| B lymphocyte stimulating factor(BLyS)is an important member of tumor necrosis family and plays an important role in the immune system of mammalian.It promotes B cell growth,differentiation,apoptosis,and regulates T cells.However,BLyS in teleosts is less reported and the disease-resistant mechanism of BLyS in vivo experiment is basically absent.In this study,the full-length cDNA sequence of BLyS in Trachinotus ovatus(TroBLyS)was cloned,and the structural characteristics of TroBLyS were investigated.Moreover,the function of TroBLyS was analyzed in vivo and in vitro experiments.The main results of this study are shown as follows:In this study,we cloned the cDNA sequence of BLyS of golden pompano.Sequence analysis showed that the structure of TroBLyS protein was similar to other mammals and fish species,and the sequence homology was high compared with those species.Moreover,TroBLyS possesses the similar functional domain and receptor binding sites,such as,a furin-like protease,a "Flap" structure,and three cysteine residues(Cys-121,Cys-211,Cys-224).TroBLyS contains no signal peptide and belongs to type ? transmembrane protein.The phylogenetic analysis showed that TroBLyS was clustered with Miichthys miiuy BLyS,Lateolabrax japonicus BLyS,and Epinephelus awoara BLyS,which belong to Perciformes.These characteristics suggested that TroBLyS may have the similar function with other teleosts.Analysis of tissues distribution performed by qRT-PCR showed that TroBLyS was ubiquitously expressed in eleven tissues(liver,head kidney,muscle,spleen,skin,blood,stomach,intestine,gill,heart,and brain)in healthy golden pompano,and more highly expressed in liver and head kidney,which are immune organs.After stimulated by Vibrio harveyi,TroBLyS was significantly upregulated in liver,spleen,and head kidney.These findings indicated that TroBLyS may be involved in the immune response against pathogens infection in T.ovatus.In order to investigate the function of TroBLyS,recombinant protein(rTroBLyS)was prepared and purified.The results indicated that rTroBLyS is able to promote lymphocyte proliferation.Moreover,to analyze the function of TroBLyS with vivo experiment,we contrasted the eukaryotic expression plasmid pTroBLyS to inject the muscle of golden pompano.The results showed that the over-expressing of TroBLyS improved significantly the macrophages activity and the acid phosphatase and alkaline phosphatase levels.After V harveyi infection,the bacteria amount in pTroBLyS-immuned group were significantly lower than the control group,which may due to the over-expressing of TroBLyS enhances the activity of macrophages and improves the ability to resist the pathogens of organism.In addation,the experiment of inhibiting TroBLyS showed that siTroBLyS could significantly reduce the expression of TroBLyS and the expression levels of TroBLyS in immune organs were significantly lower than the control group.Then,after stimulated by V.harveyi,the amount of V.harveyi was significantly increased than the control fish when inhibiting TroBLyS.Thereafter,this present study estimated the potential of pTroBLyS as the immune enhancer.Fish were immunized with pTroBLyS for three weeks,then infected with V.harveyi.The results showed that the survival rate of the control group was 25.7%,while the immune group was 71.5%,which suggested TroBLyS has the ability to significantly improve the survial rate of T.ovatus.Thus,TroBLyS has the potential as an immune enhancer.Take together,these results of this study provide evidences that TroBLyS plays a key role in the anti-bacterial immunity of T.ovatus.This study will enrich the mechanism of antibacterial immunity of T.ovatus,and provide the theoretical foundation for healthy and efficient farming of T.ovatus. |
PDF Full Text Request