Hutchinson L E, Stevens M G, Olsen S C
United States Department of Agriculture, Agriculture Research Service, Ames, IA 50010, USA.
Vet Immunol Immunopathol. 1994 Dec;44(1):13-29. doi: 10.1016/0165-2427(94)90166-x.
Bovine cytokine-specific primers and the reverse transcription-polymerase chain reaction (RT-PCR) were used to clone cDNA fragments that were specific for bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma. Specificity of the cDNA fragments was verified by sequence analysis based on known bovine IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma gene sequences. In addition, RT-PCR was used to monitor cytokine mRNA expression in concanavalin A (Con A) and lipopolysaccharide (LPS)-stimulated bovine peripheral blood mononuclear cells (PBMC), and the results were compared with those obtained by measuring PBMC cytokine secretion using biologic assays. IL-1 activity in LPS-stimulated PBMC cultures was similar at 12 h and 24 h, although the activity decreased by approximately 40% at 48 h. IL-2 and IFN-gamma activity in supernatants of Con A-stimulated PBMC cultures was low at 12 h and reached maximum levels at 48 h. RT-PCR transcript analysis detected an increase in IL-1 alpha, IL-1 beta, IL-2, and IFN-gamma mRNA expression that was usually correlated with the detection of these soluble cytokines by the bioassays. These results indicate that RT-PCR is a sensitive and effective method of obtaining cDNA probes and that this technique can be used to monitor bovine cytokine mRNA expression.
使用牛细胞因子特异性引物和逆转录-聚合酶链反应（RT-PCR）克隆牛白细胞介素-1α（IL-1α）、白细胞介素-1β（IL-1β）、白细胞介素-2（IL-2）和干扰素-γ（IFN-γ）特异性的cDNA片段。基于已知的牛IL-1α、IL-1β、IL-2和IFN-γ基因序列，通过序列分析验证cDNA片段的特异性。此外，使用RT-PCR监测伴刀豆球蛋白A（Con A）和脂多糖（LPS）刺激的牛外周血单个核细胞（PBMC）中细胞因子mRNA的表达，并将结果与通过生物学检测测量PBMC细胞因子分泌所获得的结果进行比较。LPS刺激的PBMC培养物中IL-1活性在12小时和24小时时相似，尽管在48小时时活性下降了约40%。Con A刺激的PBMC培养物上清液中的IL-2和IFN-γ活性在12小时时较低，并在48小时时达到最高水平。RT-PCR转录分析检测到IL-1α、IL-1β、IL-2和IFN-γ mRNA表达增加，这通常与通过生物学检测对这些可溶性细胞因子的检测相关。这些结果表明，RT-PCR是获得cDNA探针的一种灵敏且有效的方法，并且该技术可用于监测牛细胞因子mRNA的表达。
