Construction and Expression of MAGE-3 Target Gene Prokaryotic Recombinant Plasmid

Objective To construct MAGE-3 target gene prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 and analysis of its expression in BL21(DE3) E.coli.Methods MAGE-3 target gene was obtained by RT-PCR.After sequencing,the target gene was cloned into the expression vector pGEX-4T1 to construct MAGE-3 prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE3.The E.coli BL21(DE3) containing the expression plasmid was induced by IPTG、identified by 12%SDS-PAGE electrophoresis and Western-blot.Results MAGE-3 target gene fragment(349bp) was amplified and the prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 was correctly constructed.DNA sequencing results show that the sequence of MAGE-3 aim gene in position clones is the completely same as the sequence of the GenBank public.The 35kD fusion protein was observed in BL21(DE3) E.coli and verified that it is the very aim protein.Conclusion Prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 was successfully constructed and the fusion protein was expressed by induced.These set a foundation for providing antigen which will be used as peptide vaccine and specific diagnose reagent based on MAGE-3 target gene,and also provide experimental basis for further research.