【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。更多还原

【Abstract】 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 activi-ties of transfected HepG2 cells reduced accordingly as pcDNA3.1/HisC-XB+pGL-MDR>pcDNA3.1/HisC-XC+pGL-MDR>pcDNA3.1/HisC+pGL-MDR with dose-effects manner.Conclusion 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.更多还原