Study On The Combined Gene Vaccine Of Actinobacillus Pleuropneumoniae ApxⅣ And Pasteurella Multocida OmpH Protein

Porcine infectious pleuropneumonia caused by Actinobacillus pleuropneumoniae(App)and atrophic rhinitis and porcine pneumonia caused by Pasteurella multocida(Pm)are common respiratory diseases in pigs,and the above diseases are often mixed infections.Pigs of all ages are all susceptible and can be spread through aerosols and contact.It often presents a whole herd outbreak,causing great losses to the pig industry.In this study,the exotoxin protein ApxⅣ of Actinobacillus pleuropneumoniae and the outer membrane protein H(OmpH)of Pasteurella multocida were used to prepare a dual gene vaccine that can simultaneously prevent and control the above two diseases,and act for pleuropneumonia.The prevention and control of bacillus and Pasteurella multocida provide a theoretical basis.In this study,we used online biological software to screen the T cell and B cell epitopes of APP’s exotoxin ApxⅣ and Pm’s outer membrane protein H(OmpH)genes;after the screened dominant epitope sequences were connected by connecting peptides,new peptides were constructed Section(named New),and analyze it with online bioinformatics software;respectively construct ApxⅣ,OmpH and New gene sequences by homologous recombination method into the eukaryotic expression vector GV658,and verify the size of the target gene by double enzyme digestion;Transfect the three newly constructed recombinant plasmids into HEK293 cells,and evaluate their safety to the cells by CCK-8 experiment;RT-PCR and western blotting to verify the transcription and expression of foreign gene proteins;immunofluorescence(IFA)to verify the recombination Immune localization of foreign gene proteins in plasmids in cells.The test results show that the New peptide has good immunogenicity.The constructed recombinant plasmids GV658-ApxⅣ,GV658-OmpH,and GV658-New double enzyme digestion results are consistent with the size of the target gene,and can be transfected into HEK293 cells and successfully expressed.And there is no obvious damage to HEK293 cells.Mix the constructed 3 recombinant plasmids in equal proportions to prepare APP and Pm dual gene vaccine,and prepare GV658-ApxⅣ,GV-658-OmpH,GV658-New 3 plasmid vaccines.PBS is the control group,and SD is treated every 2 weeks.Rats were immunized for 3 consecutive times.Peripheral blood was collected 2 weeks after the last immunization and the internal organs were dissected for analysis.The results of flow cytometry showed that the values of CD3+ cells and CD4+/CD8+cells in the dual vaccine group(Mix)increased,which were significantly higher than those in the PBS group(P<0.05);the serum antibody results showed that the values of the Mix(double vaccine)group The antibody titers of APP and Pm were significantly higher than those of the PBS group(P<0.05),and were not significantly different from the antibody titers of APP,ApxⅣ,OmpH and New groups(P>0.05);organ pathological tissue sections and organs The weight ratio results showed that the experimental groups of each group were the same as the control group,and the results of tissue sections and organ weight ratios were normal;the immunofluorescence(IFA)results of liver tissue sections showed that all experimental groups except the control group were obviously visible in the liver tissue Fluorescence staining.In summary,the recombinant dual gene vaccine constructed in this project produces antibody titers consistent with the results of APP,ApxⅣ,OmpH,and New vaccines,and does not cause significant damage to the animal body.It has good safety and can be used at the same time.Two diseases,APP and Pm,produce immune protection.In this way,it provides a theoretical basis and research foundation for the research of APP and Pm.