【作者】 王少蕾；

【导师】 张春枝； 许龙；

【作者基本信息】 大连工业大学 ， 发酵工程， 2009， 硕士

【摘要】 禽流感病毒是正黏病毒科的主要成员,是单股负链分节段的RNA病毒,依赖自身的RNA聚合酶复合体催化其基因组在宿主细胞核中进行转录和复制。目前多种研究报道表明禽流感病毒能够跨种传播,给人类健康造成了巨大的威胁,因此加强禽流感病毒致病机制的研究显得格外重要。聚合酶酸性蛋白(PA)是RNA聚合酶复合体3个亚基中的重要角色,它不但参与病毒复制、病毒RNA转录、病毒粒子组装等多种病毒活动过程,还具有核酸内切酶活性和蛋白酶活性。本文以高致病性禽源H5N1亚型禽流感病毒PA原核表达及相互作用蛋白筛选为切入点,对PA蛋白的功能进行了初步研究。首先,构建了17个PA突变体的表达载体,SDS-PAGE电泳及Western blot分析突变体的表达丰度,证明PA基因的61-120bp和325-426bp可能是影响PA蛋白表达的两个重要区域,同时N端缺失长度在143-408个氨基酸残基之间的9个突变体在大肠杆菌中获得高表达。为制备各片段的特异性抗血清、验证蛋白的相互作用区域、表达得到全长PA蛋白奠定基础。其次,采用MATCHMAKER GAL4 Two-hybrid System 3(clontech公司),以H5N1型病毒的PA为诱饵,筛选人肝cDNA文库相互作用蛋白,得到8个蛋白与PA相互作用,分别为GPATCH8、GPR89A、ADH1A、ADH1C、TTR、PMSA、LBP、ETFA,并在酵母内验证这6个蛋白与H5N1型病毒的PA有相互作用。第三,根据GenBank相关序列资料设计特异引物,通过PCR的方法,扩增得到GPR89A、ADH1C、TTR、PMSA、LBP、ETFA6个全长基因。构建了6个蛋白的原核及真核表达载体,Western blot检测,除GPR89A外其余蛋白在肺腺癌细胞A549中有表达。为后续免疫共沉淀试验进一步验证蛋白相互作用奠定基础。本研究发现了影响PA原核表达的区域,并筛选出8种PA的相互作用蛋白,进一步揭示了禽流感病毒致病过程中PA蛋白的功能,促进了对流感病毒致病机理的认识,为禽流感病毒疫苗和新型抗病毒药物的研究提供依据。更多还原

【Abstract】 The molecular determinants and related mechanisms that make certain influenza viruses highly pathogenic species,including humans,remain poorly understood.Numerous studies have shown that influenza virus virulence in mammalian species is a polygenic trait.The influenza virus RNA polymerase is a complex composed of three subunits,PA,PB1, and PB2.The polymerase,together with the nucleoprotein(NP) and the viral RNA template, (RNP),which carry out viral transcription and replication.PA not only carry out viral transcription and replication,but also induces a generalized proteolytic process.In this paper, the expression of PA protein and the protein interacting with PA were studied.First,17 deletion mutants of PA protein were constructed and expressed in Ecoli.Rosseta GamiB(DE),we conclusion that the two segments of 61th-120th bp and the 325th-426th bp of PA gene may be the very fragments that affect the expression level of PA protein.At the same time,nine mutants with the deletion between the 1th bp and the 426th bp of the PA gene got higer expression.Second,we use H5N1 PA as bait protein to screen the related protein by yeast Two-hybrid System 3.Eight positive clones were identified,they are GPATCH8、GPR89A、ADH1A、ADH1C、TTR、PMSA、LBP、ETFA.The interaction of these 8 proteins with PA were tested by yeast mating.Third,the full-length of gene GPR89A、ADH1C、TTR、PMSA、LBP and ETFA are cloned and constructed into pCMV-3HA vector and pTIG-Trx-Etag vector.Protein ADH1C、TTR、PMSA、LBP and ETFA can be expressed by A549.In conclusion,based on these results,the function of PA protein and the molecular pathogenic mechanism of AIV could be greatly and deeply enderstood,and the strategy for prevention against and treatment for AIV would be formed.更多还原