摘要
目的克隆人纤溶酶原Kringle5 (K5 )区基因 ,并进行重组蛋白的表达纯化及活性鉴定。方法用PCR方法从正常成人肝cDNA库扩增出人纤溶酶原K5区基因 ,构建K5的原核表达载体pET 2 2b(+) K5 6×His,IPTG诱导蛋白表达后经Ni+ -树脂亲和层析进行纯化 ,通过血管内皮细胞增殖抑制试验检测蛋白活性。结果经PCR成功扩增出了 2 4 3bp的K5区基因 ,测序正确后克隆进大肠杆菌分泌型表达载体pET 2 2b(+) ,重组质粒在BL2 1中成功表达出分子量为 14 0 0 0的蛋白质 ,纯化后的蛋白纯度达95 % ,具有抑制血管内皮细胞增殖活性的功能 ,K5蛋白浓度为 4 μg/mL时抑制率达 5 0 %。 结论纤溶酶原K5的成功克隆。
Objective To clone human plasminogen Kringle 5 gene into pET-22b(+) vector, and then purify the recombinant protein and assay its biological activity. Methods Using PCR technique, the gene encoding human plasminogen Kringle 5 was amplified from the cDNA library of the adult human liver, and the PCR product was ligated with PCR2.1 vector. Then the correct clone of PCR2.1-K5 as the template to amplify K5 gene was added to 6 His gene. After sequencing correctly the K5-6 His gene was inserted into pET-22b(+), which was expressed in BL21 and purified by affinity chromatography through Ni 2+-resin column.The purified protein was tested by endothelial cell proliferation inhibition assay. Results The K5 gene was 243 bp,which was expressed in BL21 at a high level. SDS-PAGE and Western blot showed its molecular mass was 14 000. The purity of K5-6 His reached more than 95% by affinity chromatography. The protein exhibited the inhibitory activity of endothelial cell proliferation. Conclusion The cloning, expression and purification of human plasminogen Kringle 5 may play a role in the antiangiogenesis therapy of tumor and atherosclerosis.
出处
《上海第二医科大学学报》
CSCD
北大核心
2005年第2期151-154,共4页
Acta Universitatis Medicinalis Secondae Shanghai
基金
上海市科委重点科技项目 (99JC40 4 1 )资助
