基于蛋白免疫印迹的水蛭抗凝活性成分研究
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摘要
目的:建立Native-PAGE结合Western blot检测水蛭活性多肽与凝血酶相互作用产物的方法,探究水蛭有效抗凝成分。方法:本研究分别提取宽体金线蛭、日本医蛭两种水蛭虫体总蛋白及日本医蛭唾液蛋白,用凝血酶滴定法测定其抗凝血酶活性,Western blot检测凝血酶-活性多肽复合体。结果:研究发现两种水蛭Native-PAGE-WB均检测到凝血酶-活性多肽复合体,且SDS-PAGE-WB检测到复合体变性后产生凝血酶条带。结论:建立的Native-PAGE-WB法可有效检出水蛭活性多肽和凝血酶相互作用产物,且发现同水蛭素类似,宽体金线蛭活性抗凝多肽与凝血酶在体外也能形成稳定的蛋白复合体。
Native-PAGE can conserve the bioactivity of protein after the separation of electrophoresis.Western blot is a protein detection technique used to detect specific antigens(such as thrombin) with specific antibodies. In this research Native-PAGE-WB methods was established to detect Protein-Protein Interactions between thrombin and anticoagulant components and to explore the active anticoagulants from two leech species. In this method NP-40 lysis buffer was used to extract the total proteins from Whitmania pigra Whitman and Hirudo nipponica Whitman, then Antithrombin activity of two leech species was determined by thrombin titration. It was found that both leech species the thrombin-active peptides was detected through Native-PAGE-WB, and the SDS-PAGE-WB found that the strips produced after the degeneration of the complex were the same as that of standard thrombin.It comes to the conclusion that the anticoagulants from Whitmania pigra Whitman can form stable protein complexes with thrombin in vitro like hirudin.
Native-PAGE can conserve the bioactivity of protein after the separation of electrophoresis.Western blot is a protein detection technique used to detect specific antigens(such as thrombin) with specific antibodies. In this research Native-PAGE-WB methods was established to detect Protein-Protein Interactions between thrombin and anticoagulant components and to explore the active anticoagulants from two leech species. In this method NP-40 lysis buffer was used to extract the total proteins from Whitmania pigra Whitman and Hirudo nipponica Whitman, then Antithrombin activity of two leech species was determined by thrombin titration. It was found that both leech species the thrombin-active peptides was detected through Native-PAGE-WB, and the SDS-PAGE-WB found that the strips produced after the degeneration of the complex were the same as that of standard thrombin.It comes to the conclusion that the anticoagulants from Whitmania pigra Whitman can form stable protein complexes with thrombin in vitro like hirudin.
引文
1张和韡,王丽萍.水蛭素的研究进展.中国中西医结合肾病杂志,2013(1):76-78.
2国家药典委员会.中华人民共和国药典(一部).北京:化学工业出版社,2015:83
3 Markwardt F.Isolation and chemical characterization of hirudin.HoppeSeyler's Zeitschrift fur physiologische Chemie,1957,308(2-4):147.
4 Markwardt F.Hirudin as alternative anticoagulant--a historical review.Seminars in Thrombosis&Hemostasis,2002,28(5):405.
5 Oliveira D G L,Alvarez-Flores M P,Lopes A R,et al.Functional characterisation of vizottin,the first factor Xa inhibitor purified from the leech Haementeria vizottoi.Thromb Haemost,2012,108(3):570-578.
6 Finney S,Seale L,Sawyer R T,et al.Tridegin,a new peptidic inhibitor of factor XIIIa,from the blood-sucking leech Haementeria ghilianii.Biochem J.(1997)324,797-805
7万明.宽体金线蛭抗凝血活性寡肽研究.长沙:湖北中医药大学博士学位论文,2012.
8欧兴长,丁家欣,张玲.水蛭素的研究概况.中国药学杂志,1991,26(7):396-399.
10 Wang X,Li F,Song G,et al.Broad-spectrum four-dimensional orthogonal electrophoresis:a novel comprehensively feasible system for protein complexomics investigation.Mol Cell Proteomics,2012,11(9):786-799.
11张卫,张瑞贤,李健,等.中药水蛭品种考证及资源可持续利用发展探讨.中国中药杂志,2013,38(6):914-918.
12石萍,鲁增辉,曾纬,等.日本医蛭的研究进展.世界科学技术-中医药现代化,2016,18(11):2013-2018.
13孙倩倩.水蛭纤溶活性蛋白的分离纯化与性质研究.山东大学,2014.
15张彬,汪波,龚元,于翔,吕军仪.几种水蛭抗凝血物质提取及活性分析.中山大学学报(自然科学版),2012,51(4):92-96.
16李宝红.基于水蛭传统给药的抗凝活性组份的相关研究.广州:广州中医药大学博士学位论文,2014.
18 Zheng X,Li J,Chen Z,et al.Purification and characterization of an anticoagulant oligopeptide from Whitmania pigra Whitman.Pharmacogn Mag,2015,11(43):444-448.
19 Tan C,Wan W,Zhang R,et al.The study of the relationship of rhirudin and prethrombus.Nuclear Techniques,2000.23(5):343-346.
2国家药典委员会.中华人民共和国药典(一部).北京:化学工业出版社,2015:83
3 Markwardt F.Isolation and chemical characterization of hirudin.HoppeSeyler's Zeitschrift fur physiologische Chemie,1957,308(2-4):147.
4 Markwardt F.Hirudin as alternative anticoagulant--a historical review.Seminars in Thrombosis&Hemostasis,2002,28(5):405.
5 Oliveira D G L,Alvarez-Flores M P,Lopes A R,et al.Functional characterisation of vizottin,the first factor Xa inhibitor purified from the leech Haementeria vizottoi.Thromb Haemost,2012,108(3):570-578.
6 Finney S,Seale L,Sawyer R T,et al.Tridegin,a new peptidic inhibitor of factor XIIIa,from the blood-sucking leech Haementeria ghilianii.Biochem J.(1997)324,797-805
7万明.宽体金线蛭抗凝血活性寡肽研究.长沙:湖北中医药大学博士学位论文,2012.
8欧兴长,丁家欣,张玲.水蛭素的研究概况.中国药学杂志,1991,26(7):396-399.
10 Wang X,Li F,Song G,et al.Broad-spectrum four-dimensional orthogonal electrophoresis:a novel comprehensively feasible system for protein complexomics investigation.Mol Cell Proteomics,2012,11(9):786-799.
11张卫,张瑞贤,李健,等.中药水蛭品种考证及资源可持续利用发展探讨.中国中药杂志,2013,38(6):914-918.
12石萍,鲁增辉,曾纬,等.日本医蛭的研究进展.世界科学技术-中医药现代化,2016,18(11):2013-2018.
13孙倩倩.水蛭纤溶活性蛋白的分离纯化与性质研究.山东大学,2014.
15张彬,汪波,龚元,于翔,吕军仪.几种水蛭抗凝血物质提取及活性分析.中山大学学报(自然科学版),2012,51(4):92-96.
16李宝红.基于水蛭传统给药的抗凝活性组份的相关研究.广州:广州中医药大学博士学位论文,2014.
18 Zheng X,Li J,Chen Z,et al.Purification and characterization of an anticoagulant oligopeptide from Whitmania pigra Whitman.Pharmacogn Mag,2015,11(43):444-448.
19 Tan C,Wan W,Zhang R,et al.The study of the relationship of rhirudin and prethrombus.Nuclear Techniques,2000.23(5):343-346.