Study On Separation, Determination And Function Of Phage Endolysin Gene

With increasing of bacteria resistance to antibiotics, it is urgent to find an antimicrobial agent that is effective and uneasy to create resistant. The results show that the phage and phage-encoded lytic enzymes are the most ideal substitute of antibiotics for treatment and prevention of bacterial infection. Because they have important value on drug resistance of anti-bacterial, screening proteins with antibacterial activity from the phage is a new approach and method.In the study, a fierce bacteriophage named Enterobacteria phage WL08 was isolated from the chicken manure, and gene 23 encoding capsid protein of Enterobacteria phage WL08 was 96% similar to gene 23 of Enterobacteria phage RB69. E gene that encodes endolysis was cloned. Sequence analysis showed that this gene consisted of 474bp, encoding a 157 amino acid protein with 98% similarity with the E gene of Enterobacteria phage RB69. Then E gene was transformed: First of all, E gene and RI gene were bridged by （Gly-Gly-Gly-Gly-Ser）3 to obtain RIE gene; secondly, E gene was transformed through PCR amplification to obtain E1-87, E_34-157 and E_110-157.Then inhibition effect of RIE, E, E_1-87, E_34-157 and E_110-157 protein on the growth of E.coli was manifested by cloning RIE, E, E_1-87, E_34-157 and E_110-157 gene into the pHERD30T vectors, which contains a PBAD promoter. The result indicated that RIE, E, E_1-87, E_34-157 and E_110-157 protein showed the bacteriostatic activity against growth of E.coli. To explore a new approach of expression of E protein in E.coli 2566, E gene obtained by PCR was cloned into the vector pTYB11 to construct a fusion expression plasmid. Purification of E protein showed antimicrobial activities against E.coli Top10 and S.aureus at 1mg/ml concentration in vitro.