Molecular Cloning, Sequence Analysis And Prokaryotic Expression Of Sweetfish Macrophage Inflammatory Protein-2Gene

Chemokines are a group of small （8～10kDa）, mostly basic related molecules thatregulate cell trafficking of various types of leukocytes. It is actively involved in immune andinflammation response. The chemokines that we have found contain the following four categories.CXC, CC, C and CX3C. Macrophage inflammatory protein-2（MIP-2） is a typical CXCchemokine, which promotes neutrophils to be the sites of inflammation and eli minates theinflammatory response. In this study, we obtained the sequence of MIP-2gene from de-novotranscriptome sequencing of sweetfish macrophages. An open reading frame was composed of318nucleotides, which encoded a protein of105a mino acids with a molecular weight of11.6kDa. ItsN-ter minal19residues were the signal peptides. Sequence alignment showed that sweetfish MIP-2shared the highest homology with the MIP-2from Esox lucius with54%protein sequenceidentity and39%to41%identity to other teleosts. Otherwise, sweetfish MIP-2shared lowerhomology with mammals. Sweetfish MIP-2contained the same CXC structure with other teleosts’MIP-2, IL8and IL8-like. In healthy sweetfish, MIP-2mRNA was mainly expressed in the spleen,kidney, head and gills, weakly expressed in the liver, heart, muscle and intestine. Quantitative RT-PCR analysis showed that MIP-2transcripts were significantly up-regulated in the tested tissues,especially the kidney and muscle. The highest values were23and25times than that in thecontrol group. Since then, the trend of expression was slowing down and returned to the controlgroup expression level. MIP-2was obtained by Prokaryotic expression. Antiserum was made byimmunized mice. Our results suggested that MIP-2might play an important role in sweetfishantibacterial immunity.