丙型肝炎病毒核心蛋白酵母双杂交诱饵载体的构建及鉴定研究
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摘要
目的构建含丙型肝炎病毒(hepatitis C virus,HCV)核心(core,C)蛋白基因的酵母双杂交诱饵载体,为C蛋白与宿主蛋白相互作用机制研究奠定基础。方法聚合酶链反应(PCR)法扩增HCV 1b基因型C蛋白基因,经EcoR I和Pst I双酶切后插入到诱饵载体pGBKT7中,构建含HCV C蛋白基因的酵母双杂交诱饵载体pGBKT7-Core,经酶切分析及核酸测序分析加以鉴定。醋酸锂法将pGBKT7-Core转化入酵母菌株Y2HGold,蛋白印迹法检测C蛋白的表达,通过表型筛选检测诱饵蛋白对酵母细胞的毒性作用及对报告基因的激活作用。结果重组质粒pGBKT7-Core中的C蛋白基因为576 bp,经核酸测序分析与NCBI中注册的HCV 1b基因型C蛋白基因序列一致;转化酵母菌后检测到C蛋白的表达;HCV C蛋白对酵母菌Y2HGold无毒性,且对报告基因无激活作用。结论含HCV C蛋白基因的酵母双杂交诱饵载体构建成功,表达稳定。
Objective To construct a bait vector containing the gene encoding hepatitis C virus( HCV) core( C) protein in yeast two-hybrid system,laying the foundation for further study of the mechanism of interaction between C protein and host proteins. Methods The cDNA fragment encoding C protein of HCV 1b genotype was amplified by PCR,then digested by EcoR I /Pst I and cloned into the bait vector pGBKT7 to construct recombinant bait plasmid pGBKT7-Core. Then right fragment of recombinant plasmid pGBKT7-Core was tested by both restriction endonuclease analysis. The sequence analysis was transformed into the yeast strain Y2 HGold by LiAc method. The expression of C protein was detected by Western blot. The toxicity and autoactivation of bait protein was tested by phenotype assay. Results The gene encoding HCV C protein in the recombinant plasmid pGBKT7-Core was 576 bp and consistent with that of HCV 1b genotype registered in NCBI. The expression of C protein was detected by Western blot after being transformed into the yeast cell. HCV C protein was not toxic to the yeast strain Y2 HGold,and could not be activate the reporter genes. Conclusion The bait vector containing the gene encoding HCV C protein was constructed successfully and expressed stably.
Objective To construct a bait vector containing the gene encoding hepatitis C virus( HCV) core( C) protein in yeast two-hybrid system,laying the foundation for further study of the mechanism of interaction between C protein and host proteins. Methods The cDNA fragment encoding C protein of HCV 1b genotype was amplified by PCR,then digested by EcoR I /Pst I and cloned into the bait vector pGBKT7 to construct recombinant bait plasmid pGBKT7-Core. Then right fragment of recombinant plasmid pGBKT7-Core was tested by both restriction endonuclease analysis. The sequence analysis was transformed into the yeast strain Y2 HGold by LiAc method. The expression of C protein was detected by Western blot. The toxicity and autoactivation of bait protein was tested by phenotype assay. Results The gene encoding HCV C protein in the recombinant plasmid pGBKT7-Core was 576 bp and consistent with that of HCV 1b genotype registered in NCBI. The expression of C protein was detected by Western blot after being transformed into the yeast cell. HCV C protein was not toxic to the yeast strain Y2 HGold,and could not be activate the reporter genes. Conclusion The bait vector containing the gene encoding HCV C protein was constructed successfully and expressed stably.
引文
[1]张莉莉,冯国和.丙型肝炎病毒核心蛋白作用研究进展[J].世界华人消化杂志,2008,16(18):2019-2024.
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[2]Lee JW,Liao PC,Young KC,et al.Identification of hnRNPH1,NF45,and C14orf166 as novel host interacting partners of the mature hepatitis C virus core protein[J].J Proteome Res,2011,10(10):4522-4534.
[3]Kasprzak A,Adamek A.Role of hepatitis C virus proteins(C,NS3,NS5A)in hepatic oncogenesis[J].Hepatol Res,2008,38(1):1-26.
[4]Lee SK,Park SO,Joe CO,et al.Interaction of HCV core protein with14-3-3epsilon protein releases Bax to activate apoptosis[J].Biochem Biophys Res Commun,2007,352(3):756-762.
[5]Mohd-Ismail NK,Deng L,Sukumaran SK,et al.The hepatitis C virus core protein contains a BH3 domain that regulates apoptosis through specific interaction with human Mcl-1[J].J Virol,2009,83(19):9993-10006.
[6]Tsutsumi T,Suzuki T,Shimoike T,et al.Interaction of hepatitis C virus core protein with retinoid X receptor alpha modulates its transcriptional activity[J].Hepatology,2002,35(4):937-946.
[7]Mori Y,Moriishi K,Matsuura Y.Hepatitis C virus core protein:its coordinate roles with PA28gamma in metabolic abnormality and carcinogenicity in the liver[J].Int J Biochem Cell Biol,2008,40(8):1437-1442.
[8]Liu J,Ding X,Tang J,et al.Enhancement of canonical Wnt/β-catenin signaling activity by HCV core protein promotes cell growth of hepatocellular carcinoma cells[J].PLoS One,2011,6(11):e27496.
[9]Suter B,Kittanakom S,Stagljar I.Two-hybrid technologies in proteomics research[J].Curr Opin Biotechnol,2008,19(4):316-323.