钩端螺旋体毒性蛋白VapC核酸酶活性及其对宿主细胞毒性作用的研究

发布时间：2018-04-04 23:11

  本文选题：致病性钩端螺旋体　切入点：毒素-抗毒素系统　出处：《浙江大学》2012年硕士论文

【摘要】：目的了解钩端螺旋体毒素-抗毒素系统中毒性蛋白VapC的功能及其对宿主细胞的毒性作用。 方法以致病性问号钩体黄疸出血群赖型赖株基因组DNA为模板,采用PCR扩增全长vapB、vapC、vapBC基因并构建其原核表达系统。采用SDS-PAGE检测目的重组蛋白rVapB和rVapC表达情况,Ni-NTA亲和层析柱提纯rVapB和rVapC.检测rVapB和rVapC有无水解问号钩体赖株及THP-1细胞DNA或RNA活性。分别采用实时荧光定量RT-PCR和Western Blot试验,检测问号钩体赖株感染THP-1细胞前后vapB和vapC基因转录及表达水平的变化。采用免疫荧光激光共聚焦显微镜法检测VapB和VapC蛋白在问号钩体赖株感染THP-1细胞中表达情况。构建vapB和vapC基因真核表达载体并转染细胞,采用CCK-8试剂检测VapB和VapC蛋白对细胞活性的影响。 结果所克隆的vapB和vapC基因核苷酸及氨基酸序列与文献报道完全相同。所构建的原核表达系统能分别表达rVapB和rVapC。rVapC可水解RNA,但不水解DNA。问号钩体赖株感染THP-1细胞后,vapB和vapC基因转录及表达水平均显著上调,部分毒性蛋白VapC外分泌。问号钩体赖株感染细胞内可检出毒性蛋白VapC,转染vapC基因的人肾小管上皮细胞HEK293大量死亡 结论问号钩体赖株VapC蛋白为RNA酶,可在感染宿主细胞过程中外分泌并对细胞有明显毒性。
[Abstract]:Objective to investigate the function of VapC in leptospira toxin-antitoxin system and its toxicity to host cells.Methods based on the genomic DNA of the pathogenic Leptospira interrogans with jaundice haemorrhage, the full length of vapBvapvapBC gene was amplified by PCR and its prokaryotic expression system was constructed.The expression of recombinant protein rVapB and rVapC was detected by SDS-PAGE and purified rVapB and rVapC by Ni-NTA affinity chromatography.The activity of DNA or RNA in rVapB and rVapC were detected.Real-time quantitative RT-PCR and Western Blot tests were used to detect the changes of vapB and vapC gene transcription and expression before and after Leptospira interrogans infection in THP-1 cells.The expression of VapB and VapC proteins in THP-1 cells infected with Leptospira interrogans was detected by immunofluorescence confocal laser microscopy.The eukaryotic expression vector of vapB and vapC genes was constructed and transfected into the cells. The effects of VapB and VapC proteins on cell activity were detected by CCK-8 reagent.Results the nucleotide and amino acid sequences of vapB and vapC genes were identical to those reported in the literature.The constructed prokaryotic expression system could express rVapB and rVapC.rVapC, but not rVapC.rVapC.The transcription and expression levels of vapB and vapC genes were significantly up-regulated in THP-1 cells infected with Leptospira interrogans, and some toxic protein VapC exocrine.The virulence protein VapC was detected in the infected cells of Leptospira interrogans, and the HEK293 of human renal tubular epithelial cells transfected with vapC gene died in large numbers.Conclusion the VapC protein of Leptospira interrogans is a RNA enzyme, which can exocrine during the infection of host cells and has obvious toxicity to the cells.
【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】：R377

【共引文献】

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