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目的优化重组鲨鱼血管生成抑制因子(Shark angiogenesis inhibition factor,SAIF)功能活性区(aSAIF)蛋白的纯化工艺。方法利用离子交换层析和镍柱亲和层析对带有组氨酸标签的融合蛋白His6-SUMO-aSAIF进行纯化。纯化后的融合蛋白经SUMO蛋白酶切除小分子泛素样修饰蛋白(Small ubiquitin-related modifier,SUMO)标鉴,再经镍柱二次亲和层析获得非融合aSAIF蛋白,采用WST-8法对其进行血管生成抑制活性检测。结果优化的纯化工艺参数为:离子交换层析采用0.2 mol/L的NaCl进行洗脱;亲和层析的洗脱液为20 mmol/L磷酸盐,500 mmol/L NaCl,250 mmol/L咪唑,pH 7.4;SUMO蛋白酶与融合蛋白的最佳酶切比例为1∶480,酶切时间为1 h。最终获得的aSAIF蛋白纯度可达95%,具有较好的抑制血管内皮细胞增殖的活性,且呈剂量依赖性。结论已成功建立了aSAIF的纯化工艺,为后续大规模生产及抑制血管生成机制的研究奠定了基础。
OBJECTIVE: To optimize the purification process of recombinant saSA angiogenesis inhibition factor (SAIF) functional active region (aSAIF) protein. Methods His6-SUMO-aSAIF with histidine tag was purified by ion-exchange chromatography and nickel column affinity chromatography. The purified fusion protein was digested with SUMO protease to remove the small molecule ubiquitin-related modifier (SUMO), and the non-fusion aSAIF protein was obtained by nickel affinity chromatography. The WST-8 method was used to determine It performs the assay of angiogenesis inhibitory activity. Results The optimal purification parameters were as follows: ion exchange chromatography was performed with 0.2 mol / L NaCl; affinity chromatography eluted with 20 mmol / L phosphate, 500 mmol / L NaCl, 250 mmol / L imidazole , pH 7.4. The optimal ratio of SUMO protease and fusion protein was 1: 480, and the digestion time was 1 h. The finally obtained aSAIF protein purity of up to 95%, has better inhibition of vascular endothelial cell proliferation activity, and in a dose-dependent manner. Conclusion The purification of aSAIF has been successfully established and laid the foundation for the subsequent large-scale production and inhibition of angiogenesis mechanism.