猪博卡病毒间接ELISA方法的建立及与PCV2共感染对细胞的影响
发布时间：2018-07-24 14:15
【摘要】：猪博卡病毒(Porcine bocavirus,PBoV)是瑞典研究人员于2009年在患病猪中检测到的,常在发生仔猪多系统衰竭综合征、腹泻和呼吸道疾病的猪群中检出,国内外研究表明PBoV在患病猪群和健康猪群中均存在。目前对于PBo V致病性仍然存在争议,推测该病毒致病性较弱,需要与其他病毒协同发挥致病作用。临床上与其他病毒存在双重和多重感染,尤其与猪圆环病毒2型(Porcine Circovirus type 2,PCV2)感染检出率高,我们推测PCV2可能在该病毒致病过程中起着重要作用。本研究选择猪博卡病毒VP1的优势抗原表位进行基因克隆和原核表达,利用重组蛋白初步建立间接ELISA抗体检测方法。同时通过PBoV单独感染及与PCV2混合感染猪肠上皮细胞(IPEC-J2),初步探讨该病毒对IPEC-J2的感染能力,以及PCV2对PBoV感染的协同作用。具体研究主要有以下两部分:1.猪博卡病毒间接ELISA抗体检测方法的初步建立扩增猪博卡病毒NP08株部分VP1基因,构建重组表达质粒pET-tVP1,原核表达获得大小为56.7kDa的重组蛋白。用重组VP1蛋白作为包被抗原,初步建立PBoV的间接ELISA抗体检测方法。与Western blot结果符合率在81.25%,批内和批间重复试验变异系数均小于10%。应用本方法对12个场832份猪血清进行检测,PBoV抗体阳性率25.6%,其中腹泻猪群抗体阳性率32.65%(207/634),健康猪群抗体阳性率3.03%(6/198)。2.PBoV与PCV2共感染对细胞影响的研究(1)将PBoV、PCV2单独或混合接种猪肾细胞(PK-15),发现空白对照组、PCV2组细胞无病变,PBoV感染组及混合感染组出现不同程度堆积、脱落等细胞病变,且混合感染组比单独感染组细胞病变明显。然而,Real-time PCR检测发现PCV2并不能促进PBoV在PK-15细胞上增殖,且与感染先后顺序无关。(2)将PBoV、PCV2单独或混合接种IPEC-J2,HE染色发现感染组细胞有脱落现象,细胞病变主要发生在细胞质中,细胞质降解变稀薄、有空洞现象。病毒感染24h后收集细胞,PBoV和PCV2单独感染细胞凋亡率分别为6.70%和6.29%,而PBoV和PCV2同时感染细胞凋亡率为10.8%,明显高于单独感染组。Real-time PCR检测发现PCV2并不能促进PBoV在IPEC-J2细胞上增殖,且与感染先后顺序无关;PBoV也不能促进PCV2在IPEC-J2细胞上增殖。混合感染后四种炎性因子(IL-6、IL-8、IL-1β、TNF-α)在转录和翻译水平上均比单独感染后有极显著上调(P0.001);对IPEC-J2跨膜上皮电阻值测定:感染组在1h时TEER值极速下降后上升,在12h时又下降,12-84h期间TEER趋于平稳;进一步研究发现感染组OCLN、CLDN1、ZO-1表达显著下调(P0.01),主要发生在细胞感染早期。总之,本研究首次探讨了PCV2对PBoV在细胞水平增殖和致病性的影响,发现混合感染组比单独感染组细胞病变严重。PCV2不能促进PBoV增殖,但可以促进感染细胞炎性因子表达,对细胞屏障破坏更严重。为进一步探讨PBoV感染及致病机理提供了依据。
[Abstract]:Porcine bocavirus (Porcine bocavirus, PBoV) was detected by Swedish researchers in 2009 in sick pigs, often detected in piglets of piglets with multiple system failure syndrome, diarrhoea and respiratory diseases. Domestic and foreign studies have shown that PBoV exists in both sick pigs and healthy pigs. The pathogenicity of PBo V is still controversial, It is inferred that the virulence of the virus is weak and needs to play a pathogenic role with other viruses. There is a double and multiple infection with other viruses, especially with the Porcine Circovirus type 2 (PCV2) type 2 (PCV2) infection. We speculate that PCV2 may play an important role in the pathogenesis of the virus. The dominant antigen epitopes of VP1 were cloned and prokaryotic expression, and the indirect ELISA antibody detection method was preliminarily established by recombinant protein. At the same time, the infection ability of the virus to IPEC-J2 and the synergistic effect of PCV2 on PBoV infection were preliminarily discussed by PBoV infection and PCV2 mixed infection of pig intestinal epithelial cells (IPEC-J2). The main two parts of the study are as follows: 1. the detection method of indirect ELISA antibody for porcine Boka virus was initially established to amplify the partial VP1 gene of the porcine Boka virus strain, construct the recombinant expression plasmid pET-tVP1, and obtain the recombinant protein of 56.7kDa in the prokaryotic expression. The recombinant VP1 protein was used as the envelope antigen, and the indirect ELISA antibody detection of PBoV was preliminarily established. Method. The coincidence rate with Western blot results was 81.25%. The coefficient of variation in both batch and interbatch repeated tests was less than that of 10%. application. The positive rate of PBoV antibody was 25.6%, among which the positive rate of the antibody of diarrhea pig group was 32.65% (207/634), the positive rate of healthy pig group was 3.03% (6/198).2.PBoV and PCV2 co infected with the cells. The study (1) inoculated PBoV, PCV2 alone or mixed with pig kidney cells (PK-15), and found blank control group, PCV2 group had no pathological changes, PBoV infection group and mixed infection group had different degrees of accumulation, abscission and other cytopathic lesions, and the mixed infection group was more obvious than the single infection group. However, Real-time PCR detection found PCV2 did not promote it. The proliferation of PBoV on PK-15 cells was not related to the sequence of infection. (2) PBoV, PCV2 was inoculated separately or mixed with IPEC-J2, and HE staining found that the cells in the infected group were falling off, the cytopathic changes were mainly in the cytoplasm, the cytoplasm degradation was thinner and void. The virus infected 24h and collected the cells, PBoV and PCV2 separately infected the cell apoptosis. The rate of PBoV and PCV2 was 6.70% and 6.29%, while the rate of apoptosis was 10.8%, which was significantly higher than that of.Real-time PCR detected in the single infection group. PCV2 did not promote the proliferation of PBoV on IPEC-J2 cells, and it was not related to the sequence of infection; PBoV did not promote the proliferation of PCV2 on IPEC-J2 cells. Four inflammatory factors (IL-6, IL) after mixed infection (IL-6, IL) -8, IL-1 beta, TNF- alpha at the level of transcription and translation were significantly up-regulated (P0.001), and the resistance values of IPEC-J2 transmembrane epithelium were measured: the TEER value of the infection group decreased at 1H at the speed of TEER and decreased at 12h, and the TEER tended to be stable at 12-84h. Further studies found that the infection group was OCLN, CLDN1, and principal downregulation. In a word, the effect of PCV2 on the proliferation and pathogenicity of PBoV at the cell level was first discussed. It was found that the severe.PCV2 in the mixed infection group could not promote the proliferation of PBoV, but could promote the expression of inflammatory cytokines in infected cells, and the damage to the cell barrier was more serious. The further study of PBo V infection and pathogenesis provide a basis.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651
