Human BCCIP Protein Expression And Purification And Overexpression Of Cell Line Construction

The BCCIP protein is a BRCA2 and CDKN1A(p21Waf1/Cip1)Interacting protein,which binds to a highly conserved domian of BRCA2,and a C-terminal domain of the p21.In this study,we express and purify BCCIP protein,which immunizes mice to produce BCCIP antibody.In addition,we construct a stable transfection cell line that induced overexpression of BCCIP in MCF-7 cell line.Overexpression of BCCIP cells was confirmed by PCR,Western Blot method to detect its expression,and to study the effect of overexpression of BCCIP on the growth and proliferation of breast cancer cells.We constructed p GEX-6P-1-BCCIP? and ? recombinant plasmids,and determined the optimal inducible expression conditions to induce the expression of GST-BCCIP? and GST-BCCIP? proteins.Then a large amount of BCCIP? and BCCIP? proteins were obtained by Pre Scission Protease digestion.BCCIP protein immunized mice to prepare BCCIP antibody,and used HCT116,He La,293 T cell protein samples to verify the titer and specificity of BCCIP antibody.We determined the optimal drug concentration of G418 in the MCF-7cell line,and demonstrated that the Tet system can work in the MCF-7 cell line.The p Tet on plasmid was transiently transfected into MCF-7 cells,and MCF-7 Tet on stable cell line was successfully screened by G418.We successfully constructed p TRE2-puro-myc-BCCIP? and p TRE2-puro-myc-BCCIP? recombinant plasmid.Based on the MCF-7 Tet on stably transfected cells,we successfully selected a stable cell line that can induce the expression of BCCIP protein.we next determined the optimal drug concentration of dox and the optimal induction time.The number of induced BCCIP? and BCCIP? cells was significantly less than that of thecontrol group,indicating that BCCIP inhibited the growth and proliferation of MCF-7 cells.Conclusion:First,Induced expression of recombinant protein GST-BCCIP?/? in prokaryotic E.coli BL21(DE3),and the Pre Scission Protease was digested to obtain a large amount of BCCIP? and BCCIP? proteins,and the BCCIP antibody was successfully prepared.Second,we constructed overexpression of BCCIP in MCF-7 cell line.It was found that high expression of BCCIP can effectively inhibit cell growth and proliferation,demonstrating that BCCIP can inhibit the growth of breast cancer cells.