本文關鍵詞：IFN-γ誘導鸚鵡熱嗜衣原體持續(xù)性感染及相關基因的表達　出處：《南華大學》2014年碩士論文　論文類型：學位論文

  更多相關文章： 鸚鵡熱嗜衣原體 持續(xù)性感染 人重組干擾素-γ 基因表達

【摘要】：目的：建立IFN-γ誘導的鸚鵡熱嗜衣原體（Chlamydophila psittaci，Cps）持續(xù)性感染的細胞模型，檢測與Cps6BC持續(xù)性感染相關基因的表達變化。 方法： 體外培養(yǎng)HeLa單層細胞感染Cps6BC標準菌株，分別用5、15、25、35、45、55ng/mL的人重組干擾素-γ（recombinant hunan IFN-γ，rhIFN-γ）誘導Cps6BC持續(xù)性感染30h和用35ng/mL rhIFN-γ誘導12、24、36、48、60h，利用間接免疫熒光染色法（indirect immunofluorescence assay，IFA）檢測Cps6BC感染能力的變化。用IFA和透射電鏡檢測rhIFN-γ誘導Cps6BC持續(xù)性感染后包涵體形態(tài)的變化。rhIFN-γ誘導Cps6BC持續(xù)性感染后，去除誘導劑，補充足量的L-色氨酸，利用IFA和透射電鏡檢測Cps6BC感染能力和包涵體形態(tài)的恢復情況。用Realtime-PCR檢測Cps6BC持續(xù)性感染狀態(tài)下目的基因的mRNA表達水平的變化以及Western blot檢測CPSIT_0844、CPSIT_0594蛋白表達水平的變化。 結果： 用不同濃度的rhIFN-γ誘導Cps6BC標準株持續(xù)性感染30h后，Cps6BC的感染能力均降低，且呈明顯的劑量依賴關系。35ng/mL rhIFN-γ誘導不同時間后，，Cps6BC的感染能力也出現不同程度的下降，尤其是36、48、60h后感染能力顯著降低。IFA和透射電鏡觀察顯示，rhIFN-γ誘導后，Cps6BC包涵體體積變小，結構疏松，并可見異形網狀體，成熟子代原體數目減少，呈現典型的持續(xù)性感染特征。去除誘導劑，補充足量的L-色氨酸后，Cps6BC的感染能力及包涵體形態(tài)均可恢復到急性感染狀態(tài)。 rhIFN-γ誘導Cps6BC持續(xù)性感染后，CPSIT_0844、CPSIT_0846、CPSIT_0959、CPSIT_0594、CPSIT_0310、CPSIT_0870、CPSIT_0057、CPSIT_0208在mRNA水平的表達均發(fā)生了一定的改變。核糖體蛋白CPSIT_0870在2～36h顯著上調，而半胱氨酸脫硫酶CPSIT_0959在36h、48h顯著下調。Ⅲ型分泌系統(tǒng)效應蛋白CPSIT_0594、CPSIT_0844均在2～48h上調，且分別在36h和48h顯著上調，而CPSIT_0846在2～24h顯著上調，36h后顯著下調。膜蛋白CPSIT_0057、CPSIT_0310在2～48h上調，而CPSIT_0208在48h后下調。Westernblot結果顯示，rhIFN-γ誘導Cps6BC持續(xù)性感染后，CPSIT_0844、CPSIT_0594蛋白表達的改變與其mRNA水平的表達變化一致。 結論： 1.建立了rhIFN-γ誘導的Cps6BC持續(xù)性感染細胞模型； 2. CPSIT_0844、 CPSIT_0846、 CPSIT_0959、 CPSIT_0594、 CPSIT_0310、CPSIT_0870、CPSIT_0057、CPSIT_0208在rhIFN-γ誘導Cps6BC持續(xù)性感染過程中的表達出現一定的變化，這些基因可能與衣原體持續(xù)性感染有關。
[Abstract]:Objective: to establish a cell model of persistent infection of Chlamydophila psittacius (Cpss) induced by IFN- 緯. The expression changes of genes associated with persistent infection of Cps6BC were detected. Methods: In vitro, HeLa monolayer cells were infected with Cps6BC standard strain. Recombinant hunan IFN- 緯 was obtained with 55 ng / mL of recombinant human interferon- 緯. RhIFN- 緯) induced Cps6BC persistent infection for 30 h and 35 ng / mL rhIFN- 緯 for 48 h. Indirect immunofluorescence staining was used to detect indirect immunofluorescence assay. IFA). To detect the change of Cps6BC infection ability. To detect the changes of inclusion body morphology after Cps6BC persistent infection induced by rhIFN- 緯 by IFA and transmission electron microscopy. RhIFN- 緯 induced Cps6. After persistent BC infection. Remove the inducer and supplement a sufficient amount of L-tryptophan. IFA and transmission electron microscopy (TEM) were used to detect the ability of Cps6BC infection and the shape recovery of inclusion body. Realtime-PCR was used to detect the mR of the target gene under the condition of persistent Cps6BC infection. Changes of na expression level and Western. CPSIT_0844 was detected by blot. Changes of CPSIT_0594 protein expression level. Results: The ability of Cps6BC infection of Cps6BC standard strain was decreased after 30 hours of persistent infection induced by different concentrations of rhIFN- 緯. In a dose-dependent manner, the infection ability of Cps6BC was decreased in different time after rhIFN- 緯 was induced by 35 ng / mL rhIFN- 緯, especially 36ng / mL rhIFN- 緯. After 60 hours, the ability of infection decreased significantly. IFA and TEM observation showed that the volume of Cps6BC inclusion body decreased after rhIFN- 緯 induction, the structure was loose, and the heteromorphic reticular bodies could be seen. The number of mature progenies decreased, showing a typical persistent infection. After removing the inducer and replenishing sufficient amounts of L-tryptophan. The infection ability and inclusion body morphology of Cps6BC can be restored to acute infection state. After Cps6BC persistent infection induced by rhIFN- 緯, CPSITT: 0844 / CPSITT: 0846 / CPSIT0959 / CPSIT0594. CPSIT_0310,CPSIT_0870,CPSIT_0057. The expression of CPSIT_0208 in mRNA was changed to some extent, and ribosomal protein CPSIT_0870 was upregulated significantly at 2 ~ 36 h. The cysteine desulfurization enzyme CPSIT_0959 was significantly down-regulated at 36h and 48h. Type 鈪

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