人血管抑制素基因克隆、表达和生物活性分析

Cloning of Human Angiostatin Gene and Its Secretive Expression in Pichia pastoris

应用PCR技术从人胎肝cDNA文库中扩增了人血管抑制素基因。将克隆的基因重组进酵母质粒pPIC9K获得含该基因的重组质粒pPIC9KA3。用电激法将质粒pPIC9K转化毕节酵母GS115 ,经PCR检测获得含人血管抑制素基因的酵母工程菌GS115 (pPIC9KA3)。再用G418筛选法 ,在含不同浓度的G418平板上筛选高拷贝整合的转化子。对高拷贝整合的转化子进行发酵培养和诱导表达。SDS_PAGE及Western印迹分析显示 :表达产物约占胞外蛋白的 43% ,相当于 94mg L ,并具有免疫活性。并能抑制bFGF诱导的鸡胚尿囊膜新生血管的生成。还对用G418筛选高拷贝整合转化子的方法做了探索。

Human angiostatin gene was amplified from fetal liver cDNA library by PCR techni que and cloned into Yeast-E^coli shuttle vector pPIC9K resulting in recomb in ant plasmid pPIC9KA3 which was then transformed into Pichia pastoris GS115. The positive transformants were first determined by PCR technique and G418 meth od was used to screen the high copy integrative transformants. The human angiost atin protein was expressed and secreted into the medium after methanol induction and reacted with antibody against human plasminogen as shown by SDS-PAGE and We stern blot. The expressed product constitutes 43% of the total extracellular p rotein corresponds to 94 mg/L as estimated by Shimadzu CS-930 scanner and protei n quantitation. The recombinant angiostatin inhibits the bFGF induced CAM angiog enesis. Screening of high copy integrative transformants by G418 method was disc ussed.