| | | | | 乙型肝炎病毒前S1结合蛋白的筛选和鉴定 | | | 李丹;王小众;林纳;陈丰霖 | | | 目的利用酵母双杂合体系筛选HBV前S1结合蛋白,进一步探讨前S1蛋白在HBV感染中的作用。方法PCR扩增HBV前S1基因序列,根据酵母双杂合体系构建饵质粒pAS2-1- preS1,并测定序列;饵质粒转化入酵母菌AH109,Western印迹法证实前S1-BD融合蛋白在酵母细胞中的正确表达。pAS2-1-preS1及正常成人肝细胞cDNA文库共转化酵母细胞,通过选择性培养及β-半乳糖苷酶活性测定筛选阳性菌落,分离实验排除假阳性克隆,扩增阳性克隆的目的基因并测定序列,查询其同源序列,行交合实验进一步证实目的蛋白同前S1蛋白在酵母细胞中能否特异性结合。结果成功构建pAS2-1-preS1饵质粒,Western印迹法证实转化饵质粒的酵母细胞能正确表达前S1- BD融合蛋白,pAS2-1-preS1及正常成人肝细胞cDNA文库共转化酵母细胞后,选择性培养出101个菌落,经β-半乳糖苷酶活性测定及分离实验后筛选出1个阳性克隆,其cDNA插入片段与丝裂原活化蛋白激酶-胞外信号调节蛋白激酶(MAPK-ERK)激酶1(MEK1)基因高度同源。交合实验证实MEK1同前S1蛋白在酵母细胞中可特异性结合。结论酵母双杂合体系证实MEK1可能是一个前S1结合蛋白。
【作者单位】：福建医科大学附属协和医院消化科;福建医科大学附属协和医院消化科;福建医科大学附属协和医院消化科;福建医科大学附属协和医院消化科
【关键词】：肝炎病毒,乙型;前S1蛋白;酵母双杂合体系
【基金】：福建省自然科学基金(C0310018)
【分类号】：R373.21
【DOI】：CNKI:SUN:ZHCR.0.2007-01-007
【正文快照】：
HBV是通过逆转录复制基因组的嗜肝DNA病毒家族的一员，HBV感染可致急、慢性肝炎，并同原发性肝细胞癌(HCC)的进展密切相关田。HBV的前S基因组编码前Sl及前52蛋白，前S1蛋白参与HBV的病毒人胞、定位、复制、转录和分泌等各个环节。寻找并鉴定前Sl结合蛋白将为进一步阐明前Sl蛋白 | | |  推荐 CAJ下载  PDF下载 | | | CAJViewer7.0阅读器支持所有CNKI文件格式，AdobeReader仅支持PDF格式 | | | | Screening and identifying hepatitis B virus preS1-interactive protein | | | LI Dan WANG Xiao-zhong LIN Na CHEN Feng-lin Department of Digestive;Union Hospital of Fujian Medical University.Fuzhou 350001;China | | | Objective To screen and identify the hepatitis B virus(HBV)preS1-interactive proteins from normal human liver cDNA library by yeast two hybrid system 3 and explore the role of preS1 in the pathogenesis of HBV infection.Methods HBV proS1 gene was amplified by polymerase chain reaction(PCR),HBV preS1 bait plasmid,named pAS2-1-preS1,was constructed by yeast two hybrid system 3 and verified by auto-sequencing assay,pAS2-1-preS1 was transformed into the yeast AH109 and preS1-BD fusion protein expressed in the yeast cells was confirmed by Western blotting method.Yeast cells were cotransformed with PAS2-1-preS1 and normal human liver cDNA library were grown in selective SC/-trp-leu-his-ade2 medium.The second screen was performed withβ-gal activity detection and false positive clones were eliminated by segregation analysis.Thereafter,true positive clones were amplified,sequenced and analyzed with bioinformaties.Lastly,mating experi- ment was performed to confirm the binding of putative proteins with preS1 in the yeast cells.Results Bait plasmid pAS2-1-preS1 was successfully constructed and pAS2-1-preS1 correctly expressed preS1-BD fusion protein in the yeast AH109.One hundred and one clones grew in the selective SC/- trp-leu-his-ade2 medium,and only one clone past throughβ-gal activity detection and segregation analysis.The inserted cDNA fragment showed high homology with mitogen activated protein kinases- extracellular signal-regulated kinase kinase 1(MEK1).Furthermore,mating experiment identified that the binding of MEK1 with preS1 protein was specific.Conclusion MEK1 is a candidate protein which can interact with preS1 in vivo by yeast two hybrid system.
【Keyword】：Hepatitis B virus;PreS1 protein;Yeast two hybrid system |
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