Characterization And Function Of A Gene Tumor Necrosis Factor A Induced Protein 8(TNFAIP8) From Red Crayfish,Procambarus Clarkii

Procambarus clarkii,known as the freshwater crayfish belongs to Arthropoda,Crustacea,Decapoda,Astacidae,is an important economically cultivated freshwater species in our country.Due to the high temperature and water containing a lot of bacteria and viruses during the intensive aquaculture,diseases occurred and caused the death of the individual with major economic loss.Innate immunity is the main immune system of invertebrates to defense the invasion of pathogenic microorganisms.Clarification of the immune mechanism of invertebrates against microorganisms are useful for the improveme-nt of theory and provide insight in the breeding of disease-resistant species.Tumor necrosis factor-α-induced protein 8(TNFAIP8)is a family of proteins that contain a variety of proteins involved in autoimmune diseases,cancer as well as the regulation of multiple signaling pathways.The purpose of this study was to investigate the function TNFAIP8 in the immune response of crayfish against microorganisms.1.The ORF of TNFAIP8 was cloned from P.clarkii by PCR method.The ORF of PC-TNFAIP8 was 564 bp and this gene encoded a polypeptide of 187 amino acids.Theoretical isoelectric point and molecular mass for the protein are 9.038 and 21.4 kDa,respectively.Phylogenic analysis showed that P.clarkii TNFAIP8 was highly homologous to that of invertebrates.2.The ORF of Pc-TNFAIP8 was ligated to PGEX-4T-3 vector for protein expression.SDS-PAGE analysis demonstrated that a 40 kDa recombinant protein was successfully expressed in Escherichia coli cells.The GST column chromatography was used to purify recombinant proteins and the concentration of recombinant protein was about 1 mg/mL.3.Through the GST Pull-down assay,a hemocyanin protein interacted with TNFAIP8 protein was identified.4.The expression patterns of Pc-TNFAIP8 indifferent tissues was determined by real-time PCR using total RNAs from hemocytes,gill,gut,stomach,heart,muscle and hepatopancreas.The result showed of Pc-TNFAIP8 was expressed in all the examined tissues with higher levels in hemocytes,gill and gut.5.The expression of Pc-TNFAIP8 was significantly down-regulated in hemocytes,gill,gut after LPS and PolyI:C induction.The recombination TNFAIP8 protein induction and knockdown of Pc-TNFAIP8 gene by small interfering RNA(siRNA)transfection significantly induced the the expression of immune-related genes such as Toll,Serpin,Bcl,Lectin,TNF and Hemocyanin genes in hemocytes and gills.All together,these results suggest that Pc-TNFAIP8 play a role in the immune response in P.clarkii.