The Expression Of Rabbit Bordetella Bronchiseptica PRN Protein And The Study On Its Immunoprotection

Bordetella bronchiseptica (Bb) is an important pathogenic bacterium which can cause acute and chronic respiratory system disease. Bb can infect numerous animals, such as swine, horse, cat, canine, rabbit, mouse, cavia cobaya and so on. It always results in rhinitis and bronchial pneumonia of the infect animals. Human beings are also infected by Bb, especially children and immune deficient individuals. The high infection rate in rabbits impact rabbit product industry seriously, and affect the quality of experiment rabbits used for research.Pertactin (PRN) is the major protective antigen of Bb which exists in the outer membrane of bacteria. It was also a significant adhesion molecule, and it plays an important role during Bb colonizing. Some researchs have shown that PRN can significantly decrease the colonization level of Bb in the lung of immunized mice. We carried out the cloning and expression of prn gene, and we studied the immuno-protection of the recombinat protein.At the same time, we also used the recombinant protein PRN as coating antigen in order to establish an indirect ELISA diagnostic method for detection of Bordetella serum antibody. The main experiments and results are as follows:1. With a pair of specific primer designed to amplify prn gene of Bordetella bronchiseptica in rabbits, a fragment of the target gene was amplified by PCR. The target gene was colonized into PMD19-T Vector, and the recombinant plasmid was constructed using prokaryotic expression vector pGEX-4T-1 after being identified by PCR and restriction endonuclease analysis.The recombinant plasmid was transformed into Escherichia.coliBL21 (DE3) and the recombinant fusion proteins could be highly experessed in inclusion body form at the presence of IPTG, while at the same time a small amount of the protine could be experessed in supernatant in a soluble form. The Western blot analysis unfolded the excellent immunogenicity of the recombinant protein, which might be used as coating antigen to develop the ELISA method for Bb specific antibody diagnosis as well as a constituent of vaccine for Bb prevention.2. An indirect ELISA assay for detection of Bordetella bronchiseptica antibodie was developed with the recombinant antigen PRN in above experiment. The optimum coatedconcentrationof the recombinant protein PRN ascertained in this experiment is 500ng /mL; the best sealing buffer is 2% BSA and the sealing time is 1.5h(37℃), testing sera dilution is 1:40; the best sera reaction time is 60 min,enzyme labeledantibody dilution is 1:5000 and the reaction time is 60min. The optimal colour development time of substrate is 10min(37℃). This method was sensitive, specific, rapid, simple, accurate and reproducible, and it is capable to identify animals infected with or exposed to Bordetella bronchiseptica. Therefore, it can be a valuable diagnostic tool in both clinical diagnosis and research.3. The recombinant PRN proteins of Bordetella bronchisepticain three different concentrations weremixed and emulsified separately with Freund's adjuvant at ratio 1:1, and the ICR micewere immunized with the vaccine, meanwhile seting Bb whole-cell inactivated vaccine as positive control, the negative bacteria which contained void vector after ultrasonication as negative control and sterile PBS as blank control. During the course of immunity, through tail intravenous blood sampling the immune mice, using indirect ELISA measured its serum titers. Injecting to the abdominal cavity of the immunized mice with 2×LD50 bacilli, record the deaths of these mice after ten days, the results showed that there were no deaths in the two higher concentration protein immune group and the positive control group, so their livability were 100%,2 mice died in the lowest concentration protein immune group, the livability were 66.7%, while the negative control group mortality rate is as high as 83.4% and the blank control is 100%. The dead mice manifested as loss of appetite and lassitude before death. The typical form of Bb can be isolated from liver and lung.This study has successfully expressed the recombinant protein PRN; and of good immune protection for clean-level ICR mice; at the same time, this study also successfully applied recombinant protein PRN to set up a kindof indirect ELISA diagnostic method for detection of bronchial defeat blood Bordetella serum antibody. Thus it has laid an important foundation for the diagnosis and control of the disease, as well as the development of specific vaccine.