鸡毒支原体热休克蛋白GrpE粘附特性的鉴定

Identification of the adhesive characterization of the heat shock protein GrpE of the Mycoplasma gallisepticum

鸡毒支原体(MG)脂质相关蛋白(LAMPs)的粘附特性在感染宿主细胞中起重要的作用,本实验室前期利用快速蛋白液相色谱和质谱鉴定MG的grp E基因编码的GrpE蛋白为热休克蛋白。为进一步鉴定GrpE蛋白是否具有粘附特性,本研究通过原核表达MG株grp E基因,并利用表达纯化的重组蛋白GrpE(rGrpE)免疫BALB/c小鼠。制备抗GrpE的单克隆抗体(MAb)。通过亚细胞定位,结果显示GrpE定位于细胞表面。通过激光共聚焦显微镜观察到rGrpE对DF-1细胞具有明显的粘附作用,而且利用抗GrpE的MAb能够特异抑制MG膜蛋白对DF-1细胞的粘附。此外,夹心ELISA和流式细胞术试验结果也表明制备的MAb能够显著抑制MG膜蛋白对DF-1细胞的粘附,并呈剂量依赖关系。上述结果表明热休克蛋白GrpE是MG的一种新的具有粘附特性的蛋白。

The adhesion property of lipid-associated membrane proteins play a critical role in Mycoplasma gallisepticum to infect host cells, our previous study indicated the GrpE of M.gallisepticum belonged to heat shock protein through fast protein liquid chromatography and mass spectrometry analysis. To identify the adhesive characteristic of GrpE, the monoclonal antibody （MAb） was prepared by immunized BALB/c mouse with purified rGrpE expressed in E.coli. Then, the cellular co-localization shown that both rGrpE and the membrane proteins extracted from M.gallisepticum had the capabilities to adhere on chick embryo fibroblast （DF-1）. However, when rGrpE and the membrane protein were incubated with the MAb against GrpE, the adhesion was specifically inhibited and decreased significantly. In addition, the ELISA and flow cytometry tests also confirmed that the adhesion of the rGrpE was a specific adhesion. These results demonstrate that GrpE is the related adhesion protein of M.gallisepticum.