Screening Of Protective Antigens Against Pasteurella Multocida From Swine

Pasteurella multocida, a pathogen which causes pig lung disease and pasteurella mullocida pneumonia. It can also be one of the main pathogens causes swine atrophic rhinitis. In order to develop an effective vaccine against Pasteurella mullocida, we screened for 5 geneswhich are located on the outer membrane of bacteria, which are Nlpl, BamD, OmpH, P1pP and FabA. The amplified target gene were inserted into vector pET-28a(+), respectively, as a result, recombinant plasmids pET28N1pI, pET28BamD, pET28OmpH, pET28P1pP and pET28FabA were obtained. The recombinant plasmids were sequenced, and showed high conservation within different strains. The recombinant plasmid were then transferred into BL21 (DE3) and expressed. The result of SDS-PAGE showed that P1pP and FabA protein mainly exist in the form of soluble protein, while N1pI, BamD and OmpH mainly form inclusion body. It is found that pET28NlpI, pET28BamD, pET28OmpH, pET28P1pP and pET28FabA recombinant protein can be identified to immune serum against Pasteurella multocida from Western Blot, proving their reactionogenicity.To determin whether these recombinant protein can induce high level of specific antibodies in mice, which can provide protection in mice against challenging with same serotype of Pm. The purification of recombinant proteins were prepared to be recombinant subunit vaccine with aluminium glue mixed. Selected Km female mice randomly, and immuned with the vaccine. Detected antibody levels of mice on each 13d-post-immunization by indirect ELISA. It is found that on the second 13d-post-immunizition, besides the negative control group, all other mice's antibody level was increased, especially the inactivated Pm vaccine group, antibody level is the highest; while mice antibody from Nlpl, BamD, OmpH, P1pP and FabA vaccine group rises as well. On 14d-post-immunization, all the mice were challenged with certain doses of Pm CVCC 439 strains. Experiments showed that N1pI, BamD, OmpH, PlpP and FabA protein can provide same serotype protection in mice, which P1pP and FabA protein are the most effective.To make sure these recombinant protein have the cross protection in different capsular antigen serotypes of Pasteurella multocida. KM female mice were selected randomly, with immuned with prepared recombinant subunit vaccine mixed with freund's adjuvant. The mice were challenged with a certain doses of pasteurella multocida CVCC 444, CVCC 434 and CVCC 439 strains, respectively, on 14d-postimmune. The results showed P1pP protein provides a better cross protective, FabA can provide good cross protective for mice as well, however, other proteins can only provide weak cross protection.