Prokaryotic expression and purification of mouse macrophage migration inhibitory factor gene

Objective: To clone mouse macrophage migration inhibitory (MIF) gene, express in prokaryotic cells and purify the expressed product. Methods: The total RNA of lung tissue of mice were extracted for amplification of MIF gene by RT-PCR. The amplified MIF gene was cloned into vector pMD18-T, and the constructed recombinant plasmid pMD18-M was identified by restriction analysis and sequencing. The target gene was recovered and cloned into expression vector pET-28a (+), and the constructed recombinant plasmid pET-28a-M was transformed to E. coli BL21 (DE3) for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot, then purified by Ni2+-NTA agarose column chromatography. Results: The length of amplified MIF gene fragment was consistent with that expected, and the sequence was completely identical to that reported in Gen-Bank. The expressed recombinant MIF, with a relative molecular mass of about 15 000, existed in a form of inclusion body and contained about 40% of total somatic protein. It showed good reactogenicity and reached a purity of more than 95% after purification. Conclusion: Mouse MIF gene was successfully cloned and expressed in prokaryotic cells, and the expressed product reached a high purity, which laid a foundation of study on the function of MIF.