Development of DNA vaccine against outer membrane protein of Riemerella anatipestifer

• Main Authors: Liou, Jhong-Jie, 劉忠杰
• Other Authors: Chu, Chun-Yen
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/15530051596588838427

碩士 === 國立屏東科技大學 === 動物疫苗科技研究所 === 104 === Riemerella anatipestifer is gram-negative bacillus, non-spore forming, and distinguished twenty-one serotypes which based on different surface antigens. The infected duck showed that clinical signs include diarrhea, lethargy and respiratory disorder, called new duck disease. In addition, the systemic serositis and septicemia would be fatal so that lead to the great economic losses in the waterfowl industry and the commercial vaccine cannot provide the cross-protection. Previous studies, all of the serotypes have outer membrane protein A (ompA), furthermore, it has good immunogenicity. In this study, we isolated different serotypes of Riemerella anatipestifer and constructed ompA gene to yT&A vector which sequences are similar to 99 %. We also had cloned the ompA gene into pTCY vector and transfected to Vero cells that confirmed of the product size and antigenicity by SDS-PAGE and Western blot. We had finished the dose dependent test and combined with CpG adjuvant to immunize ducks. After two weeks, challenged by serotype 2. The result showed that 100 μg plasmid DNA is suitable. Then, we had used prime-boost strategy to evaluate the vaccine potency. The results showed that after boosting the antibody titer of all vaccinated groups were significantly higher than control group (p < 0.05) and DNA+ Subunit can elicit long-term antibody till 16 weeks. The T cell proliferation index of Subunit+ Subunit group is significantly higher than the other groups. (p < 0.05). The CD4+ of all immunized groups were significantly higher than control group (p < 0.05); the CD8+ or CD4+ / CD8+ of DNA+DNA and Subunit+ Subunit were significantly higher than the other groups (p < 0.05). Regarding to the results, Subunit+Subunit can induce better and short-term immune response, however, DNA priming and protein boosting immunization scheme is potential to elicit long-term antibody.