Development of pigeon circovirus vaccine

• Main Authors: Chang, Rui-Zhe, 張睿哲
• Other Authors: Chuang, Kuo-Pin
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/6a8gw2

碩士 === 國立屏東科技大學 === 動物疫苗科技研究所 === 105 === Pigeon circovirus (PiCV) is one of the main virus infesting pigeons. Once infected PiCV cause immunosuppression and cause secondary infection by bacteria and viruses inducing young pigeon disease syndrome (YPDS). The classical symptoms of YPDS include anorexia, depression, vomiting, diarrhea, ruffled feathers and abnormal flight. It has caused huge economic losses in pigeon fancier. Adenoviruses are often used as vectors for gene therapy and foreign gene expression. Because the adenovirus vector has the advantages of easy culture to high titer, high transduction efficiency in splitting and non-dividing cells and induce great T cell immunity respond. The study is divided into three parts: Try to culture PiCV in vitro, development of PiCV vaccine and animal immunization experiment. The recombinant adenovirus and PiCV capsid recombinant protein were produced by HEK293pTP mammalian cells using the pAdEasy system as vaccine. The research results shows, as PiCV continues to passage to LMH cell, its viral content decreases gradually. We also successfully recombined the target fragment PiCV capsid gene into BJ5183 bacteria. The recombinant vector was also transfected into HEK293pTP cells, and significant fluorescence was observed under fluorescence microscopy. In addition, we also selected a similar infection ability of the virus plaque, culturing its titer to 107.4 PFU/mL. Confirmation of protein results by western blotting also showed that antibody-specific binding was produced at the target site of 32 kDa, demonstrating that mammalian cells produced PiCV recombinant capsid protein. In order to find the optimal conditions for the production of recombinant viruses and proteins, we found that the use of less virus can recover more viruses. On the contrary, in protein recovery must use more virus. On the other hand, we also found that the use of recombinant adenovirus vector can be successfully infected DF1 and produce PiCV capsid target protein. In the animal test, heterologous prime-boost immunization regimens with recombinant adenovirus and protein were used in the pigeon, followed by enzyme-linked immunosorbent assay (ELISA) and cytokine assay to assess vaccine efficacy.