【摘要】 应用PCR方法 ,扩增人纤溶酶原cDNA基因中K4K5cDNA片段 ,与酵母表达载体 pPIC9K重组 ,获得表达质粒p9kkk 18。该质粒转化毕赤酵母菌GS115 ,用G418 YPD筛选高拷贝表型 ,PCR筛选K4K5cDNA与酵母染色体整合形成的阳性克隆 ,阳性克隆用甲醇诱导表达。表达产物r K4K5分子量约 2 1 5kD ,占分泌总蛋白 80 %以上 ,产物浓度为 15 0～ 2 5 0mg/L。初步纯化产物抑制牛毛细血管内皮 (BCE)细胞增殖与鸡胚绒毛尿囊膜 (CAM )新生血管生成。更多还原

【Abstract】 Kringles of human plasminogen expcept kringle 4 can inhibit the endothelial cell growth.To determine whether recombinant plasminogen kringle 4\|5(r\|K4K5) can inhibit the growth of bovine capillary endothelial (BCE) cell and the angiogenesis of chick embryo chorioallantoic membrane (CAM),we cloned and constructed the modified cDNA region encoding kringles 4 and 5 (K4K5) of human plasmin (ogen) and expressed it with pichia multi\|copy expression system.One clone,with the most productive expression was selected from hundreds of transformants.Our data showed that the expression product r\|K4K5 (MW 21 5kD) amounted to 150～250mg/liter,over 80% of the total secreted protein.It could,dose\|dependently,inhibit BCE cell proliferation and inhibit chick embryo CAM angiogenesis.更多还原