本文選題：豬流行性腹瀉病毒　切入點(diǎn)：進(jìn)化分析　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【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)引起的豬的一種以嘔吐、腹瀉和脫水為主要臨床癥狀的腸道傳染病。2010年以來(lái),該病在我國(guó)多個(gè)省份暴發(fā)流行,給生豬產(chǎn)業(yè)造成了巨大的經(jīng)濟(jì)損失。當(dāng)前,該病已成為制約我國(guó)養(yǎng)豬業(yè)健康發(fā)展的重要豬病之一。河南省是我國(guó)養(yǎng)豬大省,2010年以來(lái)的PED疫情給該省的養(yǎng)豬業(yè)帶來(lái)了沉重打擊和持續(xù)性影響。針對(duì)本次疫情開(kāi)展PEDV的遺傳進(jìn)化分析及免疫學(xué)檢測(cè)方法研究,對(duì)于認(rèn)識(shí)該病的流行規(guī)律、建立針對(duì)性的防控措施至關(guān)重要。PEDV的S蛋白是位于病毒粒子表面的結(jié)構(gòu)蛋白,含有PEDV的主要抗原表位,是建立PEDV診斷方法的重要靶抗原,S基因也是PEDV分子流行病學(xué)研究的主要候選基因;ORF3是PEDV唯一的一個(gè)輔助蛋白,與病毒的毒力有關(guān),可以用于區(qū)分PEDV的強(qiáng)、弱毒株。本論文開(kāi)展了基于ORF3基因和S基因的PEDV遺傳進(jìn)化分析,對(duì)于明確河南省PEDV的流行規(guī)律及分子遺傳特征具有重要意義;N蛋白是PEDV的衣殼蛋白,也是PEDV的主要結(jié)構(gòu)蛋白之一。在PEDV感染早期,豬體內(nèi)就能檢測(cè)出高水平的抗N蛋白抗體。因此,N蛋白可以作為靶蛋白用于PEDV的早期診斷;另外,PEDV作為豬的一種腸道病毒,引起的免疫應(yīng)答主要以粘膜免疫為主,IgA抗體水平更能反映病毒感染情況及疫苗免疫保護(hù)效力,建立PEDV IgA抗體檢測(cè)方法對(duì)于PEDV的疫苗免疫評(píng)價(jià)意義重大。本論文通過(guò)制備PEDV的S和N重組蛋白,分別建立了基于重組S蛋白的PEDV IgA抗體檢測(cè)方法和基于重組N蛋白的PEDV IgG抗體免疫層析快速檢測(cè)試紙。研究?jī)?nèi)容和結(jié)果如下:1.采用RT-PCR方法,對(duì)來(lái)自河南省許昌、新鄭、平頂山、鄭州、新鄉(xiāng)、鶴壁、開(kāi)封、駐馬店、周口、洛陽(yáng)、三門峽等地的116個(gè)不同規(guī)模大小的豬場(chǎng)開(kāi)展了PEDV病原學(xué)檢測(cè)。結(jié)果顯示,發(fā)病豬群糞便和乳汁中PEDV的陽(yáng)性率分別為60.8㳠和56.7㳠;以PEDV的ORF3基因和部分S基因?yàn)榘谢?對(duì)14個(gè)PEDV陽(yáng)性樣品毒株進(jìn)行克隆、測(cè)序并進(jìn)行了遺傳進(jìn)化分析。結(jié)果表明,14個(gè)PEDV毒株之間ORF3基因核苷酸序列的相似性為96.7-100.0㳠之間,氨基酸序列的相似性為94.0-100.0㳠,與參考毒株之間核苷酸序列的相似性為96.9-99.8㳠,氨基酸序列的相似性為94.5-99.3㳠;基于ORF3和部分S基因的核苷酸序列構(gòu)建的進(jìn)化樹(shù)顯示,14個(gè)樣品毒株之間以及與國(guó)內(nèi)毒株CH/ZMDZY/11、BJ-2011-1、JS-HZ2012、GD-B、CH/FJZZ-9/2012、CH/FJND-3/2011和CH/S親緣關(guān)系較近,而與SM98、LZC及CV777遺傳距離較遠(yuǎn);根據(jù)S基因推導(dǎo)的氨基酸序列分析結(jié)果顯示,14個(gè)樣品毒株在抗原表位區(qū)7-146 aa和271-278 aa存在較為集中的氨基酸變化,推測(cè)這些氨基酸的變化可能改變了PEDV流行毒株的抗原性,從而影響了疫苗的免疫效果。2.采用HE染色、免疫組化及形態(tài)學(xué)方法,研究了毒株CH/HNQX-3/14引起仔豬腸道的病理學(xué)變化,初步證實(shí)了回腸絨毛M細(xì)胞在PEDV感染中的重要性;在全基因組測(cè)序的基礎(chǔ)上,開(kāi)展了病毒基因組的序列比較、系統(tǒng)發(fā)育和重組分析。結(jié)果顯示,與CV777株相比,CH/HNQX-3/14的S基因存在4個(gè)明顯的缺失區(qū)域(nt 20,813-20,824,nt 21,058-21,063,nt 21,127-21,132和nt 22,252-22,257)。其基因組序列與CH/ZMDZY/11相似性最高(99.1㳠);重組分析發(fā)現(xiàn)CH/HNQX-3/14基因組當(dāng)中存在4個(gè)可靠性較高的潛在重組斷點(diǎn),分別位于ORF1a(nt 2,769)、S(nt 20,691和nt 22,176)和N(nt 27,252)基因中。CH/HNQX-3/14的核苷酸序列在斷點(diǎn)(nt 2,769)前和斷點(diǎn)(nt 27,252)后區(qū)域主要來(lái)自于CV777,在斷點(diǎn)nt 20,691和nt 22,176之間區(qū)域主要來(lái)自于DR13(韓國(guó)疫苗株的親本毒株)。推測(cè)CH/HNQX-3/14為嵌合式毒株,可能由CV777、DR13和CH/ZMDZY/11 3個(gè)毒株通過(guò)基因重組演化而來(lái)。3.對(duì)PEDV流行毒株(CH/HNQX-3/14)的部分S1基因(包含499-638 aa,748-755aa和756-771 aa 3個(gè)抗原表位區(qū))進(jìn)行了克隆,并構(gòu)建了重組表達(dá)質(zhì)粒pET30a(+)-pS1,轉(zhuǎn)化大腸桿菌BL21后,通過(guò)優(yōu)化表達(dá)條件,獲得了可溶性表達(dá)的重組pS1蛋白,該蛋白能夠與豬的PEDV陽(yáng)性血清發(fā)生特異性反應(yīng)。以純化后的重組pS1蛋白作為包被抗原,初步建立了PEDV IgA抗體間接ELISA檢測(cè)方法,該方法具有較好的特異性、敏感性和重復(fù)性,與韓國(guó)BIONOTE公司生產(chǎn)的PEDV IgA抗體檢測(cè)試劑盒相比,檢測(cè)血清和乳汁的符合率分別為93.6㳠和93.9㳠,可應(yīng)用于PEDV IgA抗體的臨床檢測(cè)及免疫評(píng)價(jià)。4.構(gòu)建了用于表達(dá)PEDV N蛋白的pET28a(+)-N重組質(zhì)粒,轉(zhuǎn)化大腸桿菌BL21后,通過(guò)優(yōu)化表達(dá)條件,實(shí)現(xiàn)了其可溶性表達(dá)。經(jīng)Ni-NTA親和層析和凝膠過(guò)濾層析對(duì)重組N蛋白進(jìn)行純化后,以膠體金標(biāo)記N蛋白固定在結(jié)合墊上作為檢測(cè)探針,分別用1.0mg/mL的金黃色葡萄球菌A蛋白(SPA)和2.9 mg/mL的His單抗包被硝酸纖維素膜作為檢測(cè)線和質(zhì)控線,制備組裝成免疫層析檢測(cè)試紙,該試紙與TGEV、CSFV、PRRSV、PRV及FMDV的陽(yáng)性血清無(wú)交叉反應(yīng),與商品化進(jìn)口ELISA試劑盒的符合率為94.0㳠。該試紙的研制為PEDV的早期快速診斷提供了新的技術(shù)手段。
