本文選題：噬菌體　切入點：大腸埃希菌　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:大腸埃希菌(Escherichia coli)為革蘭陰性桿菌,屬于腸桿菌科細(xì)菌,主要存在人和動物的腸道內(nèi),也可存在于自然界的環(huán)境。通常情況下,大腸埃希菌作為正常菌群成員對機(jī)體無損害作用,但在某些特殊情況如機(jī)體的免疫功能下降、通過移位侵襲進(jìn)入組織和器官后,可引起各種感染。此外,一些致病性大腸埃希菌可引起感染和爆發(fā)流行如腸產(chǎn)毒性大腸埃希菌和腸致病性大腸埃希菌等可通過糞口途徑傳播,故應(yīng)引起足夠的重視。本項研究通過分離鑒定大腸埃希菌噬菌體,來探索噬菌體作用于大腸埃希菌感染的可能性,為噬菌體療法用于細(xì)菌感染提供依據(jù)。方法:1、15株大腸埃希菌臨床分離株作為宿主,用常規(guī)方法,從污水中分離出一株裂解性噬菌體,命名為ZG49。2、采用無菌接種環(huán),對單個噬菌斑進(jìn)行挑取,然后接種到宿主菌懸浮液中,初步純化和滴定噬菌體,觀察形態(tài)3、繪制其生長曲線。記錄噬菌體生長曲線的潛伏期、暴發(fā)期和暴發(fā)量。4、制備噬菌體蛋白樣品后進(jìn)行SDS-PAGE電泳以及染色脫色5、觀察噬菌體圖譜,進(jìn)行噬菌體穩(wěn)定性試驗6、對噬菌體基因組測序和基因功能注釋結(jié)果:1、15株大腸埃希菌臨床分離株作為宿主,用常規(guī)方法,從污水中分離出一株裂解性噬菌體,命名為zg49。2、通過觀察,噬菌體zg49的形態(tài)學(xué)特征符合有尾病毒目、足尾病毒科噬菌體。噬菌體zg49的最佳感染復(fù)數(shù)為1;3、一步生長曲線表明,zg49的潛伏期為30分鐘、爆發(fā)期為30分鐘,爆發(fā)量可達(dá)到200pfu/ml;4、sds-page電泳可呈現(xiàn)4條主要蛋白帶,分子量分別為50kda、45kda、30kda和10kda;5、裂解譜分析顯示,噬菌體zg49能裂解6株大腸埃希菌臨床分離株(裂解率40%)。6、基因組測序分析表明,噬菌體zg49的基因組為雙鏈dna、大小為39208bp,g+c含量是49.86%。7、利用rast軟件首先對基因組進(jìn)行基因預(yù)測,而后對基因組做功能注釋�？偣差A(yù)測到46個蛋白編碼基因,長度在150~3888bp之間。未檢出trna和rrna。8、根據(jù)blastp和interproscan軟件對zg49基因組進(jìn)行注釋,發(fā)現(xiàn)24種已知編碼基因,包括裂解素(orf1)、引物酶(orf3)、dna多聚酶(orf5)、歸巢結(jié)合蛋白(orf8)、外切酶(orf10)、頭-尾連接蛋白(orf15)、衣殼蛋白(orf16,17)、尾管蛋白a(orf19)、尾管蛋白b(orf20)、核心蛋白(orf21,23,24,25)、尾絲蛋白(orf26)、穿孔素(orf27)、dna裝配蛋白(orf28,30)、內(nèi)肽酶(orf29)、dna指導(dǎo)的rna多聚酶(orf37)、dna連接酶(ORF 40)、宿主RNA多聚酶抑制蛋白(ORF 43)、單鏈DNA結(jié)合蛋白(ORF 44)和內(nèi)切酶(ORF 46)。ZG49全基因組序列已提交至Gen Bank,登錄號:KX669227。結(jié)論:本項研究以大腸埃希菌臨床分離株為宿主菌,從環(huán)境中分離一株裂解性噬菌體,命名為噬菌體ZG49,并對其生物學(xué)特性進(jìn)行了初步分析。根據(jù)噬菌體ZG49的形態(tài)特征,噬菌體ZG49屬于有尾病毒目、短尾病毒科噬菌體。噬菌體ZG49基因組為雙鏈DNA、長度為39,208 bp,G+C含量為49.86%。噬菌體ZG全基因組序列已提交至Gen Bank,登錄號:KX669227。
[Abstract]:Objective: Escherichia coli, a gram-negative bacterium, belongs to the Enterobacteriaceae. It mainly exists in the intestines of humans and animals, and can also exist in the natural environment. Escherichia coli, as a member of the normal flora, has no harmful effect on the body, but in some special cases, such as the decline in immune function of the body, it can cause various infections by translocation and invasion into tissues and organs. Some pathogenic Escherichia coli can cause infections and outbreaks, such as enterotoxic Escherichia coli and enterogenic Escherichia coli, which can be transmitted through feces. In order to explore the possibility of phage acting on Escherichia coli by isolation and identification of Escherichia coli phage, we should pay more attention to it. Methods A lytic bacteriophage named ZG49.2 was isolated from sewage by routine method. The single plaque was picked out, then inoculated into the suspension of the host bacteria, the phage was purified and titrated, the morphology 3 was observed, the growth curve was plotted, and