乙型肝炎病毒DNA聚合酶反式调节蛋白HBVDNAPTP1的亚细胞定位与结构预测

Subcellular localization and structure prediction of HBVDNAPTP1 transactivated by hepatitis B virus DNA polymerase

目的利用真核细胞绿色荧光表达系统分析乙型肝炎病毒DNA聚合酶反式调节蛋白HBVDNAPTP1的亚细胞定位,并用生物信息学方法对其结构进行分析。方法将HBVDNAPTP1基因片段插入pEGFP-C1载体中,分别转染HepG2细胞与COS7细胞,在荧光倒置显微镜下观察绿色荧光融合蛋白的细胞内定位,并应用生物信息学技术分析HBVDNAPTP1的结构特征。结果经RT-PCR扩增获得HBVDNAPTP1基因片段,成功构建HBVDNAPTP1基因的绿色荧光表达载体,转染细胞后,48～72h内细胞质出现明显的绿色荧光信号。结构预测结果表明HBVDNAPTP1无卷曲螺旋区域,有6个疏水区;无特殊二级结构,有2个酪蛋白激酶Ⅱ磷酸化位点、4个N端豆蔻酰基化位点和3个蛋白激酶C磷酸化位点;不具有跨膜螺旋结构,无信号肽。结论HBVDNAPTP1的亚细胞定位结果与生物信息学分析相符,为进一步研究其生物学功能及其在乙型肝炎、肝细胞癌中的作用机制奠定了基础。

Objective To explore the subcellular localization of HBVDNAPTP1 transactivated by hepatitis B virus （HBV） DNA polymerase with green fluorescent protein （GFP） expression system in eucaryofic cells, and predict the protein structure by bioinformafics. Methods The DNA fragment of HBVDNAPTP1 was amplified by RT-PCR and inserted into pEGFP-C1 vector. The ligation product was transfected into HepG2 and COS7 cells, respectively. The subcellular localization of green fluorescent fusion protein was observed with fluorescent inverted microscope. The structure of HBVDNAPTP1 protein was predicted by bioinformafics. Results The DNA fragment of HBVDNAPTPI was amplified by RT-PCR. HBVDNAPTP1 green fluorescent protein expression vector was successfully constructed. Visible green fluorescent signals appeared in cell plasma 48-72 hours after the transfection of recombined vector.Bioinlrormatics analysis of HBVDNAPTP1 showed that the protein had six hydrophobic regions in non-coil region. There was no secondary structure, but consisted of 2 casein kinase Ⅱphosphorylation sites, four N-myristoylation sites and three protein kinase C phosphorylation sites. The protein had no trans-membrane helix structure or signal peptide. Conclusions The subeellular localization of HBVDNAPTP1 is in accordance with the results of bioinformatics analysis. It may provide some clues for further study on the biological functions and mechanism of action of HBVDNAPTP1 gene in type 13 hepatitis and hepatocellular carcinoma.