[Abstract]:Porcine epidemic diarrhea (Porcine epidemic, diarrhea, PED) from porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) in pigs caused by a vomiting, diarrhea and dehydration as the main clinical symptoms of intestinal infectious diseases in.2010 years, the disease epidemic in many provinces in China, causing huge the economic losses to the pig industry. At present, the disease has become one of the important diseases restricting the healthy development of pig industry in China. Henan province is China's pig big province, has brought a heavy blow and persistent effect since 2010 PED epidemic to the province's pig industry. Research on genetic analysis and immunological detection methods of the outbreak of PEDV, the popular understanding of the law of the disease, the establishment of S protein targeted prevention and control measures are critical.PEDV structure protein located on the virion surface, the main epitope containing PEDV, is built An important target antigen PEDV diagnosis method, the main candidate S gene is PEDV molecular epidemiological study on gene; ORF3 is an auxiliary protein PEDV only, associated with virulence, PEDV can be used to distinguish the strong and weak strain. This thesis has carried out the PEDV analysis of genetic evolution because of the ORF3 gene and S based. Is of great significance for the epidemic regularity and molecular genetic characteristics of clear PEDV in Henan province; N protein is PEDV capsid protein, one of the main structural protein is PEDV. In the early stage of PEDV infection, pigs can detect high levels of anti N protein antibody. Therefore, N protein can be used as a target protein for early diagnosis of PEDV in addition, PEDV; as a pig intestinal virus, the immune response caused mainly by mucosal immunity, the antibody level of IgA could reflect the virus infection and vaccine immune protection effect, a detection of PEDV IgA antibody Method for evaluation of PEDV vaccine of great significance. This paper through the preparation of PEDV S and N recombinant protein were established PEDV IgG antibody chromatography PEDV IgA for detection of antibodies against the recombinant S protein and recombinant N protein based on fast test paper. Research contents and results are as follows: 1. using the RT-PCR method, from Henan Province, Xuchang, Xinzheng, Pingdingshan, Zhengzhou, Xinxiang, Hebi, Zhumadian, Zhoukou, Luoyang City, Sanmenxia, and other places of 116 different size farms carried out PEDV pathogen detection. The results showed that the positive rate of PEDV infected pigs feces and milk were 60.8? And 56.7?; ORF3 gene and part of the PEDV S gene as the target gene, cloning of 14 PEDV positive samples were sequenced and strain and phylogenetic analysis. The results showed that among 14 PEDV strains similarity of nucleotide sequence of ORF3 gene for the 96.7-100.0? Between the amino acid sequence similarity of 94.0-100.0?, and the similarity between the nucleotide sequences of reference strains of 96.9-99.8?, the amino acid sequence similarity of 94.5-99.3?; phylogenetic tree of nucleotide sequence of ORF3 and part of S gene to construct the display based on a sample of 14 strains between and with strains CH/ZMDZY/11, BJ-2011-1, JS-HZ2012. GD-B, CH/FJZZ-9/2012, CH/FJND-3/2011 and CH/S close genetic relationship, with