【摘要】：目的了解感染人單核細(xì)胞THP-1前后問(wèn)號(hào)鉤端螺旋體(簡(jiǎn)稱(chēng)鉤體)外膜蛋白表達(dá)變化,為選擇鉤體基因工程疫苗侯選抗原提供依據(jù)。 方法采用Triton X-114法提取感染THP-1細(xì)胞前后問(wèn)號(hào)鉤體黃疸出血群賴(lài)型賴(lài)株外膜蛋白。采用雙向電泳技術(shù)分離鉤體外膜蛋白,銀染色法檢測(cè)感染前后鉤體外膜蛋白表達(dá)差異。感染后4個(gè)表達(dá)顯著上調(diào)和4表達(dá)顯著下調(diào)的蛋白點(diǎn)進(jìn)行胰酶水解后,采用LC-MS/MS方法鑒定蛋白質(zhì)。應(yīng)用生物信息學(xué)軟件分析靶蛋白跨膜區(qū)和信號(hào)肽,采用實(shí)時(shí)熒光定量RT-PCR檢測(cè)感染細(xì)胞前后靶基因mRNA變化。采用Western blot檢測(cè)感染THP-1細(xì)胞前后外膜蛋白與相應(yīng)抗體反應(yīng)性強(qiáng)弱。 結(jié)果感染THP-1細(xì)胞60min后,鉤體外膜蛋白中Loa22、GroEL、F0F1ATP合成酶α和β亞單位表達(dá)水平均顯著升高(P0.05),FlaB2、LigB、OmpA家族蛋白和OmpA表達(dá)顯著下降(P0.05),實(shí)時(shí)熒光定量RT-PCR檢測(cè)結(jié)果與之基本一致(P0.05)。生物信息學(xué)分析結(jié)果顯示,OmpA和OmpA家族蛋白為跨膜蛋白,Loa22、LigB和OmpA家族蛋白含有信號(hào)肽。Western blot檢測(cè)結(jié)果顯示感染THP-1細(xì)胞后鉤體OMPs中Loa22與兔抗Loa22血清的反應(yīng)信號(hào)比感染細(xì)胞前較強(qiáng)。 結(jié)論感染細(xì)胞時(shí)鉤體外膜蛋白表達(dá)譜可發(fā)生明顯變化。感染后高表達(dá)的外膜蛋白、尤其是GroEL和Loa22可作為鉤體基因工程疫苗候選抗原。
[Abstract]:Objective to investigate the changes of outer membrane protein expression of Leptospira interrogans (Leptospira) before and after infection with human monocyte THP-1, and to provide evidence for selection of candidate antigen for Leptospira gene engineering vaccine. Methods Triton X-114 method was used to extract the outer membrane protein of Leptospira question mark haemorrhage strain before and after infection with THP-1 cells. The outer membrane proteins of Leptospira were isolated by two-dimensional electrophoresis. The expression of outer membrane proteins of Leptospira was detected by silver staining before and after infection. The protein was identified by LC-MS/MS method after trypsin hydrolysis of 4 protein spots which were significantly up-regulated and significantly down-regulated after infection. The transmembrane region and signal peptide of target protein were analyzed by bioinformatics software. The changes of target gene mRNA before and after infection were detected by real-time fluorescence quantitative RT-PCR. Western blot was used to detect the responsiveness of the outer membrane proteins to the corresponding antibodies in infected THP-1 cells. Results after infection with 60min of THP-1 cells, the expressions of 偽 and 尾 subunits of Loa22 GroELF0F1ATP synthase in Leptospira outer membrane protein were significantly increased (P0.05). The expression of FlaB2LigBOmpA family protein and OmpA in Leptospira cells decreased significantly (P0.05), and the results of real-time fluorescence quantitative RT-PCR were consistent with those of Leptospira cells (P0.05). The results of bioinformatics analysis showed that OmpA and OmpA family proteins were transmembrane proteins Loa22LigB and OmpA family proteins containing signal peptides. Western blot analysis showed that the response signals of Loa22 and rabbit anti-Loa22 serum in Leptospira OMPs after infection were stronger than those before infection. Conclusion the outer membrane protein expression profile of Leptospira can be changed obviously in infected cells. Highly expressed outer membrane proteins, especially GroEL and Loa22, can be used as candidate antigens of Leptospira gene engineering vaccine.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392

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