Macrophage Migration Inhibitory Factor Of Trachinotus Ovatus,is Involved In The Antimicrobial Immune Response

Macrophage migration inhibitory factor(MIF)is a class of immunoregulatory cytokines with unique structure,which is similar to the partial function of enzyme,chemokines,hormone,and can participate in the body of the cell proliferation,differentiation,cell survival and apoptosis,and other biological process,is the key to connect the nonspecific immunity and nonspecific immune signaling molecules.In view of this,this study carried out a cloning identification of the TroMIF and a preliminary study of the biological function of its participation in Trachinotus ovatus.The results of the study are as follows:(1)The results of sequence analysis showed that the open reading frame of TroMIF was345 bp,encoding 115 amino acids,and its relative molecular mass was 12.3 KDa.The homology comparison revealed that TroMIF has a high homology with other bonefish MIF(82.61% ~ 95.65%),and the highest homology with sea bass at 95.65%.Sequence analysis showed that TroMIF has oxidoreductase active site(C57ALC60),catalytic active site(K33)and isomerase active site(P2).Phylogenetic analysis showed that TroMIF were closely related with MIF of large yellow croaker(Larimichthys crocea).(2)Tissue-specific results showed that TroMIF can be expressed in 11 tissues(blood,liver,spleen,head kidney,intestine,gill,heart,brain,stomach,skin and muscle)of healthy Trachinotus ovatus,the highest expression in liver and the second in head kidney.After infection with Edwardsiella tarda,the TroMIF expression in the spleen and head kidney increased significantly,with the head kidney reaching a peak at 12 h and the spleen reaching its peak at 24 h.In this study,the TroMIF overexpression vector p CNTroMIF was constructed,and a specific si RNA(si TroMIF)also to be synthesized successfully.After we transfected it into the Trachinotus ovatus kidney cell line,E.tarda performed infection experiments.Experimental results show that the overexpression of TroMIF can significantly enhance the ability of kidney cells to resist bacterial infection,and the colony count results are significantly reduced compared with the control group.In line with this,the amount of E.tarda infection increased0 when TroMIF expression was disrupted by si TroMIF in Trachinotus ovatu kidney cell lines.Above experiments indicate that TroMIF may be involved in the body’s anti-infective immunity and play an important role in the antibacterial immune response of Trachinotus ovatus.(3)In order to study the function of TroMIF,a prokaryotic expression vector was constructed and the active recombinant protein r TroMIF was induced and purified.In vitro experiments show that r TroMIF can significantly inhibit the migration of head kidney lymphocytes(HKL)and head kidney mononuclear cells(HKM);we have also observed that r TroMIF could significantly induce the proliferation of HKL when it was incubated with HKL,and the induction was dose-dependent.In addition,we found that r TroMIF has significant oxidoreductase activity in enzyme activity testing experiments,suggesting that TroMIF may play a role in the immune response against bacterial infections through its unique enzyme activity.In summary,there results show that TroMIF can improve the body’s antibacterial immune response.The research on the antibacterial effect of TroMIF provides new ideas for the green healthy and efficient breeding of Trachinotus ovatus.