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

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.