大黄鱼MSTN成熟肽区基因的原核表达

Prokaryotic Expression of Mature Peptide Coding Gene of Pseudosciaena crocea Myostatin

根据本实验室克隆的大黄鱼肌肉生长抑制素(MSTN)基因编码序列设计引物,通过RT-PCR法扩增大黄鱼MSTN成熟肽区域的基因,将目的片段插入pMD18-T克隆质粒,并测序验证。验证后用BamHⅠ和HindⅢ限制性内切酶酶切含目的片段的pMD18-T克隆质粒和pET-28 a表达质粒,构建MSTN-pET-28 a重组表达质粒。将重组表达质粒转化至BL21菌中,经IPTG诱导后表达蛋白的大小约17 kD。

According to the myostatin（MSTN） gene of Pseudosciaena crocea,a pair of primers was designed to amplify mature peptide coding sequence of MSTN and optimized with restriction sites for two restriction endonucleases BamHⅠand Hind Ⅲ.The target fragment was amplified from total RNA using RT-PCR.The cloning plasmids pMD-18T and expression plasmids pET-28a were double digested with BamHⅠand Hind Ⅲ,respectively.The recombinant expression plasmids MSTN-pET-28a were constructed and then transformed into BL21 bacteria.Positive clones were selected,cultured,and induced by IPTG,and the molecular weight of the expressed protein is about 17 kD.