O1群El Tor型霍乱弧菌分型噬菌体VP5和其受体的研究
发布时间：2018-07-25 09:14
【摘要】： 霍乱弧菌(Vibrio cholerae)是急性致死性腹泻病——霍乱的致病菌。霍乱弧菌已发现200多种不同的O抗原血清群,其中仅01群和0139群能够引起霍乱流行。目前处于霍乱的第七次世界范围的大流行中,与第六次大流行由古典生物型霍乱弧菌引起不同,第七次大流行的致病菌株是El Tor型霍乱弧菌。目前在我国使用的分型方法是噬菌体-生物分型,可将各种来源的01群El Tor型霍乱弧菌分为32个噬菌体型和12个生物型,结合二者又将菌株分为流行株和非流行株,可作为追溯传染来源、传播途径和分析流行形式的流行病学工具之一。在实际中应用噬菌体-生物分型的结果区别应对流行株和非流行株,对于霍乱的预防控制起到了事半功倍的效果。 VP5是五个分型噬菌体之一,了解分型噬菌体的形态结构、基因组特征,噬菌体感染霍乱弧菌的机制、噬菌体敏感菌株和抗性菌株之间的差异,对于认识噬菌体-生物型别指导霍乱防治的机理、了解01群El Tor型霍乱弧菌的遗传差异和分化有重要作用。基于噬菌体-生物分型的所有流行株型别中,仅有6b型霍乱弧菌对VP5噬菌体不敏感。1998至2001年,在我国四川省出现噬菌体—生物分型为6b的流行优势菌型,区别于同时期其他省份或四川省1998年前和2001年后的流行优势菌型(1b型)。我们试图从这一特殊菌型流行的现象出发,了解对VP5噬菌体不敏感的6b型菌株能够得以流行的机制,了解分型噬菌体VP5裂解霍乱弧菌的过程。 我们通过电镜观察到分型噬菌体VP5头部呈正六边形,边长约37nm,半径约31nm,具有短尾,尾长约18nm,推测其为典型的短尾20面体结构。其基因组测序得到长度为39789bps的序列,预测了36个ORF和其中7个ORF的功能,前16个在正链上,后20个在负链上。该基因组未发现rRNA和tRNA基因。 我们选择了1998至2001年四川省流行的01群霍乱弧菌6b菌株22株和同时期四川及其他省份流行的霍乱弧菌1b菌株23株。实验证实了1998—2001年期间在四川流行的1b型和6b型菌株均为产毒株。通过对这45株霍乱弧菌的ompW基因与N16961序列比对发现6b菌株的ompW基因ORF第298-308碱基位置缺失了11个bp,而其余23株菌非6b型菌株的ompW基因ORF是完整的。在1b和6b菌型复杂的遗传背景下,这一特征可能可以作为追踪这类具有特殊遗传背景的菌型的遗传标记。N16961-dompW在低营养状态下的生长优势和更强的生物膜形成能力,提示这一菌型在环境适应中的优势。 将N16961的ompW基因以氯霉素基因替代后,N16961由VP5敏感变为VP5抗性,回补ompW基因,缺失株能恢复对VP5的敏感。表达纯化的OmpW蛋白可以使VP5噬菌体对N16961的裂解钝化,说明N16961中OmpW蛋白为霍乱弧菌分型噬菌体VP5的受体。 通过本研究,我们对01群El Tor型霍乱弧菌分型噬菌体VP5的形态结构、基因组及其受体,6b型菌株的遗传特征和分子标记,都有了初步的认识,能够帮助我们理解噬菌体-生物分型的理论基础和噬菌体对于菌株环境适应的意义。 模式生物是研究病原细菌的重要工具,常利用模式生物模拟感染或共生的过程,来研究病原细菌的毒力因子、致病机制、与宿主相互作用、生物膜形成、耐药等各个方面。目前常用于研究霍乱弧菌的有小鼠、大鼠、兔等动物,都由于操作较为复杂,价格相对昂贵,影响结果的因素多而受到局限。秀丽隐杆线虫是一种广泛应用的模式生物,便于喂养和观察,从1999年开始用于致病菌的研究。目前已在多种病原菌的研究中得到应用。 这里我们尝试将秀丽隐杆线虫应用于霍乱弧菌的研究,实验中我们首先确定秀丽隐杆线虫可以霍乱弧菌为食,与大肠杆菌OP50对比,在霍乱弧菌N16961的菌苔上秀丽隐杆线虫生长和发育进程没有明显改变。我们选择了来源于01和0139群不同血清型CT阳性或阴性的霍乱弧菌共24株,通过PCR验证这些菌株都存在文献报道的霍乱弧菌对秀丽隐杆线虫致死必须的prtV基因。以大肠杆菌OP50为对照,检测秀丽隐杆线虫在24株霍乱弧菌菌苔上的生存时间,做出生存曲线,分组比较。结果显示,实验中使用霍乱弧菌较之大肠杆菌对照,使秀丽隐杆线虫生存时间明显缩短,有致死性作用。实验中使用01群和0139群霍乱弧菌较之大肠杆菌对照都有致死性作用,但两群之间无明显差异。在本实验中,CT阳性和CT阴性的霍乱弧菌较之大肠杆菌对照都有致死性作用,两者比较CT阳性组能将线虫更快致死。 本研究是将秀丽隐杆线虫应用于霍乱弧菌研究的初步尝试。参考利用秀丽隐杆线虫研究其他病原细菌的方法,初步建立了使用秀丽隐杆线虫研究霍乱弧菌的实验方法。然而,将秀丽隐杆线虫作为霍乱弧菌的研究模型,尚有理论基础需要完善、实验细节需要摸索,这些都有待于进一步的工作去探索。
[Abstract]:Vibrio cholerae (Vibrio cholerae) is an acute fatal diarrhoea - cholera pathogenic bacteria. Vibrio cholerae has found more than 200 different O antigens serogroups, of which only 01 groups and 0139 groups can cause cholera epidemic. At present, the seventh worldwide pandemic of cholera and the sixth pandemic are induced by Vibrio cholerae. The pathogenic strain of the seventh pandemic is Vibrio cholerae El Tor. The typing method used in China is a phage biotype. The 01 group of El Tor Vibrio cholerae in various sources can be divided into 32 phagocytic and 12 biotypes, and the strains are divided into epidemic and non epidemic strains, which can be used as traceability infection. Sources, transmission routes and epidemiological tools for analysis of epidemic forms. In practice, the application of phage biotyping results in the difference in response to epidemic and non epidemic strains, which has achieved half the effort in the prevention and control of cholera.
