| 大黄鱼肌肉生长抑制素基因原核表达及多克隆抗体的制备 |
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| 引用本文： | 董海阳,叶秀丽,薛良义,Diallo Amadou.大黄鱼肌肉生长抑制素基因原核表达及多克隆抗体的制备[J].台湾海峡,2011,30(2):196-202. |
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| 作者姓名： | 董海阳 叶秀丽 薛良义 Diallo Amadou |
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| 作者单位： | 宁波大学生命科学与生物工程学院,浙江,宁波,315211 |
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| 基金项目： | 国家自然科学基金，国家863计划资助项目 |
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| 摘 要： | 肌肉生长抑制素抑制动物肌肉的生长发育.根据本实验室克隆的大黄鱼（Pseudosciaena cro-cea）肌肉生长抑制素（MSTN）基因编码序列设计引物,用RT-PCR扩增目的片段,构建MSTN-pET-28 a重组表达质粒,将其转化到大肠杆菌BL21上并用1.0 mmol/dm3IPTG诱导表达,SDS-PAGE电泳检测该目的蛋白大小约为44 kDa,与理论值大小符合.用0.25 mol/dm3KCl染色后从凝胶中切取该蛋白且回收.该回收蛋白与弗氏佐剂等量混合后注射ICR小鼠制备多克隆抗体,并用Westernblot检测该抗体.免疫印迹结果在44 kDa的位置上出现棕色条带,表明大黄鱼肌肉生长抑制素的多克隆抗体制备成功.这为今后对肌肉生长抑制素功能的研究奠定了基础.
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| 关 键 词： | 海洋生物学 肌肉生长抑制素 原核表达 多克隆抗体 免疫印迹 大黄鱼 |
Prokaryotic expression and polyclonal antibody preparation of myostatin from Pseudosciaena crocea |
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| DONG Hai-yang,YE Xiu-li,XUE Liang-yi,Diallo Amadou.Prokaryotic expression and polyclonal antibody preparation of myostatin from Pseudosciaena crocea[J].Journal of Oceanography In Taiwan Strait,2011,30(2):196-202. |
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| Authors: | DONG Hai-yang YE Xiu-li XUE Liang-yi Diallo Amadou |
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| Institution: | DONG Hai-yang,YE Xiu-li,XUE Liang-yi,Diallo Amadou(College of Life Science and Biotechnology,Ningbo University,Ningbo 315211,China) |
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| Abstract: | Myostatin（MSTN）,a member of the TGF-β superfamily,plays an important role as a negative regulator in muscle growth.Natural mutation of the MSTN gene in animals,which results in the low activity of myostatin,shows a double-muscle phenotype,and results in a considerably larger diameter or number of skeletal muscle fibers.The weight of muscle tissue in MSTN-knocked out mice increases by 2～3 times.Thus,animal breeders have increasingly paid attention to the MSTN gene and its potential application in animal breeding.In cloning the Pseudosciaena crocea MSTN gene（AY 842933） in our laboratory,one pair of primers were designed and used to amplify the complete MSTN encoding sequence by RT-PCR.The amplified sequences were cloned into pMD18-T plasmids.The recombined plasmids were transferred into E.coli DH5α and extracted after clone screening and enlargement culture.After sequencing and confirmation,the recombined pMD18-T plasmids were digested with BamH Ⅰ and Hind Ⅲ endonucleases.The obtained objective fragments were inserted into pET-28a plasmids,constructing recombined expression plasmid MSTN-pET-28a.The recombined expression plasmids were then transformed into E.coli BL21 bacteria.Expression of the inserted sequences was induced with 1.0 mmol/dm3 IPTG.The molecular weight of the recombined protein is about 44 kDa.The target protein was stained with 0.25 mol/dm3 KCl and extracted to mix with Freund＇s complete and incomplete adjuvant equally.The mixture was injected into ICR mice to obtain the polyclonal antibody,once a week,three times in total.After 3 days of the last intensive immunity,the blood was obtained from eyes of immunized mice,and kept at 37℃ for 20 min,then at 4℃ overnight.The supernatant containing MSTN antibody was obtained by centrifuging the blood at 4℃（4 000 r/min,10 min）.The antibody was detected in western blot analysis.There was a brown target band in the corresponding position,which showed that the polyclonal antibody was successfully prepared.It has been reported that MSTN antibody can promote animal muscle growth.Our preparation of the Pseudosciaena crocea MSTN antibody is a basis for future MSTN application in aquaculture. |
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| Keywords: | marine biology myostatin prokaryotic expression polyclonal antibody western blot Pseudosciaena crocea |
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