Chemotactic Effect Of ?-defensin 1 On Macrophages In Megalobrama Amblycephala

Defensin is a cysteine-rich cationic short peptide with broad-spectrum antibacterial activity.It plays an important role in the body's immune function of cell migration.This research was conducted to investigate the effects of MaBD1 in the leukocytes migration and the expression of inflammatory cytokines.The main results are as follows:1.The pET-32a-MaBD1 fusion protein had been expressed by the chemical inducer Isopropyl-?-D-1-Thiogalactopyranoside.MaBD1 fusion protein was purified by Ni²⁺affinity chromatography.The MaBD1 recombinant protein showed obvious antibacterial activity against Escherichia coli,Aeromonas hydrophila and Staphylococcus aureus.2.Percoll discontinuous density gradient centrifugation was used to separate the head kidney leukocytes,then the macrophages were isolated and purified by adherence method.The light microscope and transmission electron microscope were used to identify leukocytes.The results showed that leukocytes mainly include:neutrophils,monocytes/macrophages and lymphocyte.Adherent macrophages spreading or elongated in various shapes and the nucleus was round and oval.The nucleus was biased towards the side.The macrophage surface marker CD68 was found to be highly expressed in adherent cells.3.MaBD1 recombinant protein has obvious chemotactic effect on leukocytes,especially macrophages.To further identify the MaBD1 chemotactic function sites,the synthetic peptides were sythesized for chemotaxis experiments.The results showed that the chemotactic ability of N-terminal polypeptide?MaBDP1?was significantly higher than C-terminal polypeptide?MaBDP2?.4.MaBDP1 was injected into the peritoneal cavity to observe the chemotactic activity of MaBDP1 in vivo.The results showed that MaBDP1 had chemotactic effect on the leukocytes and macrophages in vivo.The proportion of leukocytes was significantly higher in the group treated with 500 ng/mL of MaBDP1 at 2 d and the proportion of macrophages was significantly higher in the group treated with 1000ng/mL of MaBDP1 at 3 d.The qPCR was used to detect the expression of proinflammatory cytokines?IL-1?and TNF-??in different tissues of M.amblycephala.The results showed that the expression of IL-1?in the spleen,head kidney and body kidney was significantly increased at 6 h and 72 h.The expression of IL-1?in the hepatopancreas increased significantly at 6 h.TNF-?was significantly increased at 6 h only in hepatopancreas and significantly increased in spleen,head kidney and body kidney at 72 h.These results showed that MaBDP1increases the production of IL-1?and TNF-?,thus amplifying inflammatory response.