绵羊Myostatin基因真核表达载体的构建及在其原代成纤维细胞中的瞬时表达

Construction and Transfection of Sheep Myostatin Expression Vector pAcGFP-MSTN in Sheep Fibroblasts

Myostatin(MSTN)基因是胚胎期肌肉形成和出生后骨骼肌生长的主要调控因子之一,通过抑制肌细胞的扩增和分化而调控肌肉的生长和发育。为了进一步揭示MSTN在绵羊成纤维细胞中的生物学功能,采用RT-PCR从绵羊肌肉组织中扩增MSTN基因,将其cDNA终止密码子TGA删除,采用定向克隆技术连接到带有水母绿色荧光蛋白(AcGFP)报告基因的真核表达载体pAcGFP-N1中,构建融合蛋白重组质粒,经XhoⅠ/SacⅡ双酶切、测序鉴定后,用脂质体介导质粒转染绵羊原代成纤维细胞,观测荧光表达及用RT-PCR和Western blotting方法检测基因转录、蛋白质表达情况。结果表明,成功克隆绵羊MSTN基因,通过PCR方法在MSTN阅读框两端引入了XhoⅠ和SacⅡ克隆位点,成功构建pAcGFP-MSTN融合蛋白真核表达载体,重组质粒转染绵羊成纤维细胞24h后在荧光显微镜下观察到绿色荧光,通过RT-PCR扩增出1138bp的转录产物,并用Western blotting检测到78ku目的蛋白的表达。本试验为研究MSTN基因在成纤维细胞和脂肪分化调控中的具体机制奠定基础。

Myostatin（MSTN）, an important regulator of embryonic myogenesis and adult skeletal muscle growth, regula- ted the muscle development by controlling the proliferation and differentiation of myoblast. To explore the biological function of MSTN in sheep fibroblasts, we had amplified the MSTN gene from sheep skeletal muscle by reverse transcription PCR （RT-PCR）, deleted the stop codons TGA and cloned into the eukaryotic expression vector pAcGFP-N1 by directional clone. So, the fusion protein recombinant plasmid pAcGFP-MSTN had been constructed. After the restriction enzyme digestion of Xho Ⅰ/Sac Ⅱ and sequencing, the plasmid had been transfected into the sheep fibroblasts by lipofectamine. We also observed the fluorescence expression under the microscope, examined the transcription and translation of the expression vector pAcGFP- MSTN by RT-PCR and western blotting. The results showed that we had successfully cloned sheep MSTN gene, designed the enzyme site of Xho Ⅰ_ and Sac Ⅱ at the both ends of MSTN open reading frame by PCR, and constructed the fusion protein ex- pression vector pAcGFP-MSTN. We could observe the green fluorescence after 24 h of the transfection of the recombinant plasmid pAeGFP-MSTN. We also had amplified the transcription product of 1138 bp by RT-PCR. The targeted protein 78 ku was also detected by western blotting. The results would provide the important foundation to detect the regulation mechanism of MSTN in fibroblasts and adipocyte differention.