中文摘要：

研究重组人组织激肽释放酶结合蛋白（rHKal）的高密度毕赤酵母表达、纯化以及生物学活性。以质粒pPIC9-Kal/GS115（His4）转化毕赤酵母细胞,在7.5 L培养罐内高密度细胞发酵,以1%～2%的甲醇诱导表达rHKal。发酵上清液经疏水层析、亲和层析和凝胶层析分离rHKal。通过MTT及小管形成实验评价rHKal对HUVEC的生物学活性。收获发酵上清液内rHKal的表达水平为50 mg.L 1,纯化后rHKal原液的纯度达98%以上。rHKal对HUVEC的增殖抑制活性呈剂量依赖性,但对其小管形成抑制作用的量效关系呈U型曲线。rHKal具有新生血管形成抑制活性,本研究为rHKal的生产提供了有效方法。

英文摘要：

Kallistatin （Kal） is a negative acute phase endogenous protein which can inhibit tumor angiogenesis, growth and metastasis effectively. To express and purify recombinant human kallistatin （rHKal）, and characterize its biological activity, P pastoris was transformed with pPIC9-Kal/GS115 （His4） to express rHKal. The fermentation was carried out in a 7.5 L bioreactor with high density cell culture. 1%-2% methanol was added to the medium to induce the expression of rHKal. The secretion was purified with phenyl sepharose, G-25 sepharose, heparin sepharose and Sephacryl S-100 chromatography. The biological activity of purified bulk rHKal on HUVEC was evaluated with MTT and tube formation assays. The final expression of rHKal in the supernatant reached 50 mg.L-1, the purity of bulk rHKal after purification was above 98%. A dose-dependent inhibition of rHKal on HUVEC proliferation was observed, however, a U-shaped dose-response curve of rHKal on capillary formation of HUVEC was revealed. The described protocol provides an effective means for lareoarinz rHKal that could be used for anti-angiogenesis therapy in the future.