霍亂弧菌毒力基因tcpP調(diào)控基因的篩選及LysR蛋白功能的研究
發(fā)布時間：2018-11-05 07:14
【摘要】：霍亂弧菌既是自然水生環(huán)境中的常著菌也是引起人類腹瀉疾病霍亂的病原體�；魜y弧菌的主要致病因子有ctxAB基因編碼的霍亂毒素CT; TCP致病性島,它編碼TCP菌毛,該菌毛是霍亂弧菌在宿主腸道的定殖因子,也是CTXφ的受體;toxR,一個基本的毒力調(diào)節(jié)基因,編碼CT和TCP的基因都受ToxR調(diào)控。 本文以霍亂弧菌LZV608為研究對象,通過質(zhì)粒pSC123上的Mariner轉(zhuǎn)座子的隨機插入構(gòu)建突變子文庫,用熒光素酶基因作為報告基因,篩選到影響霍亂弧菌毒力基因tcpP表達的5個突變株,經(jīng)過Arbitrary PCR擴增、DNA測序以及BLAST分析后確定轉(zhuǎn)座子插入的基因分別是aphA, aphB,與代謝途徑相關(guān)的激酶aroK,未知功能的假設(shè)蛋白以及甲基趨化性蛋白VC1405。 通過構(gòu)建VC1405的框內(nèi)缺失突變株以及VC1405回復突變菌株,發(fā)現(xiàn)VC1405缺失后tcpP的表達量降低,表現(xiàn)為報告基因lux的光值降低,而在回復突變菌株中光值可以恢復。根據(jù)報道AphA和AphB相互協(xié)同,作用于tcp操縱子正調(diào)控TcpPH,我們通過熒光素酶檢測菌株證明VC1405對tcpP的調(diào)控并不是通過轉(zhuǎn)錄調(diào)節(jié)因子aphB和aphA實現(xiàn)的。 LysR轉(zhuǎn)錄調(diào)控家族(LTTRs)是一類被深入研究的轉(zhuǎn)錄調(diào)控因子。LTTRs在細菌細胞中發(fā)揮著廣泛的調(diào)控功能,其調(diào)控的目的蛋白參與了細菌的新陳代謝、細胞分裂、群體感應、毒力、運動性、固氮和抗氧化反應等。霍亂弧菌中大約有40個LysR家族蛋白,其中有些蛋白的功能已經(jīng)被詳細的研究。本實驗以未知功能的LysR家族蛋白VC1561為研究對象。通過基因敲除和互補表達的方法證明VC1561不是霍亂弧菌生長必須的基因；通過小鼠腸道定殖實驗證明它與霍亂弧菌在小鼠腸道的定殖能力無關(guān);根據(jù)Western bolt實驗結(jié)果,我們知道VC1561與霍亂弧菌毒素產(chǎn)生無關(guān);借助熒光素酶報告基因,我們構(gòu)建了Lux檢測菌株發(fā)現(xiàn)VC1561能夠激活其下游基因VC1562的表達,并且能夠抑制其自身的表達。
[Abstract]:Vibrio cholerae is not only a common bacterium in natural aquatic environment, but also a pathogen of human diarrhea. The main pathogenic factor of Vibrio cholerae is the ctxAB gene encoded CT; TCP pathogenicity island, which encodes TCP pili, which is the colonizing factor of Vibrio cholerae in the host intestine and the receptor of CTX 蠁. ToxR, is a basic virulence regulator, and the genes that encode CT and TCP are regulated by ToxR. In this paper, the mutant library was constructed by random insertion of Mariner transposon on plasmid pSC123, and luciferase gene was used as reporter gene. Five mutants affecting tcpP expression of virulence gene of Vibrio cholerae were screened. After Arbitrary PCR amplification, DNA sequencing and BLAST analysis, it was determined that the transposon inserted gene was a hypothetical protein of the unknown function of aphA, aphB, kinase aroK, associated with metabolic pathway and a methyl chemoattractant protein VC1405., respectively. It was found that the expression of tcpP decreased after VC1405 deletion, which showed that the light value of the reporter gene lux was decreased, but the light value could be recovered in the reverse-mutated strain. It is reported that AphA and AphB cooperate with each other in the regulation of TcpPH, by tcp operon. We proved by luciferase detection that the regulation of tcpP by VC1405 is not effected by the transcription regulatory factors aphB and aphA. LysR transcriptional regulation family (LTTRs) is a kind of transcription regulator which has been studied in depth. LTTRs plays a wide regulatory role in bacterial cells. The target protein involved in bacterial metabolism, cell division, population induction, virulence. Exercise, nitrogen fixation and antioxidant reactions. There are about 40 LysR family proteins in Vibrio cholerae, some of which have been studied in detail. In this study, the unknown functional LysR family protein VC1561 was used as the research object. Gene knockout and complementary expression showed that VC1561 was not a necessary gene for the growth of Vibrio cholerae. The colonization test of mouse intestine proved that it had nothing to do with the colonization ability of Vibrio cholerae in the intestinal tract of mice, and according to the results of Western bolt test, we know that VC1561 has nothing to do with the production of Vibrio cholerae toxin. With the luciferase reporter gene, we constructed a Lux detection strain and found that VC1561 could activate the expression of its downstream gene VC1562 and inhibit its own expression.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R378
