抗VEGFR2/抗CD3双特异单链抗体的表达及初步活性检测  

Expression and immunological activity of an anti-CD3×VEGFR2 bispecific single-chain antibody

作　　者：戴丽娟[1] 鄢成伟[2] 李淑珍[2] 陈莉[2] 李新禹[2] 单瑞辉[2] 于学薇[2] 唐晓波[2] 

机构地区：[1]哈尔滨医科大学附属第二医院药学部 [2]哈尔滨医科大学药学院生物制药学教研室,黑龙江哈尔滨150081

出　　处：《细胞与分子免疫学杂志》2011年第8期883-886,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：哈尔滨市科技创新人才研究专项资金(2008RFXXS018)

摘　　要：目的:构建和表达抗血管内皮细胞生长因子受体2(VEGFR2)/抗CD3双特异单链抗体(bscVEGFR2×CD3),及亲和活性测定。方法:设计抗VEGFR2/抗CD3双特异单链抗体基因序列,由公司合成并亚克隆入真核表达载体pcDNA3.1(+)中,脂质体法转染中国仓鼠卵巢细胞(CHO),筛选高效分泌表达bscVEGFR2×CD3的克隆株。表达产物经Ni-NTA柱纯化,120 g/L SDS-PAGE电泳及Western blot鉴定。应用流式细胞术(FCM)检测生物学活性。结果:bscVEGFR2×CD3重组表达载体测序证实序列正确。bscVEGFR2×CD3能够在CHO细胞中进行分泌表达,筛选出6株高表达克隆株。120 g/L SDS-PAGE显示相对分子质量(Mr)约56 000有1条带,与预期相符,Western blot证明anti-His抗体能与这条蛋白条带发生特异性结合。FCM检测bscVEGFR2×CD3能与CD3+jurkat细胞和VEGFR2+A375细胞特异性结合。结论:成功构建和表达抗VEGFR2/抗CD3双特异单链抗体,该抗体具有与VEGFR2、CD3特异性结合的免疫学活性。AIM： To construct and express an anti-VEGFR2/anti-CD3 bispecific single-chain antibody（bscVEGFR2×CD3）and to identify its binding specificities to CD3 and VEGFR2.METHODS： The gene encoding anti-VEGFR2/anti-CD3 bispecific single-chain antibody was designed and synthesized.Bispecific single-chain antibody（bsc-Ab） DNA was subcloned into a eukaryotic expression vector pcDNA3.1（＋）,then transfected into Chinese hamster ovary（CHO） cells and stable expression cell lines were selected.Expressed Bsc-Ab was purified by His-tag affinity chromatography and confirmed by 120 g/L SDS-PAGE and Western blotting.Antigen binding activity of the bsc-Ab was analyzed by FACS.RESULTS： The plasmid DNA containing bispecific single-chain fragments were confirmed.BscVEGFR2×CD3 was secreted by CHO into the supernatant.Six stable expression cell lines were established.The molecular weight of bsc-Ab was correct indicated by SDS-PAGE and Western blotting.The bsc-Ab could specifically bind to CD3＋ jurkat cells and VEGFR2＋ A375 cells.CONCLUSION： An anti-VEGFR2/anti-CD3 bispecific single-chain antibody is successfully constructed and expressed,and the antibody has specific binding capacity to CD3 and VEGFR2.

关 键 词：双特异单链抗体 CD3 VEGFR2 

分 类 号：R392.11[医药卫生—免疫学]