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目的:构建与肝癌细胞特异性结合的单链抗体-AP融合蛋白,并诱导表达。方法:提取从大型人源噬菌体抗体库中筛选的含有与HCC特异性结合的scFv-10基因的质粒,用SfiI、NotI分别酶切,连接至经同样双酶切后的原核分泌型表达载体pDAP2上,转化和筛选后,阳性细菌经IPTG诱导表达,细胞ELISA鉴定融合蛋白活性。结果:重组质粒经PCR扩增,Eco91Ⅰ单酶切及Eco91Ⅰ/NotI双酶切鉴定,证明scFv-10基因已插入到原核分泌型表达载体pDAP2上。表达产物的定位分析表明融合蛋白主要分泌性表达到周质腔,细胞ELISA证明表达产物有与HCC结合的活性。结论:成功地构建并表达了与HCC特异性结合的单链抗体-AP融合蛋白,为其应用奠定了基础。
Objective: To construct a single-chain antibody-AP fusion protein that specifically binds to hepatocellular carcinoma cells and induce its expression. Methods: Plasmids containing scFv-10 gene specific binding to HCC, which were screened from large human phage antibody library, were extracted and digested with SfiI and NotI and ligated into prokaryotic expression vector pDAP2 After transformation, screening and screening, the positive bacteria were induced by IPTG and the activity of fusion protein was identified by ELISA. Results: The recombinant plasmids were identified by EcoRⅠⅠ digestion and Eco91Ⅰ / NotI digestion. The results showed that the scFv-10 gene was inserted into prokaryotic expression vector pDAP2. Positioning analysis of the expressed product showed that the fusion protein mainly secreted into the periplasm, and the cell ELISA proved that the expressed product had HCC-binding activity. Conclusion: The single-chain antibody-AP fusion protein that specifically binds to HCC was successfully constructed and expressed, which laid the foundation for its application.