摘要
目的构建表达抗 CEA小分子抗体 ,以利于放射免疫诊断及导向治疗。方法构建 CEA单链抗体基因 ,克隆入载体 p Kp L - 3a,在大肠杆菌中包涵体表达 ,EL ISA检测活性。将轻重链基因克隆入分泌型表达载体 p SCMH,在大肠杆菌中表达 ,EL ISA检测活性。将 CEA嵌合抗体的轻链和 Fd基因克隆入杆状病毒表达载体 p Ac U W5 1中 ,在 sf9细胞中分泌表达 ,EL ISA检测活性及测定产量 ,竞争抑制 EL ISA测定其识别的抗原表位。结果多种方法复性的包涵体及原核分泌表达的单链抗体未测到活性。昆虫细胞中表达的小分子抗体具抗体活性 ,产量为 1.7μg/m L,竞争抑制实验显示其与亲本鼠单抗 C5 0识别相同的表位。结论该 CEA抗体基因在大肠杆菌中不能表达出有功能的分子 ,而在昆虫细胞中表达了具活性抗体分子 ;某些抗体基因只有在真核细胞中才能表达出有功能的抗体分子。
ObjectiveA small molecular antibody against human carcinoembryonic antigen was constructed and expressed in order to be used for radioimmunoimage and target therapy. MethodsThe gene of single chain antibody against CEA was cloned into expression vector pKpL-3a and expressed in E.coli pop2136. The antigen binding activity was analysed by ELISA. The light and heavy chain variable region genes were cloned into secretory expression vector pSCMH whose geneⅢ had been excised, and expressed in E.coli XL1-Blue. The antibody activity was assayed by ELISA. The light chain and Fd gene of CEA chimeric antibody were cloned into baculovirus expression vector pAcUW51 and expressed in sf9 cell. The activity, yield and affinity were analysed by ELISA. ResultsAfter being renatured by various methods, the specific antigen binding activity of expression product in E.coli pop2136 was not detected, nor was the secretory expression product in E.coli XL1-Blue.The small molecular chimeric antibody expressed in sf9 cell can specifically bind human CEA antigen and had the yield of 1.7 μg/mL.Competition ELISA showed that antigen epitope recognized by the chimeric antibody was the same as the parent McAb C50 ConclusionA small molecular chimeric antibody against CEA was successfully expressed in sf9 cell. Only in eucaryotic cells can some antibody genes express functional molecules. [
出处
《免疫学杂志》
CAS
CSCD
北大核心
2001年第3期169-172,共4页
Immunological Journal
基金
国家九五攻关计划资助!项目 (96 - 90 6 - 0 1- 2 3)
