本文選題：豬流行性腹瀉病毒 + M蛋白��； 參考：《東北農業(yè)大學》2015年碩士論文

【摘要】：豬流行性腹瀉病(PED)是由豬流行性腹瀉病毒(PEDV)引起的豬的一種急性高度接觸性胃腸道疾病。其臨床特點為急性嘔吐和嚴重腹瀉,可導致新生仔豬的高死亡率。世界各地區(qū)PED廣泛流行,經濟損失影響重大。M蛋白是PEDV主要結構蛋白之一,具有高度保守性,是PEDV刺激機體產生免疫保護的重要蛋白之一,決定病毒粒子的裝配位點、病毒成熟及出芽位點。另外,抗M蛋白抗體在補體存在的情況下可中和病毒感染活性。所以M蛋白特性研究對揭示PEDV感染機制及該病的綜合防治有重要的意義。本研究成功構建PEDV M蛋白的陽性重組質粒pET-32a-PEDV-M,將其在大腸桿菌表達系統中進行誘導表達,融合蛋白大小約為41 kμ,與預期大小相符。以純化復性的M蛋白為免疫原免疫大白兔,制備兔抗多克隆抗體,通過間接ELISA測定多抗效價達到105,免疫印跡(Western Blot)和間接免疫熒光(IFA)表明其與PEDV具有很好的抗原抗體反應性。以表達純化復性后的M蛋白作為靶蛋白,利用噬菌體展示隨機十二肽庫進行篩選,4輪淘選后,挑選10個與PEDV M蛋白特異性結合力高的噬菌體單克隆,序列測定并推導其氨基酸序列為AGWYCTEVLCV、AYCTRHVCYLDN,命名為M-2、M-9多肽。合成多肽的體外抗病毒試驗中,間接免疫熒光和實時熒光定量PCR實驗證明M-2、M-9及M-2與M-9多肽聯合與PEDV作用都能夠有效抑制病毒的復制,且都呈劑量依賴性。其中M-2多肽抑制病毒作用比M-9多肽高,兩種多肽聯合抗病毒作用最好。綜上,本文為進一步研究PEDV以及M蛋白功能性配體,為今后PEDV防治及小分子治療制劑的研發(fā)提供了理論基礎和試驗依據。
[Abstract]:Porcine epidemic diarrhea disease (PED) is an acute high contact gastrointestinal disease caused by porcine epidemic diarrhea virus (PEDVV). Its clinical characteristics are acute vomiting and severe diarrhea, which can lead to high mortality of newborn piglets. PED is widely prevalent in various regions of the world, and the economic loss of .M protein is one of the main structural proteins of PEDV, which is highly conserved. It is one of the most important proteins that PEDV stimulates the body to produce immune protection and determines the assembly site of virus particles. Mature and budding sites of the virus. In addition, anti-M protein antibodies can neutralize viral infection activity in the presence of complement. Therefore, the study of M protein characteristics is of great significance to reveal the mechanism of PEDV infection and the comprehensive prevention and treatment of the disease. In this study, the recombinant plasmid pET-32a-PEDV-Mwas successfully constructed. The recombinant plasmid pET-32a-PEDV-Mwas induced and expressed in Escherichia coli expression system. The size of the fusion protein was about 41k 渭, which was consistent with the expected size. Rabbit anti-polyclonal antibody was prepared by immunizing rabbits with purified M protein. The titer of polyclonal antibody was 105 by indirect ELISA assay. Western blot and indirect immunofluorescence assay showed that it had good antigen-antibody reactivity with PEDV. Using the purified M protein as the target protein, 10 phage clones with high specific binding ability to PEDV M protein were selected by phage display random dodecapeptide library after 4 rounds of panning. The amino acid sequence was identified as AGWYCTEVLCV AYCTRHVCYLDN and named M-2C M-9 polypeptide. Indirect immunofluorescence and real-time fluorescence quantitative PCR tests showed that M-2m9 and M-2 combined with M-9 peptides combined with PEDV could effectively inhibit virus replication in a dose-dependent manner in antiviral tests of synthetic peptides in vitro. The inhibitory effect of M-2 polypeptide was higher than that of M-9 polypeptide, and the antiviral effect of two peptides was the best. In summary, this paper provides a theoretical and experimental basis for the further study of PEDV and M-protein functional ligands, and for the future research and development of PEDV prevention and treatment and small molecular therapy preparations.
【學位授予單位】：東北農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.651

【參考文獻】

相關碩士學位論文 前1條

1 王明翠;豬繁殖與呼吸綜合征病毒GP5蛋白親和肽篩選與鑒定[D];東北農業(yè)大學;2010年

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