丁型肝炎病毒HDAg抗原的基因克隆、表达及细胞内定位

摘要

Objective To identify the cellular localization of HDAg protein after the cloning and expression of HDAg gene. Methods Routine molecular cloning, mismatch amplification mutation assay, Western blot, RT-PCR and immu-nofluorescence assay were adopted. Results In this study, HDAg gene was cloned from a patient' s serum co-infected with HBV/HDV. The analysis of bioinformatics supported the evidence of genotype I from Henan strain. The entire coding sequence of HDAg was cloned into an eukaryotic vector. The expression vector was transfected into human liver HepG2 cells. Expression products of both L-HDAg and S-HDAg were observed to localizate in the nuclei by immunofluo-rescence assay. Conclusion HDAg may play an important role through its cell localization in the nucleus.%目的 克隆并表达HDAg基因,确定HDAg蛋白在细胞内定位.方法 常规分子克隆、错配PCR、Western blot、RT-PCR及免疫细胞化学等.结果 通过RT-PCR及错配PCR方法从1例HBV/HDV重叠感染患者血清中成功克隆出HDAg基因,生物信息学分析证实其来源于我国流行的基因I型河南株.将HDAg基因克隆于真核载体转染肝细胞,证实两种不同形式的HDAg主要定位在细胞核.结论 HDAg定位于细胞核可能是其发挥相应生物学作用的基础.