本文關(guān)鍵詞：雞源性大腸桿菌噬菌體的分離鑒定及其抗菌性初步研究　出處：《錦州醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 噬菌體 分離鑒定 生物學(xué)特性 抗菌實(shí)驗(yàn) 雞源性大腸桿菌

【摘要】：目的本實(shí)驗(yàn)旨在從蛋雞糞便中分離出一株強(qiáng)裂解性,寬裂解譜的大腸桿菌噬菌體,初步了解其生物學(xué)特性,建立雛雞感染大腸桿菌動(dòng)物模型,應(yīng)用該噬菌體進(jìn)行抗菌性實(shí)驗(yàn),以PCR-DGGE分析不同組別雛雞腸道菌群的變化,為今后噬菌體治療雛雞大腸桿菌病奠定一定的實(shí)驗(yàn)及理論基礎(chǔ)。方法對(duì)26株雞源性大腸桿菌進(jìn)行種系分類群鑒定及其分子間的多態(tài)性分析,根據(jù)大腸桿菌種系及親緣關(guān)系選擇具有代表性的多株大腸桿菌作為分離噬菌體的宿主菌。利用宿主菌從不同地區(qū)雞場(chǎng)糞便中分離噬菌體,對(duì)分離的噬菌體進(jìn)行純化,增殖及效價(jià)與裂解譜的確定,選擇一株高效價(jià)、裂解性強(qiáng)、裂解譜寬的噬菌體,對(duì)其熱穩(wěn)定性、p H值穩(wěn)定性、乙醚及氯仿穩(wěn)定性,最佳感染復(fù)數(shù)(MOI)和一步生長(zhǎng)曲線等生物學(xué)特性進(jìn)行測(cè)定。通過(guò)確定雛雞感染大腸桿菌動(dòng)物疾病模型致病劑量(絕對(duì)致死量),建立了動(dòng)物感染模型。應(yīng)用PCR-DGGE技術(shù)對(duì)不同組別(噬菌體作用組、大腸桿菌感染陽(yáng)性組和空白對(duì)照組)雛雞的糞便菌群16S r DNA V6區(qū)段進(jìn)行分離,得到其指紋圖譜及聚類分析關(guān)系圖,分析評(píng)價(jià)噬菌體對(duì)雛雞大腸桿菌感染的抗菌作用效果。結(jié)果根據(jù)RAPD、種系分類群結(jié)果及大腸桿菌血清型,確定了14株雞源性大腸桿菌為宿主菌。應(yīng)用宿主菌從雞場(chǎng)糞便中初步分離了9株噬菌體,分別命名為SP1~SP9。噬菌體SP2經(jīng)純化后效價(jià)最高為3.6×1010pfu/m L,噬菌斑大于2mm,邊緣比較清晰;經(jīng)交叉裂解試驗(yàn)確定SP2噬菌體的裂解率為46.1%,其中完全裂解率為33.3%,相比于另外8株所分離到的噬菌體,裂解率最高,裂解譜最廣,故選擇SP2作為后續(xù)實(shí)驗(yàn)的研究對(duì)象。對(duì)噬菌體SP2的生物學(xué)特性進(jìn)行研究,該噬菌體的最佳感染復(fù)數(shù)為0.1,感染宿主菌的潛伏期為20min,裂解期70min,裂解量為36,70℃作用60min后仍可保留活性,在p H值為7時(shí),噬菌體SP2的效價(jià)最高,酸性環(huán)境對(duì)其影響較大,乙醚及氯仿對(duì)其影響較小。電鏡觀察可見噬菌體頭部呈正二十面體,可見尾部結(jié)構(gòu)。應(yīng)用雞源性大腸桿菌(O78)建立雛雞大腸桿菌感染動(dòng)物疾病模型,絕對(duì)致死量確定為3×109CFU。DGGE圖譜顯示健康雛雞1-10日齡腸道菌群多樣性逐日增加,第10日齡最為豐富;雛雞感染大腸桿菌后,圖譜多樣性出現(xiàn)變化,菌群明顯失調(diào);噬菌體作用組圖譜顯示個(gè)別條帶恢復(fù)正常,此外,整體可見有些菌群不受大腸桿菌影響,始終存在于各組各日齡不變,聚類分析圖中顯示噬菌體作用第二天后腸道菌群多樣性開始恢復(fù)。結(jié)論應(yīng)用雞源性大腸桿菌從蛋雞糞便中分離出一株裂解性強(qiáng),噬菌譜寬的大腸桿菌噬菌體,命名為SP2。熱穩(wěn)定性良好,適宜在中性或微堿性環(huán)境生長(zhǎng)發(fā)育,具有相對(duì)較長(zhǎng)的裂解期及較大的裂解量,噬菌體SP2對(duì)雛雞大腸桿菌感染具有較好抗菌作用。
[Abstract]:Objective to isolate a Escherichia coli phage with strong cleavage and wide cleavage spectrum from the feces of laying hens, to understand its biological characteristics and to establish an animal model of Escherichia coli infection in chicks. The bacteriophage was used to analyze the changes of intestinal microflora in different chicks by PCR-DGGE. Methods 26 strains of Escherichia coli were identified and analyzed by molecular polymorphism. Several representative strains of Escherichia coli were selected as host bacteria to isolate bacteriophages according to the strains and relatives of Escherichia coli, and phage was isolated from feces of chicken farms in different areas. The isolated phage was purified, proliferated, titer and cleavage spectrum was determined. A bacteriophage with high titer, strong cleavage and wide cleavage spectrum was selected, and the thermal stability of phage was stable. The biological characteristics such as the stability of ether and chloroform, the optimum number of infected moi) and the one-step growth curve were determined. The pathogenic dose (absolute lethal dose) was determined by determining the