【摘要】：致使豬死亡的原因有很多,腹瀉是重要原因之一,給養(yǎng)豬業(yè)帶來的經(jīng)濟(jì)損失很大。近年來,新發(fā)病原在豬群中被不斷檢測到,在腹瀉豬群中檢測到的豬嵴病毒(Porcine kobuvirus,PKV),有較高的陽性檢出率。豬嵴病毒(porcine kobuvirus,PKV)屬于微小核糖核酸病毒科(picornaviridae)嵴病毒屬,是一種沒有囊膜的單股正鏈RNA病毒。目前國內(nèi)關(guān)于豬嵴病毒的研究較少,檢測方法較少,有待進(jìn)一步完善。為建立豬嵴病毒的間接ELISA檢測方法,本研究對GenBank中登錄16株豬嵴病毒的VP1基因與VP3基因進(jìn)行了序列分析,挑選出適合用來建立間接ELISA檢測方法的基因,然后建立豬嵴病毒的間接ELISA檢測方法。研究內(nèi)容包括:1.豬嵴病毒VP1基因的克隆與序列分析利用RT-PCR方法克隆了豬嵴病毒CH441株VP1基因。并對該基因進(jìn)行序列分析,核苷酸一致性分析結(jié)果顯示,豬嵴病毒CH441株VP1基因與其他15株豬嵴病毒VP1基因核苷酸相似性在81.5%~90.2%,表明VP1基因存在較為明顯的變異。氨基酸同源性分析結(jié)果顯示,氨基酸序列相似性為86.6%~96.9%,該結(jié)果明顯高于核苷酸相似性,表明VP1基因的部分變異為無義突變。2.豬嵴病毒VP3基因的克隆分析與原核表達(dá)通過RT-PCR成功克隆了豬嵴病毒CH441株VP3基因。對VP3基因進(jìn)行序列分析,核苷酸一致性分析結(jié)果顯示,豬嵴病毒CH441株VP3基因與其他15株豬嵴病毒VP3基因核苷酸相似性在84.0%~90.7%。氨基酸同源性分析結(jié)果顯示,豬嵴病毒VP3基因的氨基酸序列相似性為91.5%~99.1%。上述結(jié)果表明,豬嵴病毒CH441株VP3基因與其他15株豬嵴病毒VP3基因核苷酸、氨基酸相似性均高于VP1基因,說明VP3基因的變異比VP1基因的小,更適合作為建立診斷方法的候選蛋白。本研究應(yīng)用原核表達(dá)系統(tǒng)表達(dá)了VP3蛋白,并用純化的VP3蛋白制備了豬源高免血清。3.豬嵴病毒間接ELISA檢測方法的建立利用純化的豬嵴病毒VP3蛋白作為包被抗原,成功建立了特異性與穩(wěn)定性高的檢測豬嵴病毒抗體的間接ELISA方法,為豬嵴病毒的診斷成立了一種新的方法。通過本研究建立的VP3抗體檢測方法對臨床采集的44份腹瀉豬血清樣本進(jìn)行檢測,VP3抗體陽性率為63.64%,檢測結(jié)果顯示在甘肅地區(qū)發(fā)生腹瀉的豬群中,豬嵴病毒具有較高的陽性檢出率。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65

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1 祝俊鵬;豬嵴病毒CH441株VP3基因的原核表達(dá)及間接ELISA方法的建立[D];甘肅農(nóng)業(yè)大學(xué);2015年

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本文編號：2420191