Preparation And Immunization Of OMPs, LPS And Inactivated Vaccines From Avian Pasteurella Multocida

This paper focused on the extraction and purification of outer membrane proteins (OMPs) and lipopolysaccharide (LPS) from avian Pasteurella multocida, and the inactivated of avian Pasteurella multocida, based on these researchs, avian Pasteurella multocida outer membrane proteins vaccine, lipopolysaccharide vaccine and inactivated vaccine were prepared. The immunity of these vaccines were tested and the main research contents and results are as follows:The OMPs of avian Pasteurella multocida were extracted by lysozyme method, liquid nitrogen pestled method and ultrasonic method, the concentration of OMPs were determined with coomassie brilliant blue G-250 method, and the OMPs mapping was analysed with Quantity One after SDS-PAGE. The results showed that the concentration of OMPs extracted by the above three methods were 242μg/mL, 1013μg/mL and 1770μg/mL respectively. SDS-PAGE showed that the OMPs map of the three methods were roughly identical, and every lane contain 28.7 kD, 35.6 kD and 40.3 kD proteins, with the 35.6 kD protein majority.The LPS was prepared from avian Pasteurella multocida with hot phenol-water method and phenol-chloroform-petroleum ether method (PCP method), the concentration of polysaccharide and protein were determined with phenol-vitriolic colorimetry method and coomassie brilliant blue G-250 method respectively. The results showed that the lipopolysaccharide concentration of hot phenol-water method for 2056μg/mL, PCP method for 681μg/mL, but the protein concentration of hot phenol-water method and PCP method were 31μg/mL and 9μg/mL, accounting for 1.5% and 1.3% of the polysaccharide solution respectively.Research on the optimum formaldehyde content when avian Pasteurella multocida was inactivated,then mixed the inactivated Pasteurella multocida with Freund's adjuvant prepared for inactivated vaccine. The results show that, when the formaldehyde content was 0.2%, avian Pasteurella multocida was placed for 2 h at 37℃, Pasteurella multocida can all be inactivated. The viscosity, stability and safety of the oil emulsion inactivated vaccine all conform to the requirements.Prepared for avian Pasteurella multocida OMPs vaccine, LPS vaccines and inactivated vaccine, five groups of mice (n=16) named OMPs, LPS, inactivated vaccine, attenuated live vaccine and PBS were subcutaneously immunized three times with OMPs vaccine, LPS vaccine, inactivated vaccine, attenuated live vaccine and PBS respectively. The serum antibodies were detected by indirect ELISA. The spleen lymphocyte proliferation were tested by MTT. The mice were challenged with avian Pasteurella multocida two weeks after the third immunization, the protection rate were counted. Results of indirect ELISA showed that the levels of antibodies in OMPs group, LPS group and inactivated vaccine group were significantly higher than that of PBS group (P<0.05), and were almost high of the attenuated live vaccine. Spleen lymphocyte proliferation test indicated that the SI value in OMPs group, LPS group, inactivated vaccine group and attenuated live vaccine group were mostly higher than that in PBS group appearent (P<0.01), and there was no difference among the four vaccine groups (P>0.05). The protection rate of OMPs vaccine was equivalent to that of the attenuated live vaccine (80%), better than the LPS vaccine and inactivated vaccine (70%).