霍乱弧菌毒力基因tcpP调控基因的筛选及LysR蛋白功能的研究
发布时间：2018-11-05 07:14
【摘要】：霍乱弧菌既是自然水生环境中的常著菌也是引起人类腹泻疾病霍乱的病原体。霍乱弧菌的主要致病因子有ctxAB基因编码的霍乱毒素CT; TCP致病性岛,它编码TCP菌毛,该菌毛是霍乱弧菌在宿主肠道的定殖因子,也是CTXφ的受体;toxR,一个基本的毒力调节基因,编码CT和TCP的基因都受ToxR调控。 本文以霍乱弧菌LZV608为研究对象,通过质粒pSC123上的Mariner转座子的随机插入构建突变子文库,用荧光素酶基因作为报告基因,筛选到影响霍乱弧菌毒力基因tcpP表达的5个突变株,经过Arbitrary PCR扩增、DNA测序以及BLAST分析后确定转座子插入的基因分别是aphA, aphB,与代谢途径相关的激酶aroK,未知功能的假设蛋白以及甲基趋化性蛋白VC1405。 通过构建VC1405的框内缺失突变株以及VC1405回复突变菌株,发现VC1405缺失后tcpP的表达量降低,表现为报告基因lux的光值降低,而在回复突变菌株中光值可以恢复。根据报道AphA和AphB相互协同,作用于tcp操纵子正调控TcpPH,我们通过荧光素酶检测菌株证明VC1405对tcpP的调控并不是通过转录调节因子aphB和aphA实现的。 LysR转录调控家族(LTTRs)是一类被深入研究的转录调控因子。LTTRs在细菌细胞中发挥着广泛的调控功能,其调控的目的蛋白参与了细菌的新陈代谢、细胞分裂、群体感应、毒力、运动性、固氮和抗氧化反应等。霍乱弧菌中大约有40个LysR家族蛋白,其中有些蛋白的功能已经被详细的研究。本实验以未知功能的LysR家族蛋白VC1561为研究对象。通过基因敲除和互补表达的方法证明VC1561不是霍乱弧菌生长必须的基因；通过小鼠肠道定殖实验证明它与霍乱弧菌在小鼠肠道的定殖能力无关;根据Western bolt实验结果,我们知道VC1561与霍乱弧菌毒素产生无关;借助荧光素酶报告基因,我们构建了Lux检测菌株发现VC1561能够激活其下游基因VC1562的表达,并且能够抑制其自身的表达。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
本文编号：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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
【共引文献】
相关期刊论文 前1条
1 郝民;阚飙;;霍乱弧菌的环境生存及其监测[J];疾病监测;2010年06期
相关硕士学位论文 前4条
1 金晶;霍乱弧菌LysR家族基因vc2103的功能及其调控基因的研究[D];南京农业大学;2011年
2 李建华;霍乱弧菌外膜蛋白W和U的克隆表达及抗原性研究[D];中国人民解放军军事医学科学院;2011年
3 陈建东;霍乱弧菌体内诱导及在海水中生物膜形成相关基因的筛选与鉴定[D];南京农业大学;2008年
4 郝民;应用基于上转换发光技术的免疫层析法检测霍乱弧菌[D];中国疾病预防控制中心;2010年
，本文编号：2311307
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2311307.html
最近更新
教材专著
