猪嵴病毒CH441株VP3基因的原核表达及间接ELISA方法的建立
发布时间：2019-02-12 07:12
【摘要】：致使猪死亡的原因有很多,腹泻是重要原因之一,给养猪业带来的经济损失很大。近年来,新发病原在猪群中被不断检测到,在腹泻猪群中检测到的猪嵴病毒(Porcine kobuvirus,PKV),有较高的阳性检出率。猪嵴病毒(porcine kobuvirus,PKV)属于微小核糖核酸病毒科(picornaviridae)嵴病毒属,是一种没有囊膜的单股正链RNA病毒。目前国内关于猪嵴病毒的研究较少,检测方法较少,有待进一步完善。为建立猪嵴病毒的间接ELISA检测方法,本研究对GenBank中登录16株猪嵴病毒的VP1基因与VP3基因进行了序列分析,挑选出适合用来建立间接ELISA检测方法的基因,然后建立猪嵴病毒的间接ELISA检测方法。研究内容包括:1.猪嵴病毒VP1基因的克隆与序列分析利用RT-PCR方法克隆了猪嵴病毒CH441株VP1基因。并对该基因进行序列分析,核苷酸一致性分析结果显示,猪嵴病毒CH441株VP1基因与其他15株猪嵴病毒VP1基因核苷酸相似性在81.5%~90.2%,表明VP1基因存在较为明显的变异。氨基酸同源性分析结果显示,氨基酸序列相似性为86.6%~96.9%,该结果明显高于核苷酸相似性,表明VP1基因的部分变异为无义突变。2.猪嵴病毒VP3基因的克隆分析与原核表达通过RT-PCR成功克隆了猪嵴病毒CH441株VP3基因。对VP3基因进行序列分析,核苷酸一致性分析结果显示,猪嵴病毒CH441株VP3基因与其他15株猪嵴病毒VP3基因核苷酸相似性在84.0%~90.7%。氨基酸同源性分析结果显示,猪嵴病毒VP3基因的氨基酸序列相似性为91.5%~99.1%。上述结果表明,猪嵴病毒CH441株VP3基因与其他15株猪嵴病毒VP3基因核苷酸、氨基酸相似性均高于VP1基因,说明VP3基因的变异比VP1基因的小,更适合作为建立诊断方法的候选蛋白。本研究应用原核表达系统表达了VP3蛋白,并用纯化的VP3蛋白制备了猪源高免血清。3.猪嵴病毒间接ELISA检测方法的建立利用纯化的猪嵴病毒VP3蛋白作为包被抗原,成功建立了特异性与稳定性高的检测猪嵴病毒抗体的间接ELISA方法,为猪嵴病毒的诊断成立了一种新的方法。通过本研究建立的VP3抗体检测方法对临床采集的44份腹泻猪血清样本进行检测,VP3抗体阳性率为63.64%,检测结果显示在甘肃地区发生腹泻的猪群中,猪嵴病毒具有较高的阳性检出率。
[Abstract]:There are many causes of pig death, diarrhea is one of the important reasons, which brings great economic loss to pig industry. In recent years, the new pathogen has been continuously detected in pig herd, and the positive rate of pig ridge virus (Porcine kobuvirus,PKV) detected in diarrhea pig herd is high. Porcine Crista virus (porcine kobuvirus,PKV) belongs to the genus (picornaviridae) crest virus of the family (picornaviridae), which is a single-stranded positive strand RNA virus with no envelope. At present, there are few researches and detection methods about porcine cristae virus in China, which need to be further improved. In order to establish an indirect ELISA detection method for porcine cristae virus, the VP1 and VP3 genes of 16 GenBank strains were sequenced and the genes suitable for indirect ELISA detection were selected. Then an indirect ELISA detection method for porcine cristae virus was established. The research contents include: 1. Cloning and sequence Analysis of VP1 Gene of Porcine Ridge virus CH441 strain was cloned by RT-PCR. The nucleotide identity analysis showed that the nucleotide similarity between the VP1 gene of porcine crest virus CH441 strain and other 15 strains of porcine crest virus VP1 gene was 81.5% 90.2, which indicated that the VP1 gene had obvious variation. The results of amino acid homology analysis showed that the amino acid sequence similarity was 86.6% and 96.9%, which was significantly higher than that of nucleotide similarity, indicating that some of the mutations of VP1 gene were nonsense. 2. Cloning analysis and prokaryotic expression of porcine crest virus VP3 gene of porcine crest virus CH441 strain was successfully cloned by RT-PCR. The nucleotide identity analysis of VP3 gene showed that the nucleotide similarity of VP3 gene of porcine crest virus CH441 strain and 15 other porcine crest virus VP3 genes was 84.0 / 90.7. The results of amino acid homology analysis showed that the amino acid sequence similarity of VP3 gene of porcine cristae virus was 91.5% 99.1%. The results showed that the nucleotide similarity and amino acid similarity of VP3 gene between porcine crest virus CH441 strain and 15 other porcine crest virus VP3 genes were higher than that of VP1 gene, indicating that the variation of VP3 gene was smaller than that of VP1 gene, which suggested that VP3 gene was more suitable as a candidate protein for establishing diagnostic methods. In this study, prokaryotic expression system was used to express VP3 protein, and purified VP3 protein was used to prepare porcine serum. Establishment of indirect ELISA Detection method for Porcine Ridge virus using purified Porcine Ridge virus VP3 protein as coating antigen, an indirect ELISA method with high specificity and stability for detection of porcine cristal virus antibody was successfully established. A new method for the diagnosis of porcine crest virus was established. The VP3 antibody detection method was established in this study to detect 44 serum samples of diarrhea pigs. The positive rate of VP3 antibody was 63.64. The results showed that the positive rate of VP3 antibody was 63.64. The results showed that the positive rate of VP3 antibody was 63.64 in the pigs with diarrhea in Gansu area. The positive rate of porcine cristae virus was high.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
