水产学报2023，Vol.47Issue(12)：1-16,16.DOI:10.11964/jfc.20220713612
鲤irisin多克隆抗体的制备及应用
Preparation and application of polyclonal antibody of irisin of Cyprinus carpio
摘要
Abstract
To further study the relationship between fish glucose metabolism and irisin,the irisin antibody and its application reliability need to be prepared and tested urgently.The Rosetta-irisin expression vector was construc-ted,and the mice were immunized after protein purification,dialysis,and ultrafiltration.The corresponding poly-clonal antibodies were obtained and their specificity and antibody titers were detected.The common carp irisin detection method was established to detect the changes of irisinA and irisinB in carp serum after glucose tolerance and RNAi experiments.The immunofluorescence method was used to detect the expression of irisin in carp brain,heart,hepatopancreas,and intestine,and the changes of irisin content in these tissues after glucose tolerance.Detection of the irisin content after carp myocytes differentiation was conducted.Compared with the BL21-irisin expression vector,the expression of carp irisinA/B protein by the Rosetta-irisin expression vector increased by 2.3/1.6 times at 4 h of IPTG induction;irisinA and irisinB antibody titers were 9.0×104 and 2.7×105,respectively.And there was no cross-reaction between irisinA and irisinB polyclonal antibodies,the irisinA and irisinB can be measured respectively.IrisinA and irisinB contents showed different changes after OGTT.After RNAi,irisin was significantly decreased.Immunofluorescence results showed that irisin was the most abundant in the brain,fol-lowed by hepatopancreas,and relatively less in the heart and intestine.After OGTT,the fluorescence intensity of irisinA and irisinB in the brain,liver,heart,and gut increased significantly.The results of qPCR and immunofluor-escence showed that the FNDC5 mRNA expression and irisin decreased significantly after carp myocyte differenti-ation.In this experiment,high affinity and specific irisinA and irisinB antibodies were prepared,and their applica-tion reliability was tested.The acquisition of this antibody laid the foundation for the systematic study of carp gluc-ose metabolism.At the same time,the irisin detection methods could be widely used in the quantitative study of irisin protein levels in other fish.关键词
鲤/irisinA/irisinB/多克隆抗体/糖代谢/肌细胞Key words
Cyprinus carpio/irisinA/irisinB/polyclonal antibody/glucose metabolism/myocytes分类
生物科学引用本文复制引用
职韶阳,杨丽萍,秦超彬,闫潇,张文蕾,刘茗宇,赵梦娟,聂国兴..鲤irisin多克隆抗体的制备及应用[J].水产学报,2023,47(12):1-16,16.基金项目
国家自然科学基金(31872581,31902384,31902386) （31872581,31902384,31902386）
中原千人计划河南省中原科技领军人才(204200510025) （204200510025）
河南师范大学优秀科技创新团队支持计划(2020TD02) （2020TD02）
河南省现代农业产业技术体系专项(HARS-22-16-G2) National Natural Science Foundation of China(31872581,31902384,31902386) （HARS-22-16-G2）
Zhongyuan Thousand Talents Plan Leading Talents of Zhongyuan Science and Technology of Henan Province(204200510025) （204200510025）
Innovation Scientists and Technicians Troop Construction Projects of Henan Normal University(2020TD02) （2020TD02）
Special Fund for Henan Agriculture Research System(HARS-22-16-G2) （HARS-22-16-G2）
