血管生成抑制因子K4K5 Cdna基因的克隆及其在毕赤酵母中的表达

应用PCR方法，扩增人纤溶酶原cDNA基因中K4K5 cDNA片段，与酵母表达载体pPIC9K重组，获得表达质凿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K4K5 cDNA与酵母染色体整全形成的阳性克隆,阳性克隆用甲醇诱导表达。表达产物r-K4K5分子量约21.5kD,占分泌总蛋白80％以上，产物浓度为150－250mg/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。

Molecular Cloning and Expression of the cDNA Encoding Angiogenesis Inhibitor K4K5 with Pichia pastoris

Kringles of human plasminogen except 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 21.5 kD) amounted to 150-250 mg/liter, over 80% of the total secreted protein. It could, dose-dependently, inhibit BCE cell proliferation and inhibit chick embryo CAM angiogenesis.