无融合标签人胱抑素C重组蛋白的制备

Expression and purification of tag-free cystatin C in Escherichia coli

目的构建人胱抑素C(CysC)基因的原核表达质粒pCold TF-CysC,表达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。用该CysC免疫新西兰大白兔,制备抗血清,并用间接ELISA及Western blot鉴定。结果测序结果与Genbank中人CysC序列一致,实现了CysC可溶性高表达。纯化的无标签CysC纯度大于95%,分子质量约为13.3 ku,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,能特异性识别市售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,获得了可用作免疫诊断试剂标准品的CysC,为进一步建立CysC的免疫检测方法奠定了基础。

Objective To construct a prokaryotic expression vector of human cystatin C （CysC） gene, prepare tag-free CysC to be used 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 tag-free CysC. The antiserum was prepared and titrated by indirect ELISA and Western blot respectively. Results The result of sequencing proved that the pCold TF-CysC was correctly constructed. TF-CysC was highly expressed in a soluble form. The molecular weight of purified tag-free CysC is approximately 13. 3 ku, and the purity reached to 95%. The titers of the antiserum against CysC reached more than 1： 4 x10^6 and the reactogenicity of antiserum was confirmed. Tag-free CysC was stored in 4 °C, the concentration of the CysC was found no obvious decrease in one month. Conclusions The purified tag-free CysC was obtained, which laid a foundation of development of immunological method for detection of CysC.