人血纤维蛋白溶酶原K5环的基因合成、表达、纯化与活性鉴定

Gene Synthesis,Expression and Purification of Human Plasminogen Kringle 5 in E.coli and Its Antiangiogenesis Activity

根据大肠杆菌遗传密码的偏爱性 ,人工合成人血纤维蛋白溶酶原 K5全基因 ,并在原核系统中以硫氧还蛋白融合蛋白的形式实现了高效表达 .重组蛋白通过 Ni2 +金属螯合层析得到初步纯化 ,通过肠激酶切割去除了融合标签 .应用鸡胚尿囊膜实验检测切割后的 rh K5的生物学活性 ,发现与对照组相比 ,rh K5能明显地降低血管管径、血管总面积以及血管总面积与视野面积的比值 ,表明切割后的产物具有显著抑制新生血管生成的生物学活性 .为进一步研究和开发抗血管生成药物奠定了基础 .

Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth and a potent anti cancer drug candidate.The gene of human plasminogen kringle 5 was synthesized according to the codon usage bias of E.coli .The gene was over expressed in E.coli as a thioredoxin fusion protein.Expression product was purified to almost homogeneity by Ni chelating chromatography.The fusion tag was completely removed by enterokinase cleavage.The antiangiogenic activity of the rhK5 was estimated using a chorioallantoic membrane (CAM) model of chicken embryos.Compared with the control group,the rhK5 inhibited new embryonic blood vessel growth,as measured by the formation of avascular zone.These results laid a good foundation for further research and development of new antiangiogenic drug based on plasminogen kringle 5.