VP5 is one of the five types of phage, understanding the morphological structure of the phage, the characteristics of the genome, the mechanism of phage infection of Vibrio cholerae, the difference between the sensitive and resistant strains of phage, and the understanding of the genetic differences and differentiation of the 01 group of Vibrio cholerae by the understanding of the mechanism of phage biological type guiding cholera control. In all popular plant types based on phage - based biotyping, only 6B type Vibrio cholerae were not sensitive to VP5 phage from.1998 to 2001. In Sichuan Province, the bacteriophage - type was found to be the dominant bacteria type of 6B, which was different from that of other provinces in the same period or in Sichuan Province before and after 2001 (1b type). We try to understand the mechanism of the epidemic of VP5 phage 6B strains which are not sensitive to the bacteriophage, and understand the process of splitting the phage VP5 for the lysis of Vibrio cholerae.
We observed by electron microscopy that the head of the phage VP5 was hexagonal, with a length of about 37nm and a radius of about 31nm, with a short tail and a tail length of about 18NM, and speculated that it was a typical short tailed 20 body structure. The genome was sequenced with a sequence of 39789bps length, 36 ORF and 7 ORF functions, the first 16 on the positive chain, and the latter 20 on the negative chain. RRNA and tRNA genes were not found in the genome.
We selected 01 strains of Vibrio cholerae 6B strain 6B strain from 1998 to 2001 and 23 strains of Vibrio cholerae 1b epidemic in Sichuan and other provinces. The experiment confirmed that both 1b and 6B strains prevalent in Sichuan during the period from 1998 to 2001 were all strains. The ompW gene of Vibrio parvus was compared with N16961 sequence. The site 298-308 base position of the ompW gene ORF of the present strain of 6b is missing 11 BP, while the ompW gene ORF of the remaining 23 strains of non 6B strains is complete. In the complex genetic background of 1b and 6B bacteria, this feature may be used as a genetic marker for tracing this kind of special genetic background of the genetic marker.N16961-dompW in the low nutrition state. The long dominance and stronger biofilm forming ability suggest that this strain has an advantage in environmental adaptation.
