摘要
旨在建立从猪血中提纯纤溶酶原的高效方法。通过赖氨酸亲和层析结合凝胶过滤层析,纯化猪血中的sPLG;同时,利用大肠杆菌重组表达猪组织型纤溶酶原激活剂(st-PA)和葡激酶(SAK),并通过包涵体提取对st-PA进行纯化、镍柱亲和层析结合凝胶过滤层析纯化SAK。通过检测纤溶酶特异性底物分解曲线、纤连蛋白降解试验以及体外猪血凝块溶解试验,评估本研究中制备的sPLG及激活剂SAK、st-PA的活性。结果发现,本研究建立的方法高效纯化了sPLG,每250 mL猪血浆可获得50 mg精纯的sPLG。制备的sPLG能被st-PA或SAK激活,分解纤溶酶特异性底物、降解纤连蛋白,并在体外溶解猪血凝块。综上,研究建立了高效制备高纯度sPLG、st-PA和SAK的方法,并通过st-PA和SAK验证了sPLG具备完整的纤溶酶原活性,为sPLG在猪病原入侵机制的研究提供了原材料。
The purpose of this study is to establish an efficient method for purifying plasminogen from pig blood.In this study,sPLG was purified from pig blood using lysine affinity chromatography combined with size exclusion chromatography.Meanwhile,recombinant swine tissue plasminogen activator(st-PA)and staphylokinase(SAK)were expressed in Escherichia coli.Then,st-PA was purified by refolding of inclusion bodies,and SAK was purified by nickel column affinity chromatography combined with size exclusion chromatography.The activities of the prepared sPLG,SAK and st-PA were evaluated by a plasmin-specific substrate hydrolysis assay,a fibronectin degradation test,and an in vitro pig blood clot dissolution assay.The results showed that sPLG was highly purified by the method established by this study.Approximately 50 mg of pure sPLG was obtained from 250 mL of pig blood plasma.The purified sPLG could be activated by st-PA or SAK,and the resulting plasmin could then cleave the plasmin-specific substrate,degrade fibronectin,and lyse pig blood clots in vitro.In conclusion,this study established an efficient method for the preparation of sPLG,st-PA and SAK.In addition,the complete plasminogen activity of sPLG was verified using st-PA and SAK as well.This study provide essential materials for research on the mechanisms by which pathogens invade pigs via sPLG.
作者
马可
张磊
郝飞
谢星
张珍珍
邵国青
陈蓉
冯志新
MA Ke;ZHANG Lei;HAO Fei;XIE Xing;ZHANG Zhenzhen;SHAO Guoqing;CHEN Rong;FENG Zhixin(School of Life Sciences,Nanjing Agricultural University,Nanjing 210095,China;Key Laboratory of Veterinary Biological Products Engineering,Ministry of Agriculture and Rural Affairs,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Guotai Technology Innovation Center of Veterinary Biological Products(Taizhou),Taizhou 225300,China;College of Animal Medicine,Nanjing Agricultural University,Nanjing 210095,China)
出处
《畜牧兽医学报》
北大核心
2026年第1期454-463,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
江苏省农业科技自主创新资金项目(CX(22)3029)
国家自然科学基金面上项目(32273011)
国家自然科学基金青年项目(31800160)
江苏省科协青年托举工程项目(JSTJ-23-093)。
关键词
纤溶酶原
组织型纤溶酶原激活剂
葡激酶
血栓
蛋白纯化
plasminogen
tissue plasminogen activators
staphylokinase
thrombus
protein purification
