摘要
利用基因重组技术设计和构建了一种仿生的双功能仿生细胞外基质蛋白.其基本策略是将人血管内皮钙粘素胞外区(VE-cad)和免疫球蛋白(IgG1)Fc段进行基因融合构建真核表达载体,并利用FreeStyle293真核蛋白表达系统制备融合蛋白VE-cad-Fc,利用Western blot鉴定融合蛋白,并观察了该VE-cad-Fc融合蛋白基质对人脐静脉内皮细胞(HUVECs)粘附和形态的影响.结果显示,成功克隆了VE-cad胞外段基因,并与Fc段重组形成VE-cad-Fc融合基因.在转染24h后,利用兔抗人VE钙粘素胞外区抗体验明该融合蛋白的免疫抗原性,其分子质量约为90.3KD,进一步的结果证明VE-cad-Fc融合蛋白是以二聚体的形式存在.优化该制备体系最佳表达时间为72h.细胞试验表明,VE-cad-Fc融合蛋白可显著促进HUVECs的粘附和生长.这种工程化的多功能融合蛋白基质在组织工程、再生医学和分子生物学领域有广阔的应用前景.
A type of multifunctional biomimic extracellular matrix protein was designed and constructed by using gene recombination technology.The basic strategy was to construct a eukaryotic expression vector by gene fusion of the extracellular region of human VE-cad and IgG1 Fc fragment,prepare fusion protein VE-cad-Fc by using Free Style 293 eukaryotic protein expression system,screen fusion protein,and observe the effect of this fusion protein on adhersion and morphology of HUVECs.Results shown that the extracellular region of VE-cad was successfully cloned and formed VE-cad-Fc fusion gene with Fc regions.Immunogenicity of this fusion protein was proved using rabbit anti human VE-cad antibody which only had extracellular region,the molecular weight of this fusion protein was 90.3KD,further result confirmed this fusion protein was dimer.72 hwas the optimal time to test this system.Cell test shown that VE-cad-Fc fusion protein promoted adhersion and spreading of HUVECs.This multifunctional fusion protein is widely applicable in tissue engineering、regenerative medicine and molecular biology.
出处
《南开大学学报（自然科学版）》
CAS
CSCD
北大核心
2014年第5期48-53,共6页
Acta Scientiarum Naturalium Universitatis Nankaiensis
基金
国家自然科学基金(31070855)
国家973项目(2011CB606202)
天津市科委项目(10JCYBJC10400)
