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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。
Objective: To construct human ScFv gene library of single-chain phage antibody and select anti-lung cancer antibody from it. Methods: The adjacent non-cancerous tissues of lung cancer patients were extracted. The heavy chain variable region (VH) and light chain variable region (VL) genes were amplified by RT-PCR, and then amplified by the enzyme-linked immunosorbent assay The VH and VL linkages give the single chain antibody ScFv. The double digested ScFv gene fragment was cloned into the phage expression vector pCANTAB5E to obtain a primary phage antibody library. Antibody library of lung adenocarcinoma cell line A549 was used for “adsorption-elution-amplification” screening and enrichment. A total of 4 rounds of screening were performed to identify the performance of the antibody library. Results: The phage scFv library was successfully constructed. In the process of affinity screening, the single-chain antibody of lung cancer was enriched, the harvest rate increased by wheel, the first four rounds of 115 times. Ten clones were randomly selected and 7 of them were positive for lung cancer cells by ELISA. The positive rate was 70%. CONCLUSION: The lung-related human single-chain antibody is obtained by phage display technique. The selected single-chain antibody can specifically bind to lung adenocarcinoma A549 cells.