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  本文選題：2b蛋白　切入點：LGALS1　出處：《內(nèi)蒙古民族大學》2017年碩士論文　論文類型：學位論文

【摘要】：豬繁殖與呼吸綜合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)是一種單股正鏈RNA病毒,屬于尼多病毒目(Nido virales),動脈炎病毒科(Ateriviridae),動脈炎病毒屬(Arterivirus)。PRRSV能夠引起母豬繁殖功能障礙和新生仔豬呼吸道癥狀。2b蛋白是PRRSV中較小的結構蛋白,它可能在病毒脫殼時扮演某種離子通道的角色,與病毒感染過程有關,可以促使PRRSV粒子脫殼,釋放RNA到宿主細胞內(nèi),但對病毒的裝配不起作用。研究2b蛋白與宿主細胞蛋白的相互作用,對全面了解PRRSV感染機制與深入認識2b蛋白功能具有重要意義。在前期研究中,用含有PRRSV 2b基因構建的釣餌菌株與豬肺泡巨噬細胞(PAM)cDNA文庫進行酵母雙雜交,成功的篩選到了與其相互作用的凝集素-1(LGALS1)。本研究成功構建了pGADT7-LGALS1重組質(zhì)粒,并轉(zhuǎn)入Y187酵母感受態(tài)中,與含有2b基因的Y2H酵母菌株進行了酵母回返試驗,結果顯示在SD/-Trp-Leu-His-Ade/AbA/X-α-gal的平板上長出了藍色菌落,說明兩種蛋白間發(fā)生相互作用。在GST-pull down試驗中,構建了帶有GST標簽的重組質(zhì)粒pEGX-KG-2b和帶有His標簽的重組質(zhì)粒pET-28a-LGALS1,通過轉(zhuǎn)入大腸桿菌感受態(tài)BL21中進行原核誘導表達,蛋白經(jīng)過純化后,進行pull down試驗,結果顯示,PRRSV 2b與LGALS1之間發(fā)生相互作用。在免疫共沉淀試驗中,構建了pCMV-HA-2b與pCMV-Myc-LGALS1真核表達重組質(zhì)粒,并將其共同轉(zhuǎn)染至HEK293細胞中培養(yǎng),使細胞在非變性條件下裂解,通過免疫共沉淀的方法驗證了PRRSV 2b與LGALS1之間發(fā)生相互作用。本研究利用酵母回返試驗、GST-pull down試驗、免疫共沉淀試驗三種方法成功驗證了LGALS1與PRRSV 2b蛋白間的相互作用,為研究PRRSV感染途徑和增殖過程提供了理論基礎。
[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- 偽 -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.
【學位授予單位】：內(nèi)蒙古民族大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.651

【參考文獻】

相關期刊論文 前3條

1 劉瑩;王鳳雪;溫永俊;武華;;豬繁殖與呼吸綜合征病毒非結構蛋白2的研究進展[J];中國預防獸醫(yī)學報;2015年02期

2 田志平;薛江東;馬_郴，

本文編號：1579957