SM98, LZC and CV777 genetic distance; according to the amino acid sequence analysis of S gene is showed in a sample of 14 strains in the antigen epitope of 7-146 AA and 271-278 AA amino acid change is more concentrated, it is concluded that the change of these amino acids may change the antigenicity of PEDV strains, which affect the immune effects of.2. vaccine using HE staining, immunohistochemical and morphological methods of strain CH/ induced by HNQX-3/14 in intestine of piglets The pathological changes, preliminary confirmed the importance of ileal villi M cells in PEDV infection; on the basis of whole genome sequencing, sequence comparison was carried out of the viral genome, phylogenetic and recombination analyses. The results showed that compared with the CV777 strain, S gene CH/HNQX-3/14 4 distinct deletion regions (NT 20813-20824. NT 21058-21063, NT 21127-21132 and NT 22252-22257). The CH/ZMDZY/11 genome sequence and the highest similarity (99.1?); recombinant CH/HNQX-3/14 genome analysis found that there are 4 high reliability potential recombination breakpoints were located at ORF1a (NT, 2769), S (NT 20691 and NT 22176) and N (NT 27252) gene in.CH/HNQX-3/14 the nucleotide sequence at the breakpoint (NT 2769) before and after the breakpoint (NT 27252) area mainly comes from CV777, NT 20691 and NT 22176 in the breakpoint between regions mainly from the DR13 (Korea vaccine strain Pro This suggests that CH/HNQX-3/14 strain). For chimeric strains, probably by CV777, DR13 and CH/ZMDZY/11 3 strains by gene recombination and evolution of.3. PEDV strains (CH/HNQX-3/14) of the S1 gene (including 499-638 AA, 756-771 748-755aa and 3 AA antigen epitope) were cloned, and the recombinant the expression plasmid pET30a (+) -pS1 was transformed into E. coli BL21, by optimizing the expression conditions, the soluble expression of recombinant pS1 protein, the protein to PEDV positive serum and pig specific reaction. The purified recombinant pS1 protein as antigen to establish an indirect ELISA PEDV IgA antibody detection method, this method has good specificity, sensitivity and repeatability, compared to PEDV IgA antibody detection kit production and Korea BIONOTE company, serum and milk detection rate were 93.6? And 93.9?, can be applied to PEDV Clinical detection and evaluation of immune antibody of IgA.4. was constructed for the expression of PEDV N protein pET28a (+) -N recombinant plasmid was transformed into Escherichia coli BL21, by optimizing the expression conditions, the soluble expression. By Ni-NTA affinity chromatography and gel filtration chromatography of recombinant N protein was purified using colloidal gold labeled N protein. In combination with the fixed pad as the detection probe, respectively with 1.0mg/mL of Staphylococcus aureus protein A (SPA) and 2.9 mg/mL His monoclonal antibody coated on the nitrocellulose membrane as the test line and control line, the preparation of assembled immunochromatography test strip, the strip and TGEV, CSFV, PRRSV, no cross reaction with positive serum PRV and FMDV, and imported commercial ELISA kit with the rate of 94?. the development of test paper for rapid diagnosis provides new technical means for early PEDV.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65

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