Separation And Purification Of Four Marine Phages And The Biological Characteristics Research

The abundance of marine phages in the marine ecosystem is highest.And marine phages play an important role for material and energy flow and community diversity of marine microbial system,and its value is higher and higher.Marine phages have become a hot research topic in marine ecology, but the overall study of marine phages are still at a preliminary stage.This article aims to use the strains as host which were isolated from the gut of diseased fish(suffering from enteritis)and isolated phages from mud. Study phages biological characteristics and explore theirs properties, genetic relevance.Finally, provide a basis for future study of marine phages. The research included the following aspects:1.Isolated five dominant bacteria from the intestinal of enteritis sick fish, denoted as S1,S2,S3,S4,S5, Gram staining gram negative bacteria. 5 strains by 16 Sr DNA sequencing and phylogenetic tree analysis can be divided into Vibrio, Citrobacter, Alcaligenes, Shewanella 4genera. The growth curve showed that: S1, S4, S5 culture 14 h, S2 and S3 culture 10 h can obtain the best phage infection period.2.The above 5 strains used as host and cultured to the best infection period.Then 5 strains co-cultured with mud, finally, isolated 4 strains of phages, which takes S1 as the host culture system did not obtain phage.4 phages were purified by 3 to 5 times.After culture expansion and concentrations were higher than 109 pfu /m L, their RTD value were 103 pfu /m L ~104 pfu/m L, which showed that the lytic of the 4 phages were strong. Combining with the plaque assay can preliminary determinate the phages were different species.By electron microscopy morphology can be divided into four phage Myoviridae and Corticovirus two families.By enzymatic analysis determine the phages nucleic acid type were DNA. Explore the ways of the preservation,the effect of freeze-dried preservation was best.3.Study phages sensitivity to temperature, p H, UV irradiation, chloroform and other physical and chemical factors,render step growth curve,determine the optimal multiplicity of infection(MOI), explore the adsorption kinetics and study their lysis spectrum by using 36 bacteria.Set 24 random primers for RAPD-PCR experiment and ultimately the optimal annealing temperature was 36℃.All primers were only 5 primers could be amplified RAPD profile, the polymorphic bands accounted for 82.4% in the total bands;by analyzing the genetic factor and cluster tree found that,the kinship of the four phages was distant and the genetic similarity coefficient between 0.36~0.58;Identified by SDS-PAGE electrophoresis: the main content of the protein composition of four marine phages from more to less followed: P4, P2, P1, P3 and the major structural protein of P1, P2, P3 were 38.3KD, and P4 was 58.6KD.