摘要
目的为探讨乙型肝炎病毒(HBV)核心抗原(HBcAg)在乙型肝炎发病机制中的作用,筛选、克隆出人肝细胞中与HBcAg相互作用的未知蛋白基因C12并进一步对其功能作初步研究。方法应用酵母双杂交系统-3筛选到与肝细胞蛋白结合的蛋白编码基因C12,克隆该基因并用再次用重回交实验证实作用可靠后,将基因与荧光蛋白基因融合表达,分析该基因表达产物在哺乳动物细胞中的定位;用MTT代谢实验了解C12表达对哺乳动物细胞生长代谢的影响作用;酵母双杂交实验寻找肝细胞中与之相互作用的蛋白;基因芯片技术了解C12基因表达对其他基因表达的影响。结果成功地筛选并克隆出HBcAg的肝细胞结合蛋白C12基因,经回交实验再次证实C12基因的表达产物C12蛋白与HB-cAg确切的结合作用。该基因与荧光蛋白基因融合表达转染293细胞、L02细胞后48 h细胞呈现均匀分布的强绿色荧光信号,提示C12蛋白可能主要定位在细胞质内;通过在Bel7402、L02细胞系中进行MTT检测,发现C12在上述细胞中表达没有对细胞的生长繁殖产生明显影响;用酵母双杂交系统-3筛选出肝细胞中与C12蛋白相互作用的蛋白基因16种;基因芯片技术在1 152个基因中筛选出17个差异表达的基因。结论C12蛋白主要定位在细胞质内,对哺乳动物细胞生长繁殖无明显影响,能与肝细胞中多种蛋白结合,在细胞内的过表达引起的生物过程涉及许多不同基因表达的变化。
Objective To elucidate the biological function of hepatitis B virus core antigen （HBcAg） on pathogenesis of hepatitis B and to identify and characterize the gene coding for a novel protein interacting with HBcAg in hepatocytes. Methods The yeast two-hybrid screening was performed to isolate cDNA clones encoding a novel proteins （C12） that directly interact with HBcAg. The interaction between HBcAg and C12 was verified by re-mating. The fluorescence protein fusion gene of pEGFP-N1 -C12 was transfected into 293 cells and L02 cells and observed by fluorescent microscope. MTT assay was used to study the effect of C12 on metabolism of mammal cells. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression gene adjusted by C12. Results C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence could be observed steadily in cell plasma. MTT assay showed that the C12 expression did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray were screened out, of which 16 genes were upregulated and 1 gene were downregulated. Twenty-one colonies containing 16 different genes coding for C12 binding proteins were isolated by yeast two-hybrid, 2 new genes with unknown function. Conclusion The novel protein C12 locates in cell plasma and interacts with many proteins in hepatocytes and may involve in many process of gene expression. C12 overexpression has no significant effect on the metabolism of liver cell.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第13期1413-1416,共4页
Journal of Third Military Medical University
