基础医学与临床 ›› 2009, Vol. 29 ›› Issue (11): 1155-1160.

• 研究论文 • 上一篇    下一篇

抗人肺癌单链抗体噬菌体库的构建及特异性单链抗体的鉴定

罗弋 庞华 李少林 曹辉 李淑杰 王树斌 樊春波   

  1. 重庆医科大学放射医学教研室 重庆医科大学 附属第一医院 核医学科 重庆医科大学 放射医学教研室 重庆医科大学 放射医学教研室 重庆医科大学 放射医学教研室 重庆医科大学 放射医学教研室
  • 收稿日期:2008-10-20 修回日期:2009-03-03 出版日期:2009-11-20 发布日期:2009-11-20
  • 通讯作者: 李少林

Construction of human phage display antibody ScFv library and identification of antibody ScFv against lung adenocarcinoma

Yi LUO, Hua PANG, Shao-lin LI, Hui CAO, Shu-jie LI, Shu-bin WANG, Chun-bo FAN   

  1. Dept. of Radiation Medicine,Chongqing Medical University Dept. of Nuclear Medicine, the First Affiliated Hospital, Chongqing Medical University Dept. of Radiation Medicine,Chongqing Medical University
  • Received:2008-10-20 Revised:2009-03-03 Online:2009-11-20 Published:2009-11-20
  • Contact: Shao-lin LI,

摘要: 目的 构建人源噬菌体抗体库,并从中筛选出抗肺癌人源单链抗体。方法 提取肺癌患者癌旁淋巴结组织,通过RT-PCR扩增出重链可变区基因(VH)和轻链可变区基因(VL),再经剪切-重叠-延伸PCR(SOE-PCR)将VH 和VL连接得到单链抗体(ScFv)。将双酶切后的ScFv基因片段克隆入噬菌体表达载体pCANTAB5E,得到初级噬菌体抗体库。以肺腺癌细胞株A549为抗原对抗体库进行4轮筛选富集,鉴定抗体库性能。将得到的阳性克隆用IPTG诱导表达并进行检测。结果 成功构建噬菌体单链抗体库。经筛选富集,噬菌体收获率得到增加,第4轮是第1轮的115倍。随机选取10个克隆,通过ELISA法检测到其中7个与A549细胞呈阳性反应,阳性率为70%。SDS-PAGE及ELISA检测证实得到人源抗肺癌单链抗体。结论 成功构建人源单链抗体噬菌体库,从中获得具有较高特异性的抗人肺癌单链抗体。

关键词: 噬菌体抗体库, 单链抗体, 肺腺癌

Abstract: Objective To construct a human phage single chain-antibody library, and sieve out the antibody ScFv against lung cancer from the library. Methods The total RNA was isolated from lymph node tissue of the lung cancer patients, and was used to amplify VH and VL gene by RT-PCR. VH and VL were joined with a DNA linker by SOE-PCR to form the single chain variable fragment (ScFv) gene. ScFv gene was coloned into the phage vector pCANTAB5E. Panning against lung cancer cell line A549 were performed for four rounds. The positive clones were chosen for soluble expression. Results A recombination phage single chain-antibody library was constructed. After 4 rounds panning, the number of eluted phages increased 115 times. Positive reactions to A549 were detected in 7 of 10 random clones, The positive rate was 70%. The human ScFvs against lung cancer were produced and confirmed by SDS-PAGE and ELISA analysis. Conclusions ScFvs against lung cancer were acquired by the construction of phage single chain-antibody library. The soluble ScFvs has avidity specifically to human lung cancer cells.

Key words: phage antibody library, single chain variable fragment, lung adenocarcinoma