马源白介素受体拮抗因子的表达及其体外抗炎机制的研究  

Expression of equineinterleukin receptor antagonist factor IL-1Ra and theresearch onits anti-inflammatory mechanism in vitro

作　　者：李柄霖 李佳欣 郭兴 林跃智[1] 王晓钧[1] LI Bing-lin;LI Jia-xin;GUO Xing;LIN Yue-zhi;WANG Xiao-Jun(Equine Infectious Diseases and Lentiviral Diseases Research Group,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室/马传染病与慢病毒病创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2025年第2期185-193,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金引导项目(LH2023C049);自然科学基金面上项目(32372985)。

摘　　要：白介素受体拮抗因子(IL-1Ra)是细胞因子IL-1家族的主要成员,也是一种重要的抗炎因子。为评估马源IL-1Ra在马属动物中的抗炎潜力,本研究构建原核表达质粒p GEX-6p-1-IL-1Ra,经测序鉴定正确后转化大肠杆菌BL21(DE3),于不同温度下经IPTG诱导表达后,利用GST标签蛋白纯化试剂盒纯化后通过SDS-PAGE检测重组IL-1Ra蛋白(rIL-1Ra)的表达及纯化效果。结果显示,在16℃、25℃、37℃诱导后,均在43 ku处出现目的条带,且纯化后的目的条带单一。通过流式细胞术检测不同浓度rIL-1Ra对巨噬细胞凋亡的影响;采用单荧光素酶试验检测不同浓度rIL-1Ra在各时间点对IL-1β激活的NF-κB荧光素酶报告系统活性的影响(由各实验组与阴性对照组荧光值的比值确定);采用间接免疫荧光试验(IFA)检测rIL-1Ra对NF-κB信号通路中P65蛋白细胞内定位的影响;通过荧光定量PCR(qPCR)检测rIL-1Ra对LPS诱导炎症细胞因子转录水平的影响。流式细胞术结果显示,不同浓度rIL-1Ra作用的马巨噬细胞凋亡比例均约2%,与阴性对照组均无显著差异。证明不同浓度r IL-1Ra对马巨噬细胞的凋亡无影响。单荧光素酶试验结果显示,作用4 h后,与IL-1β+PBS组相比,IL-1β+rIL-1Ra组与阴性对照组细胞荧光值的比值均极显著降低(P<0.0001),且当rIL-1Ra浓度≥1.4 ng/m L时,与IL-1β+PBS组相比,IL-1β+rIL-1Ra组与阴性对照组细胞荧光值的比值均显著和极显著降低(P<0.05、P<0.001、P<0.0001)。IFA结果显示,与阴性对照组相比,加入IL-1β后细胞核内的红色荧光强度提高并聚集,且随着IL-1β剂量的增加荧光逐渐增强。但IL-1β+rIL-1Ra组细胞核中的荧光减弱并逐渐消失。qPCR检测结果显示,与阴性对照组相比,在LPS刺激后细胞中IL-6、IL-1β、TNF-α的转录水平均极显著升高(P<0.01、P<0.001、P<0.0001)。但在LPS作用后4 h~8 h加入不同浓度(250 ng/m L~2000 ng/m L)rIL-1Ra的细胞中上述细胞因子的转录水平均�Interleukin-1 receptor antagonist(IL-1Ra)is a major member of the IL-1 cytokine family and an important anti-inflam-matory factor.To evaluate the anti-inflammatory potential of equine-derived IL-1Ra in equids.This study constructs the prokaryotic expression plasmid pGEX-6p-1-IL-1Ra.After sequence verification,the plasmid was transformed into E.coli BL21(DE3).The recombinant IL-1Ra protein(rIL-1Ra)was expressed under IPTG induction at different temperatures(16°C,25°C,and 37℃).The rIL-1Ra was purified using a GST-tagged protein purification kit and analyzed by SDS-PAGE to assess expression and purification efficiency.The results showed that the target protein appeared at 43ku after induction at all three temperatures,and the purified protein exhibited a single distinct band.Flow cytometry was used to assess the effect of different concentrations of rIL-1Ra on macrophage apoptosis.A single-luciferase assay was conducted to evaluate the effect of various concentrations of rIL-1Ra on IL-1β-activated NF-κB luciferase reporter activity at different time points,determined by the fluorescence intensity ratio between experimental and negative control groups.An indirect immunofluorescence assay(IFA)was performed to analyze the intracellular localization of the P65 protein in the NF-κB signaling pathway upon rIL-1Ra treatment.Additionally,fluorescence quantitative PCR(qPCR)was used to measure the effect of rIL-1Ra on the transcriptional levels of inflammatory cytokines induced by lipopolysaccharide(LPS).Flow cytometry results showed that the apoptosis rate of equine macrophages treated with different concentrations of rIL-1Ra was approximately 2%,and there were no significant differences between the treatment group and the negative control group,suggesting that rIL-1Ra does not affect equine macrophage apoptosis.The single-luciferase assay demonstrated that after 4 hours of treatment,compared to the IL-1β+PBS group,the fluorescence intensity ratio of the IL-1β+rIL-1Ra group to the negative control group was signific

关 键 词：马源IL-1Ra 马属动物 抗炎作用 基因克隆 原核表达 

分 类 号：S858[农业科学—临床兽医学]