Prokaryotic Expression Of Cancer-Testis Gene SSX1

Objective: To estabilish the foundation for the research of next stage,the prokaryotic expression vector of cancer-testis gene SSX1 is constructed,then the optimal inducing condition of MBP-SSX1 fusion protien is explored and MBP-SSX1 fusion protien is expressed and initially purified.Methods: The SSX1 gene is amplified by RT-PCR from the HCC tissue. Both the SSX1 gene and the pMAL-C2 plasmid are cut by BamH I and Hind III and connected together to construct the recombinant plasmid,then the recombinant plasmid is transformed into E. coli DH5α,which is blue-white screened nextly.At last,the recombinant plasmid is identified by restriction endonuclease cutting and sequencing. The correct recombinant plasmid is transfered into E. coli Rosetta. To find out the best induction conditions of expressing the fushion protein ,the E. coli Rosetta is respectively induced in varied status such as the add oppurtunity,the final concentration,the inducing time length and the inducing temperature of IPTG. The fusion protein expressed by E. coli Rosetta is purified by the amylose-resin column and cut by factor Xa protease to get the pure purpose protein.The purpose protein is identified by protein mass chromatographic analysis. Result:The result of identification by restriction endonuclease cutting and sequencing display that cancer-testis gene SSX1 is correctly connected with the pMAL-C2 plusmid and the optimal inducing condition of expressing the MBP-SSX1 fushion protein is desided at last ,which is the host bacterium is firstly shake cultrued for 3 hours in 37℃, then for 3 hours in 30℃with the final concentration 0.5mmol/L of IPTG .The MBP-SSX1 fushion protein is cut by Factor Xa protease and the purpose production is identified to be SSX1 protien by MS-MS.Conclusion:1.The prokaryotic expression vector of cancer-testis gene SSX1 is successfully constructed . The optimal inducing condition of expressing the MBP-SSX1 fushion protein is desided .The MBP-SSX1 fushion protein is expressed and initially purified.