Generation And Characterization Of Shigella Ghosts

The pathogenicity of Shigella is very strong，which causes lots of diarrhea anddeathes in the Homo sapien and animals. Virulence of Shigella is determined by theirinvasion plasmid antigen (ipa), virulent Shigella all contains a large plasmid, Shigellashould loss pathogenecity after curing the plasmid. pathopoiesis by Shigella dependson the activated coding gene products, which release toxin to poison cells afteradhering to and invading enterocyte. Lots of reports indicated that Shigella makesnumerous animals ill, such as the calfs,the cows, the grices, the chicken, the duck, therabbits, the doggies, the foxes, the beavers the monkeys and other kinds of animals.At present, only chicken Shigella disease was researched much more. In order todiminish serious dangers of this disease to homo sapien and animals, we should payattention to the disadvantages caused by animal Shigella disease, take measures tostrengthen the study of prevention and therapy to the disease for the control.PhiX174phage lysis gene E encodes a91amino acid membrane protein.ProteinE can integrate internal and external membrane of Gram-negative bacteria, resultingin a hole-like channel formed on the surface of bacteria.The diameter of hole-likestructure of bacteria ghosts is between the range of40-200nm.Cytoplasmic ofbacterial was exhausted through this pore-like structure to form the non-cytoplasmicnon-active bacteria.In order to improve lysis rate and stability of chicken origin Shigella dysenteriaeghosts, recombinant strain of Shigella with double bacteriolytic plasmids wasconstructed and its characteristics was studied. Two recombinant bacteriolysisplasmid pBV220::E and pBBRIMCS-E were co-transformed into the Shigella strainisolated from chicken to produce recombinant strain Sd（pBV220::E/pBBRIMCS-E）and named Sd3.The recombinant strain Sd（pBV220::E/pBBRIMCS-E）was grown at28℃with shaking until mid log-phase, followed by incubation at42℃to induce theexpression of gene E and produce Shigella dysenteriae ghosts. The OD600value ofbacterial cultures was monitored over20h at30min intervals. The morphology ofchicken origin Shigella dysenteriae ghosts was observed by electron microscope. The recombinant strains Sd（pBV220::E/pBBRIMCS-E）generated chicken origin Shigelladysenteriae ghosts after inducing at42℃.The ghosts retained basic cell morphology,with the lysis rate of99.9957％. Because of plasmids was uneven distribution, whichthe plasmids was lost during the proliferation of bacterial cells,when recombinantbacteriolysis plasmid was lost, the bacterial could not incompletely lysis to was killed,existing some security risk. In order to improve this phenomenon which the plasmidswas lost. The lysis gene E was amplified from recombinant bacteriolysis plasmidpBV220::E by PCR. Temperature regulation fragment CIP was amplified fromplasmid pBV220. These two fragments were ligated to form fusion fragment CIP-Eby PCR. Using the bacterial artificial chromosome to structure recombinant plasmid.The fusion fragment CIP-E was subcloned into downstream sites BamHI and SpHI oflysis gene E to generate a recombinant plasmid pBeloBAC11-CIP-E. Thisrecombinant plasmid was transformed into host cell DH5α. Results showedrecombinant plasmid pBeloBAC11-CIP-E could lysis DH5α after the temperatureshift from28℃to42℃. The lysis rate of culture was99.9948%. The recombinantplasmid pBeloBAC11-CIP-E was transformed into the Shigella strain isolated fromchicken to produce recombinant strain Sd(pBeloBAC11-CIP-E) and named Sd4. Thelysis rate of Sd4culture was99.9937%. In order to improve the efficiency ofgeneration of bacteria ghosts, increasing the lysis rate of culture, two recombinantbacteriolysis plasmid pBeloBAC11-CIP-E and pBBRIMCS-E were co-transformedinto the Shigella strain in electrotransformation to structure recombinant strain Sd(pBeLoBAC11-CIP-E/pBBRIMCS-E) and named Sd5. Results revealed that Sd5lysis rate was the best, which can receive99.999997%. After that, the Shigella ghostswere yield vastly.The living bacteria surviving in Shigella ghosts could be completely killed aftertreatment by freezing and thawing in5%or10%NaCl solution. The Shigella ghostswere shown to be intact cells structure with contents released to extracellular region.However, as bacterial cytoplasmic flow outside of bacteria, Shigella ghosts surfaceobviously shrink. animal experiment showed that Shigella ghosts are safe to chick. Atthe same time, Shigella ghosts have good immune protective effect.We can conclude from above study that the chicken origin Shigella dysenteriaeghosts were generated successfully with single bacteriolysis plasmid and doublebacteriolysis plasmids respectively, which laid foundation for future development of bacterial ghost vaccine against Shigella infection in chicken.