After replacing the ompW gene of N16961 with the chloramphenicol gene, N16961 changed from VP5 sensitive to VP5 resistance and returned to the ompW gene. The deletion strain could restore the sensitivity to VP5. The expressed OmpW protein could make the VP5 phage cracking and passivation of N16961, indicating that OmpW protein is a receptor for Vibrio cholerae in N16961.
Through this study, we have a preliminary understanding of the morphological structure, the genome and its receptor, the genetic characteristics and molecular markers of the 01 group of El Tor Vibrio cholerae type phage VP5, which can help us understand the theoretical basis of phage biotyping and the significance of phage adaptation to the strain environment.
Pattern biology is an important tool for the study of pathogenic bacteria. It often uses model organisms to simulate infection or symbiotic processes to study the virulence factors, pathogenic mechanisms, interaction with host, biofilm formation, and drug resistance, which are commonly used in mice, rats, rabbits and other animals that are commonly used to study cholera arcus because of their operation. Complicated and relatively expensive, the factors affecting the results are limited. Caenorhabditis elegans is a widely used model creature, which is easy to feed and observe. It has been used in the study of pathogenic bacteria from 1999.
Here we try to apply the Caenorhabditis elegans in the study of Vibrio cholerae. In the experiment, we first determined that the Caenorhabditis elegans could eat Vibrio cholerae, compared with the Escherichia coli OP50, and there was no obvious change in the growth and development process of C. elegans on the N16961 of Vibrio cholerae. We chose to come from the 01 and 0139 groups. A total of 24 serotype CT positive or negative Vibrio cholerae were confirmed by PCR. The prtV genes of Vibrio cholerae reported in the literature were reported. The survival time of C. elegans in the 24 strains of Vibrio cholerae was detected and the survival curves were compared. In the experiment, Vibrio cholerae was compared with Escherichia coli, which made the survival time of C. elegans significantly shortened and fatal. In the experiment, 01 groups and 0139 groups of Vibrio cholerae were lethal than those of the Escherichia coli, but there was no significant difference between the two groups. In this experiment, CT positive and CT negative Vibrio cholerae were larger. Enterobacter control had lethal effects, and the nematodes were more lethal in CT-positive group than in CT-positive group.
This study is a preliminary attempt to apply the elegans of Caenorhabditis elegans to the study of Vibrio cholerae. With reference to the study of other pathogenic bacteria by using Caenorhabditis elegans, an experimental method for the study of Vibrio cholerae using Caenorhabditis elegans is initially established. However, there is a theoretical basis for the study of C. elegans as a model of Vibrio cholerae. Perfect, experimental details need to be explored, these are to be further work to explore.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R378
[Abstract]:Vibrio cholerae (Vibrio cholerae) is an acute fatal diarrhoea - cholera pathogenic bacteria. Vibrio cholerae has found more than 200 different O antigens serogroups, of which only 01 groups and 0139 groups can cause cholera epidemic. At present, the seventh worldwide pandemic of cholera and the sixth pandemic are induced by Vibrio cholerae. The pathogenic strain of the seventh pandemic is Vibrio cholerae El Tor. The typing method used in China is a phage biotype. The 01 group of El Tor Vibrio cholerae in various sources can be divided into 32 phagocytic and 12 biotypes, and the strains are divided into epidemic and non epidemic strains, which can be used as traceability infection. Sources, transmission routes and epidemiological tools for analysis of epidemic forms. In practice, the application of phage biotyping results in the difference in response to epidemic and non epidemic strains, which has achieved half the effort in the prevention and control of cholera.
VP5 is one of the five types of phage, understanding the morphological structure of the phage, the characteristics of the genome, the mechanism of phage infection of Vibrio cholerae, the difference between the sensitive and resistant strains of phage, and the understanding of the genetic differences and differentiation of the 01 group of Vibrio cholerae by the understanding of the mechanism of phage biological type guiding cholera control. In all popular plant types based on phage - based biotyping, only 6B type Vibrio cholerae were not sensitive to VP5 phage from.1998 to 2001. In Sichuan Province, the bacteriophage - type was found to be the dominant bacteria type of 6B, which was different from that of other provinces in the same period or in Sichuan Province before and after 2001 (1b type). We try to understand the mechanism of the epidemic of VP5 phage 6B strains which are not sensitive to the bacteriophage, and understand the process of splitting the phage VP5 for the lysis of Vibrio cholerae.
