摘要
目的:构建人源性肺癌噬菌体单链抗体库,为筛选肺癌相关抗原的抗体奠定基础。方法:提取肺癌转移淋巴结总RNA,用RT-PCR技术扩增人抗体重链可变区(VH)和轻链可变区(VL)基因,在体外将VH和VL连接成单链抗体(ScFv)基因,并克隆到噬菌粒载体pCANTAB 5E中,电转化至感受态的大肠杆菌TG1,经辅助噬菌体超感染,形成噬菌体单链抗体库,采用限制性内切酶鉴定其多样性。结果:从肺癌转移淋巴结中成功提取RNA,逆转录PCR扩增出人可变区基因,连接形成单链抗体,最终构建了库容为1.2×108的抗人肺癌单链抗体库。BstNⅠ酶切法证明构建的抗体库具有良好的多样性。结论:成功地构建了噬菌体展示的抗人肺癌单链抗体库,为进一步筛选肺癌相关蛋白的可溶性抗体奠定了基础。
Objective：To construct a library of human lung cancer specific single chain Fv antibodies via phage display and establish the foundation for screening antibodies of lung cancer-associated antigen. Methods：Total RNA was extracted from meta static lymph node of lung cancer patients. The variable regions of heavy chain （VII） and light chain （VL） of human antibodies were amplified by RT-PCR. VH and VL were linked with each other to generate scFv gene in vitro. The scFv fragments were cloned into the phagemid （pCANTAB 5E） and the recombinant phagemid was transformed to susceptible E. coli TG1 by electroporation. They were superinfected by helper phages to form phage display library of single chain Fv antibodies. Finally, the diversity of the library was identified by restriction endonuclease digestion. Results.. Total RNA was extracted successfully from lymph nodes of metastatic lung cancer patients. The variable regions of human antibodies was amplified by RT-PCR and connected with each other to form ScFv. The constructed phage display antibody library contained 1.2 × 10^8 independent clones. Result of BstN I digestion showed that it had good diversity. Conclusion：The phage display library of human lung cancer specific single chain Fv antibodies is constructed successfully. It establishes the foundation for screening the soluble antibodies of lung cancer associated protein.
出处
《肿瘤》
CAS
CSCD
北大核心
2006年第9期823-826,共4页
Tumor
基金
国家自然科学基金资助项目(编号:30571552)
郑州市科技三项费用支持项目(编号:052SGYS33210)
关键词
肺肿瘤
抗体
肿瘤
人体
单链抗体(scFv)
噬菌体展示
Lung neoplasms
Antibodies,neoplasm
Human body
Single-chain antibody library
Phage display
