体外筛选对犬具有免疫刺激活性的CpG寡脱氧核苷酸

建立并优化犬特异性寡脱氧核苷酸(CpGODN)的体外筛选条件,筛选自行设计的对犬具有免疫刺激活性的CpGODN。用CpGODN2006对犬的免疫细胞进行刺激,测定细胞增殖能力。对免疫细胞的来源、细胞铺板浓度、CpGODN的刺激时间和浓度及增值能力的测定方法等条件进行优化。并对自行设计的CpGODN进行了初步筛选。结果显示,犬脾细胞对CpGODN的反应性比外周血单个核细胞好。在犬脾细胞为6×105个/孔,37℃5%CO2条件下,经10mg/LCpGODN2006刺激48h,使脾细胞明显增生。3H-TdR掺入法测得的结果比MTT法更敏感。自行设计的CpGODNR008能显著地刺激犬脾细胞增生,且优于CpGODN2006(P0.05)。结果表明,优化了犬特异性CpGODN的体外筛选条件,并筛选出了对犬具有免疫刺激活性的CpGODN。

Optimization and application of an in vitro screening method for canine specific CpG ODNs

CpG ODN is a novel high performance adjuvant for domestic animals. However, CpG ODN is species spe-cific. So it is necessary to establish a stable optimal method for screening canine specific CpG ODNs. The canine PB-MC or splenocytes were stimulated by CpG ODN 2006, the cell proliferation was examined. The different experi-ment parameters include splenocyte numbers, stimulating time and concentration of CpG ODN. Upon these opti-mized condition, we screened our self-disigned CpG ODNs. In 96 well round bottom plate, 6 × 10~5 canine spleno-cytes/well stimulated by 10 mg/L CpG ODN 2006 for 48 hours can resulted in high stimulation index. CpG ODN R008 caused significantly splenocytes proliferation. The efficacy was stronger than that induced by CpG ODN 2006 (P<0.05). We successfully established an optimal condition for screening canine specific CpG ODNs, which lays the foundations for further development of novel CpG ODNs.