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重组斑马鱼p8蛋白的原核表达及纯化 被引量：1

Prokaryotic Expression and Purification of Recombinant p8 Protein of Zebrafish
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摘要 目的:在大肠杆菌中重组表达斑马鱼p8蛋白并纯化。方法:PCR扩增斑马鱼p8蛋白基因编码区,连接到带有6×His标签的原核表达载体pET-28a中,构建重组表达质粒pET-28a-p8并转化大肠杆菌BL21(DE3),用IPTG诱导表达;优化表达条件后用Ni2+柱纯化重组蛋白。结果:构建了pET-28a-p8重组质粒;目的蛋白在大肠杆菌中获得表达,亲和纯化后,SDS-PAGE显示相对分子质量为预期的12.8×103。结论:获得了斑马鱼p8融合蛋白,为其生物学功能研究奠定了基础。 Objective： To express and purify the recombinant p8 protein of zebrafish in E.coli BL21（DE3）. Meth- ods： The coding sequence of p8 protein of zebrafish was amplified by PCR and inserted into the prokaryotic expression vector pET-28a with 6×His tag to construct the recombinant plasmid pET-28a-pS. The recombinant plas- mid was transformed into E.coli BL21（DE3）, and the expression of fusion protein was induced by IPTG. Ni2＋ met- al chelating column was utilized for the purification of the fusion protein after the expression conditions were opti- mized. Results： Recombinant plasmid pET-28a-p8 was constructed and the recombinant protein was expressed in E.coli successfully. After being purified by affinity chromatography, SDS-PAGE showed a clear protein band with a relative molecular weight of 12.8 kDa expectedly. Conclusion： The fusion p8 protein of zebrafish was successful- ly expressed and purified, which lays the foundation of further study on its biological function.
作者 孙验玲 刘振辉 李林芳
机构地区 中国海洋大学海洋生命学院 中国海洋大学海洋生物多样性与进化研究所 教育部海洋生物遗传学与育种重点实验室
出处 《生物技术通讯》 CAS 2012年第6期804-808,共5页 Letters in Biotechnology
基金 国家863计划(2008AA092603) 教育部新世纪人才支持计划(NCET-08-0501)
关键词 斑马鱼 p8蛋白 原核表达 纯化 zebrafish p8 protein prokaryotic expression purification
分类号 Q78 [生物学—分子生物学]
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同被引文献40

  • 1Mallo GV,Fiedler F,Calvo EL,et al.Cloning and expression of the rat p8 cDNA,a new gene activated in pancreas during the acute phase of pancreatitis,pancreatic development,and regeneration,and which promotes cellular growth[J].J Biol Chem,1997,272(51):32360-32369.
  • 2Ree AH,Tvermyr M,Engebraaten O,et al.Expression of a novel factor in human breast cancer cells with metastatic potential[J].Cancer Res,1999,59(18):4675-4680.
  • 3Vasseur S,Vidal Mallo G,Fiedler F,et al.Cloning and expression of the human p8,a nuclear protein with mitogenic activity[J].Eur J Biochem,1999,259(3):670-675.
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  • 6GoruppiS,Patten RD,Force T,et al.Helix-loophelix protein p8,a transcriptional regulator required for cardiomyocyte hypertrophy and cardiac fibroblast matrix metalloprotease induction[J].Mol Cell Biol,2007,27(3):993-1006.
  • 7Vasseur S,Hoffmeister A,Garcia-Montero A,et al.Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and DNA microarray analysis of livers reveals an aberrant gene expression response[J].BMC Gastroenterol,2003,3,25.
  • 8Weis S,Schlaich TC,Dehghani F,et al.P8 Deficiency Causes Siderosis in Spleens and Lymphocyte Apoptosis in Acute Pancreatitis[J].Pancreas,2014,43(8):1277-1285.
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  • 10Barbosa-Sampaio HC,Drynda R,Liu B,et al.Reduced nuclear protein 1 expression improves insulin sensitivity and protects against diet-induced glucose intolerance through up-regulation of heat shock protein 70[J].Biochim Biophys Acta,2015,1852(5):962-969.

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生物技术通讯
2012年 第6期

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