副溶血弧菌与Hela细胞表面蛋白结合相关外膜蛋白的筛选和鉴定

Screening and identification of Hela cell surface protein-binding associated adhesive outer membrane protein of Vibrio parahaemolyticus

为了从副溶血弧菌(VP)中筛选出可与宿主细胞表面蛋白互作的外膜蛋白,并研究其与宿主细胞的黏附作用,以Hela细胞株为宿主,将其表面蛋白生物素化后作为靶蛋白,利用层析技术从VP中垂钓到可与之结合的外膜蛋白,对重组蛋白进行质谱鉴定、纯化和Western Blot检测,并采用间接免疫荧光法考察重组蛋白对Hela细胞的黏附能力.从VP中共筛选出5种可与Hela表面蛋白相结合的外膜蛋白,质谱鉴定结果表明:5种蛋白分别为Peptidase、PLP、Omp W、Omp N和Chaperonin,均为胞外蛋白.Omp W、Chaperonin和Peptidase对Hela细胞的黏附作用很强,Omp N的黏附能力次之,PLP的黏附能力最弱.

To screen the outer membrane proteins of Vibrio parahaemolyticus （VP） which can interact with host cell surface protein and explore the adhesion effect of outer membrane proteins to host cells, the Hela cells were adopted as the host, and the proteins on the surface were biotinylated and then employed as the bait to fish host binding outer membrane proteins of VP by using affinity chromatography. The candidate proteins were identified by LC-MS/MS, purified and checked by Western- Blot. The adhesion activity of recombinant proteins to Hela cells were detected by indirect immune-fluorescence assays. The results of mass spectrometry identification technique showed that five outer membrane proteins of VP which can interact with the Hela cell surface proteins, i.e. Peptidase, PLP, OmpW , OmpN and Chaperonin, were screened, and all of them were extracellular proteins. Among the five proteins, OmpW, Chaperonin and Peptidase had strong adhesion activities to the Hela cells, the activity of 0mpN was lower than those of the former three proteins, and PLP barely adhered to Hela cells.