[1]向强,张瑗,崔翔,等.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[J].陆军军医大学学报(原第三军医大学学报),2013,35(23):2536-2541.
 Xiang Qiang,Zhang Yuan,Cui Xiang,et al.Preparation of FN/CDH amphotropic recombinant protein and its capacity in promoting osteoblastic adhesion and ossification[J].J Amry Med Univ (J Third Mil Med Univ),2013,35(23):2536-2541.
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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(/HTML )
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
35卷
期数:
2013年第23期
页码:
2536-2541
栏目:
论著
出版日期:
2013-12-15

文章信息/Info

Title:
Preparation of FN/CDH amphotropic recombinant protein and its capacity in promoting osteoblastic adhesion and ossification
作者:
向强张瑗崔翔王加旭刘明华
第三军医大学西南医院急救部;第三军医大学新桥医院骨科
Author(s):
Xiang Qiang Zhang Yuan Cui Xiang Wang Jiaxu Liu Minghua
Department of Emergency, Southwest Hospital, Third Military Medical University, Chongqing, 400038; Department of Orthopaedics, Xinqiao Hospital, Third Military Medical University, Chongqing, 400047, China
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
Keywords:
fibronectin cadherin mesenchymal stem cell adhesion osteoblastic differentiation
分类号:
R322.71; R341; R394-33
文献标志码:
A
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。
Abstract:
Objective       To determine the effect of an amphotropic recombinant protein of fibronectin module Ⅲ7-10/cadherin 11 EC1-2 (rFN/CDH) on cell adhesion and differentiation in human mesenchymal stem cells (hMSCs).       Methods       FN and CDH11, as two pre-molecules of our objective recombinant protein, were analyzed bioinformatically to acquire the structural sequences and functional groups. The functional fragments of FN and CDH11 were integrated using a homology modeling strategy. The fragments of FNⅢ7-10 and CDH11 EC1-2 were spliced by SOE-PCR and then inserted into the expression plasmid of pET-22b to construct a reconstructed vector. After transforming the vector into Rsoetta-gami (DE3) strain and optimizing the experimental parameters, rFN/CDH was induced to express using IPTG and then purified. Further, Western blotting was employed to verify the protein. While, centrifugal cell adhesive assay and osteoblastic induction were used to determine the bioactivity of the protein in vitro.       Results       The prokaryotic expression vector pET22b-FNⅢ 7-10/CDH11 EC1-2 encoding the objective fusion gene was successful constructed. The rFN/CDH protein was in soluble expression, with the purity reaching 96%, and identified to have the accuracy of the individual expression of fusion protein properties. Additionally, cell centrifugal adhesive assay demonstrated that the tissue-culture treated polystyrene (TCPS) coated with rFN/CDH adhered more hMSCs with differentiation potential of osteoblasts, as compared to single FN and CDH. Alkaline phosphatase (ALP) activity and osteocalcin (OCN) gene/protein expression, as the early and late markers for osteoblastic differentiation, indicated that rFN/CDH further promoted ALP activity and OCN gene expression.       Conclusion       A prokaryotic expression vector encoding rFN/CDH fusion gene is successfully constructed, and the fusion protein is purified and identified. The obtained rFN/CDH possesses excellent capacity in promoting osteoblastic adhesion and ossification, which achieves the functional integration of FN and CDH11. It can be used as an ideal construction of biomimetic modification of molecular for biological interface.

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更新日期/Last Update: 2013-12-06