We observed by electron microscopy that the head of the phage VP5 was hexagonal, with a length of about 37nm and a radius of about 31nm, with a short tail and a tail length of about 18NM, and speculated that it was a typical short tailed 20 body structure. The genome was sequenced with a sequence of 39789bps length, 36 ORF and 7 ORF functions, the first 16 on the positive chain, and the latter 20 on the negative chain. RRNA and tRNA genes were not found in the genome.
We selected 01 strains of Vibrio cholerae 6B strain 6B strain from 1998 to 2001 and 23 strains of Vibrio cholerae 1b epidemic in Sichuan and other provinces. The experiment confirmed that both 1b and 6B strains prevalent in Sichuan during the period from 1998 to 2001 were all strains. The ompW gene of Vibrio parvus was compared with N16961 sequence. The site 298-308 base position of the ompW gene ORF of the present strain of 6b is missing 11 BP, while the ompW gene ORF of the remaining 23 strains of non 6B strains is complete. In the complex genetic background of 1b and 6B bacteria, this feature may be used as a genetic marker for tracing this kind of special genetic background of the genetic marker.N16961-dompW in the low nutrition state. The long dominance and stronger biofilm forming ability suggest that this strain has an advantage in environmental adaptation.
After replacing the ompW gene of N16961 with the chloramphenicol gene, N16961 changed from VP5 sensitive to VP5 resistance and returned to the ompW gene. The deletion strain could restore the sensitivity to VP5. The expressed OmpW protein could make the VP5 phage cracking and passivation of N16961, indicating that OmpW protein is a receptor for Vibrio cholerae in N16961.
Through this study, we have a preliminary understanding of the morphological structure, the genome and its receptor, the genetic characteristics and molecular markers of the 01 group of El Tor Vibrio cholerae type phage VP5, which can help us understand the theoretical basis of phage biotyping and the significance of phage adaptation to the strain environment.
Pattern biology is an important tool for the study of pathogenic bacteria. It often uses model organisms to simulate infection or symbiotic processes to study the virulence factors, pathogenic mechanisms, interaction with host, biofilm formation, and drug resistance, which are commonly used in mice, rats, rabbits and other animals that are commonly used to study cholera arcus because of their operation. Complicated and relatively expensive, the factors affecting the results are limited. Caenorhabditis elegans is a widely used model creature, which is easy to feed and observe. It has been used in the study of pathogenic bacteria from 1999.
Here we try to apply the Caenorhabditis elegans in the study of Vibrio cholerae. In the experiment, we first determined that the Caenorhabditis elegans could eat Vibrio cholerae, compared with the Escherichia coli OP50, and there was no obvious change in the growth and development process of C. elegans on the N16961 of Vibrio cholerae. We chose to come from the 01 and 0139 groups. A total of 24 serotype CT positive or negative Vibrio cholerae were confirmed by PCR. The prtV genes of Vibrio cholerae reported in the literature were reported. The survival time of C. elegans in the 24 strains of Vibrio cholerae was detected and the survival curves were compared. In the experiment, Vibrio cholerae was compared with Escherichia coli, which made the survival time of C. elegans significantly shortened and fatal. In the experiment, 01 groups and 0139 groups of Vibrio cholerae were lethal than those of the Escherichia coli, but there was no significant difference between the two groups. In this experiment, CT positive and CT negative Vibrio cholerae were larger. Enterobacter control had lethal effects, and the nematodes were more lethal in CT-positive group than in CT-positive group.
This study is a preliminary attempt to apply the elegans of Caenorhabditis elegans to the study of Vibrio cholerae. With reference to the study of other pathogenic bacteria by using Caenorhabditis elegans, an experimental method for the study of Vibrio cholerae using Caenorhabditis elegans is initially established. However, there is a theoretical basis for the study of C. elegans as a model of Vibrio cholerae. Perfect, experimental details need to be explored, these are to be further work to explore.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R378
【相似文献】
相关期刊论文 前10条
1 许志锦,田京慧,谢正e，
本文编号：2143346
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2143346.html
最近更新
教材专著
