本文選題：豬博卡病毒　切入點(diǎn)：檢測(cè)　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：豬博卡病毒(Porcine bocavirus,PBo V)是2009年首先在患有斷奶仔豬多系統(tǒng)衰竭綜合征的豬淋巴結(jié)中檢測(cè)到的一種單股線狀DNA病毒,在國內(nèi)外豬群中廣泛存在,可能是仔豬腹瀉病重新暴發(fā)的一個(gè)重要原因。因此,開展PBo V的研究具有重要意義。本研究對(duì)豬博卡病毒的流行情況、基因組和對(duì)豬腸上皮細(xì)胞的作用進(jìn)行了初步研究,分別在基因水平和細(xì)胞水平上闡述該病毒感染機(jī)體后對(duì)機(jī)體產(chǎn)生的可能影響,為研究該病毒的致病性提供思路。主要研究結(jié)果如下:1.PBo V的PCR檢測(cè):對(duì)2013年3月至2014年12月腹瀉豬場中的豬群樣本進(jìn)行PBo V PCR檢測(cè),523份腹瀉病料中陽性134份,檢出率為25.62%,其中血清、糞便和組織的檢出率分別為76%、49%和5%;對(duì)某屠宰場健康豬群的184份樣本進(jìn)行檢測(cè),共檢出陽性87份,檢出率為47.28%,其中糞便、腸系膜淋巴結(jié)和下頜淋巴結(jié)的檢出率分別為68%、36%和2%。2.PBo V/NP08株基因組序列分析:參考Genbank收錄的PBo V基因組全序列,根據(jù)保守位點(diǎn)設(shè)計(jì)6對(duì)引物,以PCR檢測(cè)陽性的1份腹瀉病料提取的DNA為模板,完成PBo V的全基因組測(cè)序,將該毒株命名為PBo V/NP08并提交至Gen Bank數(shù)據(jù)庫,登錄號(hào):KR014255。該毒株序列全長4745bp,與PBo V/Buk-1和PBo V/SX的核苷酸同源性均為99%,與PBo V/H18的核苷酸同源性為90%;具有3個(gè)開放閱讀框(ORFs),分別編碼NP1、NS1、VP1和VP2四種病毒蛋白。3.PBo V的基因群及VP1獨(dú)有區(qū)(VP1u)上的磷脂酶A2(PLA2)保守序列分析:根據(jù)國際病毒分類委員會(huì)關(guān)于博卡病毒的分類標(biāo)準(zhǔn),將在Genbank上發(fā)布的47株P(guān)Bo V分為6個(gè)基因群,PBo V/NP08屬于基因群Ⅴ。首次發(fā)現(xiàn)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氨基酸序列中這兩個(gè)保守基序缺失。4.豬腸上皮細(xì)胞(IPEC-J2)感染PBo V后的形態(tài)學(xué)和細(xì)胞因子表達(dá)變化:將PBo V/NP08接種IPEC-J2細(xì)胞,培養(yǎng)48h后出現(xiàn)腫脹、堆積、脫落等細(xì)胞病變。在病毒接種細(xì)胞后的12-72h,IL-1β、IL-6、IL-8、IL-12、TNF-α和IFN-γ等炎性因子的表達(dá)均上調(diào),尤其是IL-6的表達(dá)在整個(gè)過程中均極顯著上調(diào);轉(zhuǎn)化生長因子TGF-β1的表達(dá)在整個(gè)過程中也極顯著上調(diào)。
[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，

本文編號(hào)：1718176