摘要
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 investigate the function of the novel protein encoded by the 2.2 kb doubly spliced variant of hepatitis B virus genome. Methods The splicing-specific gene generated by the 2.2 kb doubly spliced variant of HBV genome was amplified by means of PCR and cloned into the mammalian expression vector pcDNA3.1/HisC. The recombinant vector, i.e., pcDNA3.1/HisC-TPds, was transfected into Huh7 hepatecytes by FuGENE6 transfection reagent and the expressed protein was detected by Western blot. Huh7 hepatocyles were co-tmnsfected by pcDNA3.1/HisC-TPds together with the β-galactosidase reporter plasmid pCMVβ and pSV-β-galactosidase, cells were lysed 48 hours post transfection, the intracellular β-galatosidase activities were monitored and calculated by SPSS11.5 software. Results The splicing-specific gene with the size of around 420 bp was successfully cloned, the protein by which encoded expressed well in Huh7 cells with the highest level in the point of 48 h after transfection. The intracellular β-galatosidase activities of Huh7 hepatoeytes co-transfected with the reporter plasmids and pcDNA3.1/HisG-TPds were higher than those of empty vector pcDNA3.1/HisC with the ratio ranging from 1：10 to 1：50 of reporter plasmid to pcDNA3.1/HisC-TPds, and demonstrated the dose-effects manner within the ratio ranging from 1 ： 10 to 1：40 of reporter plasmid to pcDNA3.1/HisC-TPds. Condusion The splicing-specific protein encoded by the 2.2 Ida doubly spliced variant of hepatitis B virus genome could transactivate the CMV immediate-early promoter and the SV40 promoter and enhancer, which suggested a possible role in the pathogenesis of HBV.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第11期985-989,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金(30300015)
福建省重大科技基金(2002F005)
福建省医学创新基金(2003-CX-5)
