T7噬菌体新成员—大肠埃希菌噬菌体ZG49的分离鉴定

发布时间：2018-03-05 20:26

本文选题：噬菌体　切入点：大肠埃希菌　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:大肠埃希菌(Escherichia coli)为革兰阴性杆菌,属于肠杆菌科细菌,主要存在人和动物的肠道内,也可存在于自然界的环境。通常情况下,大肠埃希菌作为正常菌群成员对机体无损害作用,但在某些特殊情况如机体的免疫功能下降、通过移位侵袭进入组织和器官后,可引起各种感染。此外,一些致病性大肠埃希菌可引起感染和爆发流行如肠产毒性大肠埃希菌和肠致病性大肠埃希菌等可通过粪口途径传播,故应引起足够的重视。本项研究通过分离鉴定大肠埃希菌噬菌体,来探索噬菌体作用于大肠埃希菌感染的可能性,为噬菌体疗法用于细菌感染提供依据。方法:1、15株大肠埃希菌临床分离株作为宿主,用常规方法,从污水中分离出一株裂解性噬菌体,命名为ZG49。2、采用无菌接种环,对单个噬菌斑进行挑取,然后接种到宿主菌悬浮液中,初步纯化和滴定噬菌体,观察形态3、绘制其生长曲线。记录噬菌体生长曲线的潜伏期、暴发期和暴发量。4、制备噬菌体蛋白样品后进行SDS-PAGE电泳以及染色脱色5、观察噬菌体图谱,进行噬菌体稳定性试验6、对噬菌体基因组测序和基因功能注释结果:1、15株大肠埃希菌临床分离株作为宿主,用常规方法,从污水中分离出一株裂解性噬菌体,命名为zg49。2、通过观察,噬菌体zg49的形态学特征符合有尾病毒目、足尾病毒科噬菌体。噬菌体zg49的最佳感染复数为1;3、一步生长曲线表明,zg49的潜伏期为30分钟、爆发期为30分钟,爆发量可达到200pfu/ml;4、sds-page电泳可呈现4条主要蛋白带,分子量分别为50kda、45kda、30kda和10kda;5、裂解谱分析显示,噬菌体zg49能裂解6株大肠埃希菌临床分离株(裂解率40%)。6、基因组测序分析表明,噬菌体zg49的基因组为双链dna、大小为39208bp,g+c含量是49.86%。7、利用rast软件首先对基因组进行基因预测,而后对基因组做功能注释。总共预测到46个蛋白编码基因,长度在150~3888bp之间。未检出trna和rrna。8、根据blastp和interproscan软件对zg49基因组进行注释,发现24种已知编码基因,包括裂解素(orf1)、引物酶(orf3)、dna多聚酶(orf5)、归巢结合蛋白(orf8)、外切酶(orf10)、头-尾连接蛋白(orf15)、衣壳蛋白(orf16,17)、尾管蛋白a(orf19)、尾管蛋白b(orf20)、核心蛋白(orf21,23,24,25)、尾丝蛋白(orf26)、穿孔素(orf27)、dna装配蛋白(orf28,30)、内肽酶(orf29)、dna指导的rna多聚酶(orf37)、dna连接酶(ORF 40)、宿主RNA多聚酶抑制蛋白(ORF 43)、单链DNA结合蛋白(ORF 44)和内切酶(ORF 46)。ZG49全基因组序列已提交至Gen Bank,登录号:KX669227。结论:本项研究以大肠埃希菌临床分离株为宿主菌,从环境中分离一株裂解性噬菌体,命名为噬菌体ZG49,并对其生物学特性进行了初步分析。根据噬菌体ZG49的形态特征,噬菌体ZG49属于有尾病毒目、短尾病毒科噬菌体。噬菌体ZG49基因组为双链DNA、长度为39,208 bp,G+C含量为49.86%。噬菌体ZG全基因组序列已提交至Gen Bank,登录号: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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R37

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