LGALS1与PRRSV 2b蛋白相互作用的验证研究

发布时间：2018-03-07 15:56

本文选题：2b蛋白　切入点：LGALS1　出处：《内蒙古民族大学》2017年硕士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)是一种单股正链RNA病毒,属于尼多病毒目(Nido virales),动脉炎病毒科(Ateriviridae),动脉炎病毒属(Arterivirus)。PRRSV能够引起母猪繁殖功能障碍和新生仔猪呼吸道症状。2b蛋白是PRRSV中较小的结构蛋白,它可能在病毒脱壳时扮演某种离子通道的角色,与病毒感染过程有关,可以促使PRRSV粒子脱壳,释放RNA到宿主细胞内,但对病毒的装配不起作用。研究2b蛋白与宿主细胞蛋白的相互作用,对全面了解PRRSV感染机制与深入认识2b蛋白功能具有重要意义。在前期研究中,用含有PRRSV 2b基因构建的钓饵菌株与猪肺泡巨噬细胞(PAM)cDNA文库进行酵母双杂交,成功的筛选到了与其相互作用的凝集素-1(LGALS1)。本研究成功构建了pGADT7-LGALS1重组质粒,并转入Y187酵母感受态中,与含有2b基因的Y2H酵母菌株进行了酵母回返试验,结果显示在SD/-Trp-Leu-His-Ade/AbA/X-α-gal的平板上长出了蓝色菌落,说明两种蛋白间发生相互作用。在GST-pull down试验中,构建了带有GST标签的重组质粒pEGX-KG-2b和带有His标签的重组质粒pET-28a-LGALS1,通过转入大肠杆菌感受态BL21中进行原核诱导表达,蛋白经过纯化后,进行pull down试验,结果显示,PRRSV 2b与LGALS1之间发生相互作用。在免疫共沉淀试验中,构建了pCMV-HA-2b与pCMV-Myc-LGALS1真核表达重组质粒,并将其共同转染至HEK293细胞中培养,使细胞在非变性条件下裂解,通过免疫共沉淀的方法验证了PRRSV 2b与LGALS1之间发生相互作用。本研究利用酵母回返试验、GST-pull down试验、免疫共沉淀试验三种方法成功验证了LGALS1与PRRSV 2b蛋白间的相互作用,为研究PRRSV感染途径和增殖过程提供了理论基础。
[Abstract]:Porcine reproductive and respiratory syndrome virus (PRRSVV) is a single-stranded positive strand RNA virus. It belongs to Nido viralesus, Ateriviridaeae. Arterivirus.PRRSv can cause reproductive dysfunction in sows and respiratory symptoms of newborn piglets. It is a small structural protein in PRRSV. It may play the role of an ion channel when the virus is shelled, related to the virus infection process, which can induce the PRRSV particles to unshell and release RNA into the host cells. The study of the interaction between 2b protein and host cell protein is of great significance in understanding the mechanism of PRRSV infection and understanding the function of 2b protein. The bait strain containing PRRSV 2b gene was used in yeast two-hybrid hybridization with porcine alveolar macrophage (Pam) cDNA library, and the interacting lectin -1GALS1 was successfully screened. In this study, the recombinant plasmid of pGADT7-LGALS1 was constructed and transferred into Y187 yeast. A yeast return test with Y2H yeast strain containing 2b gene was carried out. The results showed that blue colony was grown on the plate of SD-Trp-Leu-His-Ader AbArX- 伪 -gal, indicating the interaction between the two proteins. The recombinant plasmid pEGX-KG-2b with GST tag and the recombinant plasmid pET-28a-LGALS1 with His tag were constructed. The recombinant plasmid pET-28a-LGALS1 was transformed into Escherichia coli (E. coli) competent BL21 for prokaryotic expression. After purification, the protein was purified for pull down test. The results showed that there was interaction between PRRSv 2b and LGALS1. In the co-immunoprecipitation test, the eukaryotic expression plasmid of pCMV-HA-2b and pCMV-Myc-LGALS1 was constructed and co-transfected into HEK293 cells for cell lysis under the condition of non-denaturation. The interaction between PRRSV 2b and LGALS1 was verified by immunoprecipitation. The interaction between LGALS1 and PRRSV 2b protein was successfully verified by three methods: yeast return test, GST-pull down test and immunoprecipitation test. It provides a theoretical basis for the study of PRRSV infection pathway and proliferation process.
【学位授予单位】：内蒙古民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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