HBVnt453-250负性调节元件作用方式与嗜肝性的研究  

Study on the characteristics and hepatotropism of the negative regulatory element of the HBVnt453- 250

作　　者：吴莹[1] 张文露[2] 余波[2] 杨扬[2] 黄爱龙[2] 

机构地区：[1]北京中医药大学中医药抗病毒教育部重点实验室,100029 [2]重庆医科大学感染性疾病分子生物学实验室(省部共建)重庆医科大学病毒性肝炎研究所,400016

出　　处：《中华微生物学和免疫学杂志》2009年第1期16-23,共8页Chinese Journal of Microbiology and Immunology

基　　金：基金项目：国家自然科学基金资助项目（30600520）

摘　　要：目的HBVnt453-250序列为乙型肝炎病毒正链的-个新的负性调节元件，探讨其作用方式是否具有方向位点依赖性、启动子选择性以及肝细胞特异性。方法构建含有负性调节元件的质粒pHBV453-250方向和位点置换体（pHBV250-453、plucHBV453-250和plucHBV250-453），分别以荧光素酶（1uciferase）及增强型绿色荧光蛋白（EGFP）作为报告基因，转染HepG2细胞后，通过双荧光素酶体系、荧光定量PCR和Westernblot分析，检测HBVnt453-250序列对目的基因转录及表达的影响；构建启动子置换的质粒pCMV453-250（1uc），转染HepG2细胞后，双荧光素酶检测系统检测荧光素酶表达；将pHBV453—250和pHBV250-453分别转染人宫颈癌细胞（HeLa）和人肺癌细胞（A549），双荧光素酶检测系统检测荧光素酶表达。结果HBVnt453-250序列以正向或反向插入荧光素酶或EGFP基因的5’端或3’端时，均能表现对目的基因转录和表达的下调作用；在CMV启动子引导下，HBVnt453-250序列明显降低了荧光素酶的活性，为对照的36．56％；正反向的HBVnt453-250序列均能在两种非肝源性细胞系中抑制荧光素酶活性。结论HBVnt453-250序列的负性调节作用无方向及位点特异性，无启动子选择性及肝细胞特异性。Objective To analyze the characteristics and hepatotropism of this negative element HBVnt453-250 sequence. Methods pHBV453-250, pHBV250-453, plucHBV453-250 and plucHBV250- 453 were constructed, with luciferase and enhanced green fluorecence protein （EGFP） gene as the reporter gene, respectively. After transfection of HepG2 cells with these plasmids, luciferase assays, real-time PCR and Western blot assays were used to detect the gene transcription and expression level. The SV40 promoter of pGL3 control and pHBV453-250 were replaced by the cytomegalovirus early promoter, resulting in plas- raids pCMVcontrol （luc） and pCMV453-250 （luc）. Results Compared with pHBV453-250, the mutant plasmids, with the inhibitory element inserted in different site or inverted orientation, exerted similar down- regulation of luciferase gene transcription and expression. Western blot analysis demonstrated the similar re- pression when EGFP was used as the reporter gene. By transfected to HepG2 cell line, the plasmid pCMV453-250 （luc） could reduce lueiferase activity （36.56%） compared with pCMLcontrol （luc）. When the plasmids plucHBV453-250 and plucHBV250-453 were transfected to non-liver cell lines （A549, HeLa）, luciferase gene was expressed weakly, compared with that of pGL3control （P 〈 0.05 ）. Conclusion The inhibitory effect of HBVnt453-250 sequence acted in both orientation- and position-independent man- nets, and had no promoter selectivity and functioned in hepatocyte-independent manner.

关 键 词：乙型肝炎病毒 正链 负性调节元件 嗜肝性 

分 类 号：R392.1[医药卫生—免疫学]