摘要
目的采用哺乳动物细胞表面展示技术构建间质上皮转化因子(c-Met)嵌合抗体3E1D7的重链补体决定区3(CDR3)随机突变抗体库。方法用定向进化的方法在嵌合抗体3E1D7重链CDR3内部造成基因随机突变,将其通过双酶切克隆到哺乳动物细胞展示载体pSZI-CD中构建随机突变抗体库。抗体库基因转染CHO细胞,流式细胞术检测抗体基因是否在细胞表面表达。结果3E1D7重链CDR3随机突变抗体库构建成功,库容量达到5.52×10^6。随机挑选20个阳性克隆进行测序鉴定,20个克隆均在不同位置发生碱基突变且没有重复突变,编码20种不同的氨基酸序列,抗体库具有较好的多样性和随机性。流式细胞术分析抗体基因库上膜情况,均检测到全长抗体在CHO细胞表面的表达。结论通过哺乳动物细胞表面展示技术,成功构建了库容量达到5.52×10^6的c-Met嵌合抗体重链CDR3随机突变抗体库。
Objective To construct a random mutagenesis library of 3E1D7,a chimerical antibody against c-mesenchymal epithelial transition factor(c-Met),using mammalian cell surface display.Methods Antibody genes with randomly mutated complementarity-determining region 3(CDR3)part were inserted into the mammalian expression plasmid pSZI-CD to construct the random mutagenesis library using double enzyme digestion.Reconstructed plasmids were then cloned into CHO cells by transfection.The expression level of antibodies on the surface of CHO cells was checked by C6 PLUS flow cytometry.Results 3E1D7 random mutagenesis library was successfully constructed with a volume of 5.52×10^6 in diversity on gene level.Sequence analysis showed that all 20 clones randomly picked from the library coded for 20 different mutated amino acid sequences in open reading frames.After transfection,the expression of full-length antibodies on CHO cell surfaces could be detected by flow cytometry.Conclusion A random mutagenesis library of a certain anti-c-Met antibody has been successfully constructed with an exhibitable diversity of 5.52×10^6,which would be a useful platform for further screening of therapeutic antibodies.
作者
郭佳
蒋韵
滕飞
尹衍新
于丽华
付敏
蒋明
房健民
GUO Jia;JIANG Yun;TENG Fei;YIN Yanxin;YU Lihua;FU Min;JIANG Ming;FANG Jianmin(Biomedical Center,Suzhou Institute,Tongji University,Suzhou 215000;Shanghai Tongji Hospital,Shanghai 200065;School of Life Sciences and Tchnology,Tongji University,Shanghai 200092,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2019年第6期557-562,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
江苏省自然科学基金青年基金(BK20150278)
上海市科学技术委员会科研计划(19431903200)