Identification And Immunological Function Of TUSC2 Gene From Red Crayfish,Procambarus Clarkii

The crawfish,Procambarus clarkii,is an important economically cultivated freshwater species in China.Due to factors such as high-density culture and poor water quality,disease occurs in P.clarkii from time to time which causing huge economic losses.Study of the innate immunity of P.clarkii has important theoretical significance for investigating the immune response mechanism of crustaceans,as well as improving the disease-resistance and breeding.Tumor suppressor candidate 2(TUSC2)can participate in the innate immune response by regulating apoptosis,however,the molecular mechanism remains unclear.In this experiment,a TUSC2 gene was cloned from P.clarkii,and it roles in the immune responses in the P.clarkii was explored.The results obtained from this study were listed as follows:1.The ORF of TUSC2 was cloned from P.clarkii by PCR method.The ORF of Pc-TUSC2 was 303 bp and this gene encoded a polypeptide of 100 amino acids.Theoretical molecular mass for the protein was 11.47 k Da.Phylogenic analysis showed that P.clarkii TUSC2 was highly homologous to that of invertebrates.2.The recombinant Pc-TUSC2 potein was highly expressed in E.coli with a concentration of approximately 1 mg/ml,and the purified protein was used to prepare the polyclonal antibody(titer of the antibody was 1:6400).3.A hemocyanin protein interacting with TUSC2 protein was identified through the pull-down assay.4.TUSC2 was expressed in all examined tissues including hepatopancreas,gut,muscle,gill,heart,stomach and hemolymph,with the highest expression level in gill.5.The expression levels of TUSC2 ene have significant changes among hemocytes,gills and intestines of P.clarkii stimulated by LPS and poly(I:C)has significant changes,and the poly(I:C)compared with controls.The recombination TUSC2 protein induction and knockdown of Pc-TUSC2 gene by ds RNA transfection significantly induced the the expression of immune-related genes such as Cyt C、p53、Bcl、BI、Ras、Raf and Hemocyanin genes in hemocytes and gills.