重组apo(a) Kringle Ⅳ-10的纯化及生物活性  

Purification of recombinant apolioprotein (a) Kringle Ⅳ-10 and its biological activity

作　　者：彭芳芳[1] 洪嘉玲[1] 喻红[1] 汪炳华[1] 殷以礼[1] 李小明[1] 

机构地区：[1]武汉大学医学院生物化学教研室,武汉430071

出　　处：《郑州大学学报（医学版）》2002年第5期636-639,共4页Journal of Zhengzhou University(Medical Sciences)

摘　　要：目的 :通过研究重组apo(a)KringleⅣ 1 0 (KⅣ 1 0 )的赖氨酸结合能力对纤溶酶原与内皮细胞结合的影响 ,探讨apo(a)在抑制纤溶过程中的作用 ,为Lp(a)致动脉粥样硬化机制研究提供依据。 方法 :将含apo (a)野生型KⅣ 1 0 (wt KⅣ 1 0 /Trp72 )和突变型KⅣ 1 0 (mut KⅣ 1 0 /Arg72 )基因片段的重组质粒 ,分别转化至E .coliDH5α菌株中并表达含这 2个重组片段的融合蛋白 ,通过Glutathione Agarosebeads亲和层析进行分离和提纯 ,经L Lys Sepharose 4B亲和层析检测其赖氨酸结合能力。再以异硫氰酸荧光素标记的纤溶酶原为配基 ,观察这 2种基因表达片段对纤溶酶原与人脐静脉内皮细胞结合的影响。结果 :实验结果显示 :在E .coliDH5α菌株中表达的GST wt KⅣ 1 0 /Trp72 能有效抑制纤溶酶原与人脐静脉内皮细胞的结合 ;而GST mut KⅣ 1 0 /Arg72 在任一浓度范围内均无这种抑制作用。结论 :apo (a)KⅣ 1 0对纤溶的抑制作用与其赖氨酸结合能力密切相关 ;而KⅣ 1 0的赖氨酸的结合能力与其赖氨酸结合位点中的Trp72Aim:To elucidate the mechanisms of atherosclerosis caused by lipoprotein(a) and discuss the inhibition of fibrinolysis by apolipoprotein(a) (apo(a)). Methods:The recombinant plasmids designated pGEX KG/Trp 72 KⅣ 10 and pGEX KG/Arg 72 KⅣ 10 were transformed to E.coli DH5α strain, and wild type (wt) and mutant type (mut) forms of apo(a) KⅣ 10 were expressed as fusion GST proteins which were isolated and purified by affinity chromatography on Glutathione Agarose. The lysine binding capacities of the GST KⅣ 10 fusion proteins were examined by affinity chromatography on Lys Sepharose. The effect of the binding of plasminogen to human umbilical vein endothelial cells (HUVEC) for GST KⅣ 10 fusion proteins were studied by using the plasminogen labeled with FITC.Results:The results indicated that the expressed GST wt KⅣ 10/Trp 72 fusion proteins bound tightly to Lys Sepharose, but the expressed GST mut KⅣ 10/Arg 72 fusion proteins had no affinity for Lys Sepharose. GST wt KⅣ 10/Trp 72 fusion proteins could effectively inhibit the binding of plasminogen to HUVEC, while GST mut KⅣ 10/Arg 72 fusion proteins could not, even if in a higher concentration. Conclusions:The inhibition of fibrinolysis by apo(a) KⅣ 10 is associated with its capacity of lysine binding.The capacity of apo(a) KⅣ 10 is associated with Trp 72 in its lysine binding sites.

关 键 词：纯化 生物活性 内皮细胞 纤溶酶原 apo(a)KringleⅣ-10 赖氨酸结合位点 

分 类 号：Q503[生物学—生物化学]