天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (10): 1195-1198.doi: 10.11958/20160317

• 细胞与分子生物学 • 上一篇    下一篇

针对人 DNAH2 蛋白的鼠源单克隆抗体的制备

常丽贤,章婧嫽,任媛媛,孙聪聪,万扬,安文彬,张英驰,袁卫平,竺晓凡   

  1. 天津， 中国医学科学院血液病医院血液学研究所（邮编 300020）
  • 收稿日期:2016-04-19 修回日期:2016-07-08 出版日期:2016-10-15 发布日期:2016-10-21
  • 通讯作者: 竺晓凡 E-mail:clx.886@163.com
  • 作者简介: 常丽贤（1984）， 女， 医师， 博士， 主要从事范可尼贫血的相关机制研究
  • 基金资助:
    国家自然科学基金资助项目（81500156， 81170470）

The preparation of the mouse monoclonal antibodies specific for the DNAH2 protein

CHANG Lixian, ZHANG Jingliao, REN Yuanyuan, SUN Congcong, WAN Yang, AN Wenbin, ZHANG Yingchi, YUAN Weiping, ZHU Xiaofan   

  1. State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China
  • Received:2016-04-19 Revised:2016-07-08 Published:2016-10-15 Online:2016-10-21
  • Contact: ZHU Xiaofan E-mail:clx.886@163.com

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摘要： 目的 制备特异性的鼠源单克隆抗 DNAH2（Homo sapiens dynein， axonemal， heavy chain 2） 抗体。 方法首先针对人 DNAH2 蛋白 N 端 1~300 aa 序列， 构建可表达 His 标签的免疫原融合表达质粒， 在大肠杆菌内以 IPTG 诱导 His-免疫原的表达并纯化；然后免疫 BALB/c 小鼠， 分离出脾淋巴细胞， 并与骨髓瘤细胞融合， 形成杂交瘤细胞；最后以酶联免疫吸附测定（ELISA）、蛋白免疫印迹法（Western blot）筛选阳性杂交瘤细胞。 结果 利用 IPTG 有效诱导了大肠杆菌内 DNAH2 免疫原的表达； 筛选的阳性杂交瘤细胞检测出 DNAH2 蛋白条带。 结论 成功制备针对人 DNAH2 蛋白的鼠源单克隆抗体。

关键词: 抗体, 单克隆, 杂交瘤, 质粒, 重组, 遗传, DNAH2

Abstract: Objective To prepare specific mouse monoclonal antibodies against Homo sapiens dynein， axonemal， heavy chain 2 (DNAH2). Methods Firstly, recombinant plasmid encoding His tagged immunogen, targeting N-terminal sequence of DNAH2 protein (1- 300 aa), in E. coli was constructed. IPTG was used to induce the expression of Hisimmunogen, which was then purified and immunized in BALB/c mice. Hybridoma cells were obtained through the fusion between myeloma cells and splenocytes isolated from BALB/c mice. Finally, ELISA and Western blot assays were performed to screen the positive hybridoma. Results IPTG was used efficiently to induce the expression of DNAH2 immunogen in E. coli. DNAH2 protein bands were detected in screened positive hybridoma. Conclusion Mouse monoclonal antiDNAH2 antibody is prepared successfully.

Key words: antibodies, monoclonal, hybridomas, plasmids, recombination, genetic, DNAH2