| Proteus mirabilis（P. mirabilis） is an acute opportunistic pathogen that widely exists in nature P. mirabilis can infect chickens at various ages, and causes certain characteristic clinical symptoms, such as paralysis of the limbs, diarrhea, encephalomalacia and sepsis. The pathogen spreads by horizontal or vertical transmission and can easily lead to outbreaks of epidemic diseases. At present, P. mirabilis has a rising infection rate and has a significant negative impact on the poultry industry, however, few vaccines are available to prevent P.mirabilis infection. Therefore, an effective vaccine against P. mirabilis should be developed.In numerous gram-negative bacteria, Omp A represents the major portion of outer membrane proteins. Korn et al reported that Omp A of P. mirabilis（a 39 k Da protein） is a highly immunoreactive protein at least in mouse, as indicated by the strong stimulation on the proliferation of B lymphocytes. Omp A has great potential as an effective antigen component to protect animals against P. mirabilis infection. The Pichia pastoris（P. pastoris） expression system has been extensively used as a recombinant expression means because of its advantages of high production yields, genetically stable expression strains, potential of recombinant protein secretion into the culture medium and the property of performing eukaryotic post-translational modifications. Moreover, P. pastoris-expressed proteins are free of endotoxins, which is critical to the security of vaccine. In present research, we produced a recombinant protein of P.mirabilis Omp A in P. pastoris. Robinia Pseudoacacia Polysaccharide（RPPS） and used it as the adjuvant for recombinant protein vaccine. The purpose of this research was to evaluate the effects of RPPS on immune responses of chickens immunized with P. mirabilis Omp A recombinant protein vaccine.1. Expression P. mirabilis Omp A in P. Pastoris To amplify Omp A genes, a pair of primers was designed according to the Omp A gene sequence in P. mirabilis（Gen Bank Type: Ref Seq（Nucleotide） NC010554.1）. The size of amplified Omp A PCR products was 1107 bp. The PCR products were digested with Eco R I and Not I, and cloned into the p PIC9 vector, which was digested with the same endonucleases.The recombinant plasmids were named p PIC9-Omp A, transformed into E. coli DH5 a,verified by restriction digestion, and sequenced. The constructed plasmids p PIC9-Omp A were transformed into competent P. Pastoris cells by lithium chloride methods. Transformants were selected on RDB plates. Correct genomic integration at the Pichia AOX1 promoter locus was confirmed by PCR. The concentration of the expressed recombinant Omp A protein reached 8.0 μg/m L after induction for 96 h with 1.0% methanol in the culture. A molecular weight of approximately 52 k Da was observed by SDS–PAGE. In addition, Omp A protein was confirmed by western blot analysis using the antibody against Escherichia coli-expressed Omp A protein. This indicated that P. mirabilis Omp A was successfully expressed in P.Pastoris.2. Evaluation the effects of RPPS on immune responses of chickens immunized with P.mirabilis Omp A recombinant protein vaccine.In order to evaluate the effects of RPPS on immune responses of chickens immunized with P. mirabilis Omp A recombinant protein vaccine, RPPS was mixed into the recombinant Omp A protein to prepare the Omp A subunit vaccine. Then we subcutaneously inoculated this vaccine into chickens to examine the immune protective effects. A total of 360 one-day-old specific-pathogen-free（SPF） chicks were randomly divided into six groups. Groups I-VI were injected subcutaneously with 0.2 m L of 100 mg/m L TPPPS-Omp A vaccine, 200 mg/m L TPPPS-Omp A, 300 mg/m L TPPPS-Omp A, F-Omp A vaccine, Omp A-only vaccine, and physiological saline, respectively. The data showed that the antibody titers against Omp A, the concentration of IL-2, T lymphocyte proliferation rate, peripheral blood CD4+ and CD8+ cell percentage in Group II were significantly higher（P < 0.05） than those in the other groups.Moreover, a high protection rate of 88.8% was observed in this group. The results suggest that the eukaryotic P. mirabilis Omp A was an ideal candidate protein for developing an effective subunit vaccine against P. mirabilis infection, and RPPS can be developed into an adjuvant for recombinant subunit vaccine. |
