一种具有双嗜性功能融合蛋白FNCDH的制备及其促粘附、成骨生物活性.docVIP

PAGE PAGE 7 [基金项目]国家自然科学基金面上项目 [通信作者]刘明华，E-mail：jjbicu@ 一种具有双嗜性功能融合蛋白FN/CDH的制备及其促粘附、成骨生物活性鉴定 向 强1，张 瑗，崔 翔，王加旭，刘明华1 （400038 重庆，第三军医大学西南医院急救部1；400047 重庆，第三军医大学新桥医院骨科2） [摘要] 目的 构建一种具有同嗜性和异嗜性细胞粘附功能的重组融合蛋白FN/CDH，验证其对人骨髓间充质干细胞 (human mesenchymal stem cell,hMSCs)的促粘附、成骨效能。方法 以纤维连接蛋白(fibronectin, FN)和钙粘附蛋白11(CDH11)作为前体分子，通过生物信息学分析了解其结构序列与功能基团。采用“同源模建”作为分子模拟策略确定FN和CDH11功能片段的接合方式。常规PCR扩增FNⅢ7-10和CDH11 EC1-2基因片段后应用重叠延伸PCR(splice-over-extension PCR, SOE-PCR)进行基因拼接。将融合基因插入原核表达载体pET-22b构建重组载体,重组载体转化表达菌株Rsoetta-gami(DE3)，实现rFN/CDH的诱导表达，蛋白纯化。免疫印迹确定融合蛋白种属，离心促粘附实验和成骨诱导验证rFN/CDH体外生物活性。 结果 成功构建了pET22b-FNⅢ 7-10/CDH11 EC1-2融合基因原核表达载体，获得了rFN/CDH的可溶性表达，纯化后rFN/CDH的纯度达到96%。初步验证了各个融合蛋白表达属性的准确性，证实rFN/CDH具有良好的促进成骨细胞粘附的生物活性。将有成骨分化潜能的hMSCs在rFN/CDH涂层的TCPS表面进行成骨诱导培养，以早期成骨标志物Alkaline phosphatase(ALP)和晚期标志物Osteocalcin(OCN)作为评价指标，提示rFN/CDH具有良好的促进细胞成骨分化的生物活性。结论 成功进行了rFN/CDH融合蛋白的原核表达、纯化和鉴定，嵌合蛋白的粘附活性在一定程度上实现了对FN和CDH11功能的叠加，rFN/CDH具有“促粘”和“促成骨”的双重生物活性，可作为一种理想的仿生修饰分子进行生物界面的构建。 [关键词]　 纤维连接蛋白；钙粘附蛋白；间充质干细胞；粘附；成骨分化 [中图法分类号] [文献标志码] A Preparation of FN/CDH amphotropic recombinant protein and a study of its capacity in promoting osteoblastic adhesion and ossification Xiang Qiang1 ,Cui Xiang,Wang Jia Xu, Liu Ming Hua1 (Departmentof Emergency ,Southwest Hospital，Third Military University, Chongqing, 400038,1, Department of Orthopaedics ,Xinqiao Hospital2, Third Military University, Chongqing, 400047, China )相应修改 相应修改 Abstract Objective The aim of this study was to investigate the effect of a amphotropic recombinant protein of fibronectin module III7-10/cadherin 11 EC1-2（rFN/CDH）on cell adhesion and differentiation. Methods In this study, FN and CDH11, as two pre-molecules of the recombinant protein, were analysed bioinformatically to acquired the structural sequences and functional groups. The fragments of FN and CDH11 were integrated using a homology modeling strategy. The gene fragments of FNⅢ7-10 and CDH11 EC1-2 were spliced by SOE-PCR and then inserted into the expression plasmid of