中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
2期
166-171
,共6页
辛晓阳%林旭瑷%李立伟%严杰
辛曉暘%林旭璦%李立偉%嚴傑
신효양%림욱애%리립위%엄걸
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
緻病性鉤耑螺鏇體%毒素-抗毒素繫統%毒性蛋白%覈痠酶活性%細胞毒性
치병성구단라선체%독소-항독소계통%독성단백%핵산매활성%세포독성
Pathogenic Leptospira species%Toxin-antitoxin system%Toxic protein%Nuclease activity%Cytotoxicity
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.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.
目的 瞭解鉤耑螺鏇體毒素-抗毒素繫統中毒性蛋白VapC的功能及其對宿主細胞的毒性作用.方法 以緻病性問號鉤體黃疸齣血群賴型賴株基因組DNA為模闆,採用PCR擴增全長vapB、vapC、vapBC基因併構建其原覈錶達繫統.採用SDS-PAGE檢測目的重組蛋白rVapB和rVapC 錶達情況,Ni-NTA親和層析柱提純rVapB和rVapC.檢測rVapB和rVapC有無水解問號鉤體賴株及THP-1細胞DNA或RNA活性.分彆採用實時熒光定量PCR和Western blot試驗,檢測問號鉤體賴株感染THP-1細胞前後vapB和vapC基因轉錄及錶達水平的變化.構建vapB和vapC基因真覈錶達載體併轉染細胞,採用CCK-8試劑檢測VapB和VapC蛋白對細胞活性的影響.結果 所剋隆的vapB和vapC基因覈苷痠及氨基痠序列與文獻報道完全相同.所構建的原覈錶達繫統能分彆錶達rVapB 和rVapC.rVapC可水解RNA,但不水解DNA.問號鉤體賴株感染THP-1細胞後,vapB和vapC基因 轉錄及錶達水平均顯著上調,部分毒性蛋白VapC外分泌.轉染vapC基因的人腎小管上皮細胞HEK293大量死亡.結論 問號鉤體賴株VapC蛋白為RNA酶,可在感染宿主細胞過程中外分泌併對細胞有明顯毒性.
목적 료해구단라선체독소-항독소계통중독성단백VapC적공능급기대숙주세포적독성작용.방법 이치병성문호구체황달출혈군뢰형뢰주기인조DNA위모판,채용PCR확증전장vapB、vapC、vapBC기인병구건기원핵표체계통.채용SDS-PAGE검측목적중조단백rVapB화rVapC 표체정황,Ni-NTA친화층석주제순rVapB화rVapC.검측rVapB화rVapC유무수해문호구체뢰주급THP-1세포DNA혹RNA활성.분별채용실시형광정량PCR화Western blot시험,검측문호구체뢰주감염THP-1세포전후vapB화vapC기인전록급표체수평적변화.구건vapB화vapC기인진핵표체재체병전염세포,채용CCK-8시제검측VapB화VapC단백대세포활성적영향.결과 소극륭적vapB화vapC기인핵감산급안기산서렬여문헌보도완전상동.소구건적원핵표체계통능분별표체rVapB 화rVapC.rVapC가수해RNA,단불수해DNA.문호구체뢰주감염THP-1세포후,vapB화vapC기인 전록급표체수평균현저상조,부분독성단백VapC외분비.전염vapC기인적인신소관상피세포HEK293대량사망.결론 문호구체뢰주VapC단백위RNA매,가재감염숙주세포과정중외분비병대세포유명현독성.
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 NiNTA affinity chromatography.Activity of rVapB and rVapC to lyse the DNAs or RNAs from L.interrogans strain Lai and THP-1 cells 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 leptospiral 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 cells,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 ceils 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.
