| As crucial cytokines,IL-10 family members play roles not only in the proliferation,differentiation and activation of immunocytes but also in inflammation and proinflammation responces.In this study,5 of IL-10 family genes(AjIL-10 a,AjIL-10 b,AjIL-20,AjIL-22 and AjIL-26)had been cloned from Japanese eel and their structure characteristics were analysised,the results are showed as follow:1)The Japanese eel IL-10 family genes shared similar structures with homologous in other vetebrates,the ORF are all consist of 5 exons and 4 introns,while AjIL-20 and AjIL-22 contained another exon and intron in the 5‘-UTR.The IL-10 family signature were conserved in all IL-10 family members of Japanese eel.AjIL-20 completely conserved the same 6 Cysteines in mammals;AjIL-10 a and AjIL-10 b got 2 more Cysteines which are conserved only in teleosts than mammals;AjIL-22 had the same 4 Cysteines as mammals except the previous 2 are in different position;AjIL-26 had only2 Cysteines while mammal IL-26 s had 4.2)The transcriptional expression of AjIL-10 a,AjIL-22 and AjIL-26 had been detected in all tissues from Japanese eel with higher AjIL-10 a expression in blood,head kidney and middle kidney and higher AjIL-22 and AjIL-26 in mucous tissues,such as intestine,gill and stomach,and skin.What‘s more,the expression of AjIL-10 a in gill,head kidney,intestine and spleen as well as AjIL-22 and AjIL-26 in intestine are all up-regulated post intraperitoneal injection of Poly I:C or edwardsiella tarda,suggesting a role in defencing against virus and bacteria.3)TFBSs common in cytokines,such as Sp1,AP-1,Oct-1,GATA-1,C/EBP?,NF-?B and GR(AjIL-22 excepted)as well as binding site for transcrition repressor YY1 were found in the 5‘ regulatory sequences of these 5 genes.And bingding sites of transcription factors relating to interferon induction(ISGF-3,ICSBP and IRF-1)are found in the regulatory sequences of AjIL-10 a,AjIL-10 b and AjIL-22.4)The recombinant plasmids for prokaryotic expression of all these genes aforementioned had been constructed,and the recombinant plasmids of PQE30-AjIL-22 and PQE30-AjIL-26 were induced to expression,with the result showed that the two were all expressioned as inclusion bodies.Meanwhile,two type II interferons,named AjIFN-? and AjIFN-?rel separately,were characterized from Japanese eel.Similar to mammal IFN-?,AjIFN-? had a 4-exon/3-intron gene structure as well as a typical IFN-? motif and a NLS(RRRR)while AjIFN-?rel has a 3-exon/2-intron gene structure and a partial conserved IFN-? motif,and moreover,the NLS is absent.AjIFN-? transcriptional expression is detected in different tissues of Japanese eel with the highest expression level detected in liver,skin and spleen.The expression level of AjIFN-? was elevated post stimulation by Poly I:C or Edwardsiella tarda,indicating a role in defense against viruses and bacteria.The analysis of the promoter activities of these truncated constructs demonstrates that the-240/+136 region of AjIFN-? promoter is essential for the initial of AjIFN-? transcription,and the-1062/-814 region may contain positive transcriptional regulatory elements while the-1252/-1062 region negative. |
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