乙型肝炎病毒正链负性调节元件的亚元件鉴定

Identification of subelements of negative regulatory element in hepatitis B virus S-(+)-strand

目的研究乙型肝炎病毒正链负性调节元件（HBVnt453～250）中存在的重要功能亚元件。方法通过构建pHBVnt453～250质粒一系列缺失10 bp的突变体,与内参pRL-TK质粒共转染HepG2细胞,于转染后48 h检测荧光素酶活性并确定亚元件的位置和序列。构建亚元件与pGL3-control载体质粒的重组质粒,与内参pRL-TK质粒共转染HepG2细胞并检测荧光素酶活性,半定量RT-PCR检测荧光素酶的mRNA水平。结果与pHBVnt453～250对照组比较,6个缺失突变体实验组的荧光素酶活性恢复到60%～80%,鉴定出3个功能亚元件。这3个亚元件的重组质粒荧光素酶活性比pGL3-control对照组降低,半定量RT-PCR显示荧光素酶的mRNA水平降低。结论HBVnt453～250序列中含有3个重要功能亚元件,以协同的方式发挥调节抑制作用。

Objective To research and identify the functional subelements of negative regulatory element in hepatitis B virus S-（＋）-strand.Methods A series of 10 bp-deleted mutants of pHBVnt453-250 plasmid and pRL-TK plasmid using for balancing transfection efficiency were cotransfected into HepG2 cells.The sequence of subelements was determined based on the detection of luciferase activities of mutants.Recombinant plasmids of subelements and pRL-TK plasmid were cotransfected into HepG2 cells.The luciferase activities of recombinant plasmids were detected and the levels of mRNA of luciferase gene were determined using RT-PCR.Results Luciferase activities of six mutants groups were recovered to 60%-80% in comparison to the pHBVnt453-250 group and three subelements were identified.The luciferase activities of subelement recombinant plasmid groups were decreased compared to the control group.RT-PCR showed that the RNAs of luciferase gene were decreased.Conclusion The results demonstrate that HBVnt453-250 contains three functional subelements,which act in a cooperative mode.