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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-32b(+)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。
Objective To obtain an active stanniocalcin 1 (STC1) recombinant protein by prokaryotic expression. Methods The recombinant DNA fragment of STC1 was cloned into the expression vector pET-32b (+). The recombinant plasmid was transformed into E. coli to induce the expression. The inclusion bodies were purified under denaturing conditions and the soluble stanniocalcin 1 fusion Protein, thrombin digested with high affinity nickel ion resin adsorption of excess peptides to obtain the desired protein. Western blot analysis to verify the target protein immunocompetence. Animal experiments to detect the biological activity of the target protein. Results The pET-32b (+) - STC1 expression vector was constructed and the inclusion body of STC1 fusion protein was expressed and purified. The soluble protein of interest was obtained after refolding, prothrombin and purification. Western blot analysis verified correctly. Biological activity tests showed that recombinant STC1 can increase phosphate reabsorption in rat excretory system. Conclusions A stanniocalcin 1 recombinant protein with biological activity was obtained, which laid the foundation for the preparation of monoclonal antibodies against STC1 and further study on the relationship between STC1 and tumor therapy.