淡水渔业2026，Vol.56Issue(1)：56-65,10.
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
Preparation of polyclonal antibodies against IL-1β and IL-18 in Micropterus salmoides and their expression profiles in response to Nocardia seriolae infection
摘要
Abstract
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.关键词
大口黑鲈(Micropterus salmoides)/IL-1β/IL-18/多克隆抗体/鰤诺卡氏菌(Nocardia seriolae)Key words
Micropterus salmoides/IL-1β/IL-18/polyclonal antibodies/Nocardia seriolae分类
农业科技引用本文复制引用
王欢,曹小娟,高坚,王庆超,陈振威,江藕,简宇清,唐伟俊,王怀池,翟艳花,张瑞轩,赵玉华..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[J].淡水渔业,2026,56(1):56-65,10.基金项目
国家重点研发计划(2023YFD2400600) （2023YFD2400600）
农业农村部中国(广西)-东盟水产种质资源综合开发与利用重点实验室和广西水产育种与健康养殖重点实验室开放课题(GXKEYLA-2023-01-20) （广西）
