牛肌生成抑制素基因全序列的克隆及原核表达

Cloning and Prokaryotic Expression of Beef Myostatin Gene

根据GenBank上发表的牛肌生成抑制素(MSTN)基因编码序列设计引物,利用RT-PCR技术,以牛肌细胞总RNA为模板,对牛的肌生成抑制素(MSTN)基因编码序列进行扩增。经酶切鉴定、DNA测序和氨基酸序列分析证实MSTN基因克隆成功。从pMD18T-MSTN克隆中获得MSTN编码序列,将其与pET32a(+)质粒连接,构建pET32a(+)-MSTN重组质粒,重组菌经IPTG诱导在大肠杆菌中表达的MSTN蛋白是以包涵体的形式表达的,SDS-PAGE特异区带分子量为60ku。

A pair of oligo primers was designed according to the published coding sequence of cattle myostatin （MSTN） gene in Genbank. The cDNA fragment of myostatin was amplified by RT-PCR from cattle skeletal muscle, and then was cloned into pMD18-T vector and subsequently identified by restriction endonuclease digestion, PCR and sequencing. The cloning plasmid pMD18T-MSTN and pET32a（＋） were digested by restriction endonuclease BamH I and Sal I simultaneously. The prokaryotic expression plasmid pET32a（＋）-MSTN was constructed and transformed into E.coli BL2 I（DE3）, and then induced by IPTG. The mature protein coding sequence of porcine myostatin gene was expressed in the form of inclusion bodies （IB）. SDS-PAGE analysis showed that the recombinant fusion protein had a molecular weight approximately 60 ku.