豬博卡病毒間接ELISA方法的建立及與PCV2共感染對細胞的影響
發(fā)布時間：2018-07-24 14:15
【摘要】：豬博卡病毒(Porcine bocavirus,PBoV)是瑞典研究人員于2009年在患病豬中檢測到的,常在發(fā)生仔豬多系統(tǒng)衰竭綜合征、腹瀉和呼吸道疾病的豬群中檢出,國內(nèi)外研究表明PBoV在患病豬群和健康豬群中均存在。目前對于PBo V致病性仍然存在爭議,推測該病毒致病性較弱,需要與其他病毒協(xié)同發(fā)揮致病作用。臨床上與其他病毒存在雙重和多重感染,尤其與豬圓環(huán)病毒2型(Porcine Circovirus type 2,PCV2)感染檢出率高,我們推測PCV2可能在該病毒致病過程中起著重要作用。本研究選擇豬博卡病毒VP1的優(yōu)勢抗原表位進行基因克隆和原核表達,利用重組蛋白初步建立間接ELISA抗體檢測方法。同時通過PBoV單獨感染及與PCV2混合感染豬腸上皮細胞(IPEC-J2),初步探討該病毒對IPEC-J2的感染能力,以及PCV2對PBoV感染的協(xié)同作用。具體研究主要有以下兩部分:1.豬博卡病毒間接ELISA抗體檢測方法的初步建立擴增豬博卡病毒NP08株部分VP1基因,構(gòu)建重組表達質(zhì)粒pET-tVP1,原核表達獲得大小為56.7kDa的重組蛋白。用重組VP1蛋白作為包被抗原,初步建立PBoV的間接ELISA抗體檢測方法。與Western blot結(jié)果符合率在81.25%,批內(nèi)和批間重復試驗變異系數(shù)均小于10%。應用本方法對12個場832份豬血清進行檢測,PBoV抗體陽性率25.6%,其中腹瀉豬群抗體陽性率32.65%(207/634),健康豬群抗體陽性率3.03%(6/198)。2.PBoV與PCV2共感染對細胞影響的研究(1)將PBoV、PCV2單獨或混合接種豬腎細胞(PK-15),發(fā)現(xiàn)空白對照組、PCV2組細胞無病變,PBoV感染組及混合感染組出現(xiàn)不同程度堆積、脫落等細胞病變,且混合感染組比單獨感染組細胞病變明顯。然而,Real-time PCR檢測發(fā)現(xiàn)PCV2并不能促進PBoV在PK-15細胞上增殖,且與感染先后順序無關。(2)將PBoV、PCV2單獨或混合接種IPEC-J2,HE染色發(fā)現(xiàn)感染組細胞有脫落現(xiàn)象,細胞病變主要發(fā)生在細胞質(zhì)中,細胞質(zhì)降解變稀薄、有空洞現(xiàn)象。病毒感染24h后收集細胞,PBoV和PCV2單獨感染細胞凋亡率分別為6.70%和6.29%,而PBoV和PCV2同時感染細胞凋亡率為10.8%,明顯高于單獨感染組。Real-time PCR檢測發(fā)現(xiàn)PCV2并不能促進PBoV在IPEC-J2細胞上增殖,且與感染先后順序無關;PBoV也不能促進PCV2在IPEC-J2細胞上增殖�；旌细腥竞笏姆N炎性因子(IL-6、IL-8、IL-1β、TNF-α)在轉(zhuǎn)錄和翻譯水平上均比單獨感染后有極顯著上調(diào)(P0.001);對IPEC-J2跨膜上皮電阻值測定:感染組在1h時TEER值極速下降后上升,在12h時又下降,12-84h期間TEER趨于平穩(wěn);進一步研究發(fā)現(xiàn)感染組OCLN、CLDN1、ZO-1表達顯著下調(diào)(P0.01),主要發(fā)生在細胞感染早期�？傊�,本研究首次探討了PCV2對PBoV在細胞水平增殖和致病性的影響,發(fā)現(xiàn)混合感染組比單獨感染組細胞病變嚴重。PCV2不能促進PBoV增殖,但可以促進感染細胞炎性因子表達,對細胞屏障破壞更嚴重。為進一步探討PBoV感染及致病機理提供了依據(jù)。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.651
本文編號：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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.651
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