论文部分内容阅读
目的研究双剪接型2．2 kb乙型肝炎病毒(HBV)剪接变异体编码的新蛋白的功能。方法PCR扩增双剪接型2．2kb HBV剪接特异性新基因并克隆于哺乳动物细胞表达载体pcDNA3．1/ HisC。以FuGENE6将此重组载体(pcDNA3．1/HisC-TPds)转染Huh7细胞,采用融合表达的多肽表位(X- press表位)抗体,通过Western blot检测目的蛋白在不同时间段的表达。pcDNA3．1/HisC-TPds分别与β-半乳糖苷酶报告载体psv-β-galactosidase(含SV40启动子/增强子)或pCMVβ(含CMV即刻早期启动子)共转染Huh7细胞,转染后48h裂解细胞并检测胞内β-半乳糖苷酶活性,实验数据以SPSS11．5软件分析。结果克隆420bp双剪接型2．2kb HBV剪接变异体新基因,Western blot显示其在Huh7细胞中表达相应蛋白,以转染后48h为最高。在含有CMV即刻早期启动子或SV40启动子/增强子的报告载体与pcDNA3．1/HisC-TPds质量比为1：10～1：50范围内,共转染有报告载体及pcDNA3．1/HisC-TPds的Huh7细胞内β-半乳糖苷酶活性均大于相应的pcDNA3．1/HisC空白载体对照组,且在1：10～1：40范围内存在剂量-效应关系。结论双剪接型2．2kb HBV剪接变异体编码的新蛋白可反式激活CMV即刻早期启动子及SV40启动子/增强子,提示其可能与HBV致病有关。
Objective To study the function of a new protein encoded by the double-spliced 2.2 kb Hepatitis B virus splice variant. Methods The 2.2 kb double stranded DNA splice-specific gene was amplified by PCR and cloned into mammalian cell expression vector pcDNA3.1 / HisC. The recombinant vector (pcDNA3.1 / HisC-TPds) was transfected into Huh7 cells with FuGENE6, and the expression of the expressed protein was detected by Western blot at different time points by Western blot. Huh7 cells were co-transfected with pcDNA3.1 / HisC-TPds and psv-β-galactosidase (including SV40 promoter / enhancer) or pCMVβ (including CMV immediate early promoter) 48h cells were lysed and the intracellular β-galactosidase activity was measured. The experimental data were analyzed by SPSS11.5 software. Results The new gene of 2.2kb HBV splice variant with 420bp double splice was cloned and expressed in Huh7 cells by Western blot, which was the highest at 48h after transfection. The reporter vector and pcDNA3.1 / HisC were co-transfected with the vector containing the CMV immediate early promoter or SV40 promoter / enhancer and the pcDNA3.1 / HisC-TPds in a mass ratio of 1:10 to 1:50, -TPds Huh7 cells β-galactosidase activity were greater than the corresponding pcDNA3.1 / HisC blank vector control group, and in the range of 1:10 to 1:40 dose-effect relationship. Conclusion The double-splice variant 2.2kb HBV splice variant encodes a novel protein that transactivates the CMV immediate early promoter and the SV40 promoter / enhancer, suggesting that it may be involved in the pathogenesis of HBV.