猪链球菌2型FBPS的纤连蛋白结合部位的初步确定

Determination of fibronectin-binding region of FBPS of Streptococcus suis type 2

根据猪链球菌2型江苏分离株HA9801的fbps基因序列,设计合成不同的引物,用含全长fbps的pMD-T-FBPS质粒为模板,通过PCR技术,扩增不同片段fbps,并按正确的阅读框架定向克隆到表达载体pET-32a(+),构建分别表达全长7～82、7～165和87～320氨基酸FBPS的重组表达质粒pFBPS、pFBPS(7～82)、pFBPS(7～165)和pFBPS(87～320);将重组质粒转化大肠杆菌BL 21(DE)株,经IPTG诱导,表达rFBPS(7～82)、rFBPS(7～165)、rFBPS(87～320)和rFBPS(全长),分子量分别为29、34、42及83kD的融合蛋白.配基亲和Western blot试验表明,表达的融合蛋白除rFBPS(7～82)外,均可与人纤连蛋白(Fn)结合,由此可以推断SS2的纤连蛋白/血纤蛋白原结合蛋白(FBPS)N端87～165氨基酸区域为具有结合活性的线性部位.

Fusion expression plasmid pFBPS, pFBPS （7 - 82）, pFBPS （7 - 165） and pFBPS3 （87 - 320） of Streptococcus suis type strain HA9801 were generated by cloning different fragments of flops amplified from pMD-T-FBPS by PCR into plasmid pET-32a（＋）. It has been confirmed that the recombinant proteins, FBI＇S, FBPS（87- 320）, and FBPS（7- 167）, which are expressed by recombinant plasmid pFBPS, pFBPS（87- 320） and pFBPS（7 - 165）, respectively, bound human fibronectin by ligand affinity Western blot assay. The results indicate the primary fibronectinbinding domain of FBPS lies within 87 - 165 amino acid residues region.