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利用PCR技术获得人尿激酶原Kringle结构域基因,将其克隆至具有T7启动子的表达质粒pET29a中,并进而插入plasminogenKringle5一段16肽片段构建了其变体,重组质粒转化至大肠杆菌BL21中,经IPTG诱导表达,其产物以包涵体形式存在.通过体外复性,得到可溶性的prourokinaseKringle及其变体.所得蛋白质经过动物肿瘤模型测活,确定变体蛋白具有抑制肿瘤生长作用.
The human pro-urokinase Kringle domain gene was obtained by PCR and cloned into the expression plasmid pET29a with the T7 promoter. A variant of the 16-mer fragment of plasminogen Kringle5 was inserted to construct the Kringle domain. The recombinant plasmid was transformed into E. coli BL21. After being induced by IPTG, the product exists in the form of inclusion bodies. Through the renaturation in vitro, soluble prourokinase Kringle and its variants are obtained. The obtained protein is measured in an animal tumor model to determine that the variant protein has an inhibitory effect on tumor growth.