[1]刘晓丽,张岸梅,林盛,等.噬菌体展示EpCAM单链抗体及免疫原性初步研究[J].陆军军医大学学报(原第三军医大学学报),2011,33(10):977-979.
 Liu Xiaoli,Zhang Anmei,Lin Sheng,et al.Preliminary study on immunogenicity of phage-displayed mouse-anti-human EpCAM single-chain antibody[J].J Amry Med Univ (J Third Mil Med Univ),2011,33(10):977-979.
点击复制

噬菌体展示EpCAM单链抗体及免疫原性初步研究(/HTML )
分享到:

陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
33卷
期数:
2011年第10期
页码:
977-979
栏目:
论著
出版日期:
2011-05-30

文章信息/Info

Title:
Preliminary study on immunogenicity of phage-displayed mouse-anti-human EpCAM single-chain antibody
作者:
刘晓丽张岸梅林盛王欣欣廖荣霞孙建国陈正堂
第三军医大学新桥医院全军肿瘤研究所;第三军医大学基础医学部医学英语教研室
Author(s):
Liu XiaoliZhang Anmei Lin Sheng Wang XinxinLiao RongxiaSun Jianguo Chen Zhengtang
Cancer Research Center, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037; Department of Medical English, College of Basic Medical Sciences, Third Military Medical University, Chongqing, 400038, China
关键词:
单链抗体噬菌体展示EpCAM免疫肿瘤干细胞
Keywords:
single-chain antibodyphage displayEpCAMimmunitycancer stem cell
分类号:
R392.11;R394-33;R392.13
文献标志码:
A
摘要:
目的    利用噬菌体展示技术进行鼠抗人EpCAM单链抗体表达并鉴定其免疫活性,构建特异靶向上皮来源肿瘤细胞及癌干细胞的单链抗体。    方法    采用全基因合成的方法合成VL+Linker+VH的EpCAM的单链抗体(ScFv)基因,克隆到pMD19-T简易载体中,将ScFv基因酶切并纯化后克隆到噬菌体载体pCANTAB-5E,热休克法转化至感受态的大肠杆菌TG1,随机挑取5个克隆,经辅助噬菌体超感染,形成鼠抗人EpCAM噬菌体单链抗体,用ELISA(以P/N>2判定为阳性)和免疫细胞化学法测定其抗原结合活性。    结果    成功获得pCANTAB-5E/EpCAM ScFv重组克隆,经ELISA和免疫细胞化学测定,噬菌体展示阳性率为20%,展示的单链抗体能与EpCAM抗原和A549细胞特异性结合。    结论    噬菌体展示技术制备EpCAM单链抗体方法简便、省时、高效。
Abstract:
Objective    To express mouse-anti-human EpCAM single-chain antibody (ScFv) specifically targeting tumor cells or cancer stem cells of epithelial origin via phage display technology, and to identify its immunological activity.     Methods    Total gene synthesis method was used to synthesize mouse-anti-human EpCAM ScFv gene with a structure of VL+Linker+VH. The gene was cloned to pMD19-T Simple Vector and then was digested, after being purified, was cloned to phage vector pCANTAB 5E. Competent E. coli TG1 cells were transformed with pCANTAB 5E containing ScFv gene. Mouse-anti-human EpCAM ScFv was produced by helper phage M13K07 superinfection with 5 randomly selected clones, and the antigen binding activity was identified by ELISA and immunocytochemistry.     Results    Recombinant pCANTAB5E/EpCAM ScFv was constructed successfully, with a positive antigen-binding rate of 20%  proven by ELISA and immunocytochemistry. The selected ScFv could specifically bind to EpCAM antigen and human lung adenocarcinoma cell line A549.     Conclusion    Mouse-anti-human EpCAM ScFv produced via phage display technology has advantages such as simple, highly efficient, and time-saving ,which is expected to be helpful in diagnosing tumor cells and cancer stem cells of epithelial origin.

相似文献/References:

[1]庞华,罗弋,李少林.人源肺腺癌噬菌体抗体库的构建及初步筛选[J].陆军军医大学学报(原第三军医大学学报),2008,30(22):2082.
 PANG Hua,LUO Yi,LI Shao-lin.Construction of and screening for phage antibody library of human lung adenocarcinoma[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(10):2082.
[2]胡川闽,郭建秀,李鹏,等.抗肝癌单链免疫毒素HAb25(scFv)-PE40的构建及其导向作用的实验研究[J].陆军军医大学学报(原第三军医大学学报),2005,27(22):2209.
[3]陈庆峰,马中良,王笑峰,等.抗粘蛋白1单链抗体的构建及其功能初步分析[J].陆军军医大学学报(原第三军医大学学报),2010,32(16):1770.
 Chen Qingfeng,Ma Zhongliang,Wang Xiaofeng,et al.Construction and characterization of anti-mucin1 single chain Fv antibody fragment[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(10):1770.
[4]王洁,李建国,庞华,等.抗HIF-1α肺腺癌人源单链抗体的制备和放射免疫显像[J].陆军军医大学学报(原第三军医大学学报),2010,32(17):1851.
 Wang Jie,Li Jianguo,Pang Hua,et al.Preparation of human anti-HIF-1α scFv of lung adenocarcinoma and its radioimmunoimaging[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(10):1851.
[5]王洁,李建国,庞华,等.抗HIF-1α肺腺癌人源单链抗体的筛选和初步鉴定[J].陆军军医大学学报(原第三军医大学学报),2009,31(23):2319.
 WANG Jie,LI Jian-guo,PANG Hua,et al.Screening and identification of human anti-HIF-1α scFv of lung adenocarcinoma[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(10):2319.
[6]范校周,郭燕丽,涂追,等.前列腺特异性膜抗原胞外区的真核表达及其纳米抗体的淘选[J].陆军军医大学学报(原第三军医大学学报),2013,35(21):2320.
 Fan Xiaozhou,Guo Yanli,Tu Zhui,et al.Eukaryotic expression of extracellular domain of prostate-specific membrane antigen and biopanning of its nanobodies[J].J Amry Med Univ (J Third Mil Med Univ),2013,35(10):2320.
[7]于俊媛,周莹冰,张雯庆,等.金黄色葡萄球菌N315 T7噬菌体cDNA展示文库的构建[J].陆军军医大学学报(原第三军医大学学报),2014,36(13):1350.
 Yu Junyuan,Zhou Yingbing,Zhang Wenqing,et al.Construction of T7 phage display cDNA library from methicillin-resistant Staphylococcus aureus N315[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(10):1350.
[8]侍立峰,梁志清.利用噬菌体肽库技术筛选卵巢特异的VEGFR3亲和肽[J].陆军军医大学学报(原第三军医大学学报),2006,28(04):308.
[9]章容,王长松,刘友生,等.抗氨基端脂多糖结合蛋白单链抗体的生物学活性研究[J].陆军军医大学学报(原第三军医大学学报),2005,27(24):2406.
[10]孙远,杨连甲,高玉好,等.抗骨形成蛋白单链抗体的构建与表达[J].陆军军医大学学报(原第三军医大学学报),1999,21(04):0.[doi:10.16016/j.1000-5404.1999.04.020 ]
 Sun Yuan,Yang Lianjia,Gao Yuhao,et al.[J].J Amry Med Univ (J Third Mil Med Univ),1999,21(10):0.[doi:10.16016/j.1000-5404.1999.04.020 ]

更新日期/Last Update: 2011-05-12