半胱氨酸蛋白酶抑制剂C重组蛋白的制备

Preparation of cystatin C recombinant protein

目的构建半胱氨酸蛋白酶抑制剂C(CysC)原核表达载体,用以快速获取低成本、高浓度、高纯度的CysC纯化蛋白,为制备应用于临床实验室检测的CysC标准物质和质控品奠定基础。方法用Codon Adaptation Tool将人的CysC编码序列优化为大肠杆菌偏好的密码子序列,再将合成的DNA片段克隆至pET-28a(+)原核表达载体。将重组CysC表达质粒转化至E.coli BL21感受态中,优化诱导表达条件、确定最优表达条件组合及有效表达序列,并用融合标签对重组蛋白进行纯化。结果成功构建重组CysC原核表达质粒,明确CysC表达序列,确定异丙基硫代半乳糖苷(IPTG)诱导重组蛋白表达浓度、诱导温度等条件,纯化的CysC蛋白可通过临床检测,且稀释倍数与检测浓度间呈现线性关系。结论成功构建重组CysC的高效原核表达系统,重组CysC蛋白纯度大、浓度高、生产周期短,有望为CysC标准物质和质控品的制备提供物质基础。

Objective To construct a prokaryotic expression vector for human cystatin C(CysC)to obtain purified CysC protein rapidly with low cost,high concentration and high purity,which laid a foundation for the preparation of CysC reference materials and quality control materials for clinical laboratory detection.Methods The CysC coding sequence provided by GenBank was optimized by E.coli Codon Adaptation Tool,and a synthetic DNA fragment was cloned into pET-28a(+)prokaryotic expression vector to construct recombinant CysC expression plasmid.The recombinant protein was transformed into E.coli BL21 competent cell.The induced expression conditions were optimized,the optimal combination of expression conditions and effective expression sequence were determined and the recombinant protein was purified by His fusion tag.Results The recombinant CysC prokaryotic expression plasmid was successfully constructed.The expression concentration and induction temperature of the recombinant protein induced by IPTG were optimized.The purified CysC protein could be detectable in clinical laboratory,and a good linear relationship between dilution ratio and detection concentration was shown.Conclusion An efficient prokaryotic expression system of recombinant CysC was successfully constructed.The recombinant CysC protein may exhibit high purity,high concentration and short production cycle,and has potential to be a candidate of reference material for laboratory quality evaluation.