the latent period of the growth curve was recorded. The phage protein samples were prepared and decolorized by SDS-PAGE electrophoresis and staining, and the phage patterns were observed. The bacteriophage stability test 6 was carried out. The results of genomic sequencing and gene function annotation showed that 15 strains of Escherichia coli were used as hosts. A lytic phage named zg49.2 was isolated from sewage by routine method. The morphological characteristics of phage zg49 were consistent with the order of tail viridae. The optimum number of phage zg49 infection was 1: 3. The one-step growth curve showed that the incubation period was 30 minutes and the outbreak period was 30 minutes. The amount of outbreak was up to 200pf / ml / ml ~ 4N / ml SDS-page, and the molecular weight was 50 kda-45kda-30kda and 10kda-5. the cleavage spectrum analysis showed that the phage zg49 could split 6 clinical isolates of Escherichia coli (lytic rate 40%. 6%), and the genomic sequencing showed that the phage could break down 6 clinical isolates of Escherichia coli (lyse rate 40%. 6%), and the results of genomic sequencing showed that, The genome of phage zg49 is double-stranded dna.The size of the phage zg49 is 39208bpng / c 49.86. the genome is predicted by rast software, and then the genome is annotated. A total of 46 protein coding genes were predicted. Trna and rrna.8 were not detected. The genome of zg49 was annotated by blastp and interproscan software, and 24 known coding genes were found. These include lyase orf1, primer enzyme orf3, homing protein orf8, exonuclease orf10, head-tail junction protein orf15, capsid protein orf1617, caudal tube protein aorf19, caudal protein borf20, core protein orf21 23232425, tail silk protein orf26, perforin orf27f27dna assembly protein orf2830, endopeptidase protein Aorf19, caudal tube protein borf20, core protein orf21, orf26, orf2830, endopeptidase. ORF40, host RNA polymerase inhibitor, single stranded DNA binding protein (ORF44) and endonuclease ORF46, ZG49, have been submitted to Gen Bank. accession No.: KX669227. Conclusion: this study is based on Escherichia coli. The clinical isolates are host bacteria. A lytic bacteriophage named ZG49 was isolated from the environment and its biological characteristics were preliminarily analyzed. According to the morphological characteristics of phage ZG49, the phage ZG49 belongs to the order Cycloviridae. Short tail viridae phage. The phage ZG49 genome is double-stranded DNA, the length of the phage is 39,208 BP ~ (G) C is 49.86. The whole genome sequence of bacteriophage ZG has been submitted to Gen Bank, accession number: KX669227.
【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】：2017
【分類號】：R37

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