【摘要】：目的：通過分析PRRSV感染MARC-145細胞疾病模型中IKKγmRNA和蛋白表達量的變化,揭示NF-κB通路的調(diào)節(jié)因子與PRRSV之間的關(guān)系,為進一步研究通路與病毒感染之間的關(guān)系奠定基礎(chǔ)。方法：通過間接免疫熒光(IFA)法評價建立的PRRSV感染Marc-145細胞模型；通過MTT法檢測LY294002抑制率；實驗分對照組、抑制劑(LY294002)組、病毒(PRRSV)組、抑制劑(LY294002)+病毒(PRRSV)組,其中對照組為只加培養(yǎng)基,抑制劑組為只加10μMY294002的溶液,病毒組為只加100TCID50的PRRS V組,抑制劑+病毒組為加入lO^M LY294002溶液和100TCIDso的病毒稀釋液。通過qPCR和Western blot檢測不同處理組的IKKγ表達量的變化,探究IKKγ參與抗PRRSV的分子免疫機制。結(jié)果：①成功建立了PRRSV感染Marc-145細胞模型。②MTT比色法檢測P13K抑制劑LY294002對Marc-145細胞的抑制率,與正常細胞組比較均有抑制,且抑制程度隨濃度升高而增強。48h內(nèi),抑制率隨時間的延長呈遞增趨勢;當(dāng)抑制時間超過48h后,抑制下降。③熒光定量PCR結(jié)果顯示：與對照相比,PRRSV組IKKγmRNA水平上調(diào),差異顯著(P0.05),LY294002組mRNA水平下調(diào),差異極顯著(P0.01),LY293002+PRRSV組]mRNA水平下調(diào),差異極顯著(P0.01)。④Western blot結(jié)果顯示：與對照組相比,感染PRRSV后IKKγ蛋白水平水平上調(diào),加入抑制劑LY294002后IKKγ蛋白表達下調(diào),I.Y294002+PRRSV組,IKKγ蛋白表達下調(diào)。結(jié)論：①上游信號通路影響下游信號通路的信號轉(zhuǎn)導(dǎo)。②LY294002抑制劑影響了PRRSV復(fù)制轉(zhuǎn)錄過程。③IKKγ在感染PRRSV后的分子免疫協(xié)調(diào)機制中發(fā)揮作用。
[Abstract]:Aim: to analyze the changes of IKK 緯 mRNA and protein expression in MARC-145 cell disease model infected with PRRSV, and to reveal the relationship between the regulatory factors of NF- 魏 B pathway and PRRSV, so as to lay a foundation for further study on the relationship between NF- kB pathway and viral infection. Methods: the model of Marc-145 cells infected with PRRSV was evaluated by indirect immunofluorescence (IFA) assay, and the inhibition rate of LY294002 was detected by MTT assay. The experiment was divided into control group, inhibitor (LY294002) group, virus (PRRSV) group and inhibitor (LY294002) virus (PRRSV) group. The control group was supplemented with medium only, the inhibitor group was only supplemented with 10 渭 MY294002 solution, and the virus group was PRRSV group with 100TCID50 only. The inhibitor virus group was the virus diluent with Lo ^ M LY294002 solution and 100TCIDso. The expression of IKK 緯 in different treatment groups was detected by qPCR and Western blot, and the molecular immune mechanism of IKK 緯 involved in anti-PRRSV was explored. Results: 1 the Marc-145 cell model of PRRSV infection was successfully established. 2 the inhibition rate of P13K inhibitor LY294002 on Marc-145 cells was detected by MTT colorimetric assay, which was significantly higher than that of the normal cell group, and the degree of inhibition increased within 48 hours. The inhibition rate increased with the prolongation of time. When the inhibition time was more than 48 hours, the inhibition decreased. 3 the results of fluorescence quantitative PCR showed that the level of IKK 緯 mRNA in PRRSV group was significantly higher than that in control group (P 0.05), and the level of mRNA in LY294002 group was significantly lower than that in control group (P 0.01). The level of mRNA in LY293002 PRRSV group was significantly lower than that in control group (P 0.01). The results of 4Western blot showed that compared with the control group, the level of IKK 緯 protein was up-regulated after infection with PRRSV, and the expression of IKK 緯 protein was down-regulated after LY294002 was added to I.Y294002 PRRSV group, and the expression of IKK-緯 protein was down-regulated in I.Y294002 PRRSV group. The expression of IKK 緯 protein was down-regulated. Conclusion: 1 upstream signal pathway affects the signal transduction of downstream signal pathway. 2Lyn294002 inhibitor affects the process of PRRSV replication and transcription. 3IKK 緯 plays a role in the molecular immune coordination mechanism after PRRSV infection.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3

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