Isolation And Identification Of A Novel Polyvalent E. Coli Phage 285P And Study Of Functional Genomics

Antimicrobial resistance is a global puzzle and hot spot. The long-term, widespread, irrational usage and the selection pressure of antimicrobials have make the number of drug resistant strain increase and the drug fast spectra magnify. Some strains are even resistant to the drugs that haven't been used in clinic. After entering our country only for 20 years, the drug fast rate of quinolones have increased quickly. Taking ciprofloxacin as an example, the drug fast rate for Escherichia coli is 60%-70%, for enterococci and staphylococci is 20%-40%. In 1996 WHO warned that drug resistance could make tuberculosis become mortal disease again. Respecting to such reasons, it's imminent to devel the next generation neotype antibiosis preparation of high performance and cheapness.As bacterial virus, bacteriophage could infect and lysis host bacteria. After being discovered, it was located to biological antibiosis. Because of the discovering and development of antibiotics and people's ignorance of the basic characteristics of the bacteriophages, the studies almost halted, but with the durg fast phenomenon becoming more and more serious, bacteriophage for biological antibiosis shine entelechy again. Thinking of host specifity, environmental affinity, converience and continuity of bacteriophage antibiosis, it's the significant develop direction. On the moment bacteriophage have been used for biological antibiosis in medicine, agriculture, foodstuff and environmental protection.However, the host specificity has limited the application of bacteriophages in a large scale. The host spectra of some bacteriophages just confines to one division, one group, one species, or even one strain, but they have no effect on other strains. Now because the artificial reconstruction of bacteriophages can satisfy the need of increasing host spectra, it has becomed one of the hot spots.The study aims at screening a novel polyvalent E. coli bacteriophage in sewage of the hospital. Then to observe the basic biological characteristics and nucleonic acid attribute. Combining genomics, proteomics and bioinformatics, the molecular recognition mechanism of phage's polyvalence and the function would be studied for the artificial reconstruction of bacteriophages in next step. Four aspect of the study content and results could be summed up as following:1. A novel polyvalent E. coli bacteriophage was isolated in a large scale screening of the hospital sewage, and the lysis valence is above 109pfu/mL. through double-layer agar cultivation, transmission electron microscope, one step growth curve, electrophoresis of nucleic acid and measurement of spectro-luminosity, to observe the negative colony, ultrastructure, character of nucleonic acid and growth curve, the outcome shows that the diameter of the negative colony is 4-5mm, transparent, aequalis size, clear edge; The phage particle looks like microballoons and classifys to Podoviridae, the diameter of head is 40-48nm, the tail is 8×23nm; one step growth curve shows that absorption rate of five minutes is 80.3%, latent period is 21 min, average releasing amount is 88; The methods of spectro-luminosity and shear of nucleinase all confirm that the nucleonic acid is double stranded DNA(dsDNA).2. Shotgun sequencing of complete genome determined a 39270bp genome; blast analysis shows it's a novel phage; evolution analysis show phage BA14 is the most similar; 43 ORFs were determined and the function was predicted, and they are tail fiber, tail, major capsid protein, lysozyme, virus internal protein, endonuclease and so on; ten major protein were modeled by SWISS-MODEL.3. To analyse the membrane protein of phage 285P using SDS-PAGE electrophoresis, two-dimensional electrophoresis and mass spectroscopy. Three protein was determined: tail protein(gp12), tail fibre protein(gp17) and major capsid protein(gp10a).4. Emphasis was placed upon the host recognizing mechanism of tail fibre. Primary structure, secondary structure and tertiary structure were analysed by comparing with T7 subgroup phage. The cladogram, amino acids component, multi-sequence clustal, hydrophilia, tenacity, antigen index, surface possibility and so on were analysed. Transmembrane domain was estimated by TMHMM Server v. 2.0. By comparing with phage T7 and BA14, the mechanism of host recognization was concluded that the tail fiber absorb to the biosyl-residue in lipopolysaccharide adventitia. And the study establish a foundation for the clone and expression of tail fiber and exploratory development of bacteriophage.To sum up, A novel polyvalent E. coli bacteriophage was isolated in a large scale screening, and the basic biological characteristics and nucleonic acid attribute were observed; three major protein were determined and qualitated and quantitated; 39270bp genome was determined and the Emphasis was placed upon the host recognizing mechanism of tail fiber. This study aims to establish a foundation for biological antibiosis in environmental disinfection, hippiatrics, agriculture, foodstuff and so on.