Fusion Expression Of PlpB And Fur Gene From Porcine Pasteurella Multocida And Evaluation Of Immune Efficacy

Porcine Pasteurella multocida (Pasteurella multocida,Pm) can cause swineinfectious atrophic rhinitis (Atrophic rhinitis, AR), pasteurellosis suum and porcinerespiratory disease syndrome (Porcine respiratory disease complex, PRDC).Pasteurella multocida is a non-host-specific pathogenic bacteria, transmitted throughthe respiratory tract, and spread in different host species, widely exists in the world.With the rapid development of the pig industry in China, the harmfulness ofPasteurellosis has become increasingly prominent and causes great economic loss tothe pig industry. The best method to prevent and control Pasteurella multocida isvaccination. Inactivated vaccine, attenuated vaccine and subunit vaccine are the majorvaccine to prevent pasteurellosis. Because of Pasteurella multocida have moreserotypes and immune mechanism is not clear, there is still a lack of ideal vaccine.Upto now, still lack of an ideal vaccine all over the world.The PlpB protein is Pasteurella multocida outer membrane protein. Studies showthat PlpB in avian Pasteurellosis have good cross protective. Fur protein as the ferricuptake regulator protein plays an important role in the growth of bacteria. Studieshave shown that Fur protein can inhibit the formation of biofilm in order to achievethe antibacterial effect, and impact the expression of outer membrane protein. Thestudy focuses on the isolation and identification of swine Pasteurella multocida inclinical, constructs the PlpB, Fur, PlpB and Fur Escherichia coli recombinant plasmid.Using bioinformatics methods and network database for PlpB and Fur sequencinghomology comparison. The results showed that isolated Pasteurella multocida isserotype B virulent strain and highly sensitive to ciprofloxacin. The PlpB gene is771bp, encoding a lipid protein of257amino acid residues. The similarity betweenPlpB protein space structure and the3K2D_A protein of Vibrio Vulnificus was higher.Built on template3K2D_A successfully construct a reliable three-dimensionalmolecular model. The Fur gene is432bp, encoding144amino acids. The similaritybetween Fur protein space structure and the ferric uptake regulator2w57.1.A werehigher. Built on template2w57.1.A successfully construct a reliable three-dimensional molecular model. Prediction the PlpB and Fur protein epitope indicate that thesetwo proteins as antigen epitope possibility is larger, and the PlpB and Fur with notransmembrane region and signal peptide, they are relatively mature and stableproteins.Three constructed recombinant plasmid was transformed into BL21, after inductionby IPTG, expression of56ku,44ku and73ku protein, named for the recombinantprotein GST-PlpB, GST-Fur and fusion protein GST-PlpB-Fur. The GST-PlpB proteinwas soluble protein. GST-Fur protein mainly existed in inclusion bodies. Fusionprotein GST-PlpB-Fur in the cells can be expressed as soluble protein or insolubleinclusion particles. Western blotting blot detection of recombinant protein GST-PlpB,GST-Fur and fusion protein GST-PlpB-Fur showed that the three protein has goodimmunogenicity. Three kinds of protein with Freund’s adjuvant emulsified,inactivated whole cell vaccine and normal saline respectively immunized mice.Detection of serum IgG, IFN-γ and IL-4content by ELISA after immunization, andthe challenge test evaluation of three subunit vaccine and a vaccine immune effect.The results showed that whole cell inactivated vaccine immune effect is the best andextra difference with the other three subunit vaccines. GST-PlpB-Fur fusion proteinvaccine immune effect is better than that of GST-PlpB and GST-Fur recombinantprotein vaccine. Fur and PlpB proteins play a synergistic role in immune effect. Theresearch for the future development of swine Pasteurella multocida subunit vaccine,the study of virulence factor cross protection performance and the role of ironregulation of gene in cells lay the foundation of other research.