Isolation And Purification With Functional Genomics Of Lytic Avian Pathogenic E. Coli Bacteriophage

The isolation and purification of the bacteriophage from Avian pathogenic Escherichia.coli (APEC) were studied and its character was determined. About4,000parts Duck’s dung from the markets in Nanjing were centrifugated and filtered. The supernatants were got. Identifying it with the method of spot and double-deck agar,136strains of bacteriophage were isolated and identified. The following experiment content includes not only the traditional sense of morphological researches consists of its purification, host range on about40among these phages but also complete genome sequencing and the functional genomics, as well as the proteomic analysis and mass spectrometry on NJOl and HX01. Take NJO1for example, it has an elongated head like a shuttle (150×48nm), and a short, noncontractile tail (18×10nm) under electron microscope and belonged to the Podovirus. The host detection showed that NJ01was lytic on three of sixty-six Escherichia coli strains tested—DE172, NJ01and MC1061. The titer was1.33×1013pfu/ml. After diluting the bacteriophages to108PFU/mL and determining their survival number every15minutes (to4hours). One-step growth curve of phage NJ01was drawn. From the curve showed the incubation period is30min, the average amount of cracking is157PFU/cell and the MOI was100.Some key steps were improved and changed on the base of the classical λ method in the research and got a high nucleotide consent and without any rupture. Put the genomes of NJ01and HX01for sequencing on a GS-FLX. Just like NJ01, genome sequence data was assembled into one single contig of77.448kb in size as shown with a G+C content of44.1%. Analysis of the genome sequence revealed the presence of136ORFs and1tRNA annotated by using various bioinformatics tools. Clusters of functionally related putative genes were defined as the module of DNA packaging-head-tail-lysis/lysogeny-DNA replication-transcriptional regulation enzymes. After comparative genomics analyses with other two known coliphage phiEco32and KBNK135, some special genes, such as ORF52which belonged to NJ01only, some other genes (ORF45, ORF89. etc) were found to be totally disagreed. Differences in the single putative tail fiber gene correlated with the specificity determinacy of the phages were studied and a highly conserved320-amino-acid (aa) domain was found which may become a hot study spot. Comparative genomics analysis showed the NJ01and phiEco32genetic and evolutionary relationship was more close, but very diffierent in both the host range and separation, which may suggested the necessity of research on the composition of phage genome and the functional gene research to expand the host range by using comparative genomics analysis.To verify the structural proteins of NJ01,2-DE analyzed as well as mass spectrometry were used, and then13spots, which represented8different structural proteins, were successfully identified included tail, tail fiber, major head protein and DNA injection proteins. It made the phage structure composition and comparison more straightforward and stereoscopic, and the method riched the annotation of the genome.Phagotherapy for bacterial infections had a long history and APEC is an important etiologic infection among the most significant infectious diseases cause colibacillosis, airsacculitis and associated pericarditis, perihepatitis and peritonitis being most typical, resulting in severe economic losses in poultry industry worldwide. To explore the virulent phage to combat with APEC and measuret the effect of the lytic phages. This reserarch was carried out with NJ01whose host was the strain-DE172. By using the sucessful infection modle of mice for calculating the LD50. Then twice does of the LD50was injected to the mice by abdominal cavity. Test Groups included six groups which was divided by diffierent time injecting the same does of phage after infection and two diffierent blank control groups for comprehensive analysis. The LD50of DE172was1×107CFU/mL.On the first day after treament,there was no significant difference between the treatment group and control group; After three days in addition to the E, F, H group, the rest of the mice were killed, the treatment group and control group, three days after the data was statistically significant difference between treatment group and control group (P<0.01). Conclusion: this experiment for phage curative effect was a exploratory experiment, the result is not obvious, the virulent phage treatment of infected mice may have some effect, but low antigenicity, but it was a try to use phage as biological agents and provided data for anti-infection treatment.