本文選題：豬鏈球菌2型 + 缺失突變株�。� 參考：《西南交通大學(xué)》2012年碩士論文

【摘要】：豬鏈球菌(Streptococcus suis, SS)為革蘭陽性溶血性兼性厭氧球菌,根據(jù)其莢膜多糖抗原性的差異,分為35個血清型,其中以豬鏈球菌2型(SS2)中國分離株毒力最強(qiáng)、危害最嚴(yán)重、流行最廣,是一種重要的人獸共患病病原體。我國江蘇和四川部分地區(qū)曾經(jīng)在1998年和2005年暴發(fā)SS2感染的大面積流行,導(dǎo)致上萬頭生豬死亡,上百人感染發(fā)病,幾十人死亡,受到全球關(guān)注,豬鏈球菌2型感染已成為成為了公共衛(wèi)生防疫的重點(diǎn)。 雖然近年來我國對豬鏈球菌2型的毒力因子及其有關(guān)發(fā)病機(jī)制的研究取得了進(jìn)展,但該菌生理生化、遺傳特性、毒力和致病機(jī)理方面還有很多未解之謎,豬鏈球菌2型中國分離菌株如何獲得必需的金屬元素鐵及其調(diào)控機(jī)理就是其中之一。 為此,本研究通過細(xì)菌形態(tài)學(xué)觀察、革蘭氏染色鏡檢和16SrDNA序列同源性分析等方法與技術(shù),從四川成都附近某養(yǎng)豬場分離獲得一株與NCBI數(shù)據(jù)庫中05ZYH33(豬鏈球菌2型中國分離毒株)同源性達(dá)到99%的豬鏈球菌菌株SS86；對該分離菌株的七個主要毒力因子CPS, MRP, EF, Sly, GDH, FBPS和ORF2的基因PCR檢測結(jié)果表明,本實(shí)驗(yàn)分離菌株與目前報道的2型豬鏈球菌的多數(shù)致病株毒力因子分布情況一致；將分離菌株培養(yǎng)在添加濃度為250gM的鐵專一螯合劑4,4'-聯(lián)吡啶(Dipyridyl, DPD)的鏈球菌THYB培養(yǎng)基中,分離菌株SS86幾乎不能生長,在含DPD螯合劑的THYB培養(yǎng)基中,分別添加1.5μM濃度的Fe2+,1.0μM濃度的Fe3+和30μM濃度的血紅蛋白時,菌株SS86可恢復(fù)到正常生長水平,表明鐵元素為豬鏈球菌SS86生長繁殖所必需；為了今后進(jìn)一步弄清豬鏈球菌2型中國分離株鐵元素吸收和調(diào)節(jié)機(jī)理,本實(shí)驗(yàn)以分離株SS86為出發(fā)菌株,采用基因敲除缺失突變/溫度敏感自殺性質(zhì)粒/電轉(zhuǎn)化方法,構(gòu)建了金屬依賴轉(zhuǎn)錄調(diào)節(jié)基因mtsR缺失突變株。用RT-PCR檢測mtsR基因缺失對豬鏈球菌金屬離子吸收系統(tǒng)Fhu、Feo、Mut和Mts操縱子的金屬離子綁定相關(guān)基因fhuA、feoA、mutA和mtsA以及鐵吸收調(diào)節(jié)基因fur的表達(dá)影響,研究結(jié)果顯示,野生菌株和mtsR基因缺失突變株的鐵吸收操縱子金屬元素綁定基因及調(diào)節(jié)基因fur表達(dá)存在差異,研究結(jié)果確認(rèn)了豬鏈球菌SS86菌株MtsR影響豬鏈球菌鐵吸收轉(zhuǎn)運(yùn)相關(guān)基因表達(dá)。 本研究結(jié)果不僅讓我們對豬鏈球菌中國分離株的毒力基因分布、鐵元素對菌株生長繁殖影響以及金屬依賴轉(zhuǎn)錄調(diào)節(jié)基因mtsR對金屬元素吸收操縱子金屬元素綁定基因及調(diào)節(jié)基因fur表達(dá)的調(diào)節(jié)有了較為系統(tǒng)的了解,同時也為我們今后進(jìn)一步深入研究豬鏈球菌2型菌株金屬離子吸收調(diào)控機(jī)理以及揭示豬鏈球菌致病機(jī)理奠定了基礎(chǔ)。
[Abstract]:Streptococcus suis (SS2) is a Gram-positive hemolytic facultative anaerobic cocci. According to the antigenicity of its capsule polysaccharides, it is divided into 35 serotypes. Among them, Streptococcus suis type 2 (SS2) is the most virulent, most harmful and prevalent strain in China.Is an important zoonotic pathogen.In some parts of Jiangsu and Sichuan provinces in China, an outbreak of SS2 infection occurred in 1998 and 2005, resulting in the deaths of tens of thousands of live pigs, hundreds of people infected with the disease and dozens of deaths.Streptococcus suis type 2 infection has become the focus of public health epidemic prevention.Although recent progress has been made in the study of virulence factors of Streptococcus suis type 2 and its pathogenesis in China, there are still many unsolved questions about the physiological and biochemical characteristics, genetic characteristics, virulence and pathogenicity of Streptococcus suis.One of them is how to obtain essential metal element iron and its regulation mechanism of Streptococcus suis type 2 Chinese isolate.Therefore, the methods and techniques of bacterial morphology observation, Gram staining and homology analysis of 16SrDNA sequence were used in this study.A strain of Streptococcus suis SS86 with 99% homology with 05ZYH33in NCBI database was isolated from a pig farm near Chengdu, Sichuan Province.The results of PCR analysis of GDH, FBPS and ORF2 showed that,The distribution of virulence factors of most pathogenic strains of Streptococcus suis type 2 was consistent with that reported at present, and the isolated strains were cultured in the THYB medium of Streptococcus suis, which was supplemented with iron specific chelator (250gM), 4x4, dipyridyl, dipyridyl, dipyridyl, dipyridyl, dipyridyl, dipyridyl, and dipyridyl.The isolated strain SS86 could hardly grow. In the THYB medium containing DPD chelator, the strain SS86 could return to normal growth level when adding 1.5 渭 M Fe2 1.0 渭 M Fe3 and 30 渭 M hemoglobin, respectively.The results showed that iron element was necessary for the growth and reproduction of Streptococcus suis SS86, and in order to further understand the mechanism of iron absorption and regulation of Streptococcus suis type 2 Chinese isolate, the isolated strain SS86 was used as the starting strain.A metal-dependent transcription regulatory gene (mtsR) deletion mutant was constructed by gene knockout deletion / temperature-sensitive suicide plasmid / electrotransformation.RT-PCR was used to detect the effect of mtsR gene deletion on the expression of metal ion-binding genes FhuAfeo AmomutA and mtsA in the metal ion absorption system of Streptococcus suis and Mts operon, and the iron absorption regulatory gene fur.There were differences in the expression of iron absorption operon metal element binding gene and regulatory gene fur between wild strain and mtsR gene deletion mutant strain. The results confirmed that SS86 strain MtsR affected the expression of iron absorption transporter related genes of Streptococcus suis.The results of this study not only allowed us to distribute virulence genes in Chinese isolates of Streptococcus suis,The effect of iron on the growth and reproduction of the strain and the regulation of metal-dependent transcription regulator gene mtsR on the metal element absorption operon metal element binding gene and the regulation gene fur expression have been systematically understood.It also lays a foundation for further study on the regulation mechanism of metal ion absorption of Streptococcus suis type 2 strain and for revealing the pathogenic mechanism of Streptococcus suis in the future.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R346;S855.1

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