含Ⅲ型纤连蛋白域蛋白5真核表达载体的构建及初步功能研究

目的 构建含小鼠FNDC5基因的绿色荧光蛋白表达载体pEGFP-C3-FNDC5,并初步探索其对C2C12肌细胞的线粒体能量代谢的影响。方法 应用反转录-聚合酶链反应法从小鼠腓肠肌和心脏组织中扩增出两端带有HindⅢ和EcoRⅠ酶切位点的FNDC5编码片段,经回收、纯化酶切后,依次连接到质粒PMD19-T和pEGFP-C3上,获得过表达载体后转染至C2C12肌细胞,48h后以激光共聚焦显微镜拍摄线粒体荧光强度,以荧光定量PCR检测与线粒体能量代谢相关的基因PGC-1β、ATPase6、ND1和ND6的表达情况。结果 PCR及测序结果表明:重组质粒pEGFP-C3含有FNDC5段,方向及大小正确,过表达FNDC5可以增强线粒体荧光强度,并增加线粒体能量代谢相关的基因PGC-1β、ATPase6、ND1和ND6的表达。结论 成功构建了小鼠真核表达载体pEGFP-C3-FNDC5,过表达FNDC5可以增强线粒体能量代谢。
Objective To construct eukaryotic expression vector pEGFP-C3-FNDC5 and to investilate its role in regulating mitochondrial biogenesis in C2C12 myocytes. Methods The cDNA coding sequence of FNDC5 containing digest site HindⅢ and EcoRⅠ was amplified by RT-PCR from gastrocnemius muscle and heart tissue of mice, and was then inserted into PMD19-T vector and pEGFP-C3 vector. The recombined plasmid was identified by PCR and further sequenced. After transfection of vector for 48h, mitochondrial content was measured by confocal microscopy and genes regulating mitochondrial biogenesis was determined by quantitative Real-Time polymerase chain reaction(qRT-PCR). ResultsPCR and DNA sequencing results showed that the recombinant plasmid pEGFP-C3-FNDC5 contained the fragment of FNDC5. FNDC5 overexpression enhanced mitochondrial content and also increased the expression of PGC-1β, ATPase6, ND1 and ND6. Conclusion The eukaryotic expression vector pEGFP-C3-FNDC5 has been constructed successfully. FNDC5 overexpre-ssion can enhance mitochondrial biogenesis