pathogenic dose (absolute lethal dose) of chicken infected with Escherichia coli disease model. The animal model of infection was established. Different groups (phage group) were treated by PCR-DGGE technique. The 16s r DNA V6 region of fecal flora was isolated from the chicks infected with E. coli and the control group. The fingerprint and cluster analysis were obtained. Analysis and evaluation of bacteriophage bacteriophage to chicken Escherichia coli infection effect. Results according to RAPD, species classification group results and Escherichia coli serotype. Fourteen strains of Escherichia coli from chicken were identified as host bacteria and 9 strains of bacteriophage were isolated from feces of chicken farm. After purification, the titer of bacteriophage SP2 was 3.6 脳 10 ~ (10) pFu / m ~ (-1), the plaque of phage was more than 2 mm, and the edge of phage was clear. The cleavage rate of SP2 phage was 46.1, and the complete cleavage rate was 33.3%. Compared with the other 8 strains, the cleavage rate was the highest and the cleavage spectrum was the most extensive. The biological characteristics of phage SP2 were studied. The optimal number of phage infection was 0.1, and the incubation period of host bacteria was 20 minutes. The activity of bacteriophage SP2 was the highest when pH value was 7:00, and the effect of acid environment on the activity of bacteriophage SP2 was higher than that of bacteriophage SP2. The effect of ether and chloroform on it was small. The positive eicosahedron and tail structure were observed in the head of phage by electron microscope. The animal model of E. coli infection in chicks was established by using chicken Escherichia coli O78). The absolute lethal dose was determined to be 3 脳 10 9 CFU. DGGE map showed that the diversity of intestinal flora of healthy chicks increased from day 1 to day 10, and was the most abundant at the 10th day. After chickens were infected with Escherichia coli, the diversity of the map was changed, and the bacterial flora was obviously out of balance. Phage group map showed that individual bands returned to normal. In addition, some bacteria groups were not affected by Escherichia coli and remained unchanged at all ages in each group. Cluster analysis showed that the diversity of intestinal flora began to recover after the second day of bacteriophage interaction. Conclusion A bacteriophage with strong cleavage and wide spectrum of phage was isolated from the feces of laying hens by chicken Escherichia coli. It is named SP2. It has good thermal stability and is suitable for growth and development in neutral or slightly alkaline environment. It has a relatively long pyrolysis period and a large amount of pyrolysis. Bacteriophage SP2 has a good antibacterial effect on Escherichia coli infection in chicks.
【學(xué)位授予單位】：錦州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.61

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