马M2型CD163单克隆抗体的制备及其在巨噬细胞极化类型检测中的应用  

Preparation of M2-type CD163 monoclonal antibody and its application in detecting the polarization type of macrophages

作　　者：段盈伊 王欣慧 陈克伟 刘荻萩[1] 戚亭[1] 郭奎 杜承[1] 王晓钧[1] DUAN Ying-yi;WANG Xing-hui;CHEN Ke-wei;LIU Di-qiu;QI Ting;GUO Kui;DU Cheng;WANG Xiao-jun(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,ChineseAcademy ofAgricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/马传染病和慢病毒病研究创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2020年第11期1159-1166,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然基金面上项目(31772720);黑龙江省自然科学基金面上项目(C2018071)。

摘　　要：本研究分别采用M1型诱导剂LPS+IFN-γ,M2型诱导剂IL-4或者IL-10刺激马巨噬细胞后,经荧光定量PCR检测巨噬细胞中5种细胞因子和3种表面标记物的转录水平,以确定M1和M2型马巨噬细胞的极比标志物和分泌的细胞因子。为方便快捷地检测马巨噬细胞经不同病原感染后的极化表型,本实验截取马巨噬细胞极化标志物CD163基因的SRCR1~SRCR4区域,并分别经原核和真核表达系统表达该重组蛋白CD163。将原核表达的马CD163蛋白免疫小鼠制备CD163的单克隆抗体(MAb),经western blot和激光共聚焦检测该MAb的反应性。以上述实验为基础,采用荧光定量PCR检测不同病原感染巨噬细胞后的3种极化标志物和3种细胞因子基因的转录水平以确定感染后的巨噬细胞的极化表型;将不同病原感染马巨噬细胞24 h后,利用制备的CD163 MAb分别采用流式细胞术检测表达CD163蛋白的马巨噬细胞数量,以及采用western blot检测不同病原感染巨噬细胞后的CD163蛋白的表达量,以进一步鉴定马巨噬细胞的极化状态。荧光定量PCR结果表明,经LPS+IFN-γ诱导后,马巨噬细胞向M1型发生了极化,TNF-α、IL-1β、IL-12、CD80基因可以作为鉴定M1型马巨噬细胞的标记基因;而经IL-4或者IL-10诱导后,马巨噬细胞向M2型发生了极化,TGF-β、IL-10、CD206、CD163基因可以作为鉴定M2型马巨噬细胞的标记基因。MAb western blot和激光共聚焦结果显示,该MAb与真核表达的CD163蛋白以及经IL-10诱导后马巨噬细胞表达的内源性CD163蛋白均能够很好反应,可以用于马巨噬细胞极化状态的检测。荧光定量PCR结果显示,马流产沙门氏菌(S.Abortus equi)、马疱疹病毒(EHV-1)和马动脉炎病毒(EAV)感染马巨噬细胞24 h后,M1型细胞因子TNF-α、IL-1β和其表面标记物CD80基因mRNA的转录水平均显著上调(p<0.05),而EIAV感染24 h后,M2型细胞因子IL-10和其表面标记物CD206、CD163基因的mRNA的转录水平均不同程�In this study,M1-type inducer LPS+IFN-γ,M2-type inducer IL-4 or IL-10 were used to stimulate the equine macrophages,and the transcription levels of cytokines and surface markers in macrophages were detected by fluorescence quantitative PCR,respectively.By this way,the cell polarization markers and cytokines of M1 and M2 type horse peripheral blood macrophages will be identified.In order to quickly and conveniently detect the polarization phenotypes of horse macrophages during the infections with different pathogens,the SRCR1-SRCR4 regions of the polarization marker CD163 gene of horse macrophages were selected in this study,and the recombinant CD163 protein was expressed through prokaryotic and eukaryotic expression systems,respectively.Monoclonal antibody(MAb)to CD163 was prepared by immunizing mice with prokaryotic expression of horse CD163 protein,and the reactivity of the MAb was detected by western blot and laser confocal.Based on the above experiments,real time PCR was used to detect the transcription levels of 3 polarization markers and 3 cytokine genes of macrophages infected with different pathogens to determine the polarization phenotypes of the infected macrophages.After 24 hours of infection with different pathogens on horse macrophages,the prepared CD163 MAb was used to detect the number of horse macrophages expressing CD163 protein by flow cytometry,and the expression level of CD163 protein was detected by western blot,so as to further identify the polarization state of horse macrophages.Real time PCR results showed that the polarization state polarized towards the M1 after the induction of LPS+IFN-γ,TNF-α,IL-1β,IL-12 and CD80 genes were determined as marker genes to identify M1-type horse macrophages.While the polarization state polarized towards the M2 after the induction of IL-4 or IL-10,TGF-β,IL-10,CD206 and CD163 genes were determined as marker genes to identify M2-type horse macrophages.MAb western blot and laser confocal results showed that the MAb could react well with eukaryotic CD163

关 键 词：马 M2型巨噬细胞 CD163蛋白 单克隆抗体 

分 类 号：S858.21[农业科学—临床兽医学]