猪新基因CFL2真核表达载体的构建及在C2C12细胞中的瞬时表达

从猪的股二头肌中提取总RNA,利用RT-PCR技术从猪骨骼肌中获得CFL2基因的编码区序列,T-A克隆至PMD-18T载体,经HindⅢ和XhoⅠ酶切后定向克隆至真核表达载体pCDNA3.1(+)中。获得的重组质粒pCDNA3.1(+)/CFL2用限制性内切酶酶切分析和DNA测序分析鉴定,并经脂质体瞬时转染C2C12细胞,最后利用PCR检测转基因细胞中CFL2基因的存在及其表达情况。结果显示:猪CFL2基因已克隆到真核表达载体pCDNA3.1(+)中,转染的C2C12细胞中检测到细胞内较高量CFL2的表达。pCDNA3.1(+)/CFL2真核表达载体的成功构建,为深入研究CFL2基因在猪肌肉细胞中的功能奠定基础,为猪肉质品质的改良提供了新的思路。

Construction and detection of the eukaryotic expression vector of porcine CFL2 gene and its transient expression in C2C12 cells

The cDNA encoding the CFL2 region was obtained by RT-PCR from the total RNA which was isolated from the porcine skeletal muscle and inserted into PMD18-T vector,then digested with the double restriction enzymes Hind Ⅲ 和XhoⅠ.The obtained CFL2 gene was inserted into the expression vector of pCDNA3.1(+).The recombinant plasmid was identified by restriction enzymes digestion analysis and DNA sequencing,the expression of the CFL2 gene was detected by RT-PCR.The result showed that both PCR and enzyme digestion analysis confirmed constructed plasmids correct.The pCDNA3.1(+)/CFL2 gene expression vector was constructed successfully.The C2C12 cells transfected with the pCDNA3.1(+)/CFL2 plasmid expressed a high level of the CFL2 protein in the cells.The construction of the CFL2 gene expression vector lay not only the foundation for the further research into the effect of CFL2 gene on the porcine skeletal muscle cell function but also provide a new way to improve the porcine muscle qualitative.