Cloning,Identification,and Antimicrobial Immune Response Of Acid Phosphatase 5 In Sciaenops Ocellatus

Acid phosphatase(Acidphosphatase,ACP)is an enzyme that catalyzes the hydrolysis of phosphate monoesters under acidic conditions.Acid phosphatase was divided into five types at the genetic structure level: erythrocyte type,lysosomal type,prostate type,macrophage type and osteoclast type.Acid phosphatase 5(ACP5),also known as Tartrate resistant acid phosphatase(Tr ACP or Tr AP)is the fifth type of highly conserved metalloproteinase of acid phosphatase.The current research on ACP5 focuses on the research and application of bone immune diseases,bone tumors and even malignant tumors,but there are few studies and applications of ACP5 in teleost fish.In this study,the gene expression and characterization of acid phosphatase 5(SoACP5)expressed in Sciaenops ocellatus,in vitro and in vivo functions were analyzed.The results are as follows:(1)The open reading frame of SoACP5 is 1002 bp,encoding 333 amino acids,and the predicted protein size is 33.96 k Da.The results of amino acid sequence comparison show that the homology of the amino acid sequence of SoACP5 gene with other species ACP5 is as high as 68.77% ~ 96.70%,indicating that SoACP5 gene has a certain degree of conservation during the evolution of species.phylogenetic analysis showes that the relationship between American redfish SoACP5 and large yellow croaker is the closest.(2)Construction of SoACP5 prokaryotic expression vector,induction of protein,purification of inclusion bodies,protein renaturation.phosphatase activity of the renaturing protein of r SoACP5 was tested using p NPP standard enzyme activity.In this experiment,the optimum p H of SoACP5 enzyme activity is 5 and the optimum temperature is 55 ℃.The optimal auxiliary metal ion is Ca2+ ions.The phosphatase activity is dependent on the Ca2+ concentration.MTT measurement of cell proliferation shows that SoACP5 has the effect of promoting cell proliferation.(3)qPCR results show that SoACP5 was expressed in 8 tissues such as liver,spleen and kidney,among which the expression level is highest in gills,followed by spleen,heart,intestine,head kidney,brain,stomach,and lowest in liver.After 12 hours of infection with Edwardsiella tarda infection,SoACP5 expression peaked in the liver and was upregulated by 20.11 times;while 24 hours after infection in the spleen,head and kidney,the expression level was highest and the spleen was upregulated.The expression of middle was up-regulated by 20.25 times,and the expression of head and kidney was upregulated by 16.18 times.(4)Construct SoACP5 overexpression vector and interference expression vector,and study its antibacterial effect in Sciaenops ocellatus.The results show that overexpression of SoACP5 could inhibit the growth of Edwardsiella tarda;on the contrary,when the expression of SoACP5 was interfered,the number of Edwardsiella tarda infections increased significantly.In summary,SoACP5 play an important role in the antibacterial immunity of Sciaenops ocellatus.The research results provide new ideas for the antipathogen immunity of Sciaenops ocellatus and help promote the healthy and sustainable development of Sciaenops ocellatus farming.