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Cloning and Prokaryotic Expression of Human Cystatin C Gene
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- Author:
- No author available
- Journal Title:
- JOURNAL OF RADIOIMMUNOLOGY
- Issue:
- 6
- DOI:
- 10.3969/j.issn.1008-9810.2013.06.041
- Key Word:
- 胱抑素C;基因;克隆;原核表达
Abstract： 目的:研究人胱抑素C(Cycstatin C,Cys C)的原核重组表达及纯化.方法:通过RT-PCR扩增得到人Cys C的cDNA序列,并将其构建到pET-32a(+)的表达载体中.通过转化入BL21(DE3)表达宿主菌及表达条件的优化,摸索其最佳表达条件.通过镍柱纯化以及DEAE-sepharose FF离子交换层析的方法纯化得到重组的人Cys C蛋白.结果:通过RT-PCR扩增的方法得到的cDNA序列通过测序证明与预期一致.通过优化表达,Cys C的表达水平达到了160mg/L,约占总可溶蛋白的 20%,表达产物通过两步纯化后获得了纯度为95%的重组Cys C.结论:成功构建Cys C原核表达系统和蛋白纯化系统,为下一步制备多克隆抗体以及开发Cys C的免疫诊断试剂奠定了基础.
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