猪源CXCL17的原核表达及其对猪巨噬细胞的趋化作用  

Prokaryotic expression of porcine CXCL17 and its chemotactic effect on porcine macrophages

作　　者：齐佳欣 钟秋 刘芮伶 郑健 李昱辰 杨倩[1] QI Jiaxin;ZHONG Qiu;LIU Ruiling;ZHENG Jian;LI Yuchen;YANG Qian(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2024年第5期932-940,共9页Journal of Nanjing Agricultural University

基　　金：江苏省自然科学基金青年基金项目(BK20240198,BK20200536)。

摘　　要：[目的]本研究旨在探究CXC趋化因子配体17(CXC motif chemokine ligand 17,CXCL17)对巨噬细胞的趋化作用并解析其分子机制。[方法]构建表达猪CXCL17蛋白的重组菌,该菌经IPTG诱导后高效表达CXCL17蛋白,并主要以包涵体形式存在;包涵体经过变性、纯化和复性获得重组蛋白,利用SDS-PAGE和Western blot对纯化的CXCL17蛋白进行验证。通过CCK-8法筛选CXCL17蛋白对猪巨噬细胞的最佳作用浓度范围,并以此设置浓度梯度处理猪巨噬细胞,用Transwell试验检测CXCL17对巨噬细胞的趋化活性。通过免疫荧光和Western blot检测细胞微丝骨架的变化及相关通路的激活情况,以揭示CXCL17趋化巨噬细胞迁移的作用机制。[结果]重组菌在IPTG终浓度为1.0 mmol·L^(-1)及37℃诱导条件下表达7 h,获得高表达量的重组猪CXCL17蛋白。该蛋白纯化后的纯度可高达95%。重组猪CXCL17蛋白可以剂量依赖诱导猪巨噬细胞迁移,其机制为通过激活微丝骨架下游的LIMK/Cofilin信号通路引起细胞微丝骨架重排,促进细胞微丝骨架解聚、聚合,从而有利于巨噬细胞迁移。[结论]本研究重组表达了猪CXCL17蛋白,并发现CXCL17蛋白调控细胞微丝骨架诱导猪巨噬细胞迁移的分子机制,为CXCL17作为黏膜免疫增强剂提供了试验依据。[Objectives]The aim of this study was to examine the chemokine impact of CXC motif chemokine ligand 17(CXCL17)on macrophages and to elucidate its molecular mechanism.[Methods]The methods involved constructing a recombinant bacterium expressing porcine CXCL17 protein,inducing its expression with IPTG to efficiently produce CXCL17 protein primarily in the form of inclusion bodies.The recombinant protein was obtained through denaturation,purification,and renaturation of inclusion bodies,with the purified CXCL17 protein confirmed by SDS-PAGE and Western blot analysis.The study utilized the CCK-8 method to identify the optimal concentration range of CXCL17 protein for porcine macrophages,employing a concentration gradient in the treatment process.The chemotactic activity of CXCL17 on macrophages was assessed through the Transwell test.The alterations in the microfilament skeleton and activation of associated pathways were examined using immunofluorescence and Western blot techniques to elucidate the mechanism underlying CXCL17-induced macrophage migration.[Results]High expression of recombinant porcine CXCL17 protein was achieved with a final concentration of 1.0 mmol·L^(-1) IPTG at 37℃for 7 h.The purity of the protein after purification reached as high as 95%.Furthermore,the recombinant porcine CXCL17 protein had been shown to induce the migration of porcine macrophages in a dose-dependent manner.This effect was primarily mediated by inducing rearrangement of the microfilament skeleton through activation of the LIMK/Cofilin signaling pathway downstream,thereby promoting depolymerization and polymerization of the microfilament skeleton and facilitating macrophage migration.[Conclusions]This study successfully demonstrated recombinant expression of porcine CXCL17 protein and elucidated its molecular mechanism in regulating cell microfilament skeleton to induce porcine macrophage migration.These findings provide an experimental basis for considering CXCL17 as a potential mucosal immune enhancer.

关 键 词：CXC趋化因子配体17 原核表达 巨噬细胞 迁移 黏膜免疫 

分 类 号：S852.4[农业科学—基础兽医学]