牛支原体表面一种新的膜蛋白(P33)的特性研究

Function study of a new surface protein (P33) in Mycoplasma bovis

牛支原体(M.bovis)是牛支原体肺炎的病原体,能够引起犊牛肺炎等多种疾病。研究表明,牛支原体粘附蛋白可能在牛支原体致病过程中起关键作用。本研究选取了牛支原体的一个假定膜蛋白,对其基因进行PCR扩增,并连接到pET-28a载体,转化于大肠杆菌BL21(DE3)中,通过IPTG诱导获得了以可溶形式表达的重组蛋白,该重组蛋白大小约为36 ku,将其命名为P33。纯化的重组蛋白免疫新西兰大白兔制备多克隆抗体。重组蛋白与胎牛肺细胞(EBL)作用,通过激光共聚焦显微镜观察到了明显的粘附作用,而ELISA试验证明该蛋白的这种粘附为特异性粘附,从而证明该蛋白是牛支原体的一个粘附相关蛋白。

Mycoplasma bovis is one of the causative agents of pneumonia and other respiratory diseases in young caves and the surface protein of M.bovis played an important role in the entry and infection. In this study, the gene of a putative surface protein, designated P33, was amplified and cloned into pET-28a for expression in E.coli BL21 （DE3）. The SDS-PAGE analysis showed that expressed recombinant protein was approximate 36 ku, mainly in soluble form, and the antiserum was prepared in rabbit immunized with the purified P33. In addition, the confocal examination showed that P33 interacted with cellular proteins in cytoplasm of embryonic bovine lung （EBL） cells detected by indirect immunofluorescence assay. Moreover, the P33 was more likely to be a membrane binding protein, which was identified by ELISA coating with EBL cellular membrane proteins and the binding activity was efficient inhibited by P33 antiserum. The results demonstrated the P33 of M.bovis possessed the ability of adhesion to EBL cells.