| In order to study the effect of acute cold stress on the expression of the cytokines in Chinese soft-shelled turtle,il21 and il22 cDNA were cloned by using cDNA end rapid amplification and bioinformatics analysis was further carried out.Based on these,the expression patterns of il21 and il22 mRNA were detected in splenic primary cells stimulated by LPS,Con A and poly I:C in Chinese soft-shelled turtle.Moreover,the mRNA levels of il1?,tnf?,il8,il21,il22 and il23 a in spleen and intestine were determined,when Chinese soft-shelled turtles were challenged with bacterial during acute cold stress.Results:1.Cloning and bioinformatics analysis of il21 and il22 cDNAThe cDNA of il21 gene was 874 bp,and ORF length was 408 bp,encoding 135 amino acids.Secondary structures mainly include alpha helix,extended chain,random curl and beta angle,and tertiary structures include signal peptide and IL15 domain.The results of phylogenetic analysis demonstrated that it clustered with the birds' ones first,followed by mammal ones.The il22 cDNA length was 2263 bp,and ORF length was 624 bp,encoding 207 amino acids.Secondary structure mainly includes the alpha helix,extending chain,random curl and beta angle,and tertiary structure includes the signal peptide,transmembrane domain and IL22 domain structure.The phylogenetic analysis showed that it clustered with the other reptiles first,followed by birds.2.The constitutive expression of il21 and il22 mRNA in selected tissues and their inductive expression in primary spleen cells after immune stimulationTissue expression pattern analysis showed that il21 and il22 mRNA have the highest levels of expression in spleen,and followed by brain and intestine,while the expression of il22 mRNA was not higher in brain and intestine.The expression of il21 mRNA was significantly up-regulated in spleen cells at 3h(p=0.0022),6h(p=0.0022),12h(p=0.0022)and 24h(p=0.0043)after Con A stimulation,with the highest expression at 3h.The expression of il22 was significantly down-regulated in spleen cells at 6h(p=0.0043),12h(p=0.0152)and 24h(p=0.0043)after Con A stimulation.3.The effect of acute cold stress on the expression selected cytokines of Chinese soft-shelled turtleThe mRNA level of il22 in intestinal showed a trend that first increase and then decrease in the Aeromonas hydrophila group at room temperature,the highest expression of il22 mRNA was observed at 24 h.The mRNA level of il22 in intestinal showed a trend that gradually increase in the Aeromonas hydrophila group under the acute cold stress,the highest expression of il22 mRNA was observed at 72 h.The mRNA level of il23 a in spleen showed a trend that first decrease and then increase in the Aeromonas hydrophila group under the acute cold stress,the highest expression of il23 a mRNA was observed at 12 h after challenge In the normal temperature group(control),the mRNA level of il23 a in spleen showed a trend that first increase then decrease and then increase,the highest expression of il23 a mRNA was observed at 72 h after challenge.Acute cold stress reduces the expression of intestinal and spleen cytokines(il1?,tnf? and il8)at early stages of stress,and subsequently increases the expression of intestinal il1? and il8 at 24 h and tnf? at 7d after cold stress.The expression of il1? and il8 in the spleen increased at 12 h and tnf? at 24 h after cold stress.Conclusions:1.The sequences of il21 and il22 cDNA were obtained,and bioinformatic analysis demonstrated that they are mammalian counterparts in Chinese soft-shelled turtle,which laid a solid foundation for the further study.2.Immunostimulant Con A enhanced the expression level of il21 mRNA in spleen cells while weakened il22 mRNA expression,which suggested thatil21 and il22 were involved in the T-cell immune response,and the specific mechanisms needs to be further studied.3.Acute cold stress affected the immune response of cytokines in Chinese soft-shelled turtle infected by Aeromonas hydrophila.The expression of most immune molecules detected showed a phenomenon of "response delay". |
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