重组人胱抑素C的原核表达及鉴定

目的构建重组人胱抑素C（cystatinC，CysC）的原核高效表达质粒，诱导表达并纯化获得CysC重组蛋白。方法根据大肠埃希菌编码蛋白的特性设计CysC编码基因序列，人工合成目的基因克隆至pET-22b（＋）表达载体中，测序及酶切鉴定正确后诱导其在大肠埃希菌BL21中表达，所获得的包涵体蛋白经亲和层析纯化后采用SDS—PAGE及Western印迹鉴定。结果酶切结果证实构建的表达质粒结构正确；测序结果显示克隆的基因序列所编码的蛋白与GenBank中的CysC氨基酸序列相符；SDS-PAGE及Western印迹结果证实获得的重组CysC融合蛋白分子量约为16kD，经NP亲和层析纯化获得纯度大于90％的目的蛋白。结论建立了重组人CysC的原核高效表达系统并获得了CysC重组蛋白。

Prokaryotic Expression and Identification of Recombinant Human Cystatin C

Objective To construct a prokaryotic expression vector of cystatin C （Cys C） and purify recombinant Cys C protein produced by the expression system. Methods The gene of human CysC was obtained by synthesis. The cDNA fragment was cloned into prokaryotic expression vector pET-22b （ ＋ ） to construct recombinant protein expression vector pET- Cys C. The positive recom- binant clone was analyzed by restriction endonuclease digestion and DNA sequencing. The pET- Cys C plasmid was transfected into E. coli. BL 21 to induce CysC expression. The inclusion body pro- tein was purified through Ni2 ＋ affinity chromatography and identified by using SDS-PAGE and West- ern blotting. Results The restriction enzyme digestion and DNA sequencing revealed the Cys C protein sequence coded by recombinant plasmid, completely corresponding to GenBank data. SDS- PAGE and Western blotting showed the size of expressed recombinant Cys C fusion protein at about 16kD. The purified recombinant Cys C through Ni2＋ affinity chromatography was greater than 90%. Conclusion In this study, we have established C protein. a high expression system of recombinant human Cys