APOBEC3F的克隆、真核表达及其抗HBV效应的初步研究

目的克隆、体外真核表达载脂蛋白B mRNA编辑酶催化多肽样3F(APOBEC3F)基因并研究其抗病毒效应。方法应用RT-PCR的方法从人PBMC细胞中扩增APOBEC3F基因,构建HA-APOBEC3F融合蛋白真核表达载体pXFA3F,pXFA3F和具有复制能力的1.3倍HBV载体pHBV1.3共转染HepG2细胞,Western印迹检测APOBEC3F融合蛋白在HepG2细胞中的表达,ELISA方法检测细胞培养上清液中的HBsAg和HBeAg水平,实时定量PCR检测HBV相关mRNA水平变化。结果克隆了APOBEC3F基因,其经测序与GeneBank公布序列一致;构建的APOBEC3F真核表达载体pXFA3F经转染可在HepG2细胞中瞬时表达;APOBEC3F可抑制乙型肝炎表面抗原和e抗原的分泌,可使转染细胞内HBV相关mRNA水平下降。结论成功克隆并真核表达了APOBEC3F基因,其在体外具有抗HBV生物学效应。

Cloning and Eukaryotic Expression of APOBEC3F and Its Anti-HBV Effect

Objective To clone human APOBEC3F gene,express it in eukaryotic cells and preliminarily study its anti-HBV effect.Methods The APOBEC3F gene from human PBMC was expanded by RT-PCR,HA-APOBEC3F eukaryotic expression plasmid pXFA3F was constructed,replication competent 1.3-fold over length HBV plasmid pHBV1.3 and pXFA3F were co-transfected into HepG2 cells,instable expression of APOBEC3F in HepG2 cells was analysed by Western blot,levels of HBs antigen and Hbe antigen in the media of co-transfected HepG2 cells was detected by ELISA and HBV relative mRNA from total RNA was determined by real-time PCR.Results Sequence of APOBEC3F cloned was consistent with the sequence published in Genebank.HA-APOBEC3F eukaryotic expression plasmid could unstably express in HepG2 cells by transfection.In co-transfected HepG2 cells,HA-APOBEC3F could inhibit the secretion of HBs antigen and HBe antigen,and down-regulate the levels of HBV relative mRNA.Conclusion APOBEC3F gene has been successfully cloned and exepressed in eukaryotic cells and APOBEC3F has anti-HBV effect in vitro.