基于N蛋白的PEDV抗体ELISA检测方法的建立及初步应用

发布时间：2018-03-27 22:09

本文选题：猪流行性腹泻　切入点：遗传变异　出处：《西北农林科技大学》2017年硕士论文

【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)引起感染猪表现为呕吐、发热、脱水、严重腹泻的一种高度接触性肠道传染病,发病主要集中在冬季,可感染各阶段猪,但以哺乳仔猪发病和病死率最高,可达100%,给全世界养猪业造成巨大经济损失。目前没有有效的防控措施,有研究表明,接种过疫苗的猪群照样有暴发PED的情况,到2010年这一现象更为严重。因此,建立检测PEDV抗体水平的诊断方法很有必要。本试验对PEDV N基因进行了克隆和原核表达,用纯化重组N蛋白为包被抗原,建立了PEDV血清抗体水平间接ELISA检测方法并初步应用于临床,获得如下研究结果:1.采集陕西省部分地区猪场疑似感染PEDV死亡的猪小肠样品5份,用RT-PCR扩增5份样品的N、S和M基因,分别命名为1-SL、2-BJ、3-YL、4-WN和5-HZ。同源性比较分析,本省流行的5个毒株与中国现用疫苗株CV777的S、M和N基因中S基因同源相似性最低,核苷酸相似性为94.9%~99.2%,氨基酸序列相似性为94.9%~99.7%,其次是N基因,核苷酸同源相似性为95.1%~99.9%,氨基酸序列相似性为96.2%~100%,M基因同源相似性最高,氨基酸序列相似性为96.2%~100%;遗传进化树分析,5个毒株的S、M和N基因均与疫苗株CV777遗传亲缘关系相对较远;同一省份不同地区5株流行株的不同基因遗传亲缘远近不一样。2.设计N基因的一对特异性引物,提取病毒RNA,以HiScript?Q RT SuperMix for qPCR说明书反转录的cDNA为模版,PCR扩增和核酸胶回收目的基因,克隆N基因并原核表达,重组质粒pET-28a-N转化至大肠埃希菌BL21。重组蛋白分子质量约58 kD,SDS-PAGE分析为可溶性蛋白。用优质镍柱纯化裂解上清液,获得量大、纯度较高的蛋白,Western blot分析与PEDV阳性血清有良好的特异反应性。用纯化N蛋白为包被抗原,ELISA检测最优条件为:抗原包被量每孔0.5μg/mL,血清以1:200倍稀释作用1.0h,酶标二抗以1:5000稀释作用1.0 h,TMB显色时间25 min。敏感性、特异性、重复性检测均良好,初步检测临床164份血清,效果较好。
[Abstract]:Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection caused by porcine epidemic diarrhea virus (PEDVV), which is characterized by vomiting, fever, dehydration and severe diarrhea in pigs. However, the suckling piglets have the highest morbidity and fatality rate, which can reach 100, causing huge economic losses to the world pig industry. There are no effective prevention and control measures at present. Studies have shown that vaccinated pigs still have PED outbreaks. By 2010, this phenomenon is more serious. Therefore, it is necessary to establish a diagnostic method to detect the level of PEDV antibody. In this study, the PEDV N gene was cloned and expressed in prokaryotic cells, and the purified recombinant N protein was used as the coated antigen. The indirect ELISA detection method of PEDV serum antibody level was established and applied to clinical practice. The following results were obtained: 1. Five small intestine samples of pigs suspected to be infected with PEDV were collected from pig farms in some parts of Shaanxi Province, and 5 samples were amplified by RT-PCR. The homology analysis showed that the homology of S gene was the lowest, the nucleotide similarity was 94.9 ~ 99.2, amino acid sequence similarity was 94.9m ~ 99.2, amino acid sequence similarity was 94.9m ~ 99.7, followed by N gene. The homology of nucleotide sequence and amino acid sequence was 95.1% and 99.9, respectively. The similarity of amino acid sequence and amino acid sequence was the highest, and the similarity of amino acid sequence was 96.20.The genetic phylogenetic tree analysis showed that the Sm and N genes of the five virulent strains were relatively far related to the CV777 of the vaccine strain. The genetic relationship of different genes of 5 epidemic strains in the same province is different. 2. Design a pair of specific primers of N gene to extract virus RNAs and use HiScript? The reverse transcription cDNA of Q RT SuperMix for qPCR was the target gene of template PCR amplification and nucleic acid gel recovery, the N gene was cloned and expressed in prokaryotic cells. The recombinant plasmid pET-28a-N was transformed into Escherichia coli BL21. The recombinant protein was analyzed into soluble protein by SDS-PAGE with a molecular weight of about 58 kD. The supernatant was purified by high quality nickel column. Western blot analysis of protein with high purity and PEDV positive serum showed good specific reactivity. The optimal conditions for detection of purified N protein by Elisa were as follows: antigen encapsulation volume was 0.5 渭 g / mL, serum was diluted by 1: 200 times for 1.0 h, and enzyme labeled second antibody was used as Elisa method. Sensitivity of 1: 5000 diluted to 1.0 h TMB for 25 mins, The specificity and repeatability were all good, and 164 serum samples were detected preliminarily.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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