Prokaryofic Expression And Purification Of Chicken Macrophage Migration Inhibitory Factor And Its Bioinformatics Analysis

Macrophage migration inhibitory factor is one of the animal kingdom relatively conservative cytokines, with the regulation of the immune function, involved in inflammation, involved in the metabolism regulation. The recent research focused on the human and experimental animal and on poultry and livestock MIF has just started, the subject is the basic research for chicken MIF. Objective:Using bioinformatics methods to analyze the sequences of MIF gene and its protein in chicken, and predict the function of this gene. In addition, there is no commercialized chicken MIF, so preparing recombinant chicken MIF using prokaryotic expression system for better study the function of MIF. Methods:Based on NCBI25species of MIF mRNA and protein sequences, using network information repository and biology software, for the prediction of chicken molecular evolution analysis and protein structure and function.According to GenBank in the MIF cDNA sequences, using Premier5.0software design a pairs of specific primer, total RNA was extracted from chincken Liver tissue by RT-PCR cloning colony MIF cDNA fragment. After PCR amplification, product was cloned into pEASY-E1vector by T-A cloning technique, the recombinant pEASY-E1-MIF was transferred to Transl-T1Phage Resistant Chemically Competent Cell, then, it was identified with double restriction enzymes digestion analysis and DNA sequencing. The recombinant pEASY-E1-MIF which was identical with GeneBank was transformed into E. coli BL21. The expression of chicken MIF protein was analyzed by SDS-PAGE after IPTG induction. Using mass spectrometry and Western blot identify Recombinant protein. Results:Sequence analysis showed that chicken MIF gene has547nucleotide acids, encoding115amino acids; there is no signal peptide and the transmembrane domain structure; presence of potential phosphorylation sites and potential N-glycosylation sites in the sequence; secondary structure by the alpha helix (25.22%), the beta an extension chain (25.22%) and random coil (49.57%) constituted; Molecular evolution analysis revealed MIF highly conserved between species, indicating that the MIF gene is irreplaceable in the process of evolution of species The construction of prokaryotic plasmid pEASY-E1-MIF was achieved. Chicken MIF sequence was mainly identical with the GendBank. E.coli BL21expressed the fusion protein of Chicken MIF. Identification of Mass spectrometry and Western blot were identical with the ChMIF. Conclusion:And construct a based on further study the biological effects of MIF gene in particular the use of MIF improve myocardial ischemia energy metabolism.