人斯钙素1在大肠杆菌中的重组表达与活性鉴定

目的：通过原核表达的方法得到有活性的斯钙素1（ STC1）重组蛋白。方法将优化合成的STC1 DNA片段克隆到表达载体pET-32 b（＋）上，重组质粒转入大肠杆菌诱导表达，在变性条件下纯化包涵体，经透析复性得到可溶的斯钙素1融合蛋白，凝血酶酶切后用高亲和镍离子树脂吸附多余多肽获得目的蛋白。 Western印迹分析验证目的蛋白免疫活性。动物实验检测目的蛋白的生物学活性。结果构建了pET-32b（＋）-STC1表达载体，表达并纯化了STC1融合蛋白包涵体，经复性、凝血酶切和纯化后获得可溶的目的蛋白。 Western印迹分析验证正确。生物学活性检测表明重组STC1能增加大鼠排泄系统对磷酸盐的重吸收。结论获得了具有生物学活性的斯钙素1重组蛋白，为制备STC1单克隆抗体和进一步研究STC1与肿瘤治疗的关系奠定了基础。

Expression and activity identification of recombinant human stanniocalcin 1

Objective To obtain recombinant human stanniocalcin 1 （STC1）with biological activity in Escheri. coli cells expression. Methods The gene was cloned into pET32b （ ＋ ） vector by fused with thioredoxin and His tag. E. coli BL21 （DE3） competent cells were transfomed by the recombinant vector~ After renaturation, the fusion protein was digested with thrombin and intact STC1 protein was purified from the digested protein using Ni ion affinity chromatography. Recombi- nant humanSTC1 protein was confirmed by Western blot analysis using goat anti-STC1 antibody. The biological activity of STC1 in rat was assayed using standard method for assessment of renal function. Results The recombinant human STC1 fu- sion protein is successfully expressed in Escherichia coli, the fusion protein was purified by affinity chromatography from the inclusion body and renaturated. Intact hSTC1 protein was released by thrombin digestion and purified by Ni ion affinity col- umn. The intact STC1 proteins was confirmed by Western blot analysis. Rat bioassay revealed that STC1 boosted phosphate reabsorption. Conclusion Recombinant STC1 protein was successfully expressed and has native biological activities. This protein could be used as an antigen for the preparation of monoclonal antibody against humanSTC1.