本文编号：2141635
[Abstract]:Porcine bocavirus (Porcine bocavirus, PBoV) was detected by Swedish researchers in 2009 in sick pigs, often detected in piglets of piglets with multiple system failure syndrome, diarrhoea and respiratory diseases. Domestic and foreign studies have shown that PBoV exists in both sick pigs and healthy pigs. The pathogenicity of PBo V is still controversial, It is inferred that the virulence of the virus is weak and needs to play a pathogenic role with other viruses. There is a double and multiple infection with other viruses, especially with the Porcine Circovirus type 2 (PCV2) type 2 (PCV2) infection. We speculate that PCV2 may play an important role in the pathogenesis of the virus. The dominant antigen epitopes of VP1 were cloned and prokaryotic expression, and the indirect ELISA antibody detection method was preliminarily established by recombinant protein. At the same time, the infection ability of the virus to IPEC-J2 and the synergistic effect of PCV2 on PBoV infection were preliminarily discussed by PBoV infection and PCV2 mixed infection of pig intestinal epithelial cells (IPEC-J2). The main two parts of the study are as follows: 1. the detection method of indirect ELISA antibody for porcine Boka virus was initially established to amplify the partial VP1 gene of the porcine Boka virus strain, construct the recombinant expression plasmid pET-tVP1, and obtain the recombinant protein of 56.7kDa in the prokaryotic expression. The recombinant VP1 protein was used as the envelope antigen, and the indirect ELISA antibody detection of PBoV was preliminarily established. Method. The coincidence rate with Western blot results was 81.25%. The coefficient of variation in both batch and interbatch repeated tests was less than that of 10%. application. The positive rate of PBoV antibody was 25.6%, among which the positive rate of the antibody of diarrhea pig group was 32.65% (207/634), the positive rate of healthy pig group was 3.03% (6/198).2.PBoV and PCV2 co infected with the cells. The study (1) inoculated PBoV, PCV2 alone or mixed with pig kidney cells (PK-15), and found blank control group, PCV2 group had no pathological changes, PBoV infection group and mixed infection group had different degrees of accumulation, abscission and other cytopathic lesions, and the mixed infection group was more obvious than the single infection group. However, Real-time PCR detection found PCV2 did not promote it. The proliferation of PBoV on PK-15 cells was not related to the sequence of infection. (2) PBoV, PCV2 was inoculated separately or mixed with IPEC-J2, and HE staining found that the cells in the infected group were falling off, the cytopathic changes were mainly in the cytoplasm, the cytoplasm degradation was thinner and void. The virus infected 24h and collected the cells, PBoV and PCV2 separately infected the cell apoptosis. The rate of PBoV and PCV2 was 6.70% and 6.29%, while the rate of apoptosis was 10.8%, which was significantly higher than that of.Real-time PCR detected in the single infection group. PCV2 did not promote the proliferation of PBoV on IPEC-J2 cells, and it was not related to the sequence of infection; PBoV did not promote the proliferation of PCV2 on IPEC-J2 cells. Four inflammatory factors (IL-6, IL) after mixed infection (IL-6, IL) -8, IL-1 beta, TNF- alpha at the level of transcription and translation were significantly up-regulated (P0.001), and the resistance values of IPEC-J2 transmembrane epithelium were measured: the TEER value of the infection group decreased at 1H at the speed of TEER and decreased at 12h, and the TEER tended to be stable at 12-84h. Further studies found that the infection group was OCLN, CLDN1, and principal downregulation. In a word, the effect of PCV2 on the proliferation and pathogenicity of PBoV at the cell level was first discussed. It was found that the severe.PCV2 in the mixed infection group could not promote the proliferation of PBoV, but could promote the expression of inflammatory cytokines in infected cells, and the damage to the cell barrier was more serious. The further study of PBo V infection and pathogenesis provide a basis.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651
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