基础医学与临床 ›› 2012, Vol. 32 ›› Issue (6): 697-701.

• 技术与方法 • 上一篇    下一篇

无融合标签人胱抑素C重组蛋白的制备

陈特,刘宇思,谌海兰,陈曦,王虹,张雪梅,胥文春   

  1. 重庆医科大学
  • 收稿日期:2012-02-16 修回日期:2012-04-25 出版日期:2012-06-05 发布日期:2012-05-25
  • 通讯作者: 胥文春 E-mail:xuwen0303@126.com

An Expression and Purification of Tagless Cystatin C

  • Received:2012-02-16 Revised:2012-04-25 Online:2012-06-05 Published:2012-05-25

摘要: 目的:构建人胱抑素C(Cystatin C,CysC)基因的原表达质粒pCold TF-CysC,表达TF-CysC融合蛋白,并制备去标签蛋白的CysC。 方法:用人CysC的全长编码基因插入原核表达载体pCold TF并测序鉴定。将重组质粒转化到大肠杆菌BL21(DE3),经IPTG诱导、镍柱纯化后获得带有His标签的重组蛋白TF-CysC。利用GST-HRV 3C蛋白酶酶切去除该融合蛋白的TF标签,再经分子筛一步法同时去除TF标签、3C蛋白酶获得CysC,SDS-PAGE鉴定其纯度。用所获无标签CysC免疫新西兰大白兔,制备抗血清,并用间接ELISA及Western blot鉴定。结果: 测序获得序列与Genbank中人CysC序列一致,实现了CysC可溶性高表达。纯化的无蛋白标签的CysC纯度大于95%,分子量约为13.3 kD,与预期相符。免疫家兔后,获得的抗血清效价达到1:4×106以上,并能特异性识别商用CysC蛋白。该蛋白稳定性良好,在4℃保存一个月未见明显下降。结论:成功应用原核表达系统获得去融合蛋白标签的CysC,为进一步建立CysC的免疫检测方法奠定了基础。

关键词: 胱抑素C, 原核表达, 蛋白纯化, 蛋白标准品

Abstract: Objective To construct a prokaryotic expression vector of human Cystatin C(CysC)gene, express TF-CysC fusion protein; prepare tagfree CysC used as protein standard for immunodiagnosis test. Methods A cDNA fragment coding for the full-length human CysC was inserted into pCold TF expression vector, followed by sequencing analysis. The constructed recombinant plasmid was transformed to E.coli BL21(DE3) for expression under induction of IPTG. After purified by Ni-Sepharose affinity chromatography, The TF-CysC was treated with GST-HRV 3C protease, and the TF tags and GST-HRV 3C proteases were removed by size-exclusion chromatography(SEC)in one step. The prepared CysC was identified by SDS-PAGE and Western blot. New Zealand rabbits were immunized with the fusion protein and the antiserum was obtained. The titers and reactogenicity of the prepared polyclonal antibody were determined by indirect ELISA and Western blot respectively. Results Restriction analysis and sequencing proved that recombinant plasmid pCold TF-CysC was constructed correctly. Tagfree CysC was high expressed in a soluble form and its molecular weight is approximately 13.3 kD, reached a purity of 95%. The titers of the prepared antiserum against CysC reached more than 1:4×106 and the good reactogenicity of antiserum was confirmed. CysC was stored in 4 ℃, and the concentration of the CysC was found no obvious decrease in one month. Conclusion The purity tagfree CysC was obtained as protein standard for immunodiagnosis test, and the method of the protein expression and purification was available and efficient.

Key words: cystatin C, prokaryotic expression, protein purification, protein standard

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