Molecular Cloning And Expression Analysis Of NF-κB Family Genes From Golden Pompano(Trachinotus Ovatus)

Nuclear factor-kappa B(NF-κB)proteins include NF-κB1,NF-κB2,Rel A,Rel B and c-Rel subunits,which are capable of forming dimers to function as transcription factors.These subunits can bind to NF-κB binding sites on target genes and then regulate the expression of target genes,which influence a broad range of biological processes including immune response,apoptosis and cancer.Golden pompano(Trachinotus ovatus)has become an economically important farming fish in south China,and the increasing emergence of diseases results in huge economic loss in the golden pompano aquaculture industry annually.Identification and analysis of immune-related molecules,their structures and immune response in Golden pompano will provide scientific reference for developing new immune strategies for the diseases control and prevention.Based on the significance of NF-κB signaling pathway in the body,in this study we cloned the genes of NF-κB family from Trachinotus ovatus(Tro NF-κB),analyzed the structure of their coding proteins and evolutionary relationship.Additionally,the genes expression distribution in tissues and its changes after stimulation were analyzed.Finally,TroRelB recombinant protein was purified and its polyclonal antibodies were prepared.The major results are summarized as follows:1.NF-κB family genes(Tro NF-κB1,Tro NF-κB2,Tro Rel A,TroRelB and Troc-Rel)from Trachinotus ovatus were cloned by RT-PCR and RACE techniques,and their full-length c DNA were 4215 bp,3836 bp,3167 bp,2781 bp and 3501 bp with ORFs of 2826 bp,2715 bp,1866 bp,1728 bp and 1947 bp,encoding 941,904,621,575 and 648 amino acids,respectively.Tro NF-κB1,Tro NF-κB2,Tro Rel A,TroRelB and Troc-Rel proteins contained 1 RHD domain and 1 IPT domain,and other domains such as DD domain and ANK domains present in Tro NF-κB1 and Tro NF-κB2 proteins.The results of homology comparison showed that the homologies of Tro NF-κB1,Tro NF-κB2,Tro Rel A,TroRelB and Troc-Rel proteins with other fishes were 81.2-88.4%,83.8-95.1%,86.0-93.5%,62.3-87.3% and 83.5-91.2%,respectively.Phylogenetic tree analysis showed that the proteins mentioned above were clustered in one branch with their counterparts from other fishes,respectively.These results indicate that the Tro NF-κB family genes are relatively conserved during evolution and infer that their corresponding functions are also relatively conserved.2.The expression of NF-κB family genes in heart,liver,spleen,head kidney,skin,brain,gills,stomach,intestine and muscle of healthy Trachinotus ovatus was detected by q PCR.The Tro NF-κB family genes were constitutively distributed in the tissues,indicating that they were involved in basic biological activities.The expression levels of these genes was not the same in different tissues,Tro NF-κB1,TroRelB and Troc-Rel were the highest expression in the spleen.Tro NF-κB2 and Tro Rel A were the highest expressed in the liver.The minimum level of them was observed at the skin tissue.The expression of these five genes was higher in immune-related tissues,suggesting that Tro NF-κB family genes are involved in the regulation of innate immune response.3.After LPS,poly I:C and Vibrio alginolyticus challenge,the expression of five genes of Tro NF-κB family was not significantly changed in muscle tissues,while significantly up-regulated in liver,spleen and head kidney(P<0.05),the specific expression changes was as follows:(1)After challenged by LPS,the expression of Tro NF-κB1 in liver was significantly up-regulated at 6h and 24 h,the expression in spleen and head kidney were significantly up-regulated at 6 h and 24 h after challenge.The time points of Tro Rel A expression significantly up-regulated were similar to Tro NF-κB1,except that Tro Rel A expression was not significantly up-regulated in head kidney at 12 h after injection.The expression of Tro NF-κB2,TroRelB and Troc-Rel in liver,spleen and head kidney were strikingly up-regulated at 12 h after challenge.Tro NF-κB2expression in head kidney and liver were respectively increased to the peak at 6h and 24 h,Troc-Rel expression in head kidney and spleen were respectively peaked at 6 h and 24 h post injection,TroRelB expression situation is similar to Troc-Rel,except that the expression level of TroRelB in head kidney was prominently up-regulated at 24 h post stimulation.(2)After Poly I:C stimulation,Tro NF-κB1 expression in spleen and liver respectively increased to the peak at 6h and 12 h post stimulation,and that in head kidney was gradually up-regulated at 6 h and reached the highest level at 24 h.The expression of Tro NF-κB2 in liver and spleen were prominently up-regulated at 6 h after injection and further increased to the peak at 12 h,and expression in head kidney peaked at 12 h.Tro Rel A expression pattern was similar to Tro NF-κB2,except that in head kidney was also significantly up-regulated at 6 h after stimulation and reached the maximum value at 24 h.The expression of TroRelB in liver and head kidney were significantly increased to the peak at 12 h after stimulation,while that in spleen rapidly peaked at 6 h post injection,then decreased at 12 h and significantly increased at 24 h.The expression of Troc-Rel in liver,spleen and head kidney were strikingly up-regulated at 24 h after challenge,and the peak expression in liver was observed at 48 h.(3)After injection with Vibrio alginolyticus,the expression of Tro NF-κB1 in liver reached the peak at 6 h,and was significantly up-regulated in liver,spleen and head kidney at 12 h and 24 h after challenge.Tro NF-κB2 expression in head kidney,liver and spleen respectively reached the highest level at 6 h,24 h and 24 h after stimulation,and that was strikingly up-regulated in liver and head kidney at 12 h.The expression of Tro Rel A in liver,spleen and head kidney respectively were significantly up-regulated and reached the maximum value at 24 h,12 h and 24 h post stimulation.TroRelB expression profile was similar to Tro NF-κB2,except that TroRelB expression in liver was not increased at 24 h post injection.The expressions of Troc-Rel in spleen,head kidney and liver were respectively prominently up-regulated at 6h,12 h and 24 h,and the expressions were respectively increased to the peak at the next progressive time point.The Tro NF-κB genes play a key role in regulating the immune response against pathogenic microorganisms.4.The expression vector pET-32a-Rel B was constructed successfully and expressed in E.coli BL21.The fusion protein was obtained with a molecular weight of 84 k Da.The polyclonal antibodies were successfully prepared by immunizing mice with the purified protein.The antibody titer reached 1:256000tested by ELISA and the result of Western blot indicated that they can specifically bind to Rel B fusion protein.The purification of TroRelB fusion protein and preparation of anti-TroRelB polyclonal antibodies lays a foundation for studying TroRelB structure and function.