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目的:探讨心室肌球蛋白重链(vmhc)基因启动子的心肌组织特异性。方法:利用PCR技术从斑马鱼基因组中克隆了vmhc编码区5’上游大小为1952bp的调控区域,应用酶切连接方法将vmhc启动子插入pGEFP-N1质粒,成功构建pEGFP-vmhc重组载体。再应用高保真DNA聚合酶PCR扩增包含vmhc启动子序列,增强型绿色荧光蛋白(EGFP)基因序列及3’UTR序列的基因片段,经过纯化后通过显微注射将vmhc-EGFP基因片段导入斑马鱼受精卵中。结果:注射后的斑马鱼心脏中出现绿色荧光,而其他部位无荧光出现。结论:vmhc启动子能够正确有效地驱动外源基因在斑马鱼心脏中特异表达,适合应用于心血管疾病的基因功能研究,基因靶向治疗等。
Objective: To investigate the myocardial tissue specificity of the promoter of cardiac myosin heavy chain (vmhc) gene. Methods: The regulatory region of 1952 bp upstream of 5 ’in coding region of vmhc was cloned from zebrafish genome by PCR. The vmhc promoter was inserted into pGEFP-N1 plasmid by restriction enzyme digestion, and the recombinant vector pEGFP-vmhc was successfully constructed. The gene fragment containing the vmhc promoter sequence, enhanced green fluorescent protein (EGFP) gene sequence and 3’UTR sequence was amplified by PCR using high-fidelity DNA polymerase. After purification, the gene fragment of vmhc-EGFP was introduced into zebra by microinjection Fish fertilized eggs. Results: Green fluorescence was observed in the hearts of zebrafish after injection, while no fluorescence appeared in other sites. CONCLUSION: The vmhc promoter can correctly and efficiently drive the expression of foreign genes in the heart of zebrafish, which is suitable for the study of gene function and gene-targeted therapy of cardiovascular diseases.