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目的构建和原核表达抗人肝癌单链抗体HAb25scFv,为其进一步的临床应用研究奠定基础。方法采用(Gly4Ser)3为连接肽,在pUC19质粒上构建HAb25scFv单链抗体基因;序列分析证实后将HAb25scFv基因亚克隆入pGFX4T1GST融合表达载体上,SDSPAGE电泳鉴定表达产物并建立目的蛋白的纯化方法;免疫荧光竞争实验鉴定HAb25scFv单链抗体的活性。结果序列分析证实在pUC19质粒上以(Gly4Ser)3连接肽成功构建了HAb25scFv单链抗体基因;HAb25scFv单链抗体基因在pGEX4T1GST融合表达载体中获得高效表达,其表达量占菌体总蛋白的60.8%;经GST亲和层析柱,可获得纯度达95.2%的GSTHAb25scFv融合蛋白;免疫荧光竞争实验证实HAb25scFv单链抗体具有与其亲本抗体相同的抗原结合特性,并至少保留了亲本抗体39.12%的亲合力。结论获得了HAb25scFv单链抗体基因,并在原核GST融合表达系统中实现了HAb25scFv的高效表达,免疫荧光竞争实验证实HAb25scFv较好地保留了其亲本抗体的特异性和亲和力。
Objective To construct and express prokaryotic single chain antibody HAb25scFv against human hepatocellular carcinoma (HCC) and lay a foundation for its further clinical application. Methods HAb25 scFv single chain antibody gene was constructed by using (Gly4Ser) 3 as linker. HAb25scFv gene was subcloned into pGFX4T1GST fusion expression vector after sequencing analysis, and the expressed product was identified by SDSPAGE electrophoresis and the purification method of the target protein was established. Immunofluorescence competition experiments identified the activity of the HAb25 scFv single chain antibody. Results The sequence analysis confirmed that the HAb25 scFv single chain antibody gene was successfully constructed on the pUC19 plasmid with the (Gly4Ser) 3 linker. The HAb25 scFv single chain antibody gene was highly expressed in the pGEX4T1GST fusion expression vector, which accounted for 60.8% of the total bacterial protein. GSTHAb25scFv fusion protein with 95.2% purity was obtained by GST affinity chromatography. The immunofluorescent competition assay confirmed that the HAb25scFv single-chain antibody had the same antigen-binding characteristics as its parent antibody and retained at least 39.12% affinity of the parent antibody . Conclusion HAb25scFv single chain antibody gene was obtained and HAb25scFv was highly expressed in prokaryotic GST fusion expression system. The results of immunofluorescence showed that HAb25scFv retained the specificity and affinity of its parental antibody.