| Bordetella avium(B. avium), the causative agent of bordetellosis, continuously causes severe losses in the poultry industry. It mainly caused respiratory diseases and acute death of young birds. Diseased birds fail to gain weight with a low feed conversion rate. Currently, the vaccines available to prevent B. avium disease are mainly the live temperature-sensitive muntant vaccine and whole-cell cephalosporins, which only can appropriate to reduce the severity of the disease or delay the appearance of clinical symptoms. Therefore, it is important to develop a novel B. avium vaccine to prevent its infection. DNA vaccine is the third generation of vaccine, following after the traditional vaccine and the gene engineering subunit vaccine. The vaccine not only has the advantage of the inactivated vaccine, but also have not been found the risk of reverse transcriptase yet, therefore it takes more and more attentions in practice. In addition, Taishan Pinus massoniana pollen polysaccharides(TPPPS) has been studied in our laboratory since 2003, which was found to be an effective adjuvant for inactivated and subunit vaccines. However, the use of TPPPS as adjuvant of DNA vaccine has not been researched. In the current study, to develop a novel B. avium vaccine, we constructed a recombinant plasmid expressing B. avium ompA. Furthermore, to improve the immunogenicity of this DNA vaccine, TPPPS was used as the adjuvant of the DNA vaccine to examine its immune conditioning effects.To amplify the OmpA gene, a pair of primers were designed according to the ompA gene sequence of B. avium(GeneBank accession number: M96550.1). The PCR product was cloned into the pMD18-T vector, and the resultant plasmid was confirmed by sequencing. The plasmid pMD18-T-OmpA was digested with EcoR I and Xho I, and cloned into the pVAX1 vector. The recombinant plasmid was confirmed by restriction digestion and sequencing.The recombinant ompA plasmids pVAX1-OmpA were transfected into the BHK-21 cells with Lipofectamine. The transfected cells were cultured in MEM containing 1% FBS for 72 h.Then the expressed ompA in the cell culture supernatants was purified with nickel affinity chromatography according to the previous method, and verified by western blotting using an anti His-Tag monoclonal antibody. The results showed that a single protein band with an apparent molecular weight of 27 KD was obtained after the purification procedure.TPPPS was prepared in our laboratory by the method of water extraction and ethanol precipitation. The recombinant plasmids were mixed with TPPPS or Freund’s adjuvant at a ratio of 1:1. A total of 224 one-day-old SPF chickens were randomly separated into sevengroups. Each chicken in groups I-VII was injected with the low dose, middle dose, and high dose TPPPS adjuvant recombinant ompA plasmid vaccines, the Freund’s adjuvant recombinant ompA plasmid vaccine, the pure recombinant ompA plasmid, empty plasmid and PBS at 1, 7, and 14 days old, respectively. At 7, 14, 21, 28, 35, 42 and 49 days post the first inoculation, three different chickens from each group were selected randomly to determine the serum antibody, IL-2, IL-4 and IFN-γ concentrations, and CD4+ and CD8+ T lymphocytes counts in peripheral blood. After the third inoculation, 20 chickens in each group were challenged intraperitoneally with 10 LD50 B. avium LL strain. Survival status of chickens was recorded for seven successive days after the challenge. The results showed that the pure DNA vaccine obviously induced the production of antibody, the secretion of cytokines, the increase of CD4+ and CD8+ T lymphocytes counts in peripheral blood, and it provided a protection rate of 50% in B. avium challenged chickens. Notably, chickens inoculated with the TPPPS-DNA vaccine and Freund’s adjuvant DNA vaccine had higher levels of immune responses than those inoculated with the pure DNA vaccine, whereas only the DNA vaccine with 200 mg/ml of TPPPS completely protected chickens against the B. avium infection. |
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