Büttner M, Belke-Louis G, Rziha H J, McInnes C, Kaaden O R
Institute of Vaccines, Federal Research Centre for Virus Diseases of Animals, Tuebingen, Germany.
Cytokine. 1998 Apr;10(4):241-8. doi: 10.1006/cyto.1997.0284.
In canine peripheral blood mononuclear cells (PBMC) the mRNAs coding for both subunits of canine interleukin 12 (IL-12) were identified using reverse transcription polymerase chain reaction (RT-PCR). Stimulation of canine PBMC with Staphylococcus aureus strain Cowan plus Concanavalin A for 5 h resulted in significant mRNA synthesis. Likewise, inactivated vaccinia virus induced IL-12 mRNA synthesis, however with different kinetics. The complete nucleotide sequence for both IL-12 subunits was determined using rapid amplification of cDNA ends (RACE)-PCR and cloning of amplified specific cDNAs. Computer-aided amino acid (aa) sequence comparison of both canine IL-12 subunits revealed more than 80% identity with the amino acid sequences of six other mammalian species. Closest relationship was found to human, porcine, bovine and cervine IL-12. However, no reactivity was found with antibodies directed against human IL-12, when supernatants of stimulated canine PBMC were tested. Supernatants of canine PBMC stimulated for IL-12 release also induced interferon gamma (IFN-gamma) mRNA as detectable by RT-PCR; however, it was not clear whether IFN-gamma mRNA synthesis was due to an IL-12 specific effect or other stimuli. As to the stimulating effect of IL-12 on canine IFN-gamma mRNA synthesis, recombinant human IL-12 was found to be a good inducer. Since IL-12 is regarded a major regulatory molecule of T-cell-mediated immune response and cell growth our work on the cloning and sequencing of this cytokine from dogs lays the basis for future investigations on the biological and possible therapeutic role of canine IL-12.
利用逆转录聚合酶链反应（RT-PCR）在犬外周血单核细胞（PBMC）中鉴定出编码犬白细胞介素12（IL-12）两个亚基的mRNA。用金黄色葡萄球菌考恩菌株加伴刀豆球蛋白A刺激犬PBMC 5小时可导致显著的mRNA合成。同样，灭活的痘苗病毒可诱导IL-12 mRNA合成，但动力学不同。使用cDNA末端快速扩增（RACE）-PCR和扩增的特异性cDNA克隆确定了两个IL-12亚基的完整核苷酸序列。对两个犬IL-12亚基进行计算机辅助氨基酸（aa）序列比较发现，与其他六种哺乳动物的氨基酸序列有80%以上的同一性。发现与人类、猪、牛和鹿的IL-12关系最为密切。然而，当检测受刺激的犬PBMC上清液时，未发现与针对人类IL-12的抗体有反应性。受刺激释放IL-12的犬PBMC上清液也可诱导逆转录聚合酶链反应检测到的干扰素γ（IFN-γ）mRNA；然而，尚不清楚IFN-γ mRNA合成是由于IL-12的特异性作用还是其他刺激。至于IL-12对犬IFN-γ mRNA合成的刺激作用，发现重组人IL-12是一种良好的诱导剂。由于IL-12被认为是T细胞介导的免疫反应和细胞生长的主要调节分子，我们对犬这种细胞因子的克隆和测序工作为未来研究犬IL-12的生物学和可能的治疗作用奠定了基础。
