摘要
为了探究奶牛乳房炎金黄色葡萄球菌毒力因子的重组蛋白能否经上皮屏障被转运至特定的乳腺局部免疫触发位点,本研究对金黄色葡萄球菌毒力因子重组蛋白FnBPB-ClfA和Fc融合蛋白Fc-FnBPB-ClfA的跨细胞转运机制、新生儿Fc受体(FcRn)是否参与此进程进行了验证。原代奶牛乳腺上皮细胞(pbMECs)接种于Transwell嵌套内的多孔膜上,培养至极性状态且细胞屏障完整,用于胞吞转运试验。将pbMECs对重组蛋白的胞吞转运试验设置为温度处理组(37℃和4℃)、蛋白A处理组、胞转途径抑制剂处理组。结果表明,2种重组蛋白均可被转运跨过pbMECs屏障,该转运作用呈温度依赖性,可能与囊泡介导的转运路径及FcRn受体有关,排除了细胞旁路扩散的可能。蛋白A对FnBPB-ClfA的跨细胞转运及亚细胞定位无明显影响,但明显减少了Fc-FnBPB-ClfA的跨细胞转运及其在细胞膜蛋白、骨架蛋白中的数量,说明Fc-FnBPB-ClfA的Fc与FcRn的结合在胞吞转运作用中发挥重要作用。LY294002、非律平以剂量-效应关系分别降低了FnBPB-ClfA、Fc-FnBPB-ClfA的跨细胞转运。结果表明,Fc融合蛋白Fc-FnBPB-ClfA能经胞吞转运作用跨过pbMECs屏障至局部免疫触发位点,且FcRn参与并促进了Fc融合蛋白的胞吞转运作用。
The objective of present research is to investigate whether recombinant proteins against bovine mastitis-causing Staphylococcus aureus could be transported via transepithelial delivery to certain sites,in where local immune responses could be triggerred.Recombinant protein of FnBPB-ClfA anchoring virulence factors of Staphylococcus aureus and corresponding Fc-fusion format Fc-FnBPB-ClfA were used to testify their transcellular transport and the potential roles of neonatal Fc receptor(FcRn) during this process.In vitro transcytosis assays with polarized primary bovine mammary epithelial cells(pbMECs) monolayers cultivated on porous membrane inserts were performed.Alteration of intracellular localization and transcellular transport of FnBPB-ClfA and Fc-FnBPB-ClfA caused by different tempretures,interfering Fc-FnBPB-ClfA's interaction with the FcRn using staphylococal protein A and two inhibitors of common transcytosis pathways were assessed by Western blot analyses.The uptake and transcellular transport of recombinant proteins were strongly temperature-dependent,which ruled out paracellular passage indicating that binding to a receptor and vesicles seems to be involved.Protein A did not inhibit transcytosis or affected intracellular localization of FnBPB-ClfA but inhibited that of Fc-FnBPB-ClfA and reduced amounts of Fc-FnBPB-ClfA that co-extracted with proteins from membranes/organelles and cytoskeletal proteins suggesting a key role of the FcRn.LY294002 and Filipin Ⅲ reduced transcytosis of FnBPB-ClfA and Fc-FnBPB-ClfA respectively in a dose-effect relation.Taken together,recombinant protein of FnBPB-ClfA and its Fc-fusion format Fc-FnBPB-ClfA could be transported across tight barrier of pbMECs to local immunity-triggering sites via transepithelial delivery,and FcRn is involved in transcytosis of engineering-Fc fusion protein Fc-FnBPB-ClfA with enhanced delivery efficiency.
引文
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