Preliminary Study On The Function Of Grouper(epinephelus Coioides)mpeg1 Against Cryptocaryon Irritans Infection

Mpeg1 protein(also known as perforin-2)is encoded by Macrophage expressed gene 1,which contains a typical membrane complex/perforin domain(MACPF),can form pores and destroy the cell membrane of invading pathogens.What’s more,Mpeg1 acts as a specific marker of macrophage in mammals.C.irritans is a highly harmful ciliated obligate parasite,causing a large economic loss.C.irritans infection can cause an insect-resistant immune response in the fish,and many immune cells can be observed in the local infection site.However,it is unclear whether macrophages are involved in the host against C.irritans infection.Based on the above background,the following experiments were carried out.In this study,we identified two kinds of Mpeg1 s from the orange-spotted grouper(Epinephelus coioides)and named them EcMpeg1 a and EcMpeg1 b,respectively.Both EcMpeg1 s contained a signal peptide,a conserved MACPF domain,a transmembrane segment,and an intracellular region.Sequence alignment demonstrated that both EcMpeg1 s share a high sequence identity with that of other teleosts.qPCR analysis showed that EcMpeg1 s were expressed in all tissues tested in healthy grouper,with the highest expression in the head kidney and spleen.After infection with C.irritans,the expression of both EcMpeg1 s was significantly upregulated in the spleen and gills,which demonstrate these two genes may be involved in the immune response of grouper against C.irritans.Furthermore,the recombinant EcMpeg1 a showed antiparasitic and antibacterial activity(against Gram-negative and-positive bacteria),whereas EcMpeg1 b had an inhibitory effect only against Gram-positive bacteria.Rabbit anti-EcMpeg1 a polyclonal antibody was prepared,which can recognize both EcMpeg1 a and EcMpeg1 b proteins.The immunofluorescence showed that EcMpeg1 positive cells were mostly distributed in the head kidney and spleen in healthy grouper.In addition,the study also found that EcMpeg1 positive cells have phagocytic activity.Immunofluorescence and immunohistochemistry showed that EcMpeg1 positive cells increased sharply after infection of C.irritans in gill,suggesting that EcMpeg1 and its positive cells may be involved in the process of grouper against C.irritans infection.