丙型肝炎病毒F蛋白磷酸化位点突变与细胞内分布  

Intercellular distribution of hepatitis C virus F protein is associated with its mutation in phosphorylation sites

作　　者：邵圣文[1] 于建国[2] 武文斌[1] 赵平[1] 丁惠[1] 戚中田[1] 

机构地区：[1]第二军医大学微生物学教研室,军队医学微生物学重点实验室,上海200433 [2]解放军第八十八医院济南军区肝病研究所

出　　处：《微生物与感染》2007年第2期78-82,共5页Journal of Microbes and Infections

基　　金：国家高新技术研究发展计划(863计划)(2006AA02Z401);军队医药卫生"十一五"专项项目(06Z026)

摘　　要：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 phesphorylation 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 sefine（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 transt＇ected into HepG2 cells by lipesome 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 and4.48±0.22, respectively （P〈0.001, t=106.5）. Conclusions Phesphorylated 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-phesphorylated F protein exists mainly in the nucleus and may effect on gene transcription.

关 键 词：丙型肝炎病毒 F蛋白 磷酸化 

分 类 号：R373[医药卫生—病原生物学]