抗癌胚抗原单链抗体-PE40免疫毒素基因的克隆与表达  

Cloning and Expression of Immunotoxin Gene of Anti-carcinoembryonic Antigen Single-chain Antibody-PE40

作　　者：邱业峰[1] 孟锐奇[1] 李树民[1] 李铁征[1] 朱平[1] 

机构地区：[1]中国人民解放军军需大学研究所,长春130062

出　　处：《中国生物制品学杂志》2004年第3期133-135,141,共4页Chinese Journal of Biologicals

摘　　要：目的扩增抗癌胚抗原单链抗体(CEA-ScFv),与绿脓杆菌外毒素(PE40)进行重组及原核表达,旨在进一步研究重组蛋白CEA-ScFv-PFA0的靶向抗肿瘤作用。方法分别扩增抗癌胚抗原单链抗体的重链区、轻链区,用overlap PCR按照VH-VL的顺序连接,克隆至pMD18T载体中。再将VH-VL基因片段与含有.PE40的表达载体pET28a连接,转化B121(DE3),IPTG诱导表达。结果经DNA测序,其结果与设计完全相符。SDS-PAGE分析,在相对分子质量66000处可见明显表达带,表达量可占菌体蛋白总量的13％以上。免疫印迹表明重组蛋白具有PF40的抗原特异性。结论抗癌胚抗原单链抗体-PE40免疫毒素的成功克隆与表达,为进一步研究其生物活性奠定基础。Objective To amplify the gene encoding anti-carcinoembryonic antigen single-chain antibody ( CEA-ScFv), fuse with Pseudomonas aeruginosa exotoxin (PE40) gene and express in E. coli. Methods Amplify the gene encoding heavy (VH) and light (VL) variable regions of anti-carcinoembryonic antigen from plasmid pSecTag respectively, assemble them to create VH-VL gene by overlap PCR and clone into pMD18T vector. Construct a fusion gene CEA-ScFv-PE40 in expression vector pET28a and transform to E. coli BL21 (DE3) for expression under the induction of IPTG. Results The DNA sequence of CEA-ScFv-PE40 gene was fully consistent with that designed. SDS-PAGE profile showed a clear band with a relative molecular weight of 66 000. The expressed product contained more than 13% of total somatic protein and was recognized by PE antibody in Western blotting. Conclusion A fusion gene of immunotoxin of anit-carcinoembryonic antigen single-chain antibody-PE40 gene was successfully cloned and expressed in E. coli. It laid a foundation of further study on the bioactivity of the expressed protein.

关 键 词：抗癌胚抗原单链抗体 绿脓杆菌外毒素 PE40 免疫毒素 基因 克隆 表达 

分 类 号：R979.1[医药卫生—药品] Q78[医药卫生—药学]