人胱抑素C参考物质制备方法的建立

Expression and purification of recombinant cystatin C in Escherichia coli

[目的]建立重组人胱抑素C(cystatin C)表达体系,获得可溶、稳定、高纯度的胱抑素C。[方法]密码子优化并人工合成人胱抑素C编码序列,与麦芽糖结合蛋白(MBP)分别克隆至PRSF-Duet载体以构建重组人p MBP-CYSC PRSF表达质粒。使用大肠杆菌E. coli BL21(DE3) p Lys S对其进行诱导表达,SDS-PAGE鉴定蛋白纯度和分子量,全自动生化仪测定纯化蛋白浓度及稳定性。[结果]成功构建重组人p MBP-CYSC PRSF表达质粒并诱导表达,胱抑素C蛋白浓度可达500 mg/L,纯度可达90%,在4℃和-20℃储存条件下监测64 w,胱抑素C蛋浓度稳定,均在允许相对偏差5%以内。[结论]通过密码子优化,成功获得浓度为500 mg/L、纯度为90%、存储稳定性至少达到64 w的胱抑素C蛋白,可充分满足该产品诊断试剂制备的需求。

[ Objective] To construct optimized human cystatin c expression system, and obtain a soluble, stable and high purity recombinant cystatin C. [ Methods ] The optimized human cystatin C gene was synthesized and cloned into pRSF - Duet vector with MBP residue to generate pMBP - CYSC PRSF expression vector. Recombinant human cystatin C protein was expressed in E. coli BL21 （ DE3 ） pLysS, the protein purity and molecular weight were identified by SDS - PAGE, the concentration of the purified human cystatin C was analyzed by hitachi analysis system. The stability of human cystatin C protein was examined during long - term storage at 4 ℃ and - 20℃. [ Results ] The prokaryotic expression vector MBP - CYSC PRSF was successfully constructed. When expressed in E. coli BL21 （ DE3 ） pLysS, soluble MBP - CYSC was harvested by nickel affinity and identified by SDS - PAGE. The concentration of soluble cystatin C reached 500 mg/L, and the purity reached 90%. Furthermore, cystatin C protein concentration remained unchanged for at least 64 weeks at 4℃ and -20℃. [ Conclusion] The cystatin C protein with a concentration of 500 mg/L, a purity of 90% and a storage stability of at least 64 weeks was successfully obtained by co- don optimization ,which can fully meet the requirement of the preparation of reference material.