免疫学杂志
免疫學雜誌
면역학잡지
IMMUNOLOGICAL JOURNAL
2001年
3期
169-172
,共4页
赵泽国%冉宇靓%孔健%孙立新%遇垅%刘军%杨治华
趙澤國%冉宇靚%孔健%孫立新%遇垅%劉軍%楊治華
조택국%염우정%공건%손립신%우롱%류군%양치화
癌胚抗原%小分子抗体%抗体工程
癌胚抗原%小分子抗體%抗體工程
암배항원%소분자항체%항체공정
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pKpL-3a,在大肠杆菌中包涵体表达,ELISA检测活性。将轻重链基因克隆入分泌型表达载体pSCMH,在大肠杆菌中表达,ELISA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pAcUW51中,在sf9细胞中分泌表达,ELISA检测活性及测定产量,竞争抑制ELISA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1.7 μg/mL，竞争抑制实验显示其与亲本鼠单抗C50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。
目的 構建錶達抗CEA小分子抗體,以利于放射免疫診斷及導嚮治療。方法 構建CEA單鏈抗體基因,剋隆入載體pKpL-3a,在大腸桿菌中包涵體錶達,ELISA檢測活性。將輕重鏈基因剋隆入分泌型錶達載體pSCMH,在大腸桿菌中錶達,ELISA檢測活性。將CEA嵌閤抗體的輕鏈和Fd基因剋隆入桿狀病毒錶達載體pAcUW51中,在sf9細胞中分泌錶達,ELISA檢測活性及測定產量,競爭抑製ELISA測定其識彆的抗原錶位。結果 多種方法複性的包涵體及原覈分泌錶達的單鏈抗體未測到活性。昆蟲細胞中錶達的小分子抗體具抗體活性,產量為1.7 μg/mL，競爭抑製實驗顯示其與親本鼠單抗C50識彆相同的錶位。結論 該CEA抗體基因在大腸桿菌中不能錶達齣有功能的分子，而在昆蟲細胞中錶達瞭具活性抗體分子;某些抗體基因隻有在真覈細胞中纔能錶達齣有功能的抗體分子。
목적 구건표체항CEA소분자항체,이리우방사면역진단급도향치료。방법 구건CEA단련항체기인,극륭입재체pKpL-3a,재대장간균중포함체표체,ELISA검측활성。장경중련기인극륭입분비형표체재체pSCMH,재대장간균중표체,ELISA검측활성。장CEA감합항체적경련화Fd기인극륭입간상병독표체재체pAcUW51중,재sf9세포중분비표체,ELISA검측활성급측정산량,경쟁억제ELISA측정기식별적항원표위。결과 다충방법복성적포함체급원핵분비표체적단련항체미측도활성。곤충세포중표체적소분자항체구항체활성,산량위1.7 μg/mL，경쟁억제실험현시기여친본서단항C50식별상동적표위。결론 해CEA항체기인재대장간균중불능표체출유공능적분자，이재곤충세포중표체료구활성항체분자;모사항체기인지유재진핵세포중재능표체출유공능적항체분자。
Objective A small molecular antibody against humancarcinoembryonic antigen was constructed and expressed in order to be used for radioimmunoimage and target therapy. Methods The 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. Results After 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. Conclusion A small molecular chimeric antibody against CEA was successfully expressed in sf9 cell. Only in eucaryotic cells can some antibody genes express functional molecules.
