Cloning and Expression of survivin Gene in E.coli and Preparation of Its Antiserum

Objective To clone and express the human survivin gene in E.coli and prepare its antiserum. Methods The survivin cDNA was amplified from the total RNA of HL-60 cells by RT-PCR.the open reading form of survivin gene was inserted to the expression plasmid pET32 a(+),transfected into E.coil BL21(DE3)and induced with IPTG,the expressed protein was analyzed with SDS-PAGE.Then,the Balb/c mice were immunized with the purified expression protein,the antiserum titer was detected with ELISA,and the specificity was identified with Western blot and immunocytochemist ry techniques. Results 　DNA sequencing result s showed that survivin gene was exactly consistent with the sequence reported in Genbank, the survivin expressed in E. coli, the protein amounted to 30 % of total bacteria protein and the molecular weight was 37 KD. The titer of antiserum against recombinant surviving was 1∶1600, identified the recombinant protein in E. coli and the surviving in tumor cell specifically. Conclusion 　The survivin gene was cloned and expressed in E. coli, the antiserum against recombinant survivin was prepared and could be detect the target protein in tumor cell.