Abstract:

Combining the bioinformatics methods and the analysis of the nucleotide sequences of anti-carcinoembryonic antigen(CEA) single chain Fv fragment(scFv), the anti-CEA disulfide stabilized single chain fragments(scdsFv) was chemical synthesized after molecular designing and codon optimizing. Apoptin gene was linked downstream to anti-CAE scdsFv via soft linker peptide and cloned into the prokaryotic expression vector pET28a. Then the recombinant plasmid was transferred into Escherichia coli strain BL21. After induce expression by isopropyl β-D-1-thiogalactopyranoside(IPTG), the protein expression was analyzed by SDS-PAGE and Western-Blot. The results show that target protein CAtin is well expressed and the maximal production is 44.1 mg/L. Human hepatoma carcinoma cell(HCC) was used to detecting antibody affinity, cellular binding activity and cellular killing activity after the purification and renaturation of the CAtin by washing and glutathione methods. The results indicate that CAtin can bind and suppress the cells effectively. The work presents here suggest that CAtin is an anti-tumor bispecific immunological directing agent which has both specific recognition activity and specific killing activity.

Key words: Carcinoembryonic antigen(CEA), Disulfide stabilized single chain Fv fragments(scdsFv), Apoptin, Prokaryotic expression