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。提取秦川牛的骨骼肌总RNA，根据基因库中海福特牛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Bam HⅠ和Xho Ⅰ酶切位点及保护碱基,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,测序后经Bam HⅠ和Xho Ⅰ双酶切,将目的片段插入到PGEX-4T-1中,构建原核表达载体PGEX-4T-1-M,将重组表达质粒转化至Rosetta(DE3)中诱导表达。结果表明,扩增的秦川牛肌肉生长抑制素全长基因1 128 bp,克隆载体经过DNA序列测定,所得基因与GenBank上发表的一致;成功构建原核表达载体PGEX-4T-1-M，经IPTG诱导,表达出了GST-M融合蛋白,用GST标签抗体做Western blotting印记证明产物大约69 ku,与预期大小相符,并在Rosetta（DE3）菌中成功的进行融合蛋白表达。
Abstract
This experiment was conducted to obtain the recombinant myostatin by technique of gene engineering. The primers were designed and produced according to Hereford's myostatin gene sequence. Bam H Ⅰ and Xho Ⅰ enzyme cut sites and protective bases were added at the end of the primer respectively. The full fragment gene was cloned by RT-PCR from Qinchuan cattle muscle,and PCR product was cloned into the pMD18-T vector. The recombinant plasmid pMD18-T was identified with Bam H Ⅰ and Xho Ⅰ restriction enzyme, and the fragment were collected by agarose gel fraction method. After purification the fragment was ligated to the pGEX4T-1 express vector, the recombinant pGEX4T-1 express vector was constructed. After transformation, the engineered strain E.coli Rosetta (DE3)/GST-M was expressed by the induction of IPTG. Sequencing analysis showed that the nucleotide sequence was the same as the published myostatin cDNA sequence 1 128 bp. The express vector pGEX4T-1-M was successfully constructed. The specific approximate 69 ku band was obtained by analysis of SDS-PAGE and GST-tag Western-blotting. The result indicated that myostatin protein had been expressed successfully in Rosetta(DE3) expressing system.
关键词
秦川牛 /
肌肉生长抑制素 /
克隆 /
原核表达 /
蛋白印迹
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Key words
Qinchuan cattle /
myostatin /
cloning /
prokaryotic expression /
Western blot
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参考文献
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脚注
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基金
陕西省13115创新工程专项（2010ZDGC-01）；校基本科研业务费重大项目(Z109021010)
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