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