Gene construction, expression and activity identification of recombinant antiª²HER2 ScFv/FDT/caspaseª²6

    REN Junª²Lin, WANG Tao, XU Yanª²Ming, MENG Yanª²Ling,WEN Weiª²Hong, ZHANG Rui, ZHANG Wei,YANG Anª²Gang

    Department of Immunology, Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct the recombinant expression vector of antiª²HER2 ScFv/FDT/caspaseª²6 and investigate its proª²apopª²tosis activity after transfected into gastric cancer SGC7901 cells. METHODS: Antiª²HER2 ScFv (e23sFv) was attached to human recombinant caspaseª²6 (RC6) gene by recombinant PCR, and the Furin recognition site of diphtheria toxin (FDT) served as the linker to construct e23sFvª²FDTª²RC6 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 proª²apoptotic activity of recombinant plasmid was detected by indirect immunofluorescent staining. RESULTS: Restriction endonuclease digestion and DNA sequencing proved that e23sFvª²RC6 and e23sFvª²FDTª²RC6 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 pCMVª²e23sFvª²FDTª²RC6 were seen by indirect immunofluorescent staining. CONCLUSION: The expression of recombinant antiª²HER2 ScFv/FDT/caspaseª²6 gene can induce the apoptosis of HER2 positive SGC7901 cells. ......