感染人单核细胞THP-1前后钩端螺旋体外膜蛋白抗原表达差异性
发布时间：2018-08-01 17:28
【摘要】：目的了解感染人单核细胞THP-1前后问号钩端螺旋体(简称钩体)外膜蛋白表达变化,为选择钩体基因工程疫苗侯选抗原提供依据。 方法采用Triton X-114法提取感染THP-1细胞前后问号钩体黄疸出血群赖型赖株外膜蛋白。采用双向电泳技术分离钩体外膜蛋白,银染色法检测感染前后钩体外膜蛋白表达差异。感染后4个表达显著上调和4表达显著下调的蛋白点进行胰酶水解后,采用LC-MS/MS方法鉴定蛋白质。应用生物信息学软件分析靶蛋白跨膜区和信号肽,采用实时荧光定量RT-PCR检测感染细胞前后靶基因mRNA变化。采用Western blot检测感染THP-1细胞前后外膜蛋白与相应抗体反应性强弱。 结果感染THP-1细胞60min后,钩体外膜蛋白中Loa22、GroEL、F0F1ATP合成酶α和β亚单位表达水平均显著升高(P0.05),FlaB2、LigB、OmpA家族蛋白和OmpA表达显著下降(P0.05),实时荧光定量RT-PCR检测结果与之基本一致(P0.05)。生物信息学分析结果显示,OmpA和OmpA家族蛋白为跨膜蛋白,Loa22、LigB和OmpA家族蛋白含有信号肽。Western blot检测结果显示感染THP-1细胞后钩体OMPs中Loa22与兔抗Loa22血清的反应信号比感染细胞前较强。 结论感染细胞时钩体外膜蛋白表达谱可发生明显变化。感染后高表达的外膜蛋白、尤其是GroEL和Loa22可作为钩体基因工程疫苗候选抗原。
[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 伪 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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2158308
[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 伪 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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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