大口黑鲈IL-1β和IL-18多克隆抗体制备及鰤诺卡氏菌感染对其表达的影响OA

The interleukin-1(IL-1)family is a group of pro-inflammatory cytokines that regulate inflammato-ry processes involved in innate immune response.IL-1β and IL-18 are two members of the IL-1 family that are most closely linked to inflammatory reactions.Similar to that in mammals,fish IL-1β gene sequence also con-tains a segment that can be cleaved by caspase-1 to produce a mature peptide of 22 kDa,and IL-1β sequences in different fish species exhibited the conserved 3rd to 6th exons.Studies have shown that fish IL-1β can exert immunomodulatory functions by stimulating T lymphocytes,and the intake of recombinant IL-1β protein can enhance fish resistance to bacterial infection.In addition,IL-18 has also been identified in several fish species and participates in inflammatory responses.However,the lack of specific antibodies against fish IL-1β and IL-18 pro-teins has severely limited research on their related functions.Largemouth bass(Micropterus salmoides)is a highly valued freshwater aquaculture fish with significant economic importance.Nevertheless,visceral sarcoidosis caused by Nocardia seriolae infection leads to massive mortality,which seriously restricts the high-quality development of the aquaculture industry.Polyclonal antibodies against largemouth bass IL-1β and IL-18 were prepared to in-vestigate the immune responses of IL-1β and IL-18 in largemouth bass during N.seriolae infection.Meanwhile,histopathology and their protein expression levels in the liver during N.seriolae infection was evaluated.Firstly,bioinformatic analysis was conducted using largemouth bass IL-1β and IL-18 proteins,which identified the 0～259aa segment of IL-1β and the 0-133aa segment of IL-18 as the target expression segments,respectively.pET-32a-IL-1β and pET-32a-IL-18 prokaryotic recombinant plasmids were constructed after PCR amplifica-tion,double enzyme digestion,and ligation.Then,these two recombinant plasmids were transformed into BL21(DE3)competent cells,which were induced for protein expression under optimal conditions(1.0 mmol/L and 0.6 mmol/L IPTG)overnight at 16℃.The soluble recombinant IL-1β and IL-18 proteins were purified with the Ni-NTA Beams 6FF gravity column.Single bands with protein molecular weights of about 49 kDa and 40 kDa were detected by SDS-PAGE,demonstrating the successful acquisition of purified IL-1β and IL-18 proteins.These two purified proteins were then immunised into one Japanese big-eared rabbit and three Balb/C mice to obtain anti-sera,respectively.Enzyme-linked immunosorbent assay indicated that rabbit/mouse-origin anti-sera titers of IL-1β and IL-18 were 1∶1 024×104,1∶1 024×103 and 1∶1 024×104,1∶1 024×103,re-spectively.Moreover,western blotting also showed that these antibodies could specifically recognise both the re-combinant IL-1β and IL-18 proteins,and endogenous largemouth bass proteins,with molecular weights of 49 KDa/40Da and 29 KDa/23 KDa,respectively.Thus the prepared antibodies against largemouth bass IL-1β and IL-18 proteins in the present study is suitable for subsequent analysis with reasonable specificity and high ti-ters.Then largemouth bass was infected with N.seriolae to detect the regulatory role of IL-1β and IL-18.The vis-ible cuboidal hepatocytes can be detected in the liver of the control group,with round cell nuclei,a cord-like structure,and other reticular fibres and connective tissue.However,an obvious granuloma can be observed in the liver of largemouth bass after N.seriolae infection,which exhibits the typical three-layer structure and infiltra-tion of inflammatory cells.Moreover,the protein levels of IL-1β and IL-18 in the infected largemouth bass liver showed a significant increase using the polyclonal antibody prepared in this study,which was consistent with histopathological results.In conclusion,the present study successfully prepared polyclonal antibodies against largemouth bass IL-1β and IL-18,and also detected increased protein expression of these antibodies during N.seriolae infection,thus providing an important basis for further research on the inflammation of largemouth bass under bacterial infection.