牛白细胞介素-8单克隆抗体制备及应用

doi:10.16431/j.cnki.1671-7236.2025.08.031

摘要/Abstract

摘要： 【目的】白细胞介素-8(IL-8)是重要的趋化因子，参与机体免疫反应，其水平升高与多种感染性疾病、自身免疫疾病等密切相关，也被鉴定为活动性肺结核病、结核性脑膜炎、牛结核病的检测靶标。本研究旨在制备针对牛IL-8的单克隆抗体，开发牛IL-8夹心ELISA检测试剂盒，用于牛血清或血浆中IL-8的定量检测，为牛结核(bovine tuberculosis,bTB)及其他炎症相关疾病的诊断及免疫学指标检测提供工具。【方法】将牛IL-8基因分别连接至pGEX-6P-1和pcDNA3.1载体，并利用大肠杆菌与293F细胞进行表达，以GST亲和柱和镍柱纯化重组蛋白；采用真核表达的重组蛋白免疫BALB/c小鼠，利用淋巴细胞杂交瘤技术制备并筛选针对牛IL-8的单克隆抗体；通过棋盘法筛选最佳抗体对并建立夹心ELISA检测方法；定量检测排菌期结核病牛、非排菌期结核病牛和健康牛血浆中IL-8水平，评价单克隆抗体对天然牛IL-8的识别能力及其对结核病牛的检测效果。【结果】 SDS-PAGE结果显示，原核和真核表达系统制备的IL-8均以可溶形式表达，且纯化的蛋白纯度高于90%；经3轮亚克隆筛选共获得2B8、2F5等5株稳定分泌牛IL-8抗体的单克隆细胞株，亚型鉴定均为IgG1型；棋盘法筛选结果显示,4G10和3D5-HRP为最佳配对抗体；以4G10和3D5-HRP建立的ELISA方法对不同感染状态的结核病牛血浆进行定量检测，结果显示，结核病牛血浆中IL-8含量极显著高于健康牛(P＜0.01)。【结论】成功制备抗牛IL-8的单克隆抗体并建立牛IL-8夹心 ELISA检测方法，可实现对天然牛IL-8的定量检测，有潜力用于结核病牛的筛查和牛免疫学指标检测。

关键词: 牛; 白细胞介素-8; 单克隆抗体; ELISA; 牛结核病

Abstract: 【Objective】 Interleukin-8 (IL-8) was a key chemokine that plays a role in the immune response,and the increase of its level was closely linked to multiple infectious diseases and autoimmune conditions.It had been recognized as a detection target for active pulmonary tuberculosis,tuberculous meningitis,and bovine tuberculosis.This study aimed to develop monoclonal antibodies against bovine IL-8 and create a sandwich ELISA kit for the quantitative detection of IL-8 in bovine serum or plasma,offering tools for diagnosing and researching bovine tuberculosis (bTB) and other inflammation-associated diseases. 【Method】 The bovine IL-8 gene was cloned into pGEX-6P-1 and pcDNA3.1 vectors and expressed in Escherichia coli and 293F cells.Recombinant proteins were purified using GST affinity and nickel columns.Eukaryotic recombinant protein was used to immunize BALB/c mice,and monoclonal antibodies against bovine IL-8 were generated and screened via lymphocyte hybridoma technology.The optimal antibody pairs were identified through checkerboard analysis,and a sandwich ELISA detection method was established.IL-8 level was quantitatively assessed in plasma samples from shedding and non-shedding tuberculosis-infected cattle as well as healthy cattle to evaluate the method’s effectiveness in detecting tuberculosis in cattle. 【Result】 SDS-PAGE analysis showed that IL-8 produced in both prokaryotic and eukaryotic expression systems was expressed in a soluble form,with purified proteins exhibiting a purity exceeding 90%.Five monoclonal cell lines stably secreting IL-8 antibodies including 2B8,2F5,and so on, were obtained after three rounds of subcloning,all confirmed as IgG1 subtype.Checkerboard analysis revealed that 4G10 and 3D5-HRP were the optimal antibody pairs.The ELISA method established with 4G10 and 3D5-HRP was applied for quantitative detection of IL-8 level in plasma from cattle with varying infection statuses,demonstrating that IL-8 level in plasma of tuberculosis-infected cattle was extremely significantly higher than that in healthy cattle (P＜0.01). 【Conclusion】 The monoclonal antibodies against bovine IL-8 were successfully prepared and a sandwich ELISA detection method was established，which could realize the quantitative measurement of bovine IL-8,with potential applications in screening for tuberculosis in cattle and analyzing immune and infection status.

Key words: cattle; interleukin-8; monoclonal antibodies; ELISA; bovine tuberculosis

中图分类号:

S852.4^＋3

杨蕊, 靳家鑫, 张泽, 时文健, 张广智, 丁家波, 鑫婷. 牛白细胞介素-8单克隆抗体制备及应用[J]. 中国畜牧兽医, 2025, 52(8): 3830-3837.

YANG Rui, JIN Jiaxin, ZHANG Ze, SHI Wenjian, ZHANG Guangzhi, DING Jiabo, XIN Ting. Development and Application of Monoclonal Antibodies Against Bovine Interleukin-8[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3830-3837.

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