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首页> 外文期刊>World Journal of Gastroenterology >Cloning and characterization of a novel hepatitis B virus core binding protein C12.
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Cloning and characterization of a novel hepatitis B virus core binding protein C12.

机译：新型乙型肝炎病毒核心结合蛋白C12的克隆和鉴定。

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AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. 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 of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION: The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression.
机译：目的：为阐明HBV核心抗原（HBcAg）在乙型肝炎发病机理中的生物学功能，鉴定并鉴定了编码与肝细胞中HBcAg相互作用的功能未知蛋白的新型基因C12。方法：构建HBcAg诱饵质粒pGBKT7-HBcAg并转化到酵母AH109中，然后将酵母与含有肝互补DNA文库质粒的酵母Y187在2XYPDA培养基中交配。将二倍体酵母接种在含有X-alpha-gal的合成滤失营养培养基（SD / -Trp-Leu-His-Ade）和合成滤失营养培养基（SD / -Trp-Leu-His-Ade）上进行两次筛选。从蓝色菌落中提取质粒并进行测序后，我们分离了一个cDNA克隆，该克隆编码一种称为C12的新型蛋白，可直接与HBcAg相互作用。重新交配再次验证了HBcAg和C12之间的相互作用。将pEGFP-N1-C12荧光蛋白融合基因转染到293和L02细胞中，并用荧光显微镜观察。 MTT还原法用于研究C12蛋白效应对哺乳动物细胞代谢的作用。进行酵母双杂交和cDNA微阵列搜索结合蛋白和C12蛋白调控的差异表达基因。结果：通过酵母双杂交系统3对C12基因进行了筛选和鉴定。通过重新配对，进一步证实了HBcAg与新基因C12编码的新蛋白之间的相互作用。 48小时后，在293和L02细胞血浆中稳定观察到融合蛋白的荧光。在MTT分析下，我们发现C12的表达不影响肝细胞的生长。通过cDNA微阵列转染C12蛋白表达质粒的HepG2细胞中有17个差异表达基因，其中C12蛋白上调了16个基因，其中1个基因下调了。通过酵母双杂交技术分离出21个菌落，其中含有16个编码C12蛋白结合蛋白的不同基因，其中有2个功能未知的新基因。结论：新型蛋白C12位于细胞血浆中，其过表达对肝细胞代谢无明显影响。它与肝细胞中的许多蛋白质相互作用，并可能参与许多基因表达过程。

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