重组人纤溶酶原Kringle1-5的制备及其生物学活性

Preparation of Human Recombinant Kringle 1-5 and Its Bioactivity

为了研究重组人纤溶酶原Kringle15(K15)的抗血管生成活性及其对内皮细胞增殖的影响,通过PCR扩增人纤溶酶原K15cDNA,定向克隆于原核表达载体pET30a(+)中,构建重组表达载体pETK15,转化E.coliBL21(DE3),IPTG诱导表达,SDSPAGE和Western杂交检测K15的表达。鸡胚尿囊膜(CAM)实验和MTT实验分别检测重组人纤溶酶原Kringle15对鸡胚新生血管生成和内皮细胞的抑制作用。结果表明,IPTG诱导原核表达载体pETK15在E.coliBL21(DE3)中的表达量约占菌体总蛋白量的32%,K15主要以包涵体形式存在,包涵体经过洗涤、溶解、Nispin亲合柱层析纯化以及蛋白质复性等步骤后,获得了纯度约为96%的重组K15蛋白。CAM实验表明,原核表达的重组人K15能有效地按剂量依赖的方式抑制鸡胚新生血管的形成。MTT实验结果显示,重组人K15特异地抑制内皮细胞的增殖,而对非内皮细胞无抑制作用。

To investigate antiangiogenesis activity and effects on endothelial cell proliferation of human recombinant K 1-5 expressed in E. coli BL21, the cDNA of human K1-5 obtained from a cloning vector pUC57K1-5 by PCR, was inserted into an expression vector pET30（ ＋ ） to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an antiangiostatin monoclonal antibody. The expressed K1-5 accounted for approximately 32 % of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed and purified to a purity of 96% by the nickel affinity chromatography. Refoled K1-5 protein was tested on chicken CAMs,and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, human recombinant K1-5 potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrated that human recombinant K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.