人纤溶酶原饼环区5蛋白的原核表达体系的建立

Construction of Prokaryotic Expression System of Human Plasminogen Kringle 5 (hPK-5) Protein

目的 构建人纤溶酶原饼环区 5 (hPK 5 )蛋白原核表达载体 ,并进行表达和鉴定 ,为获取大量高纯度、具有生物活性的hPK 5蛋白奠定基础。方法 以纤溶酶原cDNA为模板 ,PCR扩增hPK 5基因 ,经酶切后构建表达载体pBV2 2 0 /hPK 5 ,转入大肠杆菌JM10 9进行温控诱导表达 ,表达产物经纯化、复性后 ,检测其抑制鸡胚绒毛尿囊膜 (CAM)血管新生的生物学活性。结果 带有pBV2 2 0 /hPK 5的大肠杆菌表达的目的蛋白占菌体总蛋白的34 8% ,其表达蛋白具有His tag抗原活性和野生活性。结论 已成功构建了pBV2 2 0 /hPK 5重组质粒 ,并在大肠杆菌中获得高效表达。

Objective To construct a prokaryotic expression system of human plasminogen kringle 5 (hPK-5) protein, identify the expressed product and lay a foundation of preparation of hPK-5 protein with high purity and bioactivity in a large scale.Methods hPK-5 gene was amplified by PCR using human plasminogen cDNA as a template, digested with restriction endonuclease and inserted into plasmid pBV220.The constructed recombinant plasmid pBV220/hPK-5 was transformed to E.coli JM109 for expression.The expressed product was purified, re-naturalized and detected for bioactivity of inhibiting the angiogenesis of chorioallantoic membrane (CAM).Results The expressed product contained 34.8% of total somatic protein and showed His-tag antigen activity.CAM test proved that the bioactivity of expressed product was similar to that of wild hPK-5.Conclusion Recombinant plasmid pBV220/hPK-5 was successfully constructed and expressed in E.coli.