Construction of the Plasmid Containing Multi-copies of CpG Motif and Evaluation on Its Adjuvant’s Efficacy

• Main Authors: Ming-Shiou Fang, 方明秀
• Other Authors: Hso-Chi Chaung
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/89833557611052333366

碩士 === 國立屏東科技大學 === 生物科技研究所 === 94 === DNA fragments containing unmethylated CpG oligonucleotides (CpG ODN) can enhance immunity and affect the Th1/Th2 balance in varieties of animals. CpG ODN, therefore, can be applied as a potent immunostimulating adjuvant. Recent reports have demonstrated that the efficacy of CpG ODN is positively correlated to its copy number in one plasmid. The immunostimulating effects of CpG motif have been found to be species-specific. However, no specifically highly efficient CpG motif has been designed for pigs yet. Thus, we designed the CpG-oligonucleotide (ODN-D ) for pigs as compared with a valid CpG ODN (ODN-S ) in a published document. Porcine bone marrow haematopoietic-derived dendritic cells (BMHC-imDC) and mature DC (BMHC-mDC) were used as the in vitro screening system to evaluate immunostimulating activities of phosphorothioate-backboned CpG ODN (PTO ODN) and plasmids containing various copies of CpG motif. We successfully synthesize the plasmids containing different copy numbers of CpG motif. In this study, the PTO ODN-D enhanced IFN-α mRNA about 573.79±107.00 relative fold in BMHC-imDC and elevated IL-12 production in BMHC-mDC as compored with the control (P<0.05). The plasmids with four copies of ODN-D enhanced more effectively in IFN-α gene expression in BMHC-imDC than those with different copies of ODN-S in one plasmid. Moreover, plasmids containing single copy of ODN-D tended to induce more secretion of IL-12 in BMHC-imDC than those with single copy of ODN-S. However, the efficacy of CpG ODN was not positively correlated to the copy number of CpG motif in one plasmid. Therefore, more studies for verifying the correlation between the copy number of CpG motif in one plasmid and its efficacy are still needed. Furthermore, improving the DC screening system should be able to accelerate feasibility on applying CpG ODN as an effective DNA adjuvant in domestic animals.