畜牧·兽医

LTBP1蛋白单克隆抗体的制备及鉴定

  • 滕蔓 ,
  • 迟佳琦 ,
  • 柴书军 ,
  • 罗俊
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  •  (1.河南省农业科学院 农业部动物免疫学重点实验室/河南省动物免疫学重点实验室,河南郑州 450002;2.河南省农业科学院 中英禽病国际研究中心,河南 郑州 450002; 3.北京中医药大学 东方医院,北京 100078;4.河南科技大学 动物科技学院/动物疫病与公共卫生重点实验室,河南 洛阳 471003)
滕蔓(1977-),女,河南周口人,副研究员,博士,主要从事家禽免疫抑制病与肿瘤病研究。E-mail:tm135@aliyun.com

收稿日期: 2020-06-03

  网络出版日期: 2020-12-15

基金资助

国家自然科学基金项目(U1604232);中原英才计划-中原基础研究领军人才项目(2019—2020);河南省重点研发与推广专项(科技攻关)(192102110072);河南省农业科学院杰出青年科技基金项目(2019JQ01)

Development of Monoclonal Antibodies against Chicken LTBP1 Protein

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  • (1.Key Laboratory of Animal Immunology,Ministry of Agriculture & Henan Provincial Key Laboratory of Animal Immunology,
    Henan Academy of Agricultural Sciences,Zhengzhou 450002,China; 2.UK-China Centre of Excellence for Research on Avian
    Diseases,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China; 3.Dongfang Hospital,Beijing University of
    Chinese Medicine,Beijing 100078,China; 4.Key Laboratory of Animal Disease and Public Safety,College of Animal Science
    and Technology,Henan University of Science and Technology,Luoyang 471003,China)

Received date: 2020-06-03

  Online published: 2020-12-15

摘要

为了研究鸡潜在转化生长因子结合蛋白(LTBP1)功能,构建原核表达重组质粒pET-30a-LTBP1,转化E.coli BL21感受态细胞并经IPTG诱导进行表达。对以包涵体形式表达的LTBP1蛋白进行变复性和亲和层析纯化,然后免疫Balb/C小鼠,利用细胞融合技术建立单克隆抗体杂交瘤细胞株,制备抗鸡LTBP1的单克隆抗体。通过间接酶联免疫吸附试验(iELISA) 筛选,获得了2 株杂交瘤细胞株3C11-A9和2B1-G7。以效价较高的3C11-A9制备单克隆抗体腹水,间接免疫荧光试验(IFA)和蛋白免疫印迹(Western blot)试验结果均表明,单克隆抗体3C11-A9可特异性识别鸡源细胞CEF和DF-1表达的 LTBP1蛋白,同时IFA结果还显示,3C11-A9单克隆抗体腹水可与包括人、猴、猪和鼠在内的8种常见哺乳动物细胞发生特异性染色。综上,成功制备了抗鸡 LTBP1单克隆抗体,且具有多物种广谱识别特性。

本文引用格式

滕蔓 , 迟佳琦 , 柴书军 , 罗俊 . 鸡LTBP1蛋白单克隆抗体的制备及鉴定[J]. 河南农业科学, 2020 , 49(12) : 144 -150 . DOI: 10.15933/j.cnki.1004-3268.2020.12.021

Abstract

In order to study the function of latent transforming growth factor binding protein 1(LTBP1),the recombinant eukaryotic expression plasmid,pET-30a-LTBP1,was constructed and transformed into E.coli BL21 competent cells for the IPTG-induced protein expression. The recombinant LTBP1 protein was purified by denaturation and affinity chromatography,and used to immunize Balb/C mice.Cell fusion and hybridoma technology were performed to produce hybridomas and develop the monoclonal antibodies(mAbs) against chicken LTBP1 protein. The supernatant of specific antibodies were screened by indirect enzyme-linked immunosorbent assay(iELISA),and two hybridoma cell lines named as 3C11-A9 and 2B1-G7 steadily secreting antibodies against LTBP1 protein of chicken were obtained.The cell line 3C11-A9 with higher titers was chose for the preparation and purification of mAb in mouse ascites. Both of the indirect immunofluorescence assay(IFA) and Western blot analysis indicated that the 3C11-A9 mAb specifically reacted to chicken LTBP1 protein,and the IFA also demonstrated that 3C11-A9 mAb specifically stained the LTBP1 proteins expressed in eight cell lines derived from mammalians,including human being,monkey,pig and mouse.These data indicate that the chicken LTBP1-specific mAbs have been successfully developed,with a wide usage for the LTBP1.

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