摘要
目的:提高人源化抗肝癌单链抗体hscFv25的亲和力. 方法:设计并合成人源化抗肝癌单链抗体hscFv25重链及轻链CDR3的半随机突变引物,构建突变体抗体库,竞争筛选亲和力更高的突变体抗体,对所得到的高亲和力的候选抗体,在大肠杆菌中进行可溶性表达,并采用细胞ELISA、细胞涂片免疫组织化学染色的方法,对该抗体进行初步的活性鉴定. 结果:得到了3株候选高亲和力突变体抗体,其中的一株在大肠杆菌中获得了可溶性表达后,进一步的活性检测结果表明,该抗体的相对亲和力比亲本单链抗体提高了60倍左右,同时该抗体对肝癌细胞(SMMC-7721)的免疫组织化学染色呈强阳性,着色情况与亲本抗体相一致,而对正常肝细胞(HL-02)染色呈阴性. 结论:成功地构建了人源化抗肝癌单链抗体hscFv25的突变体抗体库,并筛选到了一株亲和力更高的突变体抗体.
AIM: To improve the affinity of humanized single-chain Fv (hscFv25) against hepatocellular carcinoma (HCC). METHODS: HscFv25 mutant antibody library was constructed, from which mutant antibodies with higher affinity were competitively selected. Then the selected antibodies were expressed in Ecoli under the induction of isopropylthio-β-D-galactoside (IPTG), and Cell ELISA and immunohis tochemical staining methods were used to detect the activities of the mutant antibodies. RESULTS: Three strains of mutant antibodies were obtained, and all of them could be solubly and effectively expressed in E coli. One strain of the three mutant antibodies possessed the activity of its parental antibody and the affinity was about 60 times higher than its parental antibody. CONCLUSION: The affinity of HscFv25 mutant antibody against HCC can be successfully improved after screening.
出处
《世界华人消化杂志》
CAS
2004年第11期2568-2571,共4页
World Chinese Journal of Digestology
基金
国家自然科学基金资助
No.30171065~~