本文編號：2311307
[Abstract]:Vibrio cholerae is not only a common bacterium in natural aquatic environment, but also a pathogen of human diarrhea. The main pathogenic factor of Vibrio cholerae is the ctxAB gene encoded CT; TCP pathogenicity island, which encodes TCP pili, which is the colonizing factor of Vibrio cholerae in the host intestine and the receptor of CTX 蠁. ToxR, is a basic virulence regulator, and the genes that encode CT and TCP are regulated by ToxR. In this paper, the mutant library was constructed by random insertion of Mariner transposon on plasmid pSC123, and luciferase gene was used as reporter gene. Five mutants affecting tcpP expression of virulence gene of Vibrio cholerae were screened. After Arbitrary PCR amplification, DNA sequencing and BLAST analysis, it was determined that the transposon inserted gene was a hypothetical protein of the unknown function of aphA, aphB, kinase aroK, associated with metabolic pathway and a methyl chemoattractant protein VC1405., respectively. It was found that the expression of tcpP decreased after VC1405 deletion, which showed that the light value of the reporter gene lux was decreased, but the light value could be recovered in the reverse-mutated strain. It is reported that AphA and AphB cooperate with each other in the regulation of TcpPH, by tcp operon. We proved by luciferase detection that the regulation of tcpP by VC1405 is not effected by the transcription regulatory factors aphB and aphA. LysR transcriptional regulation family (LTTRs) is a kind of transcription regulator which has been studied in depth. LTTRs plays a wide regulatory role in bacterial cells. The target protein involved in bacterial metabolism, cell division, population induction, virulence. Exercise, nitrogen fixation and antioxidant reactions. There are about 40 LysR family proteins in Vibrio cholerae, some of which have been studied in detail. In this study, the unknown functional LysR family protein VC1561 was used as the research object. Gene knockout and complementary expression showed that VC1561 was not a necessary gene for the growth of Vibrio cholerae. The colonization test of mouse intestine proved that it had nothing to do with the colonization ability of Vibrio cholerae in the intestinal tract of mice, and according to the results of Western bolt test, we know that VC1561 has nothing to do with the production of Vibrio cholerae toxin. With the luciferase reporter gene, we constructed a Lux detection strain and found that VC1561 could activate the expression of its downstream gene VC1562 and inhibit its own expression.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R378
【共引文獻】
相關(guān)期刊論文 前1條
1 郝民;闞飆;;霍亂弧菌的環(huán)境生存及其監(jiān)測[J];疾病監(jiān)測;2010年06期
相關(guān)碩士學位論文 前4條
1 金晶;霍亂弧菌LysR家族基因vc2103的功能及其調(diào)控基因的研究[D];南京農(nóng)業(yè)大學;2011年
2 李建華;霍亂弧菌外膜蛋白W和U的克隆表達及抗原性研究[D];中國人民解放軍軍事醫(yī)學科學院;2011年
3 陳建東;霍亂弧菌體內(nèi)誘導及在海水中生物膜形成相關(guān)基因的篩選與鑒定[D];南京農(nóng)業(yè)大學;2008年
4 郝民;應用基于上轉(zhuǎn)換發(fā)光技術(shù)的免疫層析法檢測霍亂弧菌[D];中國疾病預防控制中心;2010年
，本文編號：2311307
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