猪肌肉生长抑制素基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Porcine Myostatin Gene

作　　者：鲍淑青[1] 张克英[1] 陈代文[1] 龙定彪[1] 

机构地区：[1]四川农业大学动物营养研究所,雅安625014

出　　处：《动物营养学报》2007年第5期617-621,共5页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家"973"项目"畜禽肉品质性状形成的代谢与调控机理"(NO.2004CB117506)

摘　　要：本试验旨在通过基因工程方法获得重组肌肉生长抑制素蛋白。根据基因库肌肉生长抑制素基因序列设计合成特异性引物,引物两端分别加上EcoRⅠ和SalⅠ酶切位点及保护碱基,提取汉普夏猪的骨骼肌总RNA,用RT-PCR方法扩增肌肉生长抑制素全长基因,并将其克隆到pMD18-T载体上,测序后EcoRⅠ和SalⅠ双酶切,克隆到pET-30a(+)中,构建原核表达载体pET-30a-M,将重组表达质粒转化至E.coliBL21(DE3)中,诱导表达。结果表明,成功扩增出肌肉生长抑制素全长基因;克隆载体经过DNA序列测定,所得基因与国外报道的完全一致;成功构建原核表达载体pET-30a-M;经IPTG诱导,表达出了6×His-M融合蛋白,用组氨酸标签抗体做Western印记证明产物大约48 ku,与预期大小相符,肌肉生长抑制素蛋白在大肠杆菌中成功的进行了表达。This experiment was conducted to gain the recombinant myostatin by technique for gene engineering. The specific primers were designed and produced according to myostatin gene sequence. EcoR I and Sal I restriction enzyme cut sites and protective bases were added at the ends of the primer respectively. The full fragment gene was cloned by RT-PCR from pig skeletal muscle, PCR product was cloned into the pMD18-T vector. Sequencing analysis showed that the nucleotide sequence was the same as the published myostatin cDNA sequence by Voelker. The recombinant plasmid pMD18-T-M was identified with EcoR I and Sal I restriction enzyme, the fragment were collected by agarose gel fraction method. After purification the fragment was ligated to the pET-30a express vector ,the recombinant pET-30a-M express vector was constructed. After transformation, the engineered strain E. coli BL21 （DE3）/6 ×His-M was expressed by the induction of IPTG . The specific approximate 48 ku band was obtained by analysis of SDS-PAGE and Western-blotting. The result indicated that myostatin protein had been expressed successfully in E. coli expressing system.

关 键 词：肌肉生长抑制素 克隆 原核表达 纯化 

分 类 号：S828.2[农业科学—畜牧学]