噬菌体内溶素基因分离、鉴定及功能研究

发布时间：2018-03-19 05:15

本文选题：噬菌体内溶素　切入点：E基因　出处：《内蒙古工业大学》2010年硕士论文　论文类型：学位论文

【摘要】：随着致病性细菌耐药性的加剧,寻找有效且不易造成菌株耐药性的抗菌制剂迫在眉睫。噬菌体以及噬菌体编码的内溶素,是治疗和预防细菌感染最为理想的抗生素替代物,且它们对抗耐药性细菌具有较强的杀菌性。所以,从噬菌体中筛选具有抗菌活性的蛋白或多肽是一种新的途径和方法。本研究首先从鸡粪中分离到一株烈性噬菌体,编码该噬菌体衣壳蛋白的23基因序列与大肠杆菌噬菌体RB69的23基因序列(NCBI登录号:AY303349.1)的相似度达96%,命名为大肠杆菌噬菌体WL08;并从该病毒中克隆编码内溶素的E基因,序列分析结果表明,E基因全长474bp,编码157个氨基酸,与大肠杆菌噬菌体RB69的E基因相似性达98%;随后对E基因进行改造,用三个(Gly-Gly-Gly-Gly-Ser)单元构成的桥,将RI基因和E基因桥连,获得RIE基因,PCR的方法将E基因分三段扩增:E_(1-87)、E_(34-157)和E_(110-157)。将RIE、E、E_(1-87)、E_(34-157)和E_(110-157)基因克隆到pHERD30T载体上,通过生长曲线的测定,初步确定E蛋白和改造后的RIE蛋白、E_(1-87)蛋白、E_(34-157)蛋白和E_(110-157)蛋白对大肠杆菌的抑制作用;PCR扩增E基因,克隆到pTYB11载体,并转化到大肠杆菌ER2566感受态细胞中,诱导表达E蛋白。初步测定了E蛋白纯品的体外抑菌活性,实验结果显示,在E蛋白终浓度达到1mg/ml时,E蛋白对大肠杆菌Top10和金黄色葡萄球菌两株菌都有一定的抑制作用。
[Abstract]:With the aggravation of the drug resistance of pathogenic bacteria, it is urgent to find effective antimicrobial agents that are not easy to cause bacterial resistance. Bacteriophages and endolysin encoded by bacteriophages are the most ideal antibiotic substitutes for the treatment and prevention of bacterial infections. Therefore, screening proteins or peptides with antibacterial activity from phages is a new way and method. In this study, a strong phage was isolated from chicken manure. The sequence of 23 gene encoding this phage capsid protein is similar to that of Escherichia coli phage RB69. The similarity between the gene sequence and the accession number of Escherichia coli phage RB69 is 96g, named as Escherichia coli bacteriophage WL08, and the E gene encoding endolysin is cloned from the virus. Sequence analysis showed that the E gene encoding 157 amino acids was similar to the E gene of Escherichia coli bacteriophage RB69, and then the E gene was modified by using three Gly-Gly-Gly-Gly-Gly-Gly-Serial units, and the RI gene and E gene were bridged. The E gene was amplified in three parts by using RIE gene. The E gene was amplified in three parts. The gene was cloned into the pHERD30T vector. The E protein E and the modified RIE protein Estax 34-157) were preliminarily determined by the determination of the growth curve. The inhibitory effect of E protein on Escherichia coli was amplified and cloned into pTYB11 vector. It was transformed into Escherichia coli ER2566 cells and induced to express E protein. The bacteriostatic activity of pure E protein was determined in vitro, and the results showed that, When the final concentration of E protein reached 1 mg / ml, the E protein could inhibit both Escherichia coli Top10 and Staphylococcus aureus strains.
【学位授予单位】：内蒙古工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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