肺炎链球菌候选蛋白疫苗DnaJ-△A146Ply通过PI3K/Akt和NF-κB信号通路刺激巨噬细胞诱导免疫应答  

Pneumococcal Fusion Protein Dna J-△A146Ply Induces the Immune Response of Peritoneal Macrophage through PI3K/Akt and NF-κB Signaling Pathways

作　　者：粟玉凤 李达根 邢燕[1] 姚华[1] 吴静雯[1] 张雪梅[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016 [2]重庆市长寿区人民医院检验科,重庆401220

出　　处：《基因组学与应用生物学》2016年第4期768-774,共7页Genomics and Applied Biology

基　　金：国家自然科学基金(No.81571622);重庆市科委基金(No.cstc2015jcyj A0050)共同资助

摘　　要：本实验目的是研究肺炎链球菌r Dna J-△A146Ply融合蛋白引起小鼠巨噬细胞的免疫应答机制。原核表达r Dna J-△A146Ply重组蛋白,Ni+柱纯化后去除其内毒素。r Dna J-△A146Ply刺激小鼠来源腹腔巨噬细胞6 h后,提取细胞RNA,RT-PCR检测白细胞介素6(interleukin-6,IL-6)、肿瘤坏死因子(tumor necrosis factor-α,TNF-α)m RNA表达水平。24 h后取细胞培养上清,ELISA检测IL-6和TNF-α蛋白表达水平。信号通路抑制剂预处理腹腔巨噬细胞1 h后加蛋白刺激,ELISA检测上清中IL-6、TNF-α蛋白表达水平抑制情况。取不同时间点处理的细胞进行免疫印迹(Western Blot),检测p-Akt和p-NF-κB表达水平。制备获得纯度90%以上、内毒素含量小于0.1 EU/μg的r Dna J-△A146Ply融合蛋白;r Dna J-△A146Ply可刺激腹腔巨噬细胞IL-6、TNF-αm RNA及蛋白水平表达明显上调、增强Akt和NF-κB磷酸化;Akt和NF-κB抑制剂可显著减少IL-6和TNF-α表达。因此,r Dna J-△A146Ply融合蛋白通过Akt和NF-κB信号通路刺激巨噬细胞诱导免疫应答。This experiment aimed to explore the mechanism of immune response induced by pneumococcal fusion protein r Dna J-△A146Ply about mouse peritoneal macrophage. Prokaryotic expression r Dna J-△A146Ply reconstructed protein, of which LPS was removed after Ni＋column purification. The mouse peritoneal macrophages were stimulated by r Dna J-△A146Ply for 6 hours, then RNA of cells was extracted, m RNA level of IL-6,TNF-α were detected by RT-PCR. Supernatant was cultured after 24 hours, then expression levels of IL-6 and TNF-α protein in supernatant were measured by ELISA. Peritoneal macrophages were pre-treated by signaling pathway inhibitors for 1 hour, which were stimulated by protein later, then inhibit expression of IL-6 and TNF-αwas evaluated by ELISA. The phosphorylation levels of Akt and NF-κB were detected by western blot with cells processed in different time. r Dna J-△A146Ply fusion protein with more than 90% purity and less than 0.1 EU/ug LPS was obtained. r Dna J-△A146Ply significantly enhanced both the m RNA level and the protein expression of IL-6 and TNF-α in macrophage; It also advanced the phosphorylation levels of Akt and NF-κB of macrophage;And the inhibitors of Akt and NF- κB obviously decreased the expression of IL-6 and TNF-α. Therefore,Pneumococcal fusion protein r Dna J-△A146Ply induced the immune response of peritoneal macrophage through Akt and NF-κB signaling pathways.

关 键 词：肺炎链球菌Dna J-△A146Ply 巨噬细胞 Akt NF-κB 

分 类 号：R392[医药卫生—免疫学]