乙肝病毒截短型中蛋白表达载体的构建及反式激活作用的研究

Construction of recombinator plasmids with two different length C-terminal truncated middle surface protein of the hepatitis B virus and their transactivator function

目的:构建含有不同长度的乙肝病毒(HBV)截短型中蛋白的表达载体,并研究其反式激活作用。方法:设计引物扩增两条不同长度的HBV截短型中蛋白基因片段,分别定向克隆入真核表达载体pcDNA3,构建重组表达质粒pcS2与pcTS。重组子经HindⅢ单酶切、PCR及测序鉴定。将测序正确的重组子与报告质粒pGL3-control共转染肝癌细胞系HepG2,检测荧光素酶表达强度。结果:构建了两种HBV截短型中蛋白基因的表达质粒pcS2与pcTS,通过共转染证明均具有反式激活作用,且pcS2反式激活作用强于pcTS(P<0.01)。结论:成功构建了具有反式激活作用的重组表达载体,并证明HBV截短型中蛋白发挥反式激活作用的关键区段在前S2区。

Objective: To construct recombinant plasmids with two different length C-terminal truncated middle surface protein of the hepatitis B virus (MHBSt) and investigate their transactivator function. Methods: Polymerase chain reaction (PCR)was employed to amplify the coding sequence of two different MHBSt, and the products were cloned into pcDNA3 to construct two MHBSt expression vectors, named pcS2 and pcTS respectively. The recombinant plasmids were identified with Hind , PCR and DNA sequencing. pcS2 or pcTS was co-transfected into HepG2 cell line with reporter plasmid pGL3-control, then luciferase activity was detected by dual-luciferase reporter assay system. Results: Two recombinant plasmids with different length of MHBSt were constructed successfully. After co-transfected, both pcS2 and pcTS had transactivator function. Compared with pcTS, pcS2 showed more powerful transactivator function (P<0.01). Conclusion: Two MHBst recombinant plasmids which can play transactivator role are constructed successfully. The preS2 domain is the crucial region in the transactivator function of MHBSt.