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.
出处
《生物工程学报》
CAS
CSCD
北大核心
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~~
