| Riemerella anatipestifer infection,which is a major disese confronting the duck industryall over the world,is a contagious disease of ducks, goose, turkeys and various other birds, Itis idespread around the world, the disease incidence and mortality in general for10-30%, butsome duck farms amounted to more than95%. Control of this disease mainly depend onantibiotic treatment, as the bacteria to multiple antibiotic resistance continues to increase a,vaccine development will be very urgent.Bacterial ghost is intact bacterial envelope which is produced by the expression of thelysis gene E of bacteriophage PhiX174. They are non-living bacterial envelopes, whichmaintain the cellular morphology and native surface antigenic structures, so they can be usedas vaccine. The traditional vaccines include inactivated vaccines and attenuated vaccines. Theinactivated vaccines can cause effective humoral immunity, but cellular immunity andmucosal immunity, in addition, casuing lower immunogenicity is due to antigen structurehave changed in the inactivated process. Attenuated vaccines can cause effective humoralimmunity and cellular immunity besides it easily restructure with others and own recurrenttoxic phenomenon. Subunit vaccines as new generation vaccines are often poorlyimmunogenic necessitating an appropriate adjuvant in the vaccine formulation. Depend on theintact membrane antigenic structure of bacterial ghosts, it could induce both humoralimmunity and cellular immunity compared tothe traditional inactivated vaccine. Besides,there is no nucleic acid in bacterial ghosts, so it is safe and can transport conveniently afterfreeze-dry. So it can serve as vaccine candidate and provide a new way to prevent and controlbacteriosis.This research mainly includes three aspects.1）Construction of Lysis plasmidBacterial ghost is intact bacterial envelope which is produced by the expression of thelysis gene E of bacteriophage PhiX174and the expression of gene E usually controlled by the thermosensitive λpL/pR-cI857promoter.In this study we describe a mutation(T→C) at theninth nucleotide of the OR2of theλpR promoter of the λpL/pR-cI857system by overlapPCR.The bacteriolytic experimental results show that mutations to the λpL/pR-cI857systemhave stably repressed the expression of gene E at temperatures of up to37℃,the lysisefficiency of APEC could reach99.9%.In this research we have expanded the temperaturecontrolling rang of λpL/pR-cI857by altering theλpR promoter,then provide a reference for thebacterial ghost production.2）Study on preparation method of bacterial ghostWe have used three methods to product RA bacterial ghosts. The first, the lysis gene Ewas inserted into plasmids then transformed into RA bacterial. Secondly, the lysis gene E wasinserted into genome of RA bacterial. Last, we used CPP to product RA bacterial ghosts.Finally, we have succeed in producting RA bacterial ghosts using MAP(CPP), which the finalconcentration is60μM. the bacterial ghosts observed by TEM and SEM showed that theyhave the same intact membrane antigenic structure as live bacteria but no cytoplasm.3）Evaluation of the immune effect of the RA bacterial ghostImmunization of duck with RA bacterial ghosts induced humoral immunity and cellularimmunity, regardless of addition of adjuvant or not. From the comparison of result ofdetection of the antibody level, cytokine concentration detection and lymphocyte proliferationassay, we found that RA bacterial ghosts is better than inactivated vaccines. In the injectionRA WJ4of1.53times of LD50, the rates of protection could reach to100%separately in thegroup of immunization of bacterial ghosts with adjuvant and without adjuvant, and the ratesof protection could reach to100%and75%.Through comparison of three kinds of methods, we have succeed in producting RAbacterial ghosts using MAP(CPP). They could induce better humoral immunity and providehigher ptotection than traditional inactivated vaccine, this indicate that it is possible todevelop bacterial ghost as ideal vaccine candidate. |
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