甘露糖化hLYZ/eGFP融合蛋白的表达纯化及巨噬细胞定位

目的利用毕赤酵母表达蛋白具有高甘露糖修饰的特性,在毕赤酵母中表达甘露糖化修饰的人溶菌酶(human lysozyme,hLYZ);进而利用巨噬细胞(macrophage,MR)表面的甘露糖受体(mannose receptor,MR)将甘露糖化的人溶菌酶内吞入胞,为研究可入巨噬细胞杀灭其胞内感染菌的人溶菌酶提供基础。方法为使人溶菌酶可以在酵母中产生N-甘露糖化修饰,在人溶菌酶的C端设计2个N-糖基化位点;通过在其C端融合增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP),观察人溶菌酶在巨噬细胞RAW264.7中的亚细胞定位;同时在其C端引入了His标签便于融合蛋白的纯化。毕赤酵母表达的hLYZ/eGFP与RAW264.7共孵育,借助激光共聚焦显微镜观察该融合蛋白能否进入RAW264.7;通过巨噬细胞甘露糖受体(macrophage mannose receptor,MMR)的竞争性配体甘露聚糖(mannan)的阻断实验,检测甘露糖化的人溶菌酶是否通过MMR介导内吞。结果毕赤酵母表达的hLYZ/eGFP融合蛋白是相对分子质量(Mr)为(66～97)×103的弥散条带,肽N-糖苷酶F(PNGaseF)酶切后,该融合蛋白变为Mr均一的条带,与非糖基化hLYZ/eGFP融合蛋白的理论Mr一致。该融合蛋白与RAW264.7共孵育后,可见细胞质中存在eGFP的绿色荧光,而加入甘露聚糖可以抑制这一现象发生。结论毕赤酵母表达的hLYZ/eGFP融合蛋白为高甘露糖型糖基修饰蛋白,该蛋白可以通过MMR介导内吞进入巨噬细胞。

Expression of the mannosylated hLYZ/eGFP fusion protein in Pichia pastoris and subcellular localization in macrophages

Objective To express mannosylated human lysozyme(hLYZ)and enhanced green fluorescent protein(eGFP)fusion protein in Pichia pastoris and its subcellular localization in macrophages Raw264.7.Methods The hLYZ and eGFP genes were linked by PCR.In the linker a site recognized by N-glycosylation was introduced and then cloned into vector pPICZαA.The recombinant plasmid pPICZαA-hLYZ/eGFP was linearized with SacⅠ and transformed to P.pastoris strain GS115 by electroporation.Multi-copy transformants were screened with Zeocin identified by PCR,and the positive clones were induced with methanol.Ammonium sulfate precipitation and nickel affinity chromatography purified proteins,and the fusion protein incubating with macrophages were observed under a fluorescence microscope.Results Fusion protein was successfully expressed and purified.SDS-PAGE analysis showed that the fusion protein was a diffuse band with relative molecular mass(Mr) of(66-97)×103.After PNGaseF digestion,the molecular weight decreasd to the theoretical value.When the fusion protein was incubated with RAW264.7,the green fluorescent substance could be seen in the cytoplasm of RAW264.7 by confocal microscopic image.and mannan could inhibit the occurrence of this phenomenon.Conclusion The hLYZ/eGFP fusion protein expressed in P.pastoris is high-mannose-type glycosylated protein,and the hLYZ/eGFP fusion protein can be internalized into macrophages by macrophage mannose receptor(MMR).