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乙型肝炎病毒(Hepatitis B virus,HBV)是慢性肝病的一个主要致病因素,易导致肝炎、肝硬化,并最终导致肝癌。乙型肝炎病毒聚合酶(HBV Polymerase,Pol)在HBV复制过程中起着关键作用,并有可能参与肝癌的发生。Pol的这些生理功能很有可能是通过与蛋白质之间的相互作用来行使的。本文通过免疫共沉淀(Co-immunoprecipitation)结合液相色谱串联质谱(LC-MS/MS)技术,筛选、鉴定与Pol存在潜在相互作用的蛋白。用构建好的pcDNA3.1-POL-flag重组质粒转染HeLa细胞,LC-MS/MS技术筛选能与融合flag标签的Pol发生免疫共沉淀的蛋白,共得到45个蛋白,其中11个蛋白被报道能与Pol发生相互作用。Ingenuity Pathway Analysis(IPAR○)软件进一步分析45个蛋白与Pol的功能及相应的细胞网络,发现这些蛋白涉及三个大分子网络,其中4个蛋白可能参与协助Pol在细胞网络中行使其生理功能,对这4个蛋白的进一步研究将有助于为攻克乙型肝炎病提供线索。
Abstract：Hepatitis B virus(HBV)is a major cause of chronic liver disease,and frequently results in hepatitis,cirrhosis,and ultimately hepatocellular carcinoma.HBV polymerase(Pol)is an essential viral protein that is important for HBV replication and might be involved in the development of hepatocellular carcinoma.Protein-protein interactions appears to be crucial for its role.The aim of this study was to screen and identify the proteins that interact with Pol using a co-immunoprecipitation-based LC-MS/MS identification technique.The HBV Pol gene was amplified by polymerase chain reaction(PCR)and cloned into pCDNA3.1(+).The recombinant plasmid pCDNA3.1(+)-Pol-flag was transfected into HeLa cells.Liquid chromatography and tandem mass spectrometry(LC-MS/MS)identified 45 proteins that co-immunoprecipitated with flag-tagged HBV Pol.Eleven of these have previously been reported as proteins that interact with HBV Pol.A proof-of-concept-based Ingenuity Pathway Analysis(IPAR○,www.ingenuity.com)was used to characterize the functions and pathways of these 45 identified proteins and HBV Pol.Among these proteins,four proteins may play a role in three major molecular cellular networks,and are therefore worthy of further investigation.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002577
中图分类号:R373.21
引用信息:
[1]凌梦婷,宫君原,李君武,等.LTQ-FT-MS技术结合生物信息学筛选与HBV Polymerase存在潜在相互作用的宿主蛋白[J].病毒学报,2014,30(06):636-644.DOI:10.13242/j.cnki.bingduxuebao.002577.
基金信息:
国家科技重大专项课题(2012ZX10002006-003)
2014-12-02
2014-12-02
2014-12-02