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目的:表达和纯化重组葡激酶-人HC蛋白融合蛋白,并初步鉴定其生物学活性。方法:利用重叠延伸PCR方法使基因重组获得目的基因片段,插入带有GST标签的原核高效可溶性表达载体pEGX-6P-1中,构建重组表达质粒pEGX-6P-1-SAK-HC,将重组表达质粒转化大肠杆菌B834,经IPTG诱导目的蛋白表达;对融合蛋白用谷胱甘肽琼脂糖凝胶柱(GST)亲和层析柱及DEAE离子交换柱纯化,用PreScission蛋白酶切除GST标签,SDS-PAGE分析该融合蛋白的表达量和纯度,应用纤维蛋白平板溶圈法测定评价其生物学活性。结果:构建的重组表达质粒经PCR、内切酶鉴定及基因序列测定证实,目的蛋白在大肠杆菌中获得表达,SDS-PAGE显示相对分子质量(Mr)为36 000;对表达产物进行了亲和层析纯化,从上清中获得了纯度较高的葡激酶-人HC蛋白融合蛋白,体外实验显示其纤溶活性为9.4×104IU/mg。结论:获得了可溶性的葡激酶-人HC蛋白融合蛋白,且其纤溶活性与尿激酶标准品相当。为下一步进行融合蛋白免疫原性鉴定奠定了基础。
Objective: To express and purify recombinant staphylokinase-human HC protein fusion protein and to preliminary identify its biological activity. Methods: The gene fragment was inserted into the prokaryotic expression vector pEGX-6P-1 with the GST tag by overlap extension PCR and the recombinant plasmid pEGX-6P-1-SAK-HC was constructed. The plasmid was transformed into E. coli B834 and induced by IPTG. The fusion protein was purified by glutathione sepharose column (GST) affinity chromatography and DEAE ion exchange column. The GST tag was excised with PreScission protease and SDS- PAGE was used to analyze the expression and purity of the fusion protein. The biological activity of the fusion protein was evaluated by fibrinolytic assay. Results: The constructed recombinant plasmid was confirmed by PCR, restriction endonuclease and sequence analysis. The target protein was expressed in Escherichia coli. SDS-PAGE showed that the relative molecular mass (Mr) was 36 000. The expression of the recombinant protein was confirmed by affinity Chromatography purification, obtained from the supernatant of high purity staphylokinase - human HC protein fusion protein, in vitro experiments showed that the fibrinolytic activity of 9.4 × 104IU / mg. Conclusion: Soluble staphylokinase - human HC protein fusion protein was obtained, and its fibrinolytic activity was comparable to that of urokinase. Which laid the foundation for the identification of the immunogenicity of the fusion protein in the next step.