| Summary: | 碩士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 107 === E1 protein is a cytokine which has leukocytes attracting activity. E2 protein is a multifunctional cytokine. By employing recombinant polymerase chain reaction (PCR) technique, we connected E1 cDNA and E2 cDNA with three different linkers. The DNA fragments encoding the chicken fusion cytokines were cloned into the prokaryotic expression vector pET32a and transformed into Escherichia coli BL21 (DE3) for protein expression. Afterwards, we performed various activity assays to evaluate E1 and E2 activities of the fusion cytokines. On the other hand, fusion cytokine DNA fragments were cloned into the eukaryotic expression vector pEGFP-N1 or pVAX1 and the resultant plasmids were delivered into chicken embryo fibroblast cell line DF-1 by using a certain peptide. The results showed that this peptide was able to carry the resultant plasmid constructs into DF-1 cells to express green fluorescent fusion proteins or bioactive E1-linker-E2 proteins. We used either E1-linker-E2 protein or pVAXE1-linker-E2 plus peptide as the adjuvant for avian infectious bronchitis (IB) vaccine and performed chicken animal experiment to evaluate the vaccine efficacy. In the virus neutralization assay performed on chicken embryo eggs, the results showed that E1-linker-E2 protein and pVAXE1-linker-E2/peptide effectively enhanced the humoral immune response of vaccinated chickens to provide better protection than the vaccine group. In summary, we successfully expressed bioactive fusion cytokine proteins in bacteria and delivered eukaryotic expression plasmids encoding fusion cytokines into avian cells. We found that fusion cytokines, in protein or DNA form, can elicit better virus neutralization protection than the vaccine group. The fusion cytokines have the potential to be applied as a vaccine adjuvant in poultry industry in the future.
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