Construction and Expression of MAGE-3 Prokaryotic Recombinant Expression Plasmid

Objective To construct prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 and analysis of its expression in BL21 E.coli.Methods MAGE-3 aim gene was obtained by RT-PCR.The target gene was orientating cloned into pGEM-T Easy and subclone into pGEX-4T-1 vector by DNA recombinant technical.Position clones were transformed into BL21.And then they were induced with IPTG,identified by 12% SDS-PAGE electrophoresis and Western-blot.Results MAGE-3 fragment(349bp)was amplified and the prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 was correctly constructed.DNA sequecing results showed that the sequence of MAGE-3 gene fragment in position clones is the completely same as the sequence of the GenBank public.The 35kD fusion protein was observed in BL21 E.coli and verfied that it is the target protein.Conclusion Prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 was successfully constructed and the fusion protein was expressed by induced.These lay a foundation for providing antigen which will be used as peptide vaccine and specific diagnosis reagent based on MAGE-3 aim gene,and also provide an experimental basis for further research.