【摘要】： 霍亂弧菌(Vibrio cholerae)是急性致死性腹瀉病——霍亂的致病菌�；魜y弧菌已發(fā)現(xiàn)200多種不同的O抗原血清群,其中僅01群和0139群能夠引起霍亂流行。目前處于霍亂的第七次世界范圍的大流行中,與第六次大流行由古典生物型霍亂弧菌引起不同,第七次大流行的致病菌株是El Tor型霍亂弧菌。目前在我國使用的分型方法是噬菌體-生物分型,可將各種來源的01群El Tor型霍亂弧菌分為32個(gè)噬菌體型和12個(gè)生物型,結(jié)合二者又將菌株分為流行株和非流行株,可作為追溯傳染來源、傳播途徑和分析流行形式的流行病學(xué)工具之一。在實(shí)際中應(yīng)用噬菌體-生物分型的結(jié)果區(qū)別應(yīng)對(duì)流行株和非流行株,對(duì)于霍亂的預(yù)防控制起到了事半功倍的效果。 VP5是五個(gè)分型噬菌體之一,了解分型噬菌體的形態(tài)結(jié)構(gòu)、基因組特征,噬菌體感染霍亂弧菌的機(jī)制、噬菌體敏感菌株和抗性菌株之間的差異,對(duì)于認(rèn)識(shí)噬菌體-生物型別指導(dǎo)霍亂防治的機(jī)理、了解01群El Tor型霍亂弧菌的遺傳差異和分化有重要作用�；谑删w-生物分型的所有流行株型別中,僅有6b型霍亂弧菌對(duì)VP5噬菌體不敏感。1998至2001年,在我國四川省出現(xiàn)噬菌體—生物分型為6b的流行優(yōu)勢(shì)菌型,區(qū)別于同時(shí)期其他省份或四川省1998年前和2001年后的流行優(yōu)勢(shì)菌型(1b型)。我們?cè)噲D從這一特殊菌型流行的現(xiàn)象出發(fā),了解對(duì)VP5噬菌體不敏感的6b型菌株能夠得以流行的機(jī)制,了解分型噬菌體VP5裂解霍亂弧菌的過程。 我們通過電鏡觀察到分型噬菌體VP5頭部呈正六邊形,邊長(zhǎng)約37nm,半徑約31nm,具有短尾,尾長(zhǎng)約18nm,推測(cè)其為典型的短尾20面體結(jié)構(gòu)。其基因組測(cè)序得到長(zhǎng)度為39789bps的序列,預(yù)測(cè)了36個(gè)ORF和其中7個(gè)ORF的功能,前16個(gè)在正鏈上,后20個(gè)在負(fù)鏈上。該基因組未發(fā)現(xiàn)rRNA和tRNA基因。 我們選擇了1998至2001年四川省流行的01群霍亂弧菌6b菌株22株和同時(shí)期四川及其他省份流行的霍亂弧菌1b菌株23株。實(shí)驗(yàn)證實(shí)了1998—2001年期間在四川流行的1b型和6b型菌株均為產(chǎn)毒株。通過對(duì)這45株霍亂弧菌的ompW基因與N16961序列比對(duì)發(fā)現(xiàn)6b菌株的ompW基因ORF第298-308堿基位置缺失了11個(gè)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菌型復(fù)雜的遺傳背景下,這一特征可能可以作為追蹤這類具有特殊遺傳背景的菌型的遺傳標(biāo)記。N16961-dompW在低營(yíng)養(yǎng)狀態(tài)下的生長(zhǎng)優(yōu)勢(shì)和更強(qiáng)的生物膜形成能力,提示這一菌型在環(huán)境適應(yīng)中的優(yōu)勢(shì)。 將N16961的ompW基因以氯霉素基因替代后,N16961由VP5敏感變?yōu)閂P5抗性,回補(bǔ)ompW基因,缺失株能恢復(fù)對(duì)VP5的敏感。表達(dá)純化的OmpW蛋白可以使VP5噬菌體對(duì)N16961的裂解鈍化,說明N16961中OmpW蛋白為霍亂弧菌分型噬菌體VP5的受體。 通過本研究,我們對(duì)01群El Tor型霍亂弧菌分型噬菌體VP5的形態(tài)結(jié)構(gòu)、基因組及其受體,6b型菌株的遺傳特征和分子標(biāo)記,都有了初步的認(rèn)識(shí),能夠幫助我們理解噬菌體-生物分型的理論基礎(chǔ)和噬菌體對(duì)于菌株環(huán)境適應(yīng)的意義。 模式生物是研究病原細(xì)菌的重要工具,常利用模式生物模擬感染或共生的過程,來研究病原細(xì)菌的毒力因子、致病機(jī)制、與宿主相互作用、生物膜形成、耐藥等各個(gè)方面。目前常用于研究霍亂弧菌的有小鼠、大鼠、兔等動(dòng)物,都由于操作較為復(fù)雜,價(jià)格相對(duì)昂貴,影響結(jié)果的因素多而受到局限。秀麗隱桿線蟲是一種廣泛應(yīng)用的模式生物,便于喂養(yǎng)和觀察,從1999年開始用于致病菌的研究。目前已在多種病原菌的研究中得到應(yīng)用。 這里我們嘗試將秀麗隱桿線蟲應(yīng)用于霍亂弧菌的研究,實(shí)驗(yàn)中我們首先確定秀麗隱桿線蟲可以霍亂弧菌為食,與大腸桿菌OP50對(duì)比,在霍亂弧菌N16961的菌苔上秀麗隱桿線蟲生長(zhǎng)和發(fā)育進(jìn)程沒有明顯改變。我們選擇了來源于01和0139群不同血清型CT陽性或陰性的霍亂弧菌共24株,通過PCR驗(yàn)證這些菌株都存在文獻(xiàn)報(bào)道的霍亂弧菌對(duì)秀麗隱桿線蟲致死必須的prtV基因。以大腸桿菌OP50為對(duì)照,檢測(cè)秀麗隱桿線蟲在24株霍亂弧菌菌苔上的生存時(shí)間,做出生存曲線,分組比較。結(jié)果顯示,實(shí)驗(yàn)中使用霍亂弧菌較之大腸桿菌對(duì)照,使秀麗隱桿線蟲生存時(shí)間明顯縮短,有致死性作用。實(shí)驗(yàn)中使用01群和0139群霍亂弧菌較之大腸桿菌對(duì)照都有致死性作用,但兩群之間無明顯差異。在本實(shí)驗(yàn)中,CT陽性和CT陰性的霍亂弧菌較之大腸桿菌對(duì)照都有致死性作用,兩者比較CT陽性組能將線蟲更快致死。 本研究是將秀麗隱桿線蟲應(yīng)用于霍亂弧菌研究的初步嘗試。參考利用秀麗隱桿線蟲研究其他病原細(xì)菌的方法,初步建立了使用秀麗隱桿線蟲研究霍亂弧菌的實(shí)驗(yàn)方法。然而,將秀麗隱桿線蟲作為霍亂弧菌的研究模型,尚有理論基礎(chǔ)需要完善、實(shí)驗(yàn)細(xì)節(jié)需要摸索,這些都有待于進(jìn)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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378

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本文編號(hào)：2143346