心肌α肌球蛋白重链启动子驱动的CREG蛋白表达载体的构建及鉴定

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 repressor of 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BamHI和EcoRI双酶切pcDNA3．1myc—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AseI和NheI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，Westem 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。

Construction and Identification of α-MHC Promoter-driven CREG Eukaryotic Expressive Plasmid

Objective： To construct a eukaryotic expressive plasmid for cellular repressor of E1A-stimulated genes （CREG） driven by a cardiac specific promoter α-MHC and reported by green fluorescent protein, and then to observe its expression in mouse primary cardiomyocytes in vitro. Methods： CREG fragment was released from pcDNA3.1 myc-His/hCREG ptasmid by BarnH I and EcoR I digestion enzymes, and then was subcloned into pEGFP-N1 plasmid to construct pCREG-EGFP-N1 plasmid. The α-MHC promoter sequence was synthesized according to Genebank, and was attached with Ase I and Nhe I restrictive endonuclease sites at each side. The a -MHC promoter sequence was released from pUC57-α -MHC plasmid by Ase I and Nhe I digestion enzyme, and then replace cytomegalo virus （CMV） promoter of pCREG-EGFP-N1 plasmid to construct pα -MHC-EGFP-N1 plasmid. Then, the recombinant pα -MHC-CREG-EGFP-N 1 plasmid was then transfected into mouse primary cardiomyocytes by liposome. The green fluorescent protein expression was observed by fluorescent microscopy and CREG expression was detected by Western blot. Results： pα -MHC-CREG-EGFP-N1 was identified by enzyme digestion and sequencing analysis. The results showed that length of the target genes which were inserted into the recombinant plasmid was correct. The strong expression of green fluorecent protein was observed by fluorecent microscopy, and the expression of CREG protein was detected by Western blot. These results confirmed that mouse primary cardiomyocytes were successfully transfected with plasmid. Conclusion： The pα -MHC-CREG-EGFP-N1 eukaryotic expressive plasmid was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of CREG in cardiovascular diseases development and also in producing mouse cardiomyocyte specific hyper-expressive CREG transgenic mice.