Establishment Of A Novel Preparation Method For Klebsiella Pneumoniae And Haemophilus Pamsuis Ghosts By Antimicrobial Peptide And Ultrahigh Pressure

Bacterial ghost (BG) is a new kind of vaccine, it is a empty cell body withoutcytoplasm and nucleic acid contents, its biggest feature is it retains complete internaland external membrane structure as the viable organism, it can stimulate the body toproduce specific and nonspecific immune response effectively and resist theinfestation of pathogenic microorganism. Compared with traditional inactivatedvaccine, its surface antigen gets maximum degree of retention and its immunogenicityis higher. Compared with new generation vaccine, it combines dual effects of vaccineand adjuvant, and it can become excellent antigen delivery vector for loadingexogenous antigens in its internal and outer membrane, providing a platform for thestudy of the combination vaccine. With pathogenic micro-organisms becoming moreand more difficult to control, the bacterial ghost system begin to receive closeattention from the domestic and foreign researchers’ attention.Klebsiella Pneumoniae (Kpn) distributes in nature widely, it is the pathogen ofintestinal tract and respiratory tract of human and animal, it has became the secondimportant pathogens in recent years only to E. coli. Haemophilus parasuis (Hps) is thepathogen of pigs Glasser ’s, it is a kind of symbiotic bacteria in the upper respiratorytract of pigs, it can invade the body and cause systemic disease characterized byfibrinous polyserositis, arthritis and meningitis under certain conditions. There are noeffective prevention and controlling measures on these two bacterias except forinactivated vaccine. But in the progress of inactivated, its antigenicity and rate ofprotection becomes lower, adverse events increases, so research and development ofnew vaccines has become a focus for researchers.We first used antimicrobial peptides joint ultrahigh pressure to prepared BG. Weadded different concentrations of antimicrobial peptides to the bacteria liquid culturedto the logarithmic growth phase,4℃overnight, prepared the treated bacteria indifferent pressures to bacterial ghost, studying the morphology, spliting condition, security, and immunological characteristics in order to determine the preparationconditions. The experimental results showed that we failed to detect the presence ofviable cells by the plate count assay lysis when the antimicrobial peptideconcentration was28μg/mL and pressure was200MP. Therefore,to prove that itscleavage is100%. Observing in the electron microscope, the shell structure of theprepared Haemophilus parasuis bacteria shadow and Klebsiella pneumoniae bacteriashadow maintained the integrity, the contents were all released. Immunizing micewith large doses of prepared bacteria shadow and observing growth contion, resultsshowed that mouse grew normally, there were no significant difference with negativecontrol group, showed that the prepared BG had a good safety. Serum antibody testresults showed that the mouse inoculated with BG and inactivated vaccine couldproduce higher serum antibody titers, the serum antibody titers were higher producedby mouse inoculated with BG than inoculated with inactivated vaccine, its titer couldbe up to6400times two weeks after the second immunization. Challengedexperimental results showed that BG could provide100%protection for the mouseintraperitoneal injected of it.Through the results of this study, we could see that the BG prepared byantimicrobial peptides joint ultrahigh pressure film had good safety andimmunogenicity, it also provided good protection for mice, provided a new way ofthinking and method for preparing of the BG, Laid foundation for large-scalepreparation and industrial production.