Gene Cloning, Expression And Preparation Of Specific Antibody Of Human Survivin

Objective TO construct and express recombinant human survivin protein (rhsurvivin) with high purity in E.coli,and prepare the anti-survivin specific polyclonal antibody ,this can provide basic for deep study survivin protein. Methods survivin cDNA was amplified by RT-PCR and coloned into procaryotic expression vector pBV220. Then the recombinant plasmid was expressed in E.coli host strain BL21 (Gold). The expression product was detected by western blot analysis To obtain survivin protein, DEAE Sepharose Fast Flow ion exchange chromatography and Sephacryl S-200 gel filtration were performed.. The purified protein was used as a immulogen to the Guinea pig, the efficiency of this antibody was determine by the method of ELISA. Finally, immunohistochemistry technique detected whether this polyclonal antibody could specially recognize survivin protein expressing in cancer tissue.Results survivin protein was expressed in E.coli host strain BL21(Gold) in the form of inclusion body. The expression level was more than 30% of the total cell lysate. Through ion exchange chromatography and gel filtration, The purity of recombinant protein has reached 95% above, which exhibited specific reaction with the mouse anti-human antibody. The polyclonal antibody was obtained from immunize Guinea pig, the efficiency of this antibody was 1: 1000 by ELISA detection, immunohistochemistry technique indicated that this polyclonal antibody could specially recognized survivin protein expressing in cancer tissue. Conclusion our study prepared special anti-survivin antibody by gene engineering, it can serve as screening out the expression discrepancy of survivin protein in all kindsof human tumors, and farther establish basic for study the ralation of survivin and cancer.