Production And Immunogenicity Of Bacterial Ghosts From Pathogenic Escherichia Coli Isolated From Ducklings

Avian pathogenic Escherichia coli (APEC) is an important pathogen of poultry which could cause several avian diseases. Nowadays various APEC serogroups and increasingly serious problem of bacterial antibiotic resistance has become the challenge in controlling APEC. It is necessary to develop a new and high-performance vaccine to replace traditional inactivated vaccine which could not prevent the prevalence of APEC effectively.Bacterial ghosts are produced by controlled expression of cloned PhiX174gene E in Gram-negative bacteria and result in lysis.They are similar to live bacteria to have intact cell membrane and adhesiveness, but cytoplasm is discharged by formation of the E-specific transmembrane tunnel. Depend on the intact membrane antigenic structure of bacterial ghosts such as pili and LPS, it could induce both humoral immunity and cellular immunity compared to the traditional inactivated vaccine. Besides, there is no nucleic acid in bacterial ghosts, so it is safe and can transport conveniently after freeze-dry. These properties make it popular to serve as vaccine candidate and provide a new way to prevent and control APEC.The coexpression vector for PhiX174lysis gene E and Staphylococcus nuclease A(SN) was constructed sucessfully by utilization of plasmid pBV220. SN was a secondary killing gene, its expression could cause bacterial genome degradation and improve inactivation of the bacteria.In the recombinant lysis plasmid.expression of lysis gene E and SN were started by two different promoters, and they were all controlled by temperature sensitive regulatory element CIts857. Depend on the recombinant lysis plasmid, the lysis efficiency of APEC DE17isolated from duck could reach to99.99%. And the bacterial ghosts observed by TEM and SEM showed that they have the same intact membrane antigenic structure as live bacteria but no cytoplasm.Recent research showed that cell-penetrating peptide (CPP) also could be used to produce bacterial ghosts rapidly. One of CPPs named MAP was synthesized to produce bacterial ghosts of DE17, in the suitable concentration the lysis efficiency was100%.And the bacterial ghosts observed by TEM and SEM showed that there is some extent cytoplasm discharged.Immunization of duck with APEC DE17bacterial ghosts induced better humoral immunity than immunization with DE17inactivated by formaldehyde, regardless of addition of adjuvant nor not.In the injection of50times of LD50, the rates of protection could reach to90%and80%separately in the group of immunization of bacterial ghosts with adjuvant and without adjuvant.and the rates of protection could reach to70%and40%separately in the group of immunization of bacteria inactivated by formaldehyde with adjuvant and without adjuvant.This indicate that bacterial ghosts could provide much better ptotection.The rates of protection in the group of immunization of bacterial ghosts inactivated by MAP is70%.It is equal to the group of immunization of bacteria inactivated by formaldehyde with adjuvant.The mechanism is still to be study forwardly.In this study we have successfully constructed recombinant lysis plasmid and synthesized CPP MAP. Bacterial ghosts of DE17were produced efficiently by these two methods,and they could induce better humoral immunity and provide higher ptotection than traditional inactivated vaccine,this indicate that it is possible to develop bacterial ghost as ideal vaccine candidate.