应用酵母双杂交系统筛选与流感病毒NS2蛋白相互作用的宿主蛋白

发布时间：2018-09-06 19:27

【摘要】：流感病毒的NS2蛋白能介导病毒核糖核蛋白复合体(vRNP)的核输出过程,并且能够调控病毒聚合酶的活性,参与禽流感病毒在哺乳动物宿主体内的适应过程。但相对于流感病毒其余蛋白质来讲,NS2的研究鲜有报道。经过本研究捕获到新的与流感病毒NS2相互作用的宿主蛋白,不但拓展了流感病毒与宿主因子的相互作用网络,而且为进一步探索流感病毒的复制周期和致病及免疫机制奠定了基础。酵母双杂交系统作为蛋白质相互作用的经典技术,已被应用于宿主蛋白与流感病毒蛋白的相互作用研究。本试验采用该技术为基础,以H5N1流感病毒的NS2蛋白作为“诱饵”蛋白,从含有Calu-3、A549、THP-1和U251四种细胞的混合cDNA文库中挑选出其相互作用蛋白,研究互作的生物学功能。主要研究内容如下:(1)以禽流感病毒A/Anhui/02/2005(H5N1)NS2蛋白为诱饵,从含有Calu-3、A549、THP-1和U251四种细胞的混合cDNA文库中筛选与其相互作用的蛋白将H5NS2克隆于pGBKT7中,转化酵母菌Y2HGold,检测诱饵质粒无细胞毒性以及不存在自激活现象后,将pGBKT7-H5NS2/Y2HGold与4种细胞系的Y187cDNA文库进行杂交,经过筛选最后共捕获到7种蛋白,分别为HGS、EXOSC4、ZWINT、PRDX3、IRF3、NDUFB9和RMND5B。根据Gene Ontology分析结果,这些蛋白分别参与细胞生长发育、细胞代谢和细胞定位等过程。(2)免疫共沉淀(Co-IP)验证其相互作用在实践中选择了7种蛋白中的过氧化物氧还酶3(PRDX3)进行了更为深入的研究。将H5NS2、PRDX3分别克隆于pEGFP-C1、pCAGGS中,在293T细胞中共转染pEGFP-C1和pCAGGS-myc-PRDX3或pEGFP-C1-H5NS2和pCAGGS-myc-PRDX3质粒,进行Co-IP及Western blot检测。结果显示在293T细胞中,外源表达的H5NS2蛋白和PRDX3蛋白拥有特异性的相互作用。(3)GST pull-down进一步验证其相互作用将H5NS2克隆于pGEX-6p-1中,并在大肠杆菌中表达制备GST-H5NS2重组蛋白和GST标签蛋白。以GST-H5NS2融合表达蛋白为诱饵或者作对照的GST蛋白与转染pCAGGS-myc-PRDX3质粒的293T细胞的细胞裂解液孵育,进行GSTpull-down。通过GST pull-down试验结果显示,H5NS2蛋白与PRDX3存在特异的,直接的相互作用。为进一步研究两者之间相互作用如何影响NS2蛋白功能以及病毒的复制周期奠定了基础。
[Abstract]:The NS2 protein of influenza virus can mediate the nuclear exportation process of ribonucleoprotein complex (vRNP) and regulate the activity of virus polymerase and participate in the adaptation process of avian influenza virus in mammalian host. But relative to the rest of the influenza virus protein, the study of NS2 is rarely reported. Through this study, a new host protein interacting with influenza virus NS2 was captured, which not only expanded the interaction network between influenza virus and host factor, It also lays a foundation for further exploring the replication cycle, pathogenesis and immune mechanism of influenza virus. Yeast two-hybrid system, as a classical technique of protein interaction, has been applied to study the interaction between host protein and influenza virus protein. On the basis of this technique, the interaction proteins of H5N1 influenza virus NS2 protein were selected from the cDNA library containing Calu-3,A549,THP-1 and U251 cells, and the biological function of the interaction was studied. The main contents are as follows: (1) using the A/Anhui/02/2005 (H5N1) NS2 protein of Avian Influenza virus as bait, the H5NS2 was cloned into pGBKT7 by screening the interacting proteins from the cDNA library containing four kinds of Calu-3,A549,THP-1 and U251 cells. After transforming yeast Y2H Gold. to detect the non-cytotoxicity and self-activation of the bait plasmid, the pGBKT7-H5NS2/Y2HGold was hybridized with the Y187cDNA library of four cell lines. After screening, seven proteins, HGS,EXOSC4,ZWINT,PRDX3,IRF3,NDUFB9 and RMND5B., were obtained. According to Gene Ontology analysis, these proteins are involved in cell growth and development, (2) Immunoprecipitation (Co-IP) confirmed that the interaction of the seven proteins, peroxoredoxin 3 (PRDX3), was further studied in practice. H5N1 NS2P PRDX3 was cloned into pEGFP-C1,pCAGGS and co-transfected with pEGFP-C1 and pCAGGS-myc-PRDX3 or pEGFP-C1-H5NS2 and pCAGGS-myc-PRDX3 plasmids in 293T cells for Co-IP and Western blot detection. The results showed that there was a specific interaction between exogenous H5NS2 protein and PRDX3 protein in 293T cells. (3) GST pull-down further confirmed that the H5NS2 was cloned into pGEX-6p-1 and expressed in Escherichia coli to produce GST-H5NS2 recombinant protein and GST label protein. Using GST-H5NS2 fusion protein as bait or as control, GST protein was incubated with 293T cell lysate transfected with pCAGGS-myc-PRDX3 plasmid to carry out GSTpull-down.. The results of GST pull-down test showed that there was a specific and direct interaction between H 5NS2 protein and PRDX3. It provides a basis for further study on how the interaction affects the function of NS2 protein and the replication cycle of the virus.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

【相似文献】