一种含精-甘-天冬氨酰三肽人纤溶酶原K区缺失突变体在巴斯德毕赤酵母中的表达、纯化与特性  

Purification and characterization of a kringle-deficit mutant of human plasminogen with Arg-Gly-Asp tripeptide expressed in Pichia pastorsis

作　　者：陈武[1] 吴茂材[1] 吴敬源[1] 杨健忠[1] 陈振林[1] 黄智慧[1] 张鑫涌[1] 肖郧[2] 

机构地区：[1]广东药学院生命科学与生物制药学院,广州510006 [2]湖北医药学院附属十堰人民医院,十堰442000

出　　处：《生物工程学报》2011年第5期764-772,共9页Chinese Journal of Biotechnology

基　　金：广东药学院博士基金项目(No.43555026)资助~~

摘　　要：为获得具有抗血小板聚集作用的重组人纤溶酶原(hPLG),尝试了一种含有精-甘-天冬氨酰(RGD)三肽的hPLG K区缺失突变体(RGD-hPLG-?K)。首先,从pDNR-LIB-HPLG中克隆出HPLG-?K。然后定点突变激活环内的Pro559为Asp559,形成RGD模序。构建的pPICZαA-RGD-HPLG-?K电转化巴斯德毕赤酵母GS115,甲醇诱导表达后可产生0.16 g/L培液的RGD-hPLG-?K,Ni-NTA层析后纯度可达90%以上;Western blotting证实所获RGD-hPLG-?K可与兔抗hPLG抗血清反应;其24 h尿激酶激活速率和纤溶活性与hPLG-?K无显著差别(P=0.630,n=5);经尿激酶激活后,RGD-hPLG-?K的血小板聚集抑制率(21.8%±1.57%)显著高于hPLG-?K(3.8%±0.33%)(P=0.000,n=5)。表明成功构建、表达了一种具有抗血小板聚集活性的hPLG突变体,为研究新型多功能溶栓药物奠定了基础。To obtain a recombinant human plasminogen（hPLG） with potential anti-platelet aggregation activity,we cloned the cDNA coding Pro544 to Asn791 of hPLG,a kringle-deficit derivative（hPLG--K）.The Pro559 in activation loop was then mutated into Asp559 to provide Arg-Gly-Asp（RGD） motif.The constructed pPICZαA-RGD-HPLG--K plasmid was expressed in yeast Pichia pastoris GS115,which produced RGD-hPLG--K about 0.160 g/L broth.After affinity chromatography,the purity of the recombinant protein reached above 90%.Western blotting test confirmed that it retained the immunological reaction capability as human PLG.Its urokinase activation rate in 24 hours and its fibrinolytic activity made no deference against native hPLG--K（P=0.630,n=5）.Importantly,after activation by urokinase,RGD-hPLG--K showed a significantly higher platelet aggregation inhibition rate（Ri）（21.8%±1.57%） than hPLG--K（3.8%±0.33%）（P=0.000,n=5）.These results proved that we constructed an hPLG mutant with anti-platelet aggregation activity,which made a foundation for developing innovative thrombolytic drugs with multifunction.

关 键 词：人纤溶酶原 RGD三肽 突变体 抗血小板聚集 巴斯德毕赤酵母 

分 类 号：Q78[生物学—分子生物学]