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  

Nuclease activity and cytotoxicity to host cells of toxic protein VapC produced by Leptospira species

作　　者：辛晓阳[1] 林旭瑷[1] 李立伟[1] 严杰[1] 

机构地区：[1]浙江大学医学院病原生物学系,杭州310058

出　　处：《中华微生物学和免疫学杂志》2012年第2期166-171,共6页Chinese Journal of Microbiology and Immunology

基　　金：基金项目：国家自然科学基金（81171534）

摘　　要：目的了解钩端螺旋体毒素一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rYapB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PCR和Westernblot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。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。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。Objective To determine the function of toxic protein VapC in toxin/antitoxin system of Leptospira species and the cytotoxicity to host cells of the toxic protein. Methods Using genomic DNA of pathogenic L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai as the template, several PCRs were performed to amplify entire vapB, vapC and vapBC genes. Subsequently, the prokaryotic expression systems of vapB, vapC and vapBC genes were constructed. Expression of the target recombinant proteins rVapB and rVapC was detected by SDS-PAGE and the expressed rVapB and rVapC were extracted by Ni- NTA affinity chromatography. Activity of rVapB and rVapC to lyse the DNAs or RNAs from L. interrogans strain Lai and THP-1 ceils were then determined. The changes of transcription and expression of vapB and vapC genes of L. interrogans strain Lai before and after infection of THP-1 cells were detected by real-time fluorescent quantitative RT-PCR and Western blot assay. The eukaryotic expression vectors of the vapB and vapC genes were generated for transfection of host cells and CCK-8 agent was used to detect the effect of lep- tospiral VapB and VapC proteins on activity of host cells. Results The nucleotide and putative amino acid sequences of the cloned vapB and vapC genes were completely identical with the reported corresponding genes. The constructed prokaryotic expression systems could express rVapB and rVapC, respectively, rVapC displayed RNase avtivity but did not lyse DNA. When L. interrogans strain Lai infected THP-1 ceils, the transcription and expression of vapB and vapC genes were upregulated and partial VapC protein was secreted from the leptospiral cells. The mass mortality was observed in HEK293 human renal tubular epithelial cells containing the vapC gene through transfection. Conclusion VapC protein of L. interrogans strain Lai is a RNase and is secreted during infection of host cells with obvious cytotoxicity.

关 键 词：致病性钩端螺旋体 毒素-抗毒素系统 毒性蛋白 核酸酶活性 细胞毒性 

分 类 号：R114[医药卫生—卫生毒理学]