摘要
猪肌生成抑制素(myostatin,MSTN)基因的cDNA去除信号肽后PCR扩增出成熟蛋白编码序列1.2kb片段熏将该片段与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BamHI和SalI内切酶识别序列的另一对引物进行PCR扩增,将回收的1.2kbPCR目的片段定向克隆到pET28a(+)表达载体上,成功地构建了编码猪肌生成抑制素成熟蛋白的原核表达载体。LB液体培养基中用IPTG诱导表达,SDS-PAGE显示重组菌表达的MSTN蛋白以包涵体形式存在;经薄层扫描仪扫描分析SDS-PAGE凝胶,表达的MST包涵体蛋白占菌体不溶性蛋白含量的27.9%,其相对分子质量为41451.3。所构建的表达载体中含六聚组氨酸标签HisTrap亲和柱纯化后纯度可达92.5%。该试验为获得较好的猪肌生成抑制素并制备抗体打下了良好的基础。
Myostatin(MSTN)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 investgate 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 was constructed.Than the re-combinant MPCS-MSTN-pET-28a(+)-BL21plasmid was cultured with LB medium and induced with IPTG and detected by using SDS-PAGE.The mature protein coding sequence of porcine myostatin gene was expressed in the form of inclu-sion bodies.Expression amount accounted27.9%of the total protein of the transformed host cell by analysis of thin-layer scanner.Comparing with different inducing time showed that the expression amount of recombinant MSTN protein was the highest after inducing3～4h.The expression protein of porcine myostatin.MPCS was purified by HisTrap TM for purification of histidine-tagged proteins.The purity of expressed reombinant MSTN protein reached to92.5%.The results lay a good foundation for the application of MSTN gene in molecular biological technique in animal nutrition and breeding.
出处
《生物技术通讯》
CAS
2004年第1期23-25,共3页
Letters in Biotechnology
基金
国家自然科学基金(30070563)
军队医药卫生青年基金(98Q079)
