Cloning of survivin gene and preparation its monoclonal antibodies as well as checking survivin expresson in liver carcinoma cells / 中华检验医学杂志

ABSTRACT

Objective To clone survivin gene, prepare its monoclonal antibody(McAb) and check its expression in liver carcinoma cells.Methods Survivin gene cDNA was amplified by RT-PCR from human breast carcinoma cell line MCF-7 and constructed into prokaryocytic expression vector.Fusion-protein of human Survivin was expressed and used for immunizing BALB/C mice.The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium.ELISA and Western blot were used to screen and identify the McAbs.Immunocytochemical staining was applied for survivin expression in liver carcinoma cells.Results The full survivin gene was cloned. The hybridoma cell that secret specific monoclonal antibody against human surviving was identified.The immunoglobulin subclasses of the McAbs were IgG1.Western blot showed that the McAbs against survivin can specifically react with MS-Survivin fusion protein. The positive reaction was found in hepatocarcinoma cell line HepG2 and hepatocarcinoma tissue by immunocytochemical staining.Conclusion The McAbs against the human Survivin were successfully prepared by a MS2-Survivin fusion protein expressed by E.coli. and the McAb had positive reaction with HepG2 and hepatocarcinoma tissue. It may be a useful tool to study the functions of survivin and check clinical cancer samples.