湖羊MyoG基因CDS的克隆及其真核表达载体的构建

Cloning Based on Overlapping PCR and Construction of Eukaryotic Expression Vector of MyoG Gene of Hu Sheep

参阅GenBank发表的波尔山羊肌细胞生成素(MyoG)基因序列,设计3对具有互补末端的特异性引物,分别扩增出湖羊MyoG基因的3个外显子,利用重叠PCR技术,将此3个外显子连接,并构建成重组质粒pcDNA3.0-MyoG,转染NIH-3T3细胞,RT-PCR鉴定。结果表明:成功克隆了湖羊MyoG基因的CDS,在离体细胞中MyoG基因发生了转录,这为进一步研究MyoG基因的体内外表达及生物学的活性奠定了基础。

Three exons of myogenin（MyoG）gene of Hu Sheep were amplified using three pairs of primers designed according to the MyoG gene sequence of Boer goat in GenBank,then linked by overlapping PCR,and inserted into the eukaryotic expression vector pcDNA3.0.NIH-3T3 cell was transfected with the constructed recombinant plasmid pcDNA3.0-MyoG,the expression of MyoG was detected by RT-PCR.The results showed that CDS of MyoG was successfully cloned in Hu sheep.Enzyme,PCR,RT-PCR analysis indicated that the recombinant plasmid pcDNA3.0-MyoG was successfully constructed,which laid a foundation of MyoG in vivo and in vitro and its specific biological activity research.