Human Macrophage Inhibitory Factor -1 (mic-1) Optimize The Expression In Pichia Pastoris, Protein Purification And Polyclonal Antibody Preparation

Objective: To construct and select a high expression human MIC-1 pichia pastorisyeast; to optimize the MIC-1 expression condition and purification procedure andproduce the anti-MIC-1 rabbit polyclone antibody.Method: The gene sequence of MIC-1 was obtained through RT-PCR from thehuman placenta, then seven codons of the MIC-1 gene were mutated to P.pastoris-preferred synonymous codons. Then the mutant gene was inserted into thepPIC9K plasmid, and the recombinant pPIC9K-mtMIC-1 plasmid was transformed intohost yeast strain GS 115 and a high expression recombinant yeast strain was selected byHis~+ and G418 plate. Six different methanol concertrations were tested to evaluate theeffect of methanol concertrations on the MIC-1 expression. Meanwhile, the effect ofdifferent induction time on the quantity of MIC-1 expression was observed. Theammonium sulfate precipitation, anion ion exchange chromatograph and size exclusionchromatograph were used to explore the purification techniques of MIC-1. The purifiedMIC-1 was used as antigen to immunize rabbit and produce the anti-MIC-1 polycloneantibody.Result: The recombinant pPIC9K-mtMIC-1 plasmid was constructectedsuccessfully, and a high expression recombinant MIC-1 yeast strain was obtained, withexpression yield was about 30～50mg/L. 3% methanol concentration with 72 hours wasidentified to be the best MIC-1 induction condition. The purification techniques ofMIC-1 were optimized, and SDS-PAGE gel shows the purity is above 90%. Anti-MIC-1rabbit polyclone antibody was obtained successfully and the antibody titer is1:128,000.Conclusion: We obtained the high expression recombinant MIC-1 yeast strain, byoptimized gene sequence with P. pastoris-preferred synonymous codons, the expressionyield is 5 time higher than that has reported. We optimized the induction condition,fermentation techniques and purification techniques of MIC-1, and the purity ofrecombinant MIC-1 is above 90%. We alse obtained high titer anti-MIC-1 polyclone antibody successfully. The work has laid the foundation for further functional researchand clinical application of MIC-1.