猪链球菌2型FBPS的纤连蛋白结合部位的初步确定

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BL21(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。

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

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 fbps amplified from pMD-T-FBPS by PCR into plasmid pET-32a(+). It has been confirmed that the recombinant proteins, FBPS, 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 fibronectin-binding domain of FBPS lies within 87 - 165 amino acid residues region.