Cloning And Identification Of Cathepsin B And Its Role In The Antimicrobial Immune Responses In Golden Pompano (Trachinotus Ovatus)

Cathepsin B（CTSB）,as one of the typical representatives of cysteine protease family,has the activity of both exopeptidase and endopeptidase.Despite its important role in antigen presentation,degradation,apoptosis,inflammatory response and physiological process of many diseases,there appears to be few studies on CTSB in teleost at present.In view of this,the cathepsin B（TroCTSB）gene of Trachinotus ovatus was cloned,and bioinformatics analysis and preliminary study on its biological function were carried out.Main contents and results are as follows:1.In this study,the TroCTSB gene was cloned from the liver of Trachinotus ovatus by PCR and analyzed by bioinformatics.With a relative molecular weight of 34k Da,it has a coding region of 993bp and encodes 330 amino acids which constitute 3 domains:signal peptide,precursor peptide and cysteine protease.Homology alignment showed that the amino acid sequence of TroCTSB was similar to that in other teleosts and mammals（68.69%～88.48%）.The highest homology was with Oreochromis niloticus as 88.48%.The results of phylogenetic tree analysis demonstrated that TroCTSB and O.niloticus CTSB,both Perciformes,gathered as one clade.2.Tissue distribution results showed that TroCTSB were expressed in 11 tissues（blood,liver,spleen,head kidney,intestine,gill,heart,brain,stomach,skin and muscle）of healthy T.ovatus,with the highest expression was in stomach,followed by liver,and the lowest in blood.After Edwardsiella tarda infection,expression levels of TroCTSB in liver,spleen,and head kidney were significantly up-regulated.The expression levels of TroCTSB peaked at12 h after infection in liver and head kidney,while at 6 h after infection in spleen,which was up-regulated by 36.2 times,3.2 times,and 3.3 times,respectively.3.In this study,we successfully constructed and induced the expression of TroCTSB fusion protein r TroCTSB.In vitro enzyme activity assays manifested that the optimal p H and temperature of purified recombinant protein r TroCTSB were 5.5 and 40°C.The toxicity test of metal ions showed that Fe²⁺,Cu²⁺,Ca²⁺and Zn²⁺all inhibit the activity of TroCTSB to different degrees,with Zn²⁺ranking the first.4.Subcellular localization showed that CTSB mainly existed in lysosomes in the cytoplasm;In addition,overexpression of TroCTSB profoundly inhibited the infection of E.tarda in golden pompano tissues,and the knockdown of TroCTSB remarkably promoted the reproduction of E.tarda in golden pompano tissues in vivo.In vivo interference assays showed that when the expression level of TroCTSB decreased,the expression level of apoptosis related gene Tro Blc-2 in T.ovatus was significantly up-regulated,while Tro Caspase-3 and Tro Caspase-9 were significantly down regulated.In conclusion,the results of this study indicate that TroCTSB can enhance the antibacterial immune response of fish,and serve as a certain regulatory role in cell apoptosis.This study contributes to futher elucidation on the disease resistance mechanism of TroCTSB,and also provided a new idea for the greener,healthier and more sustainable development of T.ovatus breeding industry.