牛fabp3和fabp4基因真核表达载体的构建及在小鼠成肌细胞中的表达

Construction of Eukaryotic Expression Vector of Bovine fabp3 or fabp4 Gene and Expression of the Gene in Mouse Myoblasts

【目的】分别构建牛fabp3和fabp4基因真核表达载体,并观察载体转染小鼠成肌细胞后基因的表达以及对内源fabp3和fabp4基因表达的影响,检测肉质性状形成相关基因在转基因细胞中的表达及对内源功能基因的影响。【方法】以pDsRED质粒为骨架载体,分别将人肌红蛋白启动子与目的基因相连、CMV启动子与红色荧光蛋白报告基因相连构建肌肉特异性表达fabp3基因的载体pDsHF3和表达fabp4基因的载体pDsHF4;用脂质体瞬时转染小鼠成肌细胞,72 h后用2%孕马血清诱导成肌细胞向肌管的分化;利用real-time PCR技术检测转染前后成肌细胞中外源牛fabp3和fabp4基因和内源小鼠fabp3和fabp4基因的表达量。【结果】与对照组相比,小鼠成肌细胞分别转染pDsHF3和pDsHF4表达载体,24 h后外源牛fabp3和fabp4基因均获得高水平的表达,48 h后有所回落;而内源小鼠fabp3和fabp4基因在成肌细胞和诱导分化的肌管细胞中的表达均受到不同程度的影响。【结论】构建的真核表达载体能在成肌细胞中高效表达,外源牛fabp3或fabp4基因的转入能够影响小鼠成肌细胞及肌管中内源fabp3和fabp4基因的表达。

[Objective] The objective of the study is to analyze the intrinsic expression bovine fabp3 and fabp4 genes, and the dynamic changes of fabp3 and fabp4 gene expression after transfected the eukaryotic expression vector into mice myoblasts. [Method] Eukaryotic expression vector pDsHF3 for fabp3 gene, pDsHF4 for fabp4 gene were constructed and transfected into mice myoblasts by using liposome technique. The transfected mice myoblasts were induced differentiation into myotubes by 2% of pregnant mare serum after 72 h transfection. The changes of gene expression of myoblasts and myotubes were analyzed by real-time PCR. [Result] The results showed that, bovine fabp3 or fabp4 genes were expressed in transfected myoblasts, respectively. The intrinsic expression of mouse fabp3 or fabp4 gene was affected in myoblasts and in differentiated myotubes. [ Conclusion ] Eukaryotic expression vectors pDsHF3 and pDsHF4 can highly express in proliferative myoblasts, and the expression of exogenous bovine fabp3 or fabp4 genes may affect intrinsic expression of mouse fabp3 and fabp4 genes in myoblasts and myotubes.