| Survivin is the smallest member of apoptosis inhibitor gene (inhibitor of apoptosis protein, IAP) family in mammalian cell. In addition, Survivin is a mitotic regulator and involves in cell cycle regulation as a member of Chromosomal Passenger Complex (CPC). Different domain of Survivin may perform different physiological functions. Survivin has become a new cancer drug target because of its bifunction and unique overexpression in tumor tissue. At present, anti-cancer drug designs targetting Survivin focus mainly on gene therapy. However, at the protein level, it is difficult to use small molecular or antibody againt survivin because Survivin is neither a kinase nor a cell surface protein.According to the unique biological function of Survivin, my research changed Thr117to Ala117of Survivin in order to prevent Aurora B kinase to phosphorylating this site, which is the key activtive site on cell proliferation. The main experimental results obtained are as follows:1) Thr117of Survivin was sucessfully changed to Ala117by overlapping PCR in base of provious plasmids pRSET-TmSm (T34A) and pGEMT-Survivin so that pRSET-TmSm (T34/117A) and pRSET-TmSm (T117A) were constructed.2) After three plasmads were transformed into E. coli BL21(DE3), these three proteins were expressed as conclusion bodies and accounted35%in all total bacterial protein in the shake flask. Three inconclusion bodies were dissolved in8M Urea and purified by SP Sepharose FF, refolde by SP column, dialysis and ultrafiltration.Finally three recombinant proteins that highest purify was95%and contained dimers were obtained.3) The three proteins were detected in cultured human breast cancer cells B-Cap37. MTT method was used to observe the cell proliferation. Flow cytometry assay was used to analyze cell cycle, apoptosis and mitochondrial membrane potential. Western blotting was used to determine the phosphorylation level of Histone H3, which was the substrate of Aurora B kinase, and the expression of activated-Caspase-3. |
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