Generation Of Escherichia Coli Ghost Anchoredly Expressing Bovine Pasteurella OmpH And Characterizatio

At present Pasteurella commercial vaccine such as inactivated vaccine and attenuated vaccine have partical immune protective effects.Pasteurella multocida has lots of different serotype in nature, the power of cross immunity protection is low; The cell epitope of inactivated vaccine may be disrupted by physical or chemical factors, and the immune effect was influenced. The ghost is a hollow which has no cytoplasm or nucleic acid. And its surface antigen retained intact, it can be presented effectively by presenting antigen, and the effect of protective immunity is good, also the production cost is cheap and can be produced in large quantities with good prospects for development. Studies have shown that OmpH of bovine Pasteurella multocida has the function of cross immune protection.The purpose of this experiment is to prepare bacterial ghost in which membrane bovine Pasteurella OmpH can be anchoring expressed.The whole segment of lysis gene E was amplified by PCR using the plasmid RFI of Bacterialphage phiX174 as the template. Then the production was subcloned to vector pMD18-T.The positive clones was identified and the gene E of pMD18-T-E was separated by double enzyme digestion.Then the whole segment of lysis gene E was inserted into plasmid pBV220 to construct recombinant plasmid pBV-E.The whole segment of gene OmpH was amplified by PCR using bovine Pasteurella multocida strain CVCC393 as template. The segment of gene E’was amplified by PCR using recombinant plasmid pBV-E as template. A fusion fragment E’-OmpH formed by overlap PCR with a flexible peptide (Gly4Ser) gene E’and gene OmpH connected together. The fusion fragment E’-OmpH was subcloned into the aprokaryotice expression plasmid pET28a on the site of EcoRI and SalI to construct a recombinant expression plasmid E’-OmpH-pET28a. The recombinant plasmid was transferred into the Escherichia Coli BL21(DE3) by heating, and the recombinant plasmid expression plasmid E’-OmpH-pET28a were successfully procured by the identifition of enzyme digestion. The E gene lysis cassette which contain the sequence of gene pBV- E from its repressor to terminator was subcloned into the recombinant expression plasmid E’-OmpH-pET28a on the site of SgrAI And Sphi to construct a two-way expression plasmid E’-OmpH-pET28a-ci857-E.The recombinant plasmid was transferred into the Escherichia coli BL21(DE3) by heating, and the two-way expression piasmid E’-OmpH-pET28a-ci857-E were successfully procured by the identifition of enzyme digestion. The expression of fusion fragment E’-OmpH induced by IPTG were identified by SDS-PAGE、westernblot、and the expression of lysis gene E were identified by the culture of bacterial ghost. Mice Experiment showed that the group of Pasteurella multocida, OmpH+freund’s adjuvant, bacterialghost, bacterialghost+freund’s adjuvant immune after 24 days the antibody titer significant difference between the control group (P< 0.01). We found that the rate of immune protection against strains attack about Pasteurella multocida, OmpH+freund’s adjuvant, bacterialghost,bacterialghost+freund’s adjuvant, and PBS control group were 100%、80%、75%、65%、 0%,can protect some annimalAs a conclusion, the present study successfully prepared the recombinant E.coli ghost expressing the OmpH establishing the preliminary technique to prepare and identify this novel vaccine. These results lay the foundation for research on development of the new vaccine which bacterial ghost as the vector to deliver the protein. However, there still exist many disadvantages with the ghost system in this study, such as the low level of OmpH expression and immune response and the time when and how long to indure the two express systems. So we still need following works to optimize this system.