禽多杀性巴氏杆菌外膜蛋白H受体的初步鉴定

Preliminary identification of the outer membrane protein H receptors of avian Pasteurella multocida

目的鉴定鸡胚成纤维(CEF)细胞膜禽多杀性巴氏杆菌外膜蛋白H(OmpH)受体。方法用膜蛋白提取试剂盒制备CEF细胞的膜蛋白,通过SDS-PAGE和配体印迹检测CEF细胞膜蛋白中的OmpH受体,采用MALDI-TOF质谱技术鉴定蛋白种类,并用配体印迹、ELISA和免疫荧光技术检测OmpH受体在不同宿主食管黏膜细胞表面的分布情况。结果在转印CEF细胞膜蛋白的NC膜上有一条明显的疑似受体条带,分子量约为49kDa。MALDI-TOF质谱鉴定结果表明该受体蛋白是ATP合成酶β亚基。在转印鸡和兔食管黏膜细胞膜蛋白的NC膜上检测到能够与OmpH结合的蛋白条带,但在牛和猪食管黏膜细胞膜蛋白中未见任何印迹条带,而OmpH与鸡和兔食管黏膜细胞膜蛋白的结合力高于猪和牛食管黏膜细胞膜蛋白。结论禽多杀性巴氏杆菌OmpH受体可能是CEF细胞膜表面的ATP合成酶β亚基。

To identify the receptors for the outer membrane protein H （OmpH） of avian P. multocida, the membrane pro- teins of chicken embryo fibroblast （CEF） cells were separated by SDS-PAGE and analyzed by Ligand blot. The OmpH-binding protein was identified by MALDI-TOF mass spectrometry, and its distribution in the membrane proteins of different host e- sophageal mucosal cells was detected by Ligand blot, ELISA and immunofluorescenee microscopy, respectively. Ligand blot a- nalysis showed that a 49-kDa membrane protein of CEF cells bound to recombinant OmpH, and MALDI-TOF spectral results demonstrated that the OmpH-binding protein was ATP synthase β subunit. In addition, the OmpH receptor was present in the chicken and rabbit mucosal cell membranes, but was not detected in the bovine and swine mucosal cell membranes. The above results indicate that the OmpH receptor may be CEF cell-derived ATP synthase beta subunit.