| 摘要: |
| 目的:构建人源噬菌体单链抗体ScFv基因文库,并从中筛选出抗肺癌抗体。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“吸附-洗脱-扩增”筛选富集,共进行4轮筛选,鉴定抗体库性能。结果:成功构建噬菌体单链抗体库。在亲和筛选过程中,肺癌单链抗体得到富集,收获率逐轮提高,第4轮为第1轮的115倍。随机选取10个克隆,通过ELISA法检测到其中7个与肺癌细胞呈阳性反应,阳性率为70%。结论:通过噬菌体展示技术得到肺癌相关人源单链抗体,筛选后的单链抗体能与肺腺癌细胞A549特异性结合。 |
| 关键词: 噬菌体展示 抗体库 单链抗体 肺腺癌 |
| DOI：10.3724/SP.J.1008.2009.00087 |
| 投稿时间：2008-06-05修订日期：2008-11-03 |
| 基金项目:国家自然科学基金(30370422). |
|
| Construction and screening of human phage display antibody library against lung adenocarcinoma |
| LUO Yi1,PANG Hua2,LI Shao-lin1*,CAO Hui1,PENG Zhi-ping1 |
| (1.Department of Radiation Medicine,Chongqing Medical University,Chongqing 400016,China*2.Department of Nuclear Medicine,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016) |
| Abstract: |
| Objective:To construct human single-chain variable fragment (ScFv) antibody library,and screen out antibodies against lung adenocarcinoma from the library.Methods: The total RNA was isolated from tumor adjacent lymph nodes of the lung adenocarcinoma patients and was used to amplify VH and VL genes by RT-PCR.VH and VL genes was joined with a DNA linker by SOE-PCR to form the ScFv.The gel purified ScFv gene repertoires were cloned into the phage vector pCANTAB5E to construct the primary phage library.Panning against lung adenocarcinoma cell line A549 was performed for four rounds and the phage library was identified.Results: A recombinant phage antibody library was successfully constructed.The fourth phage harvest yielded 115 times as much as that of the first one.During affinity screening, the antibody was enriched with the increase of panning rounds.Positive reactions to A549 were detected in 7 of 10 randomly selected clones,with a positive rate of 70%.Conclusion: A human phage-display antibody library has been successfully constructed.The selected ScFv fragment can specifically bind to human lung adenocarcinoma cell line A549. |
| Key words: phage display antibody library single chain variable fragment lung adenocarcinoma |
