩增MDR1基因启动子并克隆于萤火虫荧光素酶报告载体pGL3-Basi(c重组载体为pGL-MDR)。pGL-MDR分别与pcDNA3.1/HisC-XB或pcDNA3.1/HisC-XC共转染HepG2细胞,转染后48小时裂解细胞并检测胞内萤火虫荧光素酶活性,实验数据以SPSS 11.5软件分析。结果: 1、成功克隆X蛋白表达载体及MDR1报告载体。Western blot显示pcDNA3.1/HisC-XB或pcDNA3.1/HisC-XC在HepG2细胞中均能表达HBV X蛋白,以转染后48小时为最高;2、当pGL-MDR报告质粒与X基因重组载体或空载体质量比在1:2～1:4范围内,萤火虫荧光素酶在各转染细胞内的活性依次为pcDNA3.1/HisC-XB+pGL-MDR组pcDNA3.1/HisC-XC+pGL-MDR组pcDNA3.1/HisC+pGL-MDR组(对照组),且存在剂量-效应关系。结论: B、C基因型HBV X蛋白对多药耐药基因均有反式激活能力,且B基因型强于C基因型。
[Abstract]:Objective: to study the multidrug resisitance gene 1 (MDR) gene of hepatitis B virus (HBV) resisitance gene X protein (HBVX). MDR1) the difference in transactivation ability. Methods: B was amplified by PCR. C genotype HBV X gene was cloned into pcDNA3.1/HisC vector (. The recombinant vectors were pcDNA3.1/HisC-XB and pcDNA3.1 / HisC-XCn.Recombinant expression vectors were transfected into HepG2 cells by FuGENE6. The fusion expressed X-press peptide epitope antibody was used. The promoter of MDR1 gene was amplified by western blot and cloned into the luciferase report vector pGL3-Basi. C Recombinant vector pGL-MDR).pGL-MDR was cotransfected with pcDNA3.1/HisC-XB or pcDNA3.1/HisC-XC into HepG2 cells respectively. 48 hours after transfection, the cells were lysed and the luciferase activity of firefly was detected. The experimental data were analyzed by SPSS 11.5 software. Results: 1. Successful cloning of X protein expression Vector and MDR1 report Vector. Blot showed that both pcDNA3.1/HisC-XB and pcDNA3.1/HisC-XC could express HBV X protein in HepG2 cells. The highest rate was 48 hours after transfection. 2, when the mass ratio of pGL-MDR report plasmid to X gene recombinant vector or empty vector is in the range of 1: 2 and 1: 4. The luciferase activity of firefly in the transfected cells was in turn pcDNA3.1/HisC-XB pGL-MDR group pcDNA3.1/HisC-XC. PGL-MDR group pcDNA3.1/HisC pGL-MDR group (. Control group). And there is a dose-effect relationship. Conclusion: HBV X protein of genotype B C has transactivation ability to multidrug resistance genes, and genotype B is stronger than genotype C.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

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