猪流行性腹泻病毒全基因组序列测定及截短S基因的原核表达

发布时间：2018-08-02 16:10

【摘要】：猪流行性腹泻(Porcine Epidemic Diarrhea,PED)是由猪流行性腹泻病毒(Porcine Epidemic Diarrhea Virus,PEDV)引起、以呕吐、脱水、腹泻为主要临床症状的猪肠道传染病。本研究测定了两株PEDV全基因组序列,并对截短S蛋白进行表达。具体研究内容如下:1.PEDV全基因组序列分析根据PEDV经典株CV777全基因组序列,设计25对相互重叠的引物,以扩增PEDV全长。RT-PCR扩增、纯化后连接至pMD18-T载体,转化至DH5a感受态中,挑选阳性克隆进行测序,确定各片段序列后拼接为全基因组并进行分析。结果表明,JSLS-1、JS-2全基因组全长分别为27861nt、27930nt(GenBank登录号为KX534205、KX534206),基因组排列与PEDV经典毒株CV777一样,为5’-ORF1a-ORF1b-S-ORF3-E-M-N-3’。将JSLS-1、JS-2株及其他30株已测得全基因组的PEDV毒株进行比较,并使用MEGA7.0软件绘制系统发育树。结果显示,32株PEDV可分为两个大群即I群与II群;I群主要由2010年以后报道的毒株(包括美国流行株);II群主要由经典毒株组成,JSLS-1、JS-2属于II群。本研究分析比较了JSLS-1、JS-2株5‘UTR、3‘UTR区域、ORF1a、及结构蛋白S、E、M、N及非结构蛋白ORF3的遗传进化特征。与经典毒株CV777相比,JSLS-1 5’UTR发生碱基插入、缺失、突变,3‘UTR发生碱基插入、突变,ORF1a、S、E、ORF3发生碱基缺失、突变,M、N基因仅发生突变,未出现插入或缺失;JS-2株基因突变、缺失、插入情况同JSLS-1。2.PEDV部分S蛋白原核表达及高免血清制备使用生物信息学软件及在线分析网站分析PEDV JS-2株S蛋白序列,选取抗原表位集中的两个区域;同时选取PEDV S基因抗原表位区域(COE),设计带有酶切位点的引物,RT-PCR扩增出三个基因片段,将扩增出的片段克隆至pMD18-T载体上,通过测序分析正是该片段与JS-2的S基因序列一致;然后将克隆化截短S1基因亚克隆到表达载体pET32a(+),获得了重组表达质粒。将重组表达质粒转化到宿主菌BL21(DE3),经1mmol/L IPTG诱导后,成功表达三个截短蛋白,蛋白大小与预期一致。经表达形式鉴定,目的蛋白以包涵体形式存在。使用割胶纯化的方法获得高纯度重组蛋白。将蛋白与赛彼科206佐剂乳化后免疫5周龄昆明小白鼠雌鼠,十天后二免,二免十天后三免,三免十四天后采集血液、制备血清、处死小鼠。使用PEDV全病毒作为包被抗原,将所有血清进行200倍稀释后检测其中的抗体效价,ELISA检测结果表明:免疫后抗体水平明显上升,三免十四天OD450约为1,表现出较高的抗体水平。证明所选的三个S蛋白区域具有一定的抗原性。
[Abstract]:Porcine epidemic diarrhea (Porcine Epidemic) is caused by porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea) and its main clinical symptoms are vomiting, dehydration and diarrhea. In this study, the whole genome sequences of two PEDV strains were sequenced and truncated S protein was expressed. The specific contents of the study are as follows: 1. The whole genome sequence of PEDV was analyzed. According to the whole genome sequence of PEDV classic strain CV777, 25 pairs of overlapping primers were designed to amplify the whole length of PEDV. RT-PCR amplification was used to amplify the whole length of PEDV, then ligated to the pMD18-T vector and transformed into the DH5a receptive state. The positive clones were selected and sequenced. The fragments were sequenced and sequenced into the whole genome. The results showed that the whole genome length of JSLS-1 / JS-2 was 27861 NT (GenBank accession number was KX534205 (KX534206), and the genome arrangement was similar to that of the classical PEDV strain CV777, which was 5F1a-ORF1b-ORF1b-S-ORF3-E-M-N-30.The results showed that the total genome length of JSLS-1 was 27861 NT (GenBank accession number was KX534205, KX534206). A comparison was made between JSLS-1 strain JS-2 and other 30 strains of PEDV virus which had been detected in the whole genome, and the phylogenetic tree was plotted by MEGA7.0 software. The results showed that 32 strains of PEDV could be divided into two groups, I. E. group I and group II. The group II was mainly composed of classical strains, JSLS-1 and JS-2, which belonged to group II. In this study, the genetic and evolutionary characteristics of ORF1a, the structural protein Ser EMN and the non-structural protein ORF3 of JSLS-1 / JS-2 strain 5UTR3UTR3UTR region were analyzed and compared. Compared with the classical virus strain CV777, the JSLS-1 5'UTR had base insertion, deletion, mutation 3UTR, base deletion, mutated ORF1aSEOORF3, only mutation of MN gene, and no insertion or deletion mutation of JS-2 strain. The S protein sequence of PEDV JS-2 strain was analyzed by bioinformatics software and online analysis website, and two regions of antigen epitope concentration were selected. At the same time, the PEDV S epitope region (COE), was selected to design the primers with restriction endonuclease site to amplify three gene fragments, and the amplified fragments were cloned into the pMD18-T vector. The sequence analysis showed that the fragment was identical to the S gene sequence of JS-2. Then the truncated S1 gene was subcloned into the expression vector pET32a (), to obtain the recombinant expression plasmid. The recombinant expression plasmid was transformed into the host strain BL21 (DE3). After induction by 1mmol/L IPTG, three truncated proteins were successfully expressed, and the protein size was the same as expected. The target protein was identified as inclusion body by expression. The recombinant protein with high purity was obtained by the method of gel purification. The mice were immunized with the adjuvant cipika 206 after emulsification. The mice were immunized twice after 10 days, 3 after 10 days, and 14 days after three immunizations. The serum was prepared and the mice were killed. PEDV whole virus was used as the coating antigen. The antibody titers were detected by Elisa after 200 times dilution. The results showed that the antibody level was obviously increased after immunization, and the OD450 was about 1 on the 3rd day after immunization, showing a higher antibody level. It was proved that the three S protein regions were antigenicity.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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