猪流行性腹泻病毒抗原基因COE的原核表达与纯化

发布时间：2018-06-26 23:46

本文选题：猪流行性腹泻病毒 + 疫苗　；参考：《河南农业大学》2015年硕士论文

【摘要】：猪流行性腹泻病(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引发的一种肠道传染病,自从该病在我国流行以来,给我国养猪业造成了重大的经济损失。猪场主要通过日常消毒和接种疫苗来预防该病,但目前在市场上流行的疫苗并不能到达理想的免疫效果。研究表明仔猪主要通过初乳获得母源SIgA(分泌型免疫球蛋白A)产生对PEDV的被动免疫保护,血液中循环的IgG抗体不能对仔猪产生有效的保护,非经肠道黏膜免疫不能获得SIgA,因此通过肠道黏膜免疫是使仔猪获得免疫保护的重要途径,研制安全有效可以通过肠道黏膜免疫的新型疫苗尤为重要。S蛋白是由S基因编码的PEDV一个表面结构蛋白,也是PEDV的主要结构蛋白,它含有介导病毒侵入宿主细胞的受体结合域,还含有介导机体产生病毒中和抗体的抗原表位。有可以诱导机体产生中和抗体和提供黏膜免疫保护的作用。COE抗原表位是S基因中的一个重要抗原表位,是由chang等人发现并命名的,这个抗原表位位于S基因的499-638aa区域。自从COE抗原表位被发现后世界各国学者均尝试应用COE抗原表位基因研究制备PEDV的基因工程疫苗。本实验是研究PEDV主要抗原决定簇COE的原核表达与纯化,为研制新型的猪流行性腹泻病毒疫苗奠定基础。本实验首先从河南本地的疑似得猪流行性腹泻病的病猪小肠分离出PEDV,然后根据GenBank发表的PEDV-COE序列设计出一对引物,用PCR的方法扩增出长度为420bp的COE目的片段。将目的片段连接在载体PMD19-T上,然后转化到JM109大肠杆菌中,涂板过夜生长,挑菌接种到液体LB中培养扩增、提质粒,鉴定正确后送公司测序,将测序结果与GenBank公布的最相近的4个PEDV-COE序列做比较,发现河南本地PEDV发生了一定的突变,与河南周边地区的毒株具有一定的差异。将COE基因片段连接至PET-32a载体上构建原核表达载体PET-32a-COE,鉴定正确后将表达载体转化到BL21(DE3)pLysS大肠杆菌中。为了探索最佳的诱导条件,我们对IPTG诱导的浓度和时间进行了优化,结果显示0.6mmol/L的IPTG诱导4h能够得到最大量的目的蛋白。我们将诱导表达目的蛋白的大肠杆菌经超声破碎、经镍株蛋白纯化试剂盒纯化,透析复性,最后得到了HIS-COE目的蛋白。HIS-COE目的蛋白的成功表达与纯化为制备能够预防在本地区流行的PEDV的新型疫苗提供了参考,对PEDV基因工程疫苗的研究与制备也具有重要意义。
[Abstract]:Porcine epidemic diarrhea (PED) is a kind of intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). Pig farm mainly through daily disinfection and vaccination to prevent the disease, but the current popular vaccine in the market can not reach the ideal immune effect. The results showed that maternal Siga (secretory immunoglobulin A) produced passive immune protection against PEDV in piglets, and circulating IgG antibody in blood could not protect piglets effectively. Siga can not be obtained without intestinal mucosal immunization, so it is an important way for piglets to get immunity protection through intestinal mucosal immunization. It is particularly important to develop a new vaccine that can be immunized safely and effectively through intestinal mucosa. S protein is a surface structural protein encoded by S gene and the main structural protein of PEDV. It contains a receptor binding domain that mediates the invasion of the virus into host cells. It also contains antigenic epitopes that mediate the production of viral neutralizing antibodies. Coe epitope is an important antigen epitope in S gene which was discovered and named by chang et al. It is located in the 499-638aa region of S gene. Since the discovery of Coe epitopes, researchers all over the world have tried to prepare PEDV genetic engineering vaccine by using Coe epitope gene research. This study was aimed to study the prokaryotic expression and purification of PEDV major antigen determinant Coe, which laid a foundation for the development of a novel porcine epidemic diarrhea virus vaccine. A pair of primers were designed according to the PEDV-COE sequence published in GenBank to amplify the Coe fragment with length of 420bp. The target fragment was ligated into the vector PMD19-T, then transformed into E. coli JM109, and then grew overnight on the coated plate, inoculated into liquid LB, cultured and amplified, extracted the plasmid, and identified correctly and sent to the company for sequencing. The sequencing results were compared with the four most similar PEDV-COE sequences published by GenBank. It was found that the local PEDV mutation in Henan Province was different from that in the surrounding regions of Henan Province. Coe gene fragment was ligated into PET-32a vector to construct the prokaryotic expression vector PET-32a-COE, and the expression vector was transformed into BL21 (DE3) pLysS Escherichia coli. In order to explore the optimal induction conditions, we optimized the concentration and time of IPTG induction. The results showed that 0.6 mmol / L IPTG induction for 4 h could obtain the maximum amount of target protein. We will induce the expression of the target protein of Escherichia coli by ultrasonic fragmentation, purification of nickel strain protein purification kit, dialysis renaturation, Finally, the successful expression and purification of HIS-COE target protein 路HIS-COE target protein provided a reference for the preparation of new PEDV vaccine, which could prevent PEDV from becoming prevalent in our region. It is also of great significance for the research and preparation of PEDV gene engineering vaccine.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.651

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