B、C基因型乙型肝炎病毒X蛋白对MDR1基因反式激活能力的比较  

Comparative Analysis of Transactivating Capacities on MDR1 Gene between X Proteins of Genotype B and C in Hepatitis B Virus

作　　者：林万松[1] 徐晓[1] 陈金烟[1] 林旭[1] 

机构地区：[1]福建医科大学分子医学研究中心福建省高等学校感染与肿瘤重点实验室,福州350004

出　　处：《福建医科大学学报》2007年第6期487-490,共4页Journal of Fujian Medical University

基　　金：福建省科技计划重大项目(2005K045);福建省重大科技基金资助项目(2002F005)

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(MDR1)反式激活能力的差别。方法PCR扩增B、C基因型HBVX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Westernblot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-Basic(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48h裂解细胞并检测胞内萤火虫荧光素酶活性。实验数据以SPSS11.5软件分析。结果成功克隆X蛋白表达载体及MDR1报告载体。Westernblot显示pcDNA3.1/HisC-XB或pcDNA3.1/HisC-XC在HepG2细胞中均能表达HBVX蛋白,以转染后48h为最高。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HBVX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。Objective To investigate the differences of transactivating capacity on multidrug resistance gene-1（MDR1） between genotype B and C of HBV X proteins. Methods Genotype B and C of HBV X gene were amplified by means of PCR and cloned to the pcDNA3.1/HisC to construct the recombinant expression vector pcDNA3.1/HisC-XB and pcDNA3.1/HisC-XC separately. The recombinant vectors were separately transfected to HepG2 cells by FuGENE6 transfection reagent, and the expressed protein was detected by Western-blot using the antibody against the fused X-press epitopes. The promoter region of MDR1 gene was amplified and cloned to the firefly luciferase reporter plasmid pGL3-basic to construct the recombinant vector as pGL-MDR. HepG2 cells were co-transfected by pGL-MDR together with pcDNA3.1/HisC-XB or pcDNA3.1/HisC-XC, cells were lyses 48 hours post transfection, the intracellular firefly luciferase activities were monitored and calculated by paired-samples t test. Results Recombinant HBV X gene expression vectors and the MDR1 reporter plasmid were successfully constructed. HBV X proteins were expressed well in HepG2 cells with the highest level in the point of 48 hours post transfection, as demonstrated by western blot analysis. While the ratio of pGL-MDR reporter plasmid to either HBV X gene construct or pcDNA3.1/HisC ranged from 1： 2 to 1 ： 4. Intracellular luciferse activities of transfected HepG2 cells reduced accordingly as pcDNA3. 1/HisC-XBq-pGL-MDR〉 pcDNA3. 1/ HisC-XC＋pGL-MDR〉pcDNA3. 1/HisC＋pGL-MDR with dose-effects manner. Oonclusion Both of genotype B and genotype C of HBV X protein could transactivate the MDR1 gene in transcriptional level, and the transactivation competence of genotype B was higher than that of genotype C.

关 键 词：肝炎病毒 乙型 癌 肝细胞 基因 病毒 反式激活(遗传学) 病毒蛋白质类 基因 MDR 药物耐受性 

分 类 号：R346[医药卫生—基础医学]