Gene construction, expression and activity identification of recombinant anti-HER2 ScFv/FDT/caspase-6

AIM: To construct the recombinant expression vector of anti-HER2 ScFv/FDT/caspase-6 and investigate its pro-apop- tosisactivity after transfected into gastric cancer SGC7901 cells. METHODS: Anti-HER2 ScFv (e23sFv) was attached to human recombinant caspase-6 (RC6) gene by recombinant PCR, and the Furin recognition site of diphtheria toxin (FDT) served as the linkerto construct e23sFv-FDT-RC6 recombinant gene. The recombinant gene was cloned into the vector pCMV, and the recombinant plasmid was transiently transfected into HER2 positive SGC7901 cells and HER2 negative HeLa cells via lipofectamine mediation. MTT assay was used to show how the cell living status was affected by the gene transfection. The pro-apoptotic activity of recombinant plasmid was detected by indirect immunofluorescent staining. RESULTS: Restriction endonuclease digestion and DNA sequencing proved that e23sFv-RC6 and e23sFv-FDT-RC6 genes had been cloned into vector pCMV. After transfection, the expression of fusion protein was found through Western Blot. MTT assays showed that the growth of SGC7901 cells was inhibited. Typical apoptotic changes of the SGC7901 cells transfected with pCMV-e23sFv-FDT-RC6 were seen by indirect immunofluorescent staining. CONCLUSION: The expression of recombinant anti-HER2 ScFv/FDT/caspase-6 gene can induce the apoptosis of HER2 positive SGC7901 cells.