猪MSTN前肽定点诱变载体在C2C12细胞中的表达

摘要

This research intended to construct eukaryotic expression vector with a site-directed mu-tation of porcine MSTN propeptide gene, and verify its expression efficacy in C2C12 cells. By means of PCR amplification with DNA extracted from blood samples of Tongcheng pigs as tem-plates, MSTN propeptide gene containing the first intron was cloned and then suquenced. It was then subcloned into vector pEGFP-N1 without EGFP fragment and site-directed mutagenesis was perfomed which successfully converted Asp into Ala at the 76th amino acid site in propeptide se-quence. After being confirmed by R. E digestion and sequencing, the recombinant plasmid pEGFP (-)-N1-ProMstnD76A was transferred into C2C12 cells. After 48 h, the expression of target gene was detected at transcriptional level using RT-PCR and Real-time PCR. The results showed that the recombinant expression plasmid pEGFP(-)-N1-ProMstnD76A was constructed successfully. After being transiently transfected into C2C12 cells, the high expression level of target mRNA was detected and the primary mRNA transcript was found spliced correctly. The present research will lay a good foundation for further studying the functions of porcine MSTN propeptide gene in vivo and provide useful molecular materials for preparation of transgenic pigs.%本研究旨在克隆通城猪含有第1个内含子的MSTN前肽基因,构建真核定点诱变载体,并通过转染C2C12细胞验证载体表达的有效性.以通城猪血液样品中提取的DNA作为模板,PCR扩增得到含第1个内含子的MSTN前肽基因,扩增产物进行克隆、测序.再将目的片段定向克隆到去除了EGFP基因的pEGFP-N1表达载体上.通过定点诱变方法将前肽编码序列第76位天冬氨酸突变为丙氨酸,获得定点诱变载体pEGFP(-)-N1-ProMstnD76A,并瞬时转染C2C12细胞.转染48 h后,通过RT-PCR和Real-time PCR方法检测目的基因的表达情况.结果表明:本研究成功克隆了通城猪含第1个内含子的MSTN前肽基因,并对该基因进行定点诱变修饰,构建了真核表达载体,转染后其前体mRNA能在C2C12细胞中进行正确剪接,目的基因mRNA表达水平较高.这为猪MSTN前肽基因在体内表达的研究奠定基础,并为制备转基因猪提供有用的分子材料.