本文選題：致病性鉤端螺旋體　切入點(diǎn)：毒素-抗毒素系統(tǒng)　出處：《浙江大學(xué)》2012年碩士論文

【摘要】：目的了解鉤端螺旋體毒素-抗毒素系統(tǒng)中毒性蛋白VapC的功能及其對宿主細(xì)胞的毒性作用。 方法以致病性問號鉤體黃疸出血群賴型賴株基因組DNA為模板,采用PCR擴(kuò)增全長vapB、vapC、vapBC基因并構(gòu)建其原核表達(dá)系統(tǒng)。采用SDS-PAGE檢測目的重組蛋白rVapB和rVapC表達(dá)情況,Ni-NTA親和層析柱提純r(jià)VapB和rVapC.檢測rVapB和rVapC有無水解問號鉤體賴株及THP-1細(xì)胞DNA或RNA活性。分別采用實(shí)時(shí)熒光定量RT-PCR和Western Blot試驗(yàn),檢測問號鉤體賴株感染THP-1細(xì)胞前后vapB和vapC基因轉(zhuǎn)錄及表達(dá)水平的變化。采用免疫熒光激光共聚焦顯微鏡法檢測VapB和VapC蛋白在問號鉤體賴株感染THP-1細(xì)胞中表達(dá)情況。構(gòu)建vapB和vapC基因真核表達(dá)載體并轉(zhuǎn)染細(xì)胞,采用CCK-8試劑檢測VapB和VapC蛋白對細(xì)胞活性的影響。 結(jié)果所克隆的vapB和vapC基因核苷酸及氨基酸序列與文獻(xiàn)報(bào)道完全相同。所構(gòu)建的原核表達(dá)系統(tǒng)能分別表達(dá)rVapB和rVapC。rVapC可水解RNA,但不水解DNA。問號鉤體賴株感染THP-1細(xì)胞后,vapB和vapC基因轉(zhuǎn)錄及表達(dá)水平均顯著上調(diào),部分毒性蛋白VapC外分泌。問號鉤體賴株感染細(xì)胞內(nèi)可檢出毒性蛋白VapC,轉(zhuǎn)染vapC基因的人腎小管上皮細(xì)胞HEK293大量死亡 結(jié)論問號鉤體賴株VapC蛋白為RNA酶,可在感染宿主細(xì)胞過程中外分泌并對細(xì)胞有明顯毒性。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R377

【共引文獻(xiàn)】

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本文編號：1712117