Molecular Cloning,Characterization And Expression Analysis Of MIF And Cathepsin B From Oriental Finless Eel(Monopterus Albus)in Response To Challenge With Aeromonas Hydrophila

Aeromonas hydrophila is a pathogen that causes sepsis and is responsible for great economic losses in Asian finless eel(Monopterus albus)aquaculture.Macrophage migration inhibitory factor(MIF)is well known as a pivotal regulator of innate immunity,which play vital roles in various signaling cascades,including cell proliferation,and activation of immune responses against infections.Cathepsin B belong to lysosomal cysteine protease of the papain family,which play an important role in intracellular protein degradation,normal metabolism for the maintenance of cellular homeostasis and indirect participation of immune response.However,we know little about the MIF and Cathepsin B in Asian finless eel,up to now.In our experiments,we obtained and analysised the open reading frame(ORF)sequence of MIF and cathepsin B in finless eel using homology cloning.In addition,we analysised the expression of MIF and cathepsin B in intestine,liver,muscle,skin,spleen,kidney and heart of healthy Asian finless eel through qRT-PCR.We challenged the Asian finless eel with A.hydrophila,andanalysised the expression of MIF and cathepsin B in liver and skin at Oh,4h,8h,12h,24h and 48h.The results indicated that MIF and Cathepsin B play an important role in hemorrhagic septicemia of Asian finless eel.The MIF ORF is 348bp long,encoding a protein that is 115 amino acids(aa)long,which has been deposited in Genebank with Accession No.KY318074.The cathepsin B ORF is 993bp long and encodes a 330 aa long protein,which has been deposited in Genebank with Accession No.KY318075.Gene similarity analysis shows that MIF and cathepsin B had high similarity with the sequences from other fish species at the both nucleic acid and putative amino acid sequence level.The phylogenetic trees of MIF and cathepsin B classified the proteins into two primary groups,one containing the proteins from fish,while the other was comprised of mammalian,amphibian,reptilian and avian proteins,which confirms the developmental relationship among different groups.MIF was observed in all the tissues examined,with the highest levels in liver andkidney,moderate levels in heart,spleen,intestine and muscle,and lowest level in the skin.cathepsin B expression was highest in liver,spleen,kidney,followed by intestine,mucle,heart and skin.In general,MIF and cathepsin B expression increased after challenge with A.hydrophila.MIF and cathepsin B expression was increased at 4h and 8 h in skin,liver,kidney and spleen significantly,reaching the highest levels at 12 h.The results suggested that increased expression of MIF and cathepsin B in tissues are due to a protective function for MIF and Cathepsin B against sepsis.