心肌特异性hVEGF165真核表达载体的构建及鉴定

目的：构建MLC-2v启动子驱动的真核表达载体并鉴定特异性。方法：用MLC-2v启动子基因片段替代质粒pIRES2-EGFP的CMV启动子，而保留其增强子序列，并将目的基因hVEGF165分别克隆到两种启动子驱动的质粒，构建MEG-2v／pIRES2-EGFP-hVEGF165和pIRES2-EGFP-hVEGF165 2种质粒，瞬时转染心肌、内皮、平滑肌和成纤维细胞。结果：转染pIRES2-EGFP-hVEGF165，4种细胞均表达、目的基因及产物；而转染MLC-2v／plRES2-EGFP-hvEGF165，仅心肌细胞表达GFP和目的基因产物。结论：MLC-2v启动子驱动的质粒可心肌特异表达目的基因及蛋白。

Construction of Cardiac-specific Eukaryotic Expression Vector and Its Identification in vitro

Objective:To construct and identify a cardiac-specific eukaryotic expression vector Methods:To construct two eukaryotic expression vectors . The hVEGF165 DNA fragment was inserted into a enhanced green fluorescent protein fusion vector with CMV promoter and the other CMV promoter was replaced with MLC-2v promoter preserving CMV enhancer, the latter is cardiac-specific promoter. The two different vectors were transfected into endothelial cells , cardiomyocytes. smooth cells and fibroblast cells with liposome. Results: The four cultivated cells were successfully transfected with pIRES2-EGFP-hVEGF165, which confirmed by fluoroscopy, biochemistry and immunohistochemistry, but the vector with MLC-2v promoter only express the target gene in cardiomyocytes. Conclusion: the eukaryotic expression vector with MLC-2v promoter is a cardiac-specific expression vector.