BACKGROUND Interleukin-17 (IL-17), the characteristic cytokine secreted by T helper 17 lymphocytes (Th17 cells), plays a pivotal role in host defense and many inflammatory or autoimmune diseases. The aim of this study was to obtain purified protein caprine IL-17A (cIL-17A) as an antigen for preparing an IL-17A-specific monoclonal antibody (mAb). RESULTS The coding sequence (CDS) region of cIL-17A was cloned from the peripheral blood mononuclear cells (PBMCs) of dairy goats and then inserted into the expression vector PET 32a and transformed into competent TransB (DE3) cells. Recombinant fusion protein obtained under optimized conditions was used to immunize BALB/c mice for preparing monoclonal antibodies. Finally, the supernatants of two hybridoma cell lines showing positive reaction with the recombinant fusion protein and negative reaction with fusion tags of PET 32a were collected for western blot, immunofluorescence (IF) and immunohistochemistry (IHC) analysis. Our results showed that the maximum amount of soluble protein could be obtained directly in the supernatant when the recombinant expression cells were induced by isopropyl-β-d-thiogalactoside (IPTG) at a concentration of 0.3 mmol/L at 16 °C for 42 h. Western blot analysis showed that the mAb H8 could recognize the eukaryotically expressed cIL-17A in the supernatant of transfected HEK293T cells. Immunofluorescence and immunohistochemistry assays showed that mAb H8 could strongly recognize both the eukaryotically expressed and natural cIL-17A. CONCLUSIONS The monoclonal antibody mAb H8 prepared in this study may be a potential tool for the detection of cIL-17A and beneficial for investigating the pathogenesis of various IL-17-associated diseases.

中文翻译：

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新型抗鼠白介素-17A单克隆抗体的制备及其在免疫荧光和免疫组织化学分析中的应用。

背景技术白细胞介素17（IL-17）是T辅助细胞17淋巴细胞（Th17细胞）分泌的特征性细胞因子，在宿主防御和许多炎性或自身免疫性疾病中起关键作用。本研究的目的是获得纯化的蛋白山羊IL-17A（cIL-17A）作为制备IL-17A特异性单克隆抗体（mAb）的抗原。结果从乳山羊的外周血单个核细胞（PBMC）中克隆了cIL-17A的编码序列（CDS）区域，然后将其插入表达载体PET 32a中，并转化为感受态TransB（DE3）细胞。在优化条件下获得的重组融合蛋白用于免疫BALB / c小鼠以制备单克隆抗体。最后，收集两个杂交瘤细胞系的上清液，分别与重组融合蛋白呈阳性反应，与PET 32a融合标记呈阴性反应，进行蛋白印迹，免疫荧光（IF）和免疫组化（IHC）分析。我们的结果表明，当异丙基-β-d-硫代半乳糖苷（IPTG）以0.3 mmol / L的浓度在16°C诱导42 h时，重组表达细胞可以直接在上清液中获得最大量的可溶性蛋白。 。蛋白质印迹分析表明，mAb H8可以识别转染的HEK293T细胞上清中的真核表达cIL-17A。免疫荧光和免疫组织化学分析表明，mAb H8可以强烈识别真核表达的cIL-17A和天然的cIL-17A。