Isolation,identification And Preliminary Application Of E.coli Bacteriophage

Escherichia coli is one of the most important infectious diseases that endanger a variety of livestock and poultry,causing serious economic losses to livestock production.Clinical use of antibiotics to prevent and treat E.coli disease,an increase of production costs,while causing antibiotic residues and resistant strains of the emergence.The study of new methods of prevention and control of E.coli can not only reduce the use of antibiotics,reduce production costs,reduce the harm to animals;and can reduce drug residues,is conducive to human health and ecological environment of harmonious development.There are E.coli bacteriophage for the treatment of Escherichia coli disease.Therefore,this experiment will have important clinical application value,which is beneficial to the reduction and efficiency of breeding field,and conforms to the concept of healthy breeding.In this study,two strains of E.coli bacteriophage Hab-EA and Hab-EB were successfully isolated from the dairy farms near Harbin by double-layer agar plate method using E.coli K12 as host bacteria.After expansion,the titers of two strains of E.coli bacteriophage could reach about 1010pfu/mL.Electron transmission microscopy showed that Hab-EA belonged to the Caudovivirales and Myoviridas;Hab-EB belonged to the Caudovivirales and Siphoviridas.The results showed that the lysis rate was 9/16 and 14/16,respectively.Hab-EA with wide host range was selected as the study object.The biological characteristics of the phage Hab-EA were evaluated by two-layer agar plate method and isometric dilution method,including the determination of one-step growth curve,pH and temperature tolerance.These experimental results showed that:Hab-EA had a pH tolerance of 5.0-10.0,and was not tolerant to high temperature.According to the one-step growth curve,the latent period and the burst period were 40 and 80 min,respectively.The whole genome of phage Hab-EA was sequenced and the functional prediction of the encoded protein was analyzed.The position of Lys-EA was 78th ORF sequence from 39403bp to 39900bp,total length of 498bp,166 Amino acids.Meanwhile,the results showed that Hab-EA was rV5-like viruses.In addition,two strains of pathogenic E.coli as the attack strain with the Hab-EA at the same time were intraperitoneally injected to mice,the results showed that Hab-EA have a certain therapeutic effect for infected mice.Simulated sewage purification system,in days as a unit,within a week to detect the purification system E.coli titer,7d after E.coli titer decreased by 4 orders of magnitude.Using the nucleic acid database to analyze the bacteriophage lysin nucleic acid sequence,the degenerate primers and expression primers were designed.The nucleic acid sequence of Hab-EA lysin Lys-EA was amplified in vitro by Hab-EA genome.The recombinant bacteriophage lysin protein rHis-Lys-EA was expressed in prokaryotic expression system at 25KD.The results showed that rHis-Lys-EA had a good cleavage effect on some specific pathogenic E.coli strains for 20h.