猪鼻支原体P37蛋白相互作用蛋白的筛选与鉴定

Isolation and Identification of a Protein Interacting With P37 Protein of Mycoplasma hyorhinis

既往工作表明 ,胃癌组织中有较高的猪鼻支原体感染率 ,猪鼻支原体的主要膜蛋白P37能够诱导外周血单核细胞释放肿瘤坏死因子 (TNF) .为了深入研究P37的作用机制 ,利用酵母双杂交系统筛选与P37蛋白相互作用的蛋白质分子 .首先 ,将编码P37全长的cDNA克隆到 pGBKT7载体中 ,构建“诱饵”表达载体 pGBKT7 p37,以此筛选人胎盘组织cDNA表达文库 .在 2 6× 10 6个克隆中 ,筛选到一株能与P37相互作用的阳性克隆 ,序列测定表明 ,该阳性克隆编码人视网膜色素上皮细胞蛋白 (Norpeg蛋白 ) .在此基础上经GST PullDown实验 ,进一步证实Norpeg蛋白确能与P37相互作用 ,为进一步研究P37对细胞的作用机制奠定了基础 .

High ratio infection of Mycoplasma hyorhinis in gastric tumor tissues suggests a possible association between mycoplasma infection and cancer. P37, an outer-membrane bacterial protein from Mycoplasma hyorhinis, increases neoplastic invasivity and metastasis and induces TNF-alpha secretion from human peripheral blood mononuclear cells (PBMC). To investigate the functional mechanism of P37, yeast two-hybrid system was used to isolate proteins interacted with P37 from a human placenta cDNA library. Among the 2.6 X 10(6) transformant clones, one positive clone was obtained. Sequence analysis revealed that the clone is a cDNA fragment from Norpeg and encode a carboxy terminal protein. The interaction between P37 and Norpeg was further confirmed by ELISA and GST-Pull down assay. This result would be useful to study the function of P37 further.