| Summary: | 碩士 === 國立屏東科技大學 === 動物疫苗科技研究所 === 106 === The porcine reproductive and respiratory syndrome virus (PRRSV) causes acute respiratory disease and persistent infection in piglets and reproductive failure in pregnant sows, which can lead to serious economic losses to the swine industry worldwide. PRRSV-infected pigs develop a delayed appearance of neutralizing antibodies and a weak cell-mediated immune response. The heterodimer of glycoprotein (GP5) and non-glycosylated matrix protein (M) are the leading targets for the development of new generation of vaccines against PRRSV infection. Many studies have been corroborated that co-expressing GP5 and M proteins as a fusion protein trigger better immunogenicity than that expressing GP5 or M protein alone. The GP5/M protein fused with porcine granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrated that GM-CSF fused with GP5 and M protein of PRRSV could significantly enhance the humoral and cellular immune responses and provide protection against PRRSV challenge in pigs. Adenoviruses are often used as vectors for gene therapy and foreign gene expression due to the reason that 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 response. The recombinant adenovirus and GP5/M-GM-CSF recombinant protein were produced by HEK293pTP mammalian cells using the AdEasy system as vaccine. We also successfully recombined the target fragment GP5/M-GM-CSF gene into BJ5183 bacteria. The recombinant vector was also transfected into HEK293pTP cells, and significant fluorescence was observed under fluorescence microscopy. culturing Virus titer to 108 TCID50*/ml. Confirmation of protein results by western blotting also showed that antibody-specific binding was produced at the target site of 21 kDa、24 kDa、61 kDa, demonstrating that mammalian cells produced GP5/M-GM-CSF recombinant capsid protein. The vaccine efficacy will be revealed by animal testing via Intramuscular injection and intranasal immunization. Moreover, the immunoassay was tested by ELISA and neutralizing antibodies test.
|
|---|
