目的 探讨丙型肝炎病毒(HCV)F蛋白磷酸化位点突变对其在细胞内分布位置及功能的影响。方法 应用NetPhos2.0Server软件预测F蛋白的磷酸化位点,据此设计重叠引物,利用引物之间相互延伸获得突变型HCVf基因,该基因第13、14、114、121和124位密码子由野生型f基因的TCT突变为GAT,对应氨基酸由丝氨酸(S)突变为天冬氨酸(D)。将突变型f基因及野生型f基因定向克隆至pEGFP-N1,得到pEGFP-mf和pEGFP-f重组子。采用脂质体法将重组子转染HepG2细胞,再用激光共聚焦显微镜观察突变型F蛋白以及野生型F蛋白在细胞内的分布情况,拍照并进行半定量分析。结果 在HepG2细胞中,突变型F蛋白主要分布在细胞核内(90%),细胞质内有少量分布(10%),平均荧光强度分别为63.70±3.20和7.06±0.34,两者差异有统计学意义(P<0.001,t=99.2);野生型F蛋白主要分布在细胞质内(94.9%),细胞核内有少量分布(5.1%),平均荧光强度分别为83.34±4.07和4.48±0.22,两者差异有统计学意义(P<0.001,t=106.5)。结论 onclick="SelectDisplayDiv('ChDivSummaryMoreSummary','ChDivSummaryMore');DisplaySpanDiv('ChDivSummaryHuanYuan')"> HCVF蛋白在细胞内存在磷酸化和非磷酸化2种形式,其功能也不同。细胞质内以磷酸化F蛋白为主,非磷酸化F蛋白主要位于细胞核内。磷酸化F蛋白可能参与HCV复制调节,非磷酸化F蛋白可能影响细胞核内基因转录。

Objective To determine the relationship between phosphorylation site mutation and intercellular distribution of hepatitis C virus (HCV) F protein in HepG2 cells.Methods According to the phosphorylation sites of HCV F protein as predicted by NetPhos2.0 Server software, overlapping primers were designed and used to synthesize the HCV mutated f (mf) gene, in which the codons 13, 14, 114, 121 and 124 were mutated from TCT to GAT. Consequently, the amino acids shifted from serine(S) to aspartate (D). The recombinant pEGFP-mf and pEGFP-f were constructed by cloning the mf gene and f gene into pEGFP-N1, respectively. The resulting recombinant plasmids were transfected into HepG2 cells by liposome transfection method, and the distribution of mutant F protein or wild-type F protein in HepG2 cells was examined by laser confocal microscope.Results In HepG2 cells, the mutant F protein was located mainly in the nucleus (90%) and only 10% was in the cytoplasm. The mean fluorescence intensity of mutated F protein was significantly different between the nucleus and cytoplasm 〔63.70±3.20 and 7.06±0.34, respectively; (P<0.001,t=99.2)〕. On the contrary, the wild-type F protein was located mainly in the cytoplasm(94.9%) and only a small fraction was in the nucleus (5.1%). The mean fluorescence intensities for the wild-type F protein in the cytoplasm and nucleus were 83.34±4.07 and 4.48±0.22, respectively (P<0.001, t=106.5). Conclusions Phosphorylated and non-phosphorylated forms of the HCV F protein have different cellular distributions. The phosphorylated F protein lies mainly in cytoplasm and is involved in the regulation of HCV replication whereas the non-phosphorylated F protein exists mainly in the nucleus and may effect on gene transcription. if(document.getElementById('ChDivSummary').innerHTML!=""){CutSpan('ChDivSummary',500);DisplaySpanDiv('ChDivSummary');ClearSummaryOnLoad('SummaryLinkChID','SummaryLinkEnID');}else{CutSpan('EnDivSummary',1000);DisplaySpanDiv('EnDivSummary');ClearSummaryOnLoad('SummaryLinkEnID','SummaryLinkChID');}