河南省猪流行性腹泻病毒遗传进化分析及免疫学检测方法的建立

发布时间：2018-03-14 02:11

本文选题：猪流行性腹泻病毒　切入点：进化分析　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

【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)引起的猪的一种以呕吐、腹泻和脱水为主要临床症状的肠道传染病。2010年以来,该病在我国多个省份暴发流行,给生猪产业造成了巨大的经济损失。当前,该病已成为制约我国养猪业健康发展的重要猪病之一。河南省是我国养猪大省,2010年以来的PED疫情给该省的养猪业带来了沉重打击和持续性影响。针对本次疫情开展PEDV的遗传进化分析及免疫学检测方法研究,对于认识该病的流行规律、建立针对性的防控措施至关重要。PEDV的S蛋白是位于病毒粒子表面的结构蛋白,含有PEDV的主要抗原表位,是建立PEDV诊断方法的重要靶抗原,S基因也是PEDV分子流行病学研究的主要候选基因;ORF3是PEDV唯一的一个辅助蛋白,与病毒的毒力有关,可以用于区分PEDV的强、弱毒株。本论文开展了基于ORF3基因和S基因的PEDV遗传进化分析,对于明确河南省PEDV的流行规律及分子遗传特征具有重要意义;N蛋白是PEDV的衣壳蛋白,也是PEDV的主要结构蛋白之一。在PEDV感染早期,猪体内就能检测出高水平的抗N蛋白抗体。因此,N蛋白可以作为靶蛋白用于PEDV的早期诊断;另外,PEDV作为猪的一种肠道病毒,引起的免疫应答主要以粘膜免疫为主,IgA抗体水平更能反映病毒感染情况及疫苗免疫保护效力,建立PEDV IgA抗体检测方法对于PEDV的疫苗免疫评价意义重大。本论文通过制备PEDV的S和N重组蛋白,分别建立了基于重组S蛋白的PEDV IgA抗体检测方法和基于重组N蛋白的PEDV IgG抗体免疫层析快速检测试纸。研究内容和结果如下:1.采用RT-PCR方法,对来自河南省许昌、新郑、平顶山、郑州、新乡、鹤壁、开封、驻马店、周口、洛阳、三门峡等地的116个不同规模大小的猪场开展了PEDV病原学检测。结果显示,发病猪群粪便和乳汁中PEDV的阳性率分别为60.8㳠和56.7㳠;以PEDV的ORF3基因和部分S基因为靶基因,对14个PEDV阳性样品毒株进行克隆、测序并进行了遗传进化分析。结果表明,14个PEDV毒株之间ORF3基因核苷酸序列的相似性为96.7-100.0㳠之间,氨基酸序列的相似性为94.0-100.0㳠,与参考毒株之间核苷酸序列的相似性为96.9-99.8㳠,氨基酸序列的相似性为94.5-99.3㳠;基于ORF3和部分S基因的核苷酸序列构建的进化树显示,14个样品毒株之间以及与国内毒株CH/ZMDZY/11、BJ-2011-1、JS-HZ2012、GD-B、CH/FJZZ-9/2012、CH/FJND-3/2011和CH/S亲缘关系较近,而与SM98、LZC及CV777遗传距离较远;根据S基因推导的氨基酸序列分析结果显示,14个样品毒株在抗原表位区7-146 aa和271-278 aa存在较为集中的氨基酸变化,推测这些氨基酸的变化可能改变了PEDV流行毒株的抗原性,从而影响了疫苗的免疫效果。2.采用HE染色、免疫组化及形态学方法,研究了毒株CH/HNQX-3/14引起仔猪肠道的病理学变化,初步证实了回肠绒毛M细胞在PEDV感染中的重要性;在全基因组测序的基础上,开展了病毒基因组的序列比较、系统发育和重组分析。结果显示,与CV777株相比,CH/HNQX-3/14的S基因存在4个明显的缺失区域(nt 20,813-20,824,nt 21,058-21,063,nt 21,127-21,132和nt 22,252-22,257)。其基因组序列与CH/ZMDZY/11相似性最高(99.1㳠);重组分析发现CH/HNQX-3/14基因组当中存在4个可靠性较高的潜在重组断点,分别位于ORF1a(nt 2,769)、S(nt 20,691和nt 22,176)和N(nt 27,252)基因中。CH/HNQX-3/14的核苷酸序列在断点(nt 2,769)前和断点(nt 27,252)后区域主要来自于CV777,在断点nt 20,691和nt 22,176之间区域主要来自于DR13(韩国疫苗株的亲本毒株)。推测CH/HNQX-3/14为嵌合式毒株,可能由CV777、DR13和CH/ZMDZY/11 3个毒株通过基因重组演化而来。3.对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个抗原表位区)进行了克隆,并构建了重组表达质粒pET30a(+)-pS1,转化大肠杆菌BL21后,通过优化表达条件,获得了可溶性表达的重组pS1蛋白,该蛋白能够与猪的PEDV阳性血清发生特异性反应。以纯化后的重组pS1蛋白作为包被抗原,初步建立了PEDV IgA抗体间接ELISA检测方法,该方法具有较好的特异性、敏感性和重复性,与韩国BIONOTE公司生产的PEDV IgA抗体检测试剂盒相比,检测血清和乳汁的符合率分别为93.6㳠和93.9㳠,可应用于PEDV IgA抗体的临床检测及免疫评价。4.构建了用于表达PEDV N蛋白的pET28a(+)-N重组质粒,转化大肠杆菌BL21后,通过优化表达条件,实现了其可溶性表达。经Ni-NTA亲和层析和凝胶过滤层析对重组N蛋白进行纯化后,以胶体金标记N蛋白固定在结合垫上作为检测探针,分别用1.0mg/mL的金黄色葡萄球菌A蛋白(SPA)和2.9 mg/mL的His单抗包被硝酸纤维素膜作为检测线和质控线,制备组装成免疫层析检测试纸,该试纸与TGEV、CSFV、PRRSV、PRV及FMDV的阳性血清无交叉反应,与商品化进口ELISA试剂盒的符合率为94.0㳠。该试纸的研制为PEDV的早期快速诊断提供了新的技术手段。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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