| 猪IL-10的原核表达及其单克隆抗体的制备 |
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| 引用本文： | 刘婷婷,王兴龙,李玉峰,宋艳华,张国龙,姜平. 猪IL-10的原核表达及其单克隆抗体的制备[J]. 中国预防兽医学报, 2010, 32(4) |
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| 作者姓名： | 刘婷婷 王兴龙 李玉峰 宋艳华 张国龙 姜平 |
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| 作者单位： | 南京农业大学,农业部动物疫病诊断与免疫重点开放实验室,江苏,南京,210095 |
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| 基金项目： | 国家自然科学基金，国家科技支撑计划，江苏省科技攻关项目，教育部高等学校博士学科点专项科研基金 |
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| 摘 要： | 为了制备猪IL-10的单克隆抗体(MAb),本研究根据GenBank登录的猪白细胞介素10(PIL-10)基因序列设计两对特异性引物,利用套式PCR扩增PIL-10成熟蛋白编码基因(490bp),构建原核表达载体pET28a-PIL-10,并转化至大肠杆菌BL21,经IPTG诱导,SDS-PAGE和western blot分析鉴定,证明PIL-10重组蛋白获得表达。提取纯化该重组蛋白包涵体,免疫BALB/c小鼠,经过3次细胞融合,间接ELISA方法筛选,获得一株能稳定分泌抗猪IL-10MAb的杂交瘤细胞,将其命名为2F4。Western blot结果证明该MAb能够与重组蛋白发生特异性反应。间接ELISA测定细胞上清抗体效价为1∶1024,腹水效价为1∶128000,该MAb为IgG3亚型,轻链为λ型。杂交瘤细胞连续传代20代,分泌抗体的效价基本一致,从而为进一步建立猪IL-10的检测方法奠定了物质基础。
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| 关 键 词： | 猪IL-10 原核表达 单克隆抗体 |
Preparation of monoclonal antibody against porcine interleukin-10 protein expressed in E.Coli |
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| LIU Ting-ting,WANG Xing-long,LI Yu-feng,SONG Yan-hua,ZHANG Guo-long,JIANG Ping. Preparation of monoclonal antibody against porcine interleukin-10 protein expressed in E.Coli[J]. Chinese Journal of Preventive Veterinary Medicine, 2010, 32(4) |
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| Authors: | LIU Ting-ting WANG Xing-long LI Yu-feng SONG Yan-hua ZHANG Guo-long JIANG Ping |
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| Affiliation: | LIU Ting-ting,WANG Xing-long,LI Yu-feng,SONG Yan-hua,ZHANG Guo-long,JIANG Ping(Key Laboratory of Animal Diseases Diagnostic , Immunology,Ministry of Agriculture,Nanjing Agricultural University,Nanjing 210095,China) |
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| Abstract: | The cDNA of porcine interleukin-10(PIL-10) was amplified with nested PCR and cloned to construct recombinant plasmid pET-28a-PIL-10,then transformed into E.coli BL21. Recombinant bacteria was induced by IPTG and expression of PIL-10 was confirmed by SDS-PAGE and western blot. Monoclonal antibody(MAb) specific to PIL-10 protein was prepared by fusion spleen cells of BALB/c mice immunized with purified PIL-10 inclusion body. Positive hybridoma cell line was screened by indirect ELISA and confirmed by western ... |
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| Keywords: | porcine IL-10 prokaryotic expression MAb |
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