Screening And Researching Of Phage Receptor-binding Protein Against Its Host Strain Escherichia Coli

Escherichia coli is the common opportunistic pathogen in nosocomial infections that can lead to various local and systemic infectious diseases,including cystitis,pyelonephritis,enteritis,pneumonia,meningitis,bacteremia and so on.In recent years,as the drug resistance of E.coli continues to aggravate and spread,available antibiotics are on the verge of depletion.For this reason,the research and development of new alternative medicines and antimicrobial agents become urgent and necessary.Bacteriophage,the predators of bacteria,is considered the most abundant and diverse biological entities in nature.Since the 1st International Oxford Bacteriophages Conference was held in 2011,scientists have achieved the fruitful research in the application of phage against bacteria,especially multi-drug resistant bacterial infections.Perhaps phage will be promising in responding to the increasingly serious bacterial resistance.The studies of our previous work showed that bacteriophage v B_EcoM_ep3 isolated and preserved by our laboratory had a remarkable lysis effect on E.coli O78-6 with multi-drug resistance(?-lactams,aminoglycosides and quinolone antibiotics)compared to the sensitive strains.However,the mechanism of interaction between phage vB_EcoM_ep3 and E.coli O78-6 is not fully understood.The purpose of this research is to screen phage receptor binding protein(RBP)and study its function,which provides a theoretical basis for the mechanisms of interaction between phage and its host strains.The genome annotation reveals that vB_EcoM_ep3(gene ID: KM360178.1)has 56 ORFs.In order to effectively screen out the targets that can interact with the host bacteria in numerous phage proteins,we first established an E.coli library that randomly expressed phage proteins and tested its accuracy and molecular diversity.The constructed library was screened and identified by using E.coli O78-6 as a target.The positive clones were finally validated by gene sequencing and sequence alignment.Two proteins may be involved: phage major capsid protein and putative tail fiber protein.It is widely acknowledged that tail fiber protein,as one of the main RBPs,plays a vital role at the stage of infection.To confirm the function of tail fiber protein Dpo41,we constructed a prokaryotic expression vector for this protein and purified it.Mice were immunized with Dpo41 as an immunogen to obtain a large amount of hyperimmune serum.IgG was further isolated and purified by ammonium sulfate precipitation.The result of western blot showed that the purity and specificity of Dpo41 polyclonal antibody was good.The experiments of antibodies Dpo41 additive indicated that the Dpo41 polyclonal antibody could effectively block the Dpo41,thereby significantly decreasing the adsorption and lysis efficiency of phage vB_EcoM_ep3 on the strain O78-6.It is illuminated preliminary that tail fiber protein promotes phage lysis by efficiently adsorbing to the surface of the hosts in the process of phage infection.However,the key residues of Dpo41 and its mechanism remains to be further demonstrated.