鲤irisin多克隆抗体的制备及应用

Preparation and application of polyclonal antibody of irisin of Cyprinus carpio

为进一步研究鱼类糖代谢与鸢尾素(irisin)之间的关系,亟需制备irisin抗体并检测其应用可靠性。本实验通过构建Rosetta-irisin表达载体,经蛋白纯化、透析、超滤后免疫小鼠,获得相应多克隆抗体并检测其特异性和抗体效价。建立irisin检测方法,检测口服葡萄糖耐量(OGTT)、RNAi实验后鲤血清irisinA和irisinB的含量变化。免疫荧光检测鲤脑、肠道、心脏、肝胰脏irisin的表达及OGTT后,检测上述组织irisin含量变化。检测鲤肌细胞分化前后FNDC5 mRNA表达差异及irisin含量变化。结果显示,Rosetta-irisin表达载体在IPTG诱导4 h对鲤irisinA/B蛋白表达较BL21-irisin表达载体提高2.3/1.6倍;irisinA和irisinB抗体效价分别为9.0×10^(4)和2.7×10^(5),不存在交叉反应,可分别对鲤血清irisinA和irisinB进行测定;OGTT后,irisinA和irisinB呈现出不同的合成变化;RNAi后,irisin含量显著降低;免疫荧光结果显示,irisin在脑中表达最高,肝胰脏次之,心脏、肠道中相对较少;OGTT后,脑、肠道、心脏和肝胰脏中irisinA和irisinB荧光强度显著增加。荧光定量表达分析与免疫荧光结果显示,鲤肌细胞分化后,FNDC5和irisin含量显著降低。综上,本实验制备了高亲和力和特异性的鲤irisinA和irisinB抗体,并检测其应用可靠性。该抗体的获得为系统研究irisin对鲤糖代谢的调控机制奠定基础。同时,鲤irisin检测方法可普遍用于其他鱼类irisin蛋白质水平的定量研究。

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 9.0×10^(4) and 2.7×10^(5),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 differentiation.In this experiment,high affinity and specific irisinA and irisinB antibodies were prepared,and their application 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.