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目的:构建心肌特异性α-肌球蛋白重链(α-myosin heavy chain,α-MHC)启动子启动E1A基因阻遏子(cellular repressorof E1A-stimulated genes,CREG)和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合的真核表达载体。绿色荧光蛋白作为报告基因,方便在心肌细胞中直接观察CREG蛋白的表达,为心肌特异性转CREG基因动物模型制备提供载体。方法:用BamH I和EcoR I双酶切pcDNA3.1 myc-His/hCREG质粒得到CREG基因,亚克隆入增强绿色荧光蛋白表达质粒pEGFP-N1中,构建pCREG-EGFP-N1;根据Genebank中公布的α-MHC基因的启动子序列,人工合成pUC57-α-MHC启动子基因序列,经Ase I和Nhe I双酶切得到启动子α-MHC,亚克隆入pCREG-EGFP-N1中替代原CMV启动子,构建pα-MHC-CREG-EGFP-N1,测序鉴定。用脂质体法将该质粒转染体外培养的小鼠原代心肌细胞,荧光显微镜下观测绿色荧光蛋白的表达;Western blot检测CREG蛋白的表达。结果:成功构建pα-MHC-CREG-EGFP-N1质粒,酶切及测序结果正确;成功转染入原代培养小鼠心肌细胞,在荧光显微镜下可见绿色荧光蛋白的表达,Western blot检测到CREG蛋白的表达。结论:重组质粒pα-MHC-CREG-EGFP-N1体外转染入原代培养小鼠心肌细胞后,目的基因能够在心肌细胞中有效表达,检测方法简便可靠,为下一步建立心肌细胞特异性表达CREG的过表达转基因小鼠、深入探讨CREG在心肌疾病发生中的生物学功能研究奠定了基础。
OBJECTIVE: To construct a cell-specific repressor of E1A-stimulated genes (CREG) and enhanced green fluorescent protein (α-MHC) fluorescent protein, EGFP) fusion eukaryotic expression vector. As a reporter gene, green fluorescent protein facilitates the direct observation of CREG protein expression in cardiomyocytes and provides a carrier for the preparation of cardiac-specific CREG gene animal model. Methods: The pcDNA3.1 myc-His / hCREG plasmid was digested with BamH I and EcoR I to obtain the CREG gene, subcloned into the enhanced green fluorescent protein expression plasmid pEGFP-N1 to construct pCREG-EGFP-N1. According to the published genebank α-MHC gene promoter sequence, synthetic pUC57-α-MHC promoter gene sequence, Ase I and Nhe I by double digestion of the promoter α-MHC, subcloned into pCREG-EGFP-N1 instead of the original CMV start The pα-MHC-CREG-EGFP-N1 was constructed and sequenced. The plasmids were transfected into mouse primary cardiomyocytes cultured in vitro by Lipofectamine 2000. The expression of green fluorescent protein (GFP) was observed under a fluorescence microscope. The expression of CREG protein was detected by Western blot. Results: Plasmid pα-MHC-CREG-EGFP-N1 was constructed successfully, and the result of restriction enzyme digestion and sequencing was correct. The recombinant plasmid was successfully transfected into cardiomyocytes of primary culture and the expression of green fluorescent protein was observed under fluorescence microscope. Protein expression. Conclusion: The recombinant plasmid pα-MHC-CREG-EGFP-N1 transfected into primary cultured mouse cardiomyocytes in vitro, the target gene can be effectively expressed in cardiomyocytes, the detection method is simple and reliable for the next step to establish cardiomyocyte-specific expression CREG overexpression transgenic mice, in-depth study of CREG in the pathogenesis of myocardial disease in the study of biological function laid the foundation.