| Bordetellosis is an upper respiratory tract and infectious disease, which is caused by Bordetella avium(B. avium). This disease could spread not only through horizontal transmission but also through vertical transmission. It mainly caused high death rate of embryo, low hatchability, and acute death of young birds. It also caused upper respiratory tract symptoms of adult birds, such as labored breathing, sneezing, oculonasal discharges, and diseased birds failed to gain weight with a low feed conversion rate, which continues to be an economic problem in the poultry industry of China. In addition, it was also described in previous studies as an opportunistic human pathogen. Currently, the use of antibiotics in controlling diseases has not been recommended as they possess health hazards to consumers and B. avium has developed drug resistance. About the prevention of bordetellosis, only the the study of vaccine on turkey has been conducted abroad. Currently, the temperature-sensitive muntant vaccine and whole-cell cephalosporins used to prevent bordetellosis cannot limit the infection and spread of B. avium, although they offer protection against severe diseases and delay the appearance of clinical symptoms. They can not completely prevent the spread of this disease. Therefore, it is important to develop a novel B. avium vaccine to prevent its infection. Adjuvant is believed to establish high and long-lasting immune responses by activating the innate immune system, thereby enhancing the adaptive immune response to an administered antigen. Therefore the application of adjuvant in vaccination is indispensable because adjuvant enhances the effects of vaccines. In our previous studies, we found that Taishan Pinus massoniana pollen polysaccharides(TPPPS) is an effective adjuvant for the inactivated and subunit vaccines. However, little is known about the effects of TPPPS on the recombinant outer membrane protein A(ompA) based on eukaryotic expression system. Herein, we constructed a recombinant Pichia pastoris(P. pastoris) transformant capable of expressing the recombinant ompA of B. avium to prepare the recombinant ompA subunit vaccine and evaluated its immune effects. Furthermore, to improve the immunogenicity of this subunit vaccine, TPPPS was used as adjuvant and its immunomodulation effects on this subunit vaccine were investigated.To amplify the ompA gene, a pair of primers was designed according to the ompA gene sequence of B. avium(GenBank accession number: M96550.1). The PCR product was cloned into the pMD18-T vector, and the resultant plasmid was confirmed by sequencing. After sequencing, the resultant plasmid was digested with EcoR I and Not I restriction enzyme. The product was cloned into expression vector pPIC9 which was also digested with EcoR I and Not I restriction enzyme. Then the resultant expression vector was transformed into competent P. pastoris GS115 to obtain a transformant P. pastoris pPIC9-ompA after linearization. The transformant P. pastoris pPIC9-ompA was selected through RDB media, and then confirmed by PCR and sequencing. At the same time, blank plasmid pPIC9 was transformed into P. pastoris as a negative control.The methanol-induced expression of ompA in P. pastoris GS115 was performed following the manufacturer’s instructions. At 24, 48, 72 and 96 h post methanol induction, the culture supernatants of the P. pastoris transformed with the recombinant pPIC9-omp A plasmid and the P. pastoris transformed with blank pPIC9 plasmid were harvested through centrifugation, respectively. SDS-PAGE and Western blot analysis were carried out to identify the ompA as described in previous study. After confirmation, a novel protein band corresponding to 21 k Da in the culture supernatant of the recombinant pPIC9-omp A transformant was observed by SDS-PAGE. The protein’s molecular weight was identical to that of the foreign ompA, but this band did not appear in the culture supernatant of the control pPIC9(blank plasmid) transformant. After purifition using ProteinIso? Ni-NTA Resin kit, only the target protein band with a molecular weight of 21 kDa was observed in the result of SDS-PAGE. After western blot analysis, only one band with a 21 kDa molecular weight was observed, which was corresponded to the band in SDS-PAGE, thereby indicating the existence of the recombinant ompA and its specific immune response characteristic.A total of 180 one-day-old SPF white leghorn chickens were randomly divided into six sterilized isolators(groups I to VI), with 30 chickens in each. The chickens were allowed to acclimatize for three days before the start of the experiments. Each chicken in groups I-VI was subcutaneously inoculated with 0.2 mL of the 20, 40, and 60 mg/mL TPPPS adjuvant recombinant ompA vaccines, the Freund’s incomplete adjuvant recombinant ompA vaccine, the pure recombinant ompA vaccine, and PBS respectively at 0, 7, and 14 days post the first vaccination(dpv). At 3, 7, 14, 21, 28, 35, 42, and 49 dpv, three chickens from each group were selected randomly to determine the serum antibody titers, serum IL-4 concentrations, CD4+ and CD8+ T lymphocytes counts in the peripheral blood, and T-lymphocyte transformation rate. One week after the third vaccination(at 21 dpv), 20 chickens from each group(except from the control group) were placed into a new isolator and challenged intranasally with 10 LD50 B. avium LL strain. Clinical manifestation and survival status of the chickens were recorded for seven days after challenge. The results showed that the pure omp A vaccine induced the production of anti-omp A antibody, the secretion of IL-4, the increase of CD4+ T-lymphocytes counts and lymphocyte transformation rate in the peripheral blood. Moreover, the pure ompA vaccine provided a protection rate of 71.67 % after the B. avium challenge. Notably, TPPPS adjuvant vaccines induced higher levels of immune responses than the pure ompA vaccine, and 60 mg/m L TPPPS adjuvant vaccine showed optimal immune effects. Our findings indicated that this recombinant B. avium ompA subunit vaccine combined with TPPPS had high immunostimulatory potential. Results provided a new perspective for B. avium subunit vaccine research. |
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