本文编号：2420191
[Abstract]:There are many causes of pig death, diarrhea is one of the important reasons, which brings great economic loss to pig industry. In recent years, the new pathogen has been continuously detected in pig herd, and the positive rate of pig ridge virus (Porcine kobuvirus,PKV) detected in diarrhea pig herd is high. Porcine Crista virus (porcine kobuvirus,PKV) belongs to the genus (picornaviridae) crest virus of the family (picornaviridae), which is a single-stranded positive strand RNA virus with no envelope. At present, there are few researches and detection methods about porcine cristae virus in China, which need to be further improved. In order to establish an indirect ELISA detection method for porcine cristae virus, the VP1 and VP3 genes of 16 GenBank strains were sequenced and the genes suitable for indirect ELISA detection were selected. Then an indirect ELISA detection method for porcine cristae virus was established. The research contents include: 1. Cloning and sequence Analysis of VP1 Gene of Porcine Ridge virus CH441 strain was cloned by RT-PCR. The nucleotide identity analysis showed that the nucleotide similarity between the VP1 gene of porcine crest virus CH441 strain and other 15 strains of porcine crest virus VP1 gene was 81.5% 90.2, which indicated that the VP1 gene had obvious variation. The results of amino acid homology analysis showed that the amino acid sequence similarity was 86.6% and 96.9%, which was significantly higher than that of nucleotide similarity, indicating that some of the mutations of VP1 gene were nonsense. 2. Cloning analysis and prokaryotic expression of porcine crest virus VP3 gene of porcine crest virus CH441 strain was successfully cloned by RT-PCR. The nucleotide identity analysis of VP3 gene showed that the nucleotide similarity of VP3 gene of porcine crest virus CH441 strain and 15 other porcine crest virus VP3 genes was 84.0 / 90.7. The results of amino acid homology analysis showed that the amino acid sequence similarity of VP3 gene of porcine cristae virus was 91.5% 99.1%. The results showed that the nucleotide similarity and amino acid similarity of VP3 gene between porcine crest virus CH441 strain and 15 other porcine crest virus VP3 genes were higher than that of VP1 gene, indicating that the variation of VP3 gene was smaller than that of VP1 gene, which suggested that VP3 gene was more suitable as a candidate protein for establishing diagnostic methods. In this study, prokaryotic expression system was used to express VP3 protein, and purified VP3 protein was used to prepare porcine serum. Establishment of indirect ELISA Detection method for Porcine Ridge virus using purified Porcine Ridge virus VP3 protein as coating antigen, an indirect ELISA method with high specificity and stability for detection of porcine cristal virus antibody was successfully established. A new method for the diagnosis of porcine crest virus was established. The VP3 antibody detection method was established in this study to detect 44 serum samples of diarrhea pigs. The positive rate of VP3 antibody was 63.64. The results showed that the positive rate of VP3 antibody was 63.64. The results showed that the positive rate of VP3 antibody was 63.64 in the pigs with diarrhea in Gansu area. The positive rate of porcine cristae virus was high.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
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1 祝俊鹏;猪嵴病毒CH441株VP3基因的原核表达及间接ELISA方法的建立[D];甘肃农业大学;2015年
，本文编号：2420191
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