猪调节性T细胞分子标记物FoxP3单克隆抗体的制备与鉴定  

Preparation and identification of monoclonal antibodies of porcine regulatory T cells molecular markers FoxP3

作　　者：蒋丹 林佳琦 胡鹏程 郭博伦 林新 马燕梅[1] JIANG Dan;LIN Jiaqi;HU Pengcheng;GUO Bolun;LIN Xin;MA Yanmei(College of Animal Sciences,Fujian Agriculture and Forestry University/Key Laboratory of Animal Pathogen Infection and Immunology of Fujian Province,Fuzhou 350002,China)

机构地区：[1]福建农林大学动物科学学院/福建省畜禽病原感染与免疫学重点实验室,福建福州350002

出　　处：《畜牧与兽医》2025年第1期43-49,共7页Animal Husbandry & Veterinary Medicine

基　　金：福建省自然科学基金项目(2021J01082);福建农林大学科技创新专项基金项目(KFb22066XA);福建农林大学2022年校级大学生创新创业训练计划项目(X202210389094)。

摘　　要：调节性T细胞(Treg)是具有免疫调节活性的淋巴细胞,通过抑制各种效应性淋巴细胞来诱导自身免疫耐受。FoxP3是CD4^(+)CD25^(+)Treg细胞重要的转录因子,对其分化发育和功能维持有着重要的调控作用,因此联合FoxP3抗原可以更好地识别CD4^(+)CD25^(+)Treg细胞。为了制备出特异性的猪Treg细胞表面标记物FoxP3的单克隆抗体,本研究选择抗原性较强的基因编码片段,构建了FoxP3抗原表位区段(1~840 bp)的原核表达质粒,诱导表达并纯化后,用目的蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞与骨髓瘤细胞进行融合,通过间接ELISA、Western blot和间接免疫荧光的方法筛选能稳定分泌抗体的阳性杂交瘤细胞。结果:筛选到了5株效价较高的FoxP3单克隆抗体产生细胞(F4B9、F4A9、F1E12、F1A11和F4A10),均具有良好的特异性,能识别内源性和外源性FoxP3蛋白,且能稳定地分泌抗体,为定性或定量检测猪Treg细胞以及验证Treg细胞的功能奠定了基础。Regulatory T cells(Tregs)are a type of lymphocyte that has immunomodulatory activity,and they induce autoimmune tolerance by inhibiting various effector lymphocytes.FoxP3 gene is an important regulatory gene in the differentiation,development and function main-tenance of CD4^(+)CD25^(+)Treg cells,and it is an important transcription factor.The combination of FoxP3 antigen on the basis of CD4^(+)CD25^(+)can better recognize CD4^(+)CD25^(+)Treg cells.In order to produce specific monoclonal antibody against porcine regulatory T cell surface marker FoxP3,a prokaryotic expression plasmid of FoxP3 epitope(1-840 bp)was constructed here by selecting a gene coding fragment with strong antigenicity.After induction and purification,BALB/c mice were immunized with the target protein,and spleen cells of the immunized mice were fused with myeloma cells,and positive hybrioma cells that could secrete antibodies stably were screened using the indirect ELISA,Western blot and indirect immunofluorescence methods.The results showed that five FoxP3 monoclonal cells with high titer(F4B9,F4A9,F1E12,F1A11 and F4A10)were finally screened and they had good specificity;all of the cells could recognize endogenous and exogenous proteins and secrete antibodies stably,which laid a foundation for qualitative or quantitative detection and verification of the function of Tregs in pigs.

关 键 词：猪 TREG细胞 FOXP3 抗体制备 单克隆抗 

分 类 号：S852[农业科学—基础兽医学]