本文選題：銅綠假單胞菌 + 噬菌體��； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：銅綠假單胞菌是一種在環(huán)境中廣泛存在的革蘭氏陰性致病菌。該菌可以引起水貂嚴(yán)重的出血性肺炎，給水貂養(yǎng)殖業(yè)帶來了巨大的損失。近些年，由于多重耐藥銅綠假單胞菌的出現(xiàn)，使該菌引起感染的控制變得越來越困難。噬菌體是可以特異性感染細(xì)菌的病毒，可以作為一種新型的治療制劑，在治療多重耐藥菌株感染方面具有很大的應(yīng)用潛力。 本研究以實驗室前期在貂場患病水貂體內(nèi)分離的致病性銅綠假單胞菌菌株D9作為宿主菌，從污水中分離獲得了一株新的噬菌體，將其命名為YH6。對噬菌體YH6的生物特性進(jìn)行了研究，表明該噬菌體是可以特異性的裂解銅綠假單胞菌的烈性噬菌體，噬菌斑清晰透亮、無暈環(huán)；該噬菌體屬于有尾病毒目短尾病毒科，頭部呈正二十面體，頭部直徑大約是65nm，尾部較短，，大約是25nm；其潛伏期為30min，裂解周期為60min；最佳感染復(fù)數(shù)為0.01-0.001，噬菌體滴度能達(dá)到1010PFU/mL；該噬菌體對乙醚、氯仿不敏感；在溫度40°C、50°C環(huán)境中和pH值6.0~10.0時保持穩(wěn)定；YH6可以裂解82.61%的銅綠假單胞菌，表現(xiàn)出了廣譜的裂解活性。 對噬菌體YH6進(jìn)行了全基因組序列測序和分析，表明該噬菌體的遺傳物質(zhì)為雙鏈線性DNA，長度為73,050bp，具有90個開放閱讀框（ORFs），G+C%含量為54.88%，其基因編碼率為92.64%，與銅綠假單胞菌的噬菌體LUZ7，vB_PaeP_C2-10_Ab09，PA26和LIT1的基因組同源性較近。YH6基因組中不含有細(xì)菌毒力和溶源性相關(guān)的ORF，更利于該噬菌體作為治療制劑應(yīng)用。通過序列比對和分析確定了噬菌體YH6的裂解酶基因（LysYH6），對該基因進(jìn)行表達(dá)和純化，活性測定實驗表明裂解酶LysYH6在EDTA（25mM/L）存在的條件下表現(xiàn)出顯著的殺菌活性。 動物實驗表明，對小鼠進(jìn)行鼻腔接種銅綠假單胞菌D9（2×108CFU），能夠成功構(gòu)建小鼠的出血性肺炎模型。治療實驗表明一次性鼻腔應(yīng)用噬菌體YH6（2×107PFU，MOI=0.1）可以有效保護(hù)感染銅綠假單胞菌造成的出血性肺炎小鼠模型。對治療組小鼠血液、脾臟、肺臟進(jìn)行了菌落計數(shù)，表明噬菌體YH6應(yīng)用10h以后可以使臟器內(nèi)的菌含量降低1.5個對數(shù)單位，2d以內(nèi)可以將體內(nèi)的銅綠假單胞菌完全清除；與此相反地，未治療組臟器內(nèi)菌含量升高4個對數(shù)單位，并在2d內(nèi)引起死亡。另外，對治療組小鼠的肺臟進(jìn)行了組織病理學(xué)切片觀察，表明YH6能有效的改善銅綠假單胞菌造成的小鼠肺損傷。 綜上，本研究所分離到的噬菌體YH6在治療銅綠假單胞菌引起的小鼠出血性肺炎模型方面表現(xiàn)出很大的應(yīng)用潛力。為后續(xù)研究將該噬菌體應(yīng)用到水貂感染銅綠假單胞菌的治療奠定了堅實的基礎(chǔ)。
[Abstract]:Pseudomonas aeruginosa is a gram-negative pathogenic bacteria widely found in the environment.The bacteria can cause severe hemorrhagic pneumonia in minks and bring huge losses to mink breeding.In recent years, the emergence of multidrug resistant Pseudomonas aeruginosa makes it more and more difficult to control the infection caused by Pseudomonas aeruginosa.Bacteriophage is a virus that can infect bacteria specifically. It can be used as a new therapeutic agent and has great application potential in the treatment of multidrug resistant strains infection.In this study, a new bacteriophage was isolated from waste water of pathogenic Pseudomonas aeruginosa strain D9, which was isolated from mink farm in laboratory and named YH6.The biological characteristics of bacteriophage YH6 were studied. The results showed that the bacteriophage was a potent bacteriophage capable of specific cleavage of Pseudomonas aeruginosa, and the plaque of phage was clear and without halo ring, and the phage belonged to the family Caudoviridae.The head has a positive icosahedron, the head diameter is about 65 nm, the tail is short (about 25 nm), the incubation period is 30 min, the cleavage period is 60 min, the optimal number of infection is 0.01-0.001, the phage titer can reach 1010 PFU / mL, the phage is insensitive to ether and chloroform.At the temperature of 40 擄C ~ (50 擄C) and pH value of 6.0 ~ 10.0, the stable YH6 could decompose 82.61% of Pseudomonas aeruginosa, showing broad-spectrum pyrolysis activity.The whole genome sequence of phage YH6 was sequenced and analyzed.There was no ORF associated with bacterial virulence and lysogenesis in the group, which was more favorable for the application of the bacteriophage as a therapeutic agent.The lyase gene of bacteriophage YH6, LysYH6, was identified by sequence alignment and analysis, and the gene was expressed and purified. The activity test showed that the lyase LysYH6 exhibited significant bactericidal activity in the presence of EDTA 25 mm / L.Animal experiments showed that inoculation of Pseudomonas aeruginosa D9 2 脳 10 8 CFU through nasal cavity could successfully construct hemorrhagic pneumonia model in mice.The therapeutic experiment showed that the one-off nasal cavity application of phage YH6(2 脳 107 PFU moi 0.1) could effectively protect the hemorrhagic pneumonia model of mice infected with Pseudomonas aeruginosa.Colony count in blood, spleen and lung of mice in treatment group showed that bacteriophage YH6 could completely eliminate Pseudomonas aeruginosa within 2 days after application of bacteriophage YH6.In the untreated group, the content of bacteria in viscera increased by 4 logarithmic units and caused death within 2 days.In addition, histopathological sections of the lungs of the treated mice showed that YH6 could effectively improve the lung injury induced by Pseudomonas aeruginosa.In conclusion, the phage YH6 isolated in this study showed great potential in the treatment of hemorrhagic pneumonia model induced by Pseudomonas aeruginosa in mice.The results laid a solid foundation for further study on the application of this phage to the treatment of mink infected Pseudomonas aeruginosa (Pseudomonas aeruginosa).
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉悅;李菁華;史紅艷;溫劍平;孫延波;;腸出血性大腸埃希菌O157噬菌體的生物學(xué)特性[J];吉林大學(xué)學(xué)報(醫(yī)學(xué)版);2012年01期

2 萬學(xué)勤;唐冬生;劉愛平;譚淑儀;李萬可蘭;鄺佳;李宏鳴;;幽門螺桿菌毒性噬菌體分離純化及模擬治療研究[J];南方醫(yī)科大學(xué)學(xué)報;2011年02期

3 李曉玲;王sビ

本文編號：1742265