本文關(guān)鍵詞：BVDV與IBRV感染MDBK細(xì)胞的病毒復(fù)制與細(xì)胞凋亡研究　出處：《黑龍江八一農(nóng)墾大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

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【摘要】：牛傳染性鼻氣管炎病毒(IBRV)和牛病毒性腹瀉病毒(BVDV)是兩種重要的牛病毒性傳染病毒。兩種病毒均可造成機(jī)體的免疫抑制。臨床中存在BVDV與IBRV共同感染、BVDV與其它呼吸道病原混合感染或繼發(fā)感染的情況。本研究在體外進(jìn)行了兩種病毒感染MDBK細(xì)胞后的病毒復(fù)制和凋亡研究,為研究病毒感染的分子機(jī)制和開發(fā)疫苗奠定了基礎(chǔ)。本研究首先進(jìn)行了CP型的BVDV與IBRV的增殖,然后選擇0.2 MOI、1.0MOI、2.0 MOI的BVDV感染MDBK細(xì)胞,50%細(xì)胞病變時再分別感染IBRV(0.1 MOI),同時設(shè)單獨(dú)感染BVDV、IBRV的對照組和空白細(xì)胞對照。共感染24h后收集細(xì)胞提取病毒核酸,采用實時熒光定量PCR和間接免疫熒光等方法進(jìn)行了病毒感染、載量和細(xì)胞活性等檢測,確定了MDBK細(xì)胞感染兩種病毒后的病毒復(fù)制和細(xì)胞活性。應(yīng)用CCK-8細(xì)胞增殖試驗、TUNEL檢測法、流式細(xì)胞儀等進(jìn)行了病毒定位與凋亡等檢測,確定病毒共感染對細(xì)胞凋亡的影響。結(jié)果表明,當(dāng)BVDV 0.2MOI、1.0MOI時,IBRV的病毒拷貝數(shù)與IBRV單獨(dú)感染組差異不顯著。當(dāng)BVDV 2.0 MOI時,IBRV病毒拷貝數(shù)明顯低于IBRV單獨(dú)感染組。間接免疫熒光檢測顯示BVDV與IBRV可以共同感染MDBK細(xì)胞并造成細(xì)胞凋亡,且隨細(xì)胞感染BVDV病毒量的增加,細(xì)胞病變逐漸加劇。細(xì)胞活性檢測結(jié)果顯示,兩個病毒共同感染后對細(xì)胞造成了較大影響,隨著病毒量的增多,細(xì)胞活性降低,且共同感染組細(xì)胞活性低于單獨(dú)BVDV感染組細(xì)胞活性。流式細(xì)胞儀檢測感染率與凋亡率結(jié)果均表明,BVDV與IBRV共感染細(xì)胞的感染率均高于BVDV單獨(dú)感染的細(xì)胞,且此試驗中的BVDV對IBRV的復(fù)制有顯著影響。綜上所述,研究表明感染BVDV的MDBK細(xì)胞再次感染IBRV時,兩個病毒均可在細(xì)胞中進(jìn)行復(fù)制,但高感染復(fù)數(shù)的BVDV會加劇MDBK細(xì)胞的病變,進(jìn)而影響IBRV的復(fù)制。BVDV與IBRV共同感染引起細(xì)胞活性明顯降低,對細(xì)胞造成的損傷大于病毒單獨(dú)感染,并造成更嚴(yán)重的細(xì)胞凋亡。
[Abstract]:Bovine infectious rhinotracheitis virus (IBRV) and bovine viral diarrhea virus (BVV). It is an important bovine viral infection virus. Both viruses can cause immunosuppression. There are co-infection of BVDV and IBRV in clinic. In this study, the replication and apoptosis of two viruses infected with MDBK cells were studied in vitro. In order to study the molecular mechanism of virus infection and develop vaccine, we first carried out the proliferation of CP type BVDV and IBRV, and then selected 0.2 MOI 1.0 moi. 2. 0 MOI BVDV infected 50% of MDBK cells with cytopathic changes, and IBRV(0.1 moi was infected separately, and only BVDV was infected. IBRV control group and blank cell control group. After 24 hours of co-infection, the virus nucleic acid was extracted from the cells, and the virus infection was carried out by real-time fluorescence quantitative PCR and indirect immunofluorescence. The viral replication and cell activity of MDBK cells infected with two viruses were determined by the assay of load and cell activity. CCK-8 cell proliferation test and Tunel assay were used to detect the viral replication and cell activity. The virus localization and apoptosis were detected by flow cytometry to determine the effect of virus co-infection on cell apoptosis. The results showed that when BVDV 0.2 MOI was 1.0 MOI. The number of viral copies of IBRV was not significantly different from that of IBRV alone, when BVDV 2.0 MOI. The copy number of IBRV virus was significantly lower than that of IBRV alone. Indirect immunofluorescence assay showed that BVDV and IBRV could co-infect MDBK cells and induce apoptosis. With the increase of BVDV virus infection, the cytopathic changes were gradually aggravated. The results of cell activity test showed that the co-infection of the two viruses had a great impact on the cells, with the increase of the amount of virus. The cell activity of the co-infected group was lower than that of the group of BVDV infection alone. The results of flow cytometry showed that the infection rate and apoptosis rate were higher than those in the co-infection group. The infection rate of BVDV and IBRV co-infected cells was higher than that of BVDV alone, and the BVDV in this test had a significant effect on the replication of IBRV. Studies have shown that BVDV infected MDBK cells re-infected with IBRV, the two viruses can be replicated in the cells, but highly infected plural BVDV will aggravate the pathological changes of MDBK cells. Furthermore, the co-infection of IBRV, BVDV and IBRV resulted in a significant decrease in cell activity, which caused more damage to cells than virus infection alone, and resulted in more severe cell apoptosis.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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