發(fā)布時間：2018-08-02 16:10

【摘要】：豬流行性腹瀉(Porcine Epidemic Diarrhea,PED)是由豬流行性腹瀉病毒(Porcine Epidemic Diarrhea Virus,PEDV)引起、以嘔吐、脫水、腹瀉為主要臨床癥狀的豬腸道傳染病。本研究測定了兩株P(guān)EDV全基因組序列,并對截短S蛋白進(jìn)行表達(dá)。具體研究內(nèi)容如下:1.PEDV全基因組序列分析根據(jù)PEDV經(jīng)典株CV777全基因組序列,設(shè)計(jì)25對相互重疊的引物,以擴(kuò)增PEDV全長。RT-PCR擴(kuò)增、純化后連接至pMD18-T載體,轉(zhuǎn)化至DH5a感受態(tài)中,挑選陽性克隆進(jìn)行測序,確定各片段序列后拼接為全基因組并進(jìn)行分析。結(jié)果表明,JSLS-1、JS-2全基因組全長分別為27861nt、27930nt(GenBank登錄號為KX534205、KX534206),基因組排列與PEDV經(jīng)典毒株CV777一樣,為5’-ORF1a-ORF1b-S-ORF3-E-M-N-3’。將JSLS-1、JS-2株及其他30株已測得全基因組的PEDV毒株進(jìn)行比較,并使用MEGA7.0軟件繪制系統(tǒng)發(fā)育樹。結(jié)果顯示,32株P(guān)EDV可分為兩個大群即I群與II群;I群主要由2010年以后報(bào)道的毒株(包括美國流行株);II群主要由經(jīng)典毒株組成,JSLS-1、JS-2屬于II群。本研究分析比較了JSLS-1、JS-2株5‘UTR、3‘UTR區(qū)域、ORF1a、及結(jié)構(gòu)蛋白S、E、M、N及非結(jié)構(gòu)蛋白ORF3的遺傳進(jìn)化特征。與經(jīng)典毒株CV777相比,JSLS-1 5’UTR發(fā)生堿基插入、缺失、突變,3‘UTR發(fā)生堿基插入、突變,ORF1a、S、E、ORF3發(fā)生堿基缺失、突變,M、N基因僅發(fā)生突變,未出現(xiàn)插入或缺失;JS-2株基因突變、缺失、插入情況同JSLS-1。2.PEDV部分S蛋白原核表達(dá)及高免血清制備使用生物信息學(xué)軟件及在線分析網(wǎng)站分析PEDV JS-2株S蛋白序列,選取抗原表位集中的兩個區(qū)域;同時選取PEDV S基因抗原表位區(qū)域(COE),設(shè)計(jì)帶有酶切位點(diǎn)的引物,RT-PCR擴(kuò)增出三個基因片段,將擴(kuò)增出的片段克隆至pMD18-T載體上,通過測序分析正是該片段與JS-2的S基因序列一致;然后將克隆化截短S1基因亞克隆到表達(dá)載體pET32a(+),獲得了重組表達(dá)質(zhì)粒。將重組表達(dá)質(zhì)粒轉(zhuǎn)化到宿主菌BL21(DE3),經(jīng)1mmol/L IPTG誘導(dǎo)后,成功表達(dá)三個截短蛋白,蛋白大小與預(yù)期一致。經(jīng)表達(dá)形式鑒定,目的蛋白以包涵體形式存在。使用割膠純化的方法獲得高純度重組蛋白。將蛋白與賽彼科206佐劑乳化后免疫5周齡昆明小白鼠雌鼠,十天后二免,二免十天后三免,三免十四天后采集血液、制備血清、處死小鼠。使用PEDV全病毒作為包被抗原,將所有血清進(jìn)行200倍稀釋后檢測其中的抗體效價,ELISA檢測結(jié)果表明:免疫后抗體水平明顯上升,三免十四天OD450約為1,表現(xiàn)出較高的抗體水平。證明所選的三個S蛋白區(qū)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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.651

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