Construction And Expression Of Prokaryotic Vectors For M-CSF, MCM7 And The Functional Domain Mutants Of MCM7

Objective : To construct prokaryotic vectors and expression system which can stably express M-CSF,MCM7 and functional domain mutants of MCM7. To express M-CSF,MCM7 and Functional Domain Mutants of MCM7 in E Coli. BL21 DE3.Methods: An active M-CSF, MCM7 and functional domain mutants of MCM7 was respectively amplified by Polymerase chain reaction(PCR) and subcloned to a prokaryotic expression vector (pGEX-4T-2) to construct expression vectors, including M-CSF expression vector (pGEX-4T-2/M-CSF), MCM7 expression vector (pGEX-4T-2/MCM7/DM1-719), and functional domain mutants of MCM7 expression vector (pGEX-4T-2/MCM7/DM1-331, pGEX-4T-2/MCM7/DM332-538 and pGEX-4T-2/MCM7/DM538-719) by gene recombination. The recombinant gene was identified by agarose gel electrophoresis and sequencing. All recombinant genes were stably transfected into E Coli. BL21 DE3 using the mothod of Calcium chloride, after screening with ampicillin,the M-CSF, MCM7 and functional domain mutants of MCM7 was expressed by IPTG in E Coli. BL21 DE3.Results:The results from SDS-PAGE showed that the size of inserted fragment in the recombinant gene corresponded to that of M-CSF,MCM7 and functional domain mutants of MCM7, and that molecular weight of expressing protein was matched with that our expect after induced by IPTG.The result from sequencing experiment indicated that there was no reading frame shifts and mutations in recombinant.Conclusion: The prokaryotic expression vectors for M-CSF,MCM7 and functional domain mutants of MCM7 were successfully constructed. M-CSF , MCM7 and Functional Domain Mutants of MCM7 were successfullyexpressed in E Coli. BL21 DE3.