猪博卡病毒NP08株的基因组序列分析及其对猪肠上皮细胞的影响

发布时间：2018-04-06 05:27

  本文选题：猪博卡病毒　切入点：检测　出处：《华中农业大学》2015年硕士论文

【摘要】：猪博卡病毒(Porcine bocavirus,PBo V)是2009年首先在患有断奶仔猪多系统衰竭综合征的猪淋巴结中检测到的一种单股线状DNA病毒,在国内外猪群中广泛存在,可能是仔猪腹泻病重新暴发的一个重要原因。因此,开展PBo V的研究具有重要意义。本研究对猪博卡病毒的流行情况、基因组和对猪肠上皮细胞的作用进行了初步研究,分别在基因水平和细胞水平上阐述该病毒感染机体后对机体产生的可能影响,为研究该病毒的致病性提供思路。主要研究结果如下:1.PBo V的PCR检测:对2013年3月至2014年12月腹泻猪场中的猪群样本进行PBo V PCR检测,523份腹泻病料中阳性134份,检出率为25.62%,其中血清、粪便和组织的检出率分别为76%、49%和5%;对某屠宰场健康猪群的184份样本进行检测,共检出阳性87份,检出率为47.28%,其中粪便、肠系膜淋巴结和下颌淋巴结的检出率分别为68%、36%和2%。2.PBo V/NP08株基因组序列分析:参考Genbank收录的PBo V基因组全序列,根据保守位点设计6对引物,以PCR检测阳性的1份腹泻病料提取的DNA为模板,完成PBo V的全基因组测序,将该毒株命名为PBo V/NP08并提交至Gen Bank数据库,登录号:KR014255。该毒株序列全长4745bp,与PBo V/Buk-1和PBo V/SX的核苷酸同源性均为99%,与PBo V/H18的核苷酸同源性为90%;具有3个开放阅读框(ORFs),分别编码NP1、NS1、VP1和VP2四种病毒蛋白。3.PBo V的基因群及VP1独有区(VP1u)上的磷脂酶A2(PLA2)保守序列分析:根据国际病毒分类委员会关于博卡病毒的分类标准,将在Genbank上发布的47株PBo V分为6个基因群,PBo V/NP08属于基因群Ⅴ。首次发现基因群Ⅰ-Ⅳ的VP1u氨基酸序列中含有“HDXXY”和“YXGXF”保守基序,但基因群Ⅴ和基因群Ⅵ的VP1u氨基酸序列中这两个保守基序缺失。4.猪肠上皮细胞(IPEC-J2)感染PBo V后的形态学和细胞因子表达变化:将PBo V/NP08接种IPEC-J2细胞,培养48h后出现肿胀、堆积、脱落等细胞病变。在病毒接种细胞后的12-72h,IL-1β、IL-6、IL-8、IL-12、TNF-α和IFN-γ等炎性因子的表达均上调,尤其是IL-6的表达在整个过程中均极显著上调;转化生长因子TGF-β1的表达在整个过程中也极显著上调。
[Abstract]:Porcine bocavirus virus (PBo V) is a single-stranded DNA virus first detected in the lymph nodes of piglets with multiple system failure syndrome in weaning piglets in 2009.It may be an important reason for the recurrence of diarrhea in piglets.Therefore, the research of PBo V is of great significance.In this study, the epidemic situation of porcine Boca virus, its genomes and its effects on porcine intestinal epithelial cells were preliminarily studied, and the possible effects of the virus infection on the body were discussed at the gene level and the cellular level, respectively.In order to study the pathogenicity of the virus.The main results are as follows: 1. PCR detection of PBOV: from March 2013 to December 2014, PBo V PCR was used to detect 134 out of 523 diarrhoeal disease samples, the detection rate was 25.62%.The detectable rates of feces and tissues were 76% and 5%, respectively, and 184 samples of healthy pigs from a slaughterhouse were tested, 87 positive samples were detected, and the positive rate was 47.28%.The detection rates of mesenteric lymph nodes and mandibular lymph nodes were 68% and 36%, respectively. Genomic sequence analysis of 2%.2.PBo V/NP08 strain: referring to the whole sequence of PBo V genome included in Genbank, 6 pairs of primers were designed according to the conservative loci.The whole genome of PBo V was sequenced using DNA extracted from a sample of diarrhea disease positive by PCR. The virus strain was named PBo V/NP08 and submitted to Gen Bank database (accession number: KR014255).The nucleotide homology of the strain with PBo V/Buk-1 and PBo V/SX is 99 and 90 with that of PBo V/H18, and there are three open reading frames, encoding NP1 NS1VP1 and VP2 four viral proteins. 3. The gene group and VP1 unique region of NP1 NS1VP1 and VP2. 3.Analysis of the conserved sequence of phospholipase A2 (PLA2) on VP1u: according to the International Committee on viral Classification (ICV) criteria for the classification of Boca virus,The 47 strains of PBo V published on Genbank were divided into 6 groups: PBO V/NP08 belongs to gene group V.The conserved motifs of "HDXXY" and "YXGXF" were found in the VP1u amino acid sequence of group 鈪，

本文编号：1718176