| 摘要: |
| 猪肌生成抑制素基因myostatin (MSTN)的cDNA在去除信号肽后,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,并测序分析，结果表明其与设计序列完全一致。将该克隆载体的质粒DNA用带有BamHⅠ和SalⅠ内切酶识别序列的另1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。对成功构建的表达载体阳性克隆在LB液体培养基中用异丙基硫代-β-D-半乳糖苷(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分子质量为41.451 3 ku。因为所构建的表达载体中含六聚组氨酸标签，用His-trap亲和柱进行纯化后,纯度可达92.5%。 |
| 关键词: 猪肌生成抑制素基因 成熟蛋白编码序列 表达 纯化 |
| DOI： |
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| 基金项目:国家自然科学基金项目(30070563)；军队医药卫生青年基金项目(98Q079) |
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| Expression and purification of mature protein coding sequence of porcine myostatin gene in prokaryotic expression vector |
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| Abstract: |
| Myostatin is a negative regulator of skeletal muscle growth.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 double muscling.In order to investigate the relationship between myostatin and high lean meat rate and plump-hipped trait,the expression vectors of mature protein coding sequence (MPCS) of porcine myostatin (MSTN) were constructed.Then the recombinant MPCS-MSTN-pET28a(+)-BL21 plasmid 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 accounted 27.9% of the total protein of the transformed host cell by analysis of thin-layer scanner.Comparison with different inducing time showed that the expression amount of recombinant MSTN protein was the highest after being induced for 3-4 hours.The expression protein of porcine myostatin(MSTN) was purified by HisTrapTM for purification of histidine-tagged proteins (Pharmacia Biotech).The purity of expressed reombinant MSTN protein reached to 92.5%. |
| Key words: porcine myostatin gene mature protein coding sequence expression purification |
