Molecular Cloning Of Foxp3a And Foxp3b CDNA From Zebrafish And Analysis Of Their Expression Profiles In Response To Bacterial Challenge

Regulatory T(Treg)cells are T cell subsets of CD4+T that control the damage to the body caused by the self immune system.FOXP3 plays a central role in the development and function of Treg cells.Teleost is a lower vertebrate with both acquired and innate immunity,and fish have an immune system similar to mammals,which make it valuable in studying the evolution of immune molecules.It has been reported that bony fish have Tregs with functions similar to mammals,and the homologous molecules of FOXP3 have been characterized in many bony fish,such as pufferfish(Tetraodon nigroviridis),Nile tilapia(Oreochromis niloticus),rainbow trout(Oncorhynchus mykiss),Atlantic salmon(Salmo salar)and zebrafish(Danio rerio)(zebrafish foxp3 with predicted 5’-UTR and 3’-UTR).However,most of the study are limited to spatiotemporal distribution of m RNA level and protein level.There are few studies on the function of FOXP3 in immune response in teleost fish,especially different FOXP3 homologs within a species due to an extral whome genome duplication.To enrich the study of FOXP3 in teleost,the c DNA of the zebrafish foxp3 a and foxp3 b genes(including 5’-UTR and 3’-UTR)were cloned,and bioinformatics analysis was carried out.Then we selected the zebrafish foxp3 a and foxp3 b specific fragments to construct a recombinant prokaryotic expression plasmid carrying the HIS fusion tag,and then expressed and purified the recombinant fusion protein.Rabbit polyclonal antiserum against FOXP3 A and FOXP3 B was prepared after 4 times immunization.The titres and specificity of anti-FOXP3 A and anti-FOXP3 B polyclonal antiserum were respectively determined by indirect ELISA and Western blot.The temporal and spatial expression patterns of foxp3 a and foxp3 b in zebrafish were further studied by semi-quantitative PCR and in situ hybridization(ISH).Moreover,the expression pattern of foxp3 a and foxp3 b in whole kidney was studied by q PCR when zebrafish were intraperitoneally injected with Aeromonas hydrophila.The results shown that:The full length of foxp3 a c DNA of zebrafish is 1819 bp with a 1260 bp ORF.The length of foxp3 b c DNA of zebrafish is 1316 bp with a 1185 bp ORF.The predicted proteins contain the FOXP3 family signature zinc finger structure and forkhead domain.Multiple sequence alignment revealed that FOXP3 A and FOXP3 B amino acids in zebrafish contained zinc finger domain,leucine zipper domain and forkhead domain,which were highly conserved.Phylogenetic analysis indicated that zebrafish FOXP3 A and FOXP3 B first clustered with fish,and then clustered with mammalian.This suggested that zebrafish FOXP3 A and FOXP3 B are orthologous molecules of mammalian FOXP3.With semi-quantitive PCR it was identified that the expression levels of foxp3 a and foxp3 b m RNA changed during the development of embryos.Both foxp3 a and foxp3 b were expressed from 12 hpf,and the expression level of foxp3 a m RNA gradually increases,while the expression level of foxp3 b m RNA was highest at 24 hpf,and then gradually decreased.It was showed that foxp3 a and foxp3 b m RNA were widely distributed in the selected tissues of zebrafish.Both lymphoid and non-lymphoid tissues were found to express foxp3 a and foxp3 b m RNA.WISH was further applied in determined the expression pattern of zebrafish foxp3 a and foxp3 b at 12 and 24 hpf.The specific segments of 1415 bp at the 3’ end of foxp3 a gene and 615 bp at the 3’ end of foxp3 b gene were selected and cloned into p GEM-T-easy vector.Digoxigenin-labeled antisense RNA probes were generated by in vitro transcription.The results of WISH showed that foxp3 a and foxp3 b were widely distributed in the embryonic development at 12 hpf.Their expression gradually concentrated to the mesoderm and the main position of the head.Zebrafish was challenged with Aeromonas hydrophila infection and the results showed that the expression level of il-1β m RNA in zebrafish kidneys was increased compared with PBS group during bacterial infection,showing significant(P=0.026)or extremely significant difference(P=0.002),which demonstrated that it was a successful challengen.The expression level of foxp3a m RNA was significantly increased at 24 h(P=0.017)after bacterial infection,and the expression level at 7 d was significantly lower than that at 24 h(p=0.026);However,the expression level of foxp3 b m RNA at 24 h(P=0.675)and 7 d(P=0.238)after bacterial infection was not significantly different from that in PBS group.A 51.7 k Da S-FOXP3A-HIS fusion protein was formed in a large amount in the form of inclusion bodies with a concentration of 0.5 mmol/L IPTG at 16 °C for 10 hours,and A 49.5 k Da S-FOXP3B-HIS protein was expressed in the supernatant.Rabbits were immunized with the recombinant proteins to produce antiserum.The titers of polyclonal antiserum were all above 1.6×105 which were determined with indirect ELISA.Furthermore,antibody specificity was confirmed by Western blot.In this study,the full-length c DNA of zebrafish foxp3 a and foxp3 b were successfully cloned and the expression patterns of m RNA levels in lymphoid and non-lymphoid tissues were detected.The conserved m RNA level expression pattern of foxp3 m RNA between teleosts and mammalian suggests that it may have a conserved biological function.Moreover,the results of zebrafish foxp3 a and foxp3 b m RNA expression pattern after bacterial infection suggested that foxp3 a may be a functional homolog of mammalian foxp3.