Molecular Cloning And Expression Regulation Of Porcine MIF Gene

Macrophage migration inhibitory factor(MIF) was discovered as the first cytokine that inhibited the random migration of macrophages,and it is an important pro-inflammatory cytokine,participating in inflammatory and immune responses. Meishan pig MIF genes were cloned by RT-PCR and introns of MIF were amplified from DNA by PCR,and chromosome mapping of procine MIF by RH.Meishan pig MIF gene structures were analyzed by bioinformation and biology.By RealTime-PCR, we analyzed the tissue distribution of Meishan pig MIF.We construct recombinant pIRES2-MIF vector and its expression in vitro.The promoter of procine MIF was cloned and had promoter activity by using Dual-Luciferase(?) Reporter Assay System. NIH3T3 cells were exposed to different concentration of rosiglitazone for 24 h;or plasmid pcDNA-PPARγ2 were transfected into NIH3T3 cells.24 h later,the cells were collected and used to determine the MIF mRNA expression by RealTime-PCR. The results showed:(1)Meishan pig MIF gene located within a 761 bp genomic fragment of chromosome 14,transcribed into a 466 bp mRNA including 118 bp 3'untranslated region(UTR) and 348 bp open reading frame(ORF);(2)Distribution of procine MIF mRNA were broad,among of which MIF mRNA was the best abundance in stomach,while the lowest in heart and muscle,respectively;(3) Construction recombinant pIRES2-MIF vector was successful and its expression were highly efficient in NIH3T3;(4)We have cloned the procine MIF promoter containing a 2kb fragment of 5'-flanking region by specific primers.The promoter inserted pGL3-Basic vector(pGL3-pMIF) successfully,and had promoter activity;(5)As a result,PPARγ2 activated by rosiglitazone may result in up-regulated MIF expression in NIH3T3 cells.