体外筛选对鸡具有免疫刺激活性的CpG寡脱氧核苷酸

Screening CpG oligodeoxynucleotides(ODNs)with strong immunostimulatory activity on chicken in vitro by lymphocyte transformation assay

分别用有丝分裂原和CpG寡脱氧核苷酸 (CpGODN)对SPF鸡全血 ,经低速离心分离的外周血单个核细胞(PBMC)和脾脏细胞 ,用淋巴细胞分离液分离的PBMC和脾脏细胞进行刺激 ,用3 H TdR掺入法测定并比较上述鸡免疫细胞在不同培养温度和细胞浓度下的转化效果 ,从而建立了适用于大批量筛选对鸡具有免疫刺激活性CpGODN的淋巴细胞转化方法。进一步用该方法筛选自行设计合成的 38条ODN。结果表明 ,用低速离心法分离的鸡PBMC ,在细胞含量为10 7mL-1、 37℃、 5 %CO2 条件下 ,经CpGODN刺激 6 4～ 6 6h后 ,可以得到稳定的高效转化 ;不含CpG模体的ODN不具有免疫刺激活性 ;CpGODN 10、 11和 14具有最佳的免疫刺激活性 ,刺激指数大于 10。

The SPF chicken whole blood,peripheral blood mononuclear cells(PBMCs)and splenic lymphocytes from low-speed centrifugation of heparinized blood,PBMCs and splenic lymphocytes by Ficoll gradient centrifugation procedure,were prepared and stimulated by mitogens and CpG ODN 10 and 11,respectively.The stimulated lymphocytes were cultured at different temperatures and different cell concentrations.The transformation effect of the stimulated lymphocytes was further measured by using 3H-TdR incorporation assay to determine and compare the stimulation index(SI)of the stimulated lymphocytes from different procedures and conditions.And the chicken lymphocyte transformation assay based on plastic flat with 96 wells and suitable for large scale screening of CpG ODNs with strong immunostimulatory activity was established.And the library of 38 CpG ODNs designed and synthesized were screened by using the above established assay.The results showed as follows: The chicken PBMCs at the concentration of 10 7 mL -1 by low-speed centrifugation procedure can respond effectively and stably to the stimulation of CpG ODN 10 and 11 for 64 66 h at 37 ℃ 5% CO 2 incubation ;The ODNs without CpG motif have no stimulation activity on chicken lymphocytes; CpG ODNs 10,11,14 have the stronger stimulation activity,SI>10.