Enhancing the toolbox to study IL-17A in cattle and sheep.
Enhancing the toolbox to study IL-17A in cattle and sheep.
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DOI：
10.1186/s13567-017-0426-5
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发表时间：
2017-04-08
影响因子：
4.4
通讯作者：
Entrican G
中科院分区：
文献类型：
--
作者：
Wattegedera SR;Corripio-Miyar Y;Pang Y;Frew D;McNeilly TN;Palarea-Albaladejo J;McInnes CJ;Hope JC;Glass EJ;Entrican G
通过T细胞亚群检测反刍动物细胞因子表达的方法的发展是战略性开发用于预防传染病的新型家畜疫苗的基础。在反刍动物中检测T细胞中IFN-γ、IL-4和IL-10的表达已经有很多年的历史，但检测IL-17A表达的方法相对有限。为了弥补这一缺陷，我们克隆了牛和羊的IL-17A cdna，并在中国仓鼠卵巢（CHO）细胞中表达了具有生物活性的重组蛋白。我们使用转染的CHO细胞筛选市售抗体，以检测细胞内和培养上代细胞中IL-17A表达的能力。我们证明了牛IL-17A的ELISA检测到本地羊IL-17A。此外，ELISA中的组成多克隆抗体（pabs）通过ELISpot对牛、羊外周血表达IL-17A的单核细胞（PBMC）进行计数。我们通过流式细胞术鉴定了两种单克隆抗体（mabs），可以检测CHO细胞中重组细胞内IL-17A。其中一个单抗被用于检测PBMC细胞内IL-17A的表达，并与细胞表面表型单抗[CD4+ve， CD8+ve和Workshop Cluster 1 (WC-1)+ve γ - δ (γδ)]结合。我们发现，牛（定义为CD4+ve， CD8+ve或WC-1+ve）和羊（定义为CD4+ve或WC-1+ve）的不同T细胞亚群在激活后可以表达IL-17A。这些新技术为研究反刍动物IL-17A表达和定义特异性CD4+ve T细胞亚群激活提供了坚实的基础。本文的在线版本（doi:10.1186/s13567-017-0426-5）包含补充材料，可供授权用户使用。
The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants. The online version of this article (doi:10.1186/s13567-017-0426-5) contains supplementary material, which is available to authorized users.