Selection Of The Cross-protective Antigens From Bovine Pasteurella Multocida And Identification Of Its Fusion Protein Immunoprotection

Pasteurella multocida is the most important pathogenic member in genus Pasteurella multocida is Gram-negative bacteria and it has a broad host spectrum includeing many wild and domestic animal. P.multocida isolates are classified five capsular serogroups A、B、D、E or F and based on their lipopolysaccharide (LPS) P.multocida isolates further into 16 serovars. In the past bovine P. multocida serogroup B is the main type. But since 2008 it is epidemic in china and showed a rising trend. At present the vaccines of bovine P. multocida is only against P. multocida serogroup B, not for P. multocida serogroup A and B. Immediately although bacterins vaccines are played an important role but vaccines may do not protect hosts against heterologous challenge.These disadvantages require us to develop new type vaccines for P. multocida. So it is important to select the potential cross-protective protein antigens for development of new subunit vaccine.In this study we selected six kinds protective antigens of bovine P. multocida for research:ompA, ompH, plpE, pm0979, P6 and pfhB cloning and expression those six kinds protective antigen gene to identify the cross-protective antigens of bovine P. multocida. Thus we ues these six kinds of antigen gene in the same condition as a research object for the following research:1. Selection the cross-protective antigens of bovine Pasteurella multocidaThe candidate genes were respectively amplified by PCR with the genomic DNA of bovine P.multocida serotype A strain CQ2 as template, then the six kinds protective antigens gene were cloned into pET-30a/pET-32a (pET-30a-ompA、pET-30a-ompH、 pET-30a-PlpE、pET-30a-pm0979、pET-32a-P6 and pET-30a-pfhB) for expression in Escherichia coli BL21(DE3) and the expression of recombinant protein were induced with IPTG. Those Recombinant fusion proteins were purified and formulated with adjuvants. The purified recombinant proteins were made into subunit vaccines to immunize the mice by subcutaneous injection, the detection of the serum production and cross protection were conducted at post-immunized 21 d. The results showed that the recombinant proteins vaccine rPM0979, rPLPE and rOMPH Provide different degrees of cross-protection when challenged with 2 LD50 of P. multocida strain CQ2 or type B in mice. Among of them the recombinant protein rPLPE provide 70% or 30% cross-protection respectively. Therefore recombinant protein rPM0979, rPLPE and rOMPH protein can be used as a potential vaccine proteins.2. Expression of the fusion gene pm0979-ompH of Pasteurella multocida and its immunoprotectionAnalysis the advantage epitope of pm0979 and ompH gene then construct the fusion gene pm0979-ompH with three different length linker. Those genes were cloned into pET-30a vector for protein expression in Escherichia coli BL21 (DE3). We purified and immunized the mice with the recombinant protein. In addition rPM0979-L10-OMPH and rPM0979-L15-OMPH were able to produce protection rate of 70% or 30% against the challenging with 2 LD50 of P. multocida type A strain CQ2 or P. multocida type B in mice. ELIS A revealed that a high level of antibody was detected in the mice immunized with the recombinant protein rPM0979-L10-OMPH and rPM0979-L15-OMPH. Thus the recombinant vaccine rPM0979-OMPH provides good cross-protective immunity; furthermore this study also showed that vaccine with different length linker has influence on the protection.3. Expression of the fusion gene pm0979-PlpE of Pasteurella multocida and its immunoprotectionSelected the cross-protective antigens PlpE and pm0979 and analyzed the advantage epitope, in order to use the Gene-SOEing construct the fusion gene pm0979-PlpE with a Hydrophobic Linker. The fusion gene was ligated into PET-30a to get the expressing vector which was then transformed into Escherichia coli BL21 (DE3). We purified and immunized the mice with the recombinant protein. The results were shown that the recombinant protein rPM0979-PLPE was able to produce protection rate of 80% or 40% against the challenging with 2 LD50 of P. multocida type A strain CQ2 or P. multocida type B in mice. Thus the fusion protein rPM0979-PLPE has pm0979 and Pipe dual activity. In addition the recombinant vaccine rPM0979-PLPE provides good cross-protective immunity.