猪流行性腹泻病毒M蛋白亲和多肽筛选与功能鉴定

发布时间：2018-05-03 01:20

本文选题：猪流行性腹泻病毒 + M蛋白　；参考：《东北农业大学》2015年硕士论文

【摘要】：猪流行性腹泻病(PED)是由猪流行性腹泻病毒(PEDV)引起的猪的一种急性高度接触性胃肠道疾病。其临床特点为急性呕吐和严重腹泻,可导致新生仔猪的高死亡率。世界各地区PED广泛流行,经济损失影响重大。M蛋白是PEDV主要结构蛋白之一,具有高度保守性,是PEDV刺激机体产生免疫保护的重要蛋白之一,决定病毒粒子的装配位点、病毒成熟及出芽位点。另外,抗M蛋白抗体在补体存在的情况下可中和病毒感染活性。所以M蛋白特性研究对揭示PEDV感染机制及该病的综合防治有重要的意义。本研究成功构建PEDV M蛋白的阳性重组质粒pET-32a-PEDV-M,将其在大肠杆菌表达系统中进行诱导表达,融合蛋白大小约为41 kμ,与预期大小相符。以纯化复性的M蛋白为免疫原免疫大白兔,制备兔抗多克隆抗体,通过间接ELISA测定多抗效价达到105,免疫印迹(Western Blot)和间接免疫荧光(IFA)表明其与PEDV具有很好的抗原抗体反应性。以表达纯化复性后的M蛋白作为靶蛋白,利用噬菌体展示随机十二肽库进行筛选,4轮淘选后,挑选10个与PEDV M蛋白特异性结合力高的噬菌体单克隆,序列测定并推导其氨基酸序列为AGWYCTEVLCV、AYCTRHVCYLDN,命名为M-2、M-9多肽。合成多肽的体外抗病毒试验中,间接免疫荧光和实时荧光定量PCR实验证明M-2、M-9及M-2与M-9多肽联合与PEDV作用都能够有效抑制病毒的复制,且都呈剂量依赖性。其中M-2多肽抑制病毒作用比M-9多肽高,两种多肽联合抗病毒作用最好。综上,本文为进一步研究PEDV以及M蛋白功能性配体,为今后PEDV防治及小分子治疗制剂的研发提供了理论基础和试验依据。
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.651

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