猪肌生成抑制素基因成熟蛋白编码序列的表达与纯化

 猪肌生成抑制素基因myostatin (MSTN)的cDNA去除信号肽后对成熟蛋白编码序列PCR扩增出1.2 kb片段,将该片段与pMD18-T载体连接,转化JM109受体菌细胞,筛选阳性克隆测序分析,结果表明与设计序列完全一致。将该克隆载体的质粒DNA用带有BamH I和Sal I内切酶识别序列的另一对引物进行PCR扩增,将回收的1.2 kb PCR目的片段定向克隆到pET28a（+）表达载体上,成功地构建了猪肌生成抑制素成熟蛋白编码的原核表达载体。对成功构建表达载体阳性克隆在LB液体培养基中用IPTG诱导表达, SDS-PAGE凝胶电泳显示,重组菌表达的MSTN蛋白是以包涵体的形式表达的;SDS-PAGE凝胶经薄层扫描仪扫描分析,表达的MSTN包涵体蛋白占菌体不溶性蛋白含量的27.9%,表达的MSTN分子量为41 451.3D. 因为所构建的表达载体中含六聚组氨酸标签,则用His-trap亲和柱进行纯化后,纯度可达92.5%. 该试验为获得较好的猪肌生成抑制素基因抗原、制备抗体打下了良好的基础。

Expression and Purification of Mature Protein Coding Sequence of Porcine Myostatin Gene in Prokaryotic Expression Vector

Myostatin is a negative regulator for the growth of the skeletal muscle. The skeletal muscle of mutant animals with null or low activity of myostatin would show significantly larger diameter or more quantity of fiber, which was termed as double muscling. In order to investigate the influence of myostatin for the lean meat rate and the plump-hipped trait, the expression vector of mature protein coding sequence(MPCS) of porcine myostatin(MSTN) was constructed. The recombinant MPCS-MSTN-pET-28a(+) plasmid was transformed into E.coli BL21,which then was cultured with Laria Broth(LB)medium and induced with IPTG and detected by SDS-PAGE. The mature protein coding sequence of porcine myostatin gene was expressed in the form of inclusion bodies(IB). Expression amount is equal to the 27.9% of the total proteins in the transformed host cell showed in thin-layer scanner analysis. The different inducing time showed the different expression level of recombinant MSTN protein with the highest level when 3～4 h inducing time. The expressed histidine-tagged porcine myostatin(MSTN) protein was purified by HisTrap^TM affinity chromagraphic column (Pharmacia Biotech). The purity of expressed reombinant MSTN protein was as high as to 92.5%. The results lay a good foundation for the application of MSTN molecular biological technique in animal nutrition and breeding.