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目的:构建抗肺腺癌的单链抗体(scFv),研究其表达条件,为将这一小分子抗体应用于临床奠定基础.方法:将抗人肺腺癌单克隆抗体的重链及轻链可变区基因插入表达载体pFUW80、pTH、pTF,分别在大肠杆菌XL1-Blue、Top10、GI698/GI724中诱导表达,得到噬菌体抗体或包涵体.用SDS-PAGE和ELISA鉴定检测活性.结果:SDS-PAGE的结果表明,在pTH和pTF的表达产物中,有一条43kD的特异条带,其分子量与预期相符,单链抗体以包涵体形式出现,其表达量达细菌总蛋白的18.6%.ELISA的结果表明,噬菌体抗体和经复性处理的包涵体具有与亲本单抗相同的特异性.结论:成功地构建和在大肠杆菌中表达了抗肺腺癌单链抗体.
OBJECTIVE: To construct single-chain antibody (scFv) against lung adenocarcinoma and study its expression conditions. This will lay a foundation for the application of this small-molecule antibody in clinical practice. Methods: The anti-human lung adenocarcinoma monoclonal antibody heavy chain and light chain The variable region gene was inserted into the expression vectors pFUW80, pTH, and pTF and induced in Escherichia coli XL1-Blue, Top10, and GI698/GI724, respectively, to obtain phage antibodies or inclusion bodies. The activity was detected by SDS-PAGE and ELISA. Results: SDS The results of -PAGE showed that there was a 43kD specific band in the expression product of pTH and pTF, and its molecular weight was in agreement with the expectation. Single-chain antibody appeared in the form of inclusion body, and its expression amount was 18.6% of total bacterial protein. The results showed that the phage antibody and the renatured inclusion bodies had the same specificity as the parent monoclonal antibody. Conclusion: The anti-lung adenocarcinoma scFv was successfully constructed and expressed in E. coli.