蛋白核小球藻凝集素的分离纯化及部分性质研究
PURIFICATION AND PARTIAL CHARACTERIZATION OF CHLORELLA PYRENOIDOSA LECTIN
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摘要: 蛋白核小球藻藻粉的PBS抽提液经硫酸铵二步分级沉淀,再经DEAE-Sepharose和Sephadex G-100层析,从中分离纯化得到蛋白核小球藻凝集素(CPL).经测定,该凝集素为单个亚基的蛋白质,相对亚基分子量为1.4×102-1.5×102,分子中不含糖.在氨基酸组成中,苯丙氨酸(Phe)的含量最高,其次是天冬氨酸(Asp)和谷氨酸(Glu),不含组氨酸(His).CPL能够凝集兔、绵羊及鸽子红细胞,其中对兔红细胞的凝集活性最大,最低浓度为6.88μg/mL,对鸡、鸭及人红细胞(A型、O型及B型)无凝集活性.卵黏蛋白和7种单糖对CPL的凝血活性具有抑制作用.CPL具有很好的热稳定性,在90℃处理10min不失活.Abstract: The freeze-drying powder of a fresh microalga, Chlorella pyrenoidosa, was extracted overnight by PBS at 4℃ and a tow-step precipitate with solid ammonium sulfate(25% and 75%) was performed for the supernatant after centrifugation. The crude protein obtained was purified by DEAE-Sepharosa ion exchange, followed by Sephdex G-100 filtration. The activity of the lectin was tested wih fresh rabbit erythrocyte in each step of above procedure and protein concentration was determined using Coomassie Brilliant G250. This purified lectin(CPL) dose not contain neutral saccharide but is a monomeric protein with a relative molecular weight of 14,000-15,000, estimated by SDS-PAGE and Sephdex G-100 filtration. In its amino acid composition, Phe has the highest content, followed by Asp and Glu, but there is a lack of His. Assays for sugar or glycoproteiin inhibition and erythrocyte agglutination were done in microtiter plates. The result indicated that CPL could agglutinated erythrocytes from rabbit, sheep and pigeon but not from duck, chicken and human A, B or O blood cell groups and the highest activity was found in the agglutination with rabbit erythrocytes at 6.88μg/mL. The agglutinic activity of CPL was inhibited by seven types of monosaccharides when its concentration was at 13.75μg/mL and by ovalbumin at a range of 6.88-432μg/mL. CPL was tolerant of high temperature and its activity could be maintained even when the lectin solution was heated to 90℃ for 10min.
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Keywords:
- Chlorella pyrenoidosa /
- Lectin /
- Purification /
- Characterization
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[1] Liener I E. Phytohemagglutinins(Phygolectins) [J]. Ann Rev Physiol, 1997, 27:291-319[2] Zhang D Y, Zhu Z P. Plant Molecular Biology and Biotechnology [M]. Beijing: Science Press, 1991, 145-158. [张德颐,朱治平,植物分子生物学与生物工程. 北京:科学出版社,1991,145-158][3] Rogers D J, Hori. K. Marine algal lectin: new developments [J]. Hydrobiologia. 1993,260/261:589-593[4] Alvarez-Hernandez S, Lara-Isassi G, Arreyuin-Espinoza R. Isolation and partial characterization of griffin, a lectin from the Mexican endemic alga Codium giraffa silva [J]. Bot Mar. 1999,42:573-580[5] Hori K, Ogate T, Kamiga H, et al. Lectin-like compounds and lectin receptors in marine microalgae: hemagglutination and reactivity with purified lectins [J]. J. Phycol. 1996,32:819-825[6] Zhao Y F. Principle and Application of Biochemical Technique [M]. Hubei: Wuhan Univ. Press, 1994, 287-295. [赵永芳. 生物化学技术原理及其应用. 湖北:武汉大学出版社,1994,287-295][7] Dubosis M, Gilles KA, Hamiltion JK, et al. Colorimetric method for determination of sugars and related substance [J]. Anal Chem,1957,28:350-356[8] Zeng Z K, Wu Q Q, Bao J K. Purification and characterization of cotton lectin [J]. Acta Bot Sin,1995,37(3):204-211. [曾仲奎,吴洽庆,鲍锦库. 棉花凝集素的纯化及性质研究,植物学报,1995,37(3):204-211][9] Zeng Z K, Wu Q Q. The physiological role of plantlectin in plant [J]. Natural product. research and devlopment. 1993,5(3):69-76. [曾仲奎,吴洽庆. 植物凝集素在植物体内的生理作用. 天然产物研究与开发,1993,5(3):69-76][10] Suen C, Zhu Z, Mo HQ. Lectin [M]. Beijing: Science Press, 1986,65-66. [孙册,朱政,莫汉庆. 凝集素. 北京:科学出版社,1986,65-66][11] Hori K, Miyazawa K, Ito K. Some common properties of lectins from marine algae [J]. Hydrobiologia. 1990, 203/205:561-566[12] Hou X W, Wu B L, Zeng Z K. Purification and characterization of a lectin from Alocasia eucullata [J].1998,19(2):106-111. [侯学文,吴伯良,曾仲奎,等. 尖尾芋凝集素的纯化及性质研究[J]. 华南农业大学学报,1998,19(2):106-111][13] Kakita H, Gukuoka S, Obika G, et al. Purification and properties of a high molecular weight hemagglutinin from the red alga, Gracilaria verrucosa [J]. Bot mar. 1997, 40:241-247[14] Zheng Y, Yu P, Liu Y R. Partial properties and cell agglutination of Gracilaria bursa-pastoris lectin [J]. 2002,8(1):66-70. [郑怡,余萍,刘燕如. 脆江蓠凝集素的部分性质及细胞凝集作用. 应用与环境生物学报,2002,8(1):66-70] Liener I E. Phytohemagglutinins(Phygolectins) [J]. Ann Rev Physiol, 1997, 27:291-319[2] Zhang D Y, Zhu Z P. Plant Molecular Biology and Biotechnology [M]. Beijing: Science Press, 1991, 145-158. [张德颐,朱治平,植物分子生物学与生物工程. 北京:科学出版社,1991,145-158][3] Rogers D J, Hori. K. Marine algal lectin: new developments [J]. Hydrobiologia. 1993,260/261:589-593[4] Alvarez-Hernandez S, Lara-Isassi G, Arreyuin-Espinoza R. Isolation and partial characterization of griffin, a lectin from the Mexican endemic alga Codium giraffa silva [J]. Bot Mar. 1999,42:573-580[5] Hori K, Ogate T, Kamiga H, et al. Lectin-like compounds and lectin receptors in marine microalgae: hemagglutination and reactivity with purified lectins [J]. J. Phycol. 1996,32:819-825[6] Zhao Y F. Principle and Application of Biochemical Technique [M]. Hubei: Wuhan Univ. Press, 1994, 287-295. [赵永芳. 生物化学技术原理及其应用. 湖北:武汉大学出版社,1994,287-295][7] Dubosis M, Gilles KA, Hamiltion JK, et al. Colorimetric method for determination of sugars and related substance [J]. Anal Chem,1957,28:350-356[8] Zeng Z K, Wu Q Q, Bao J K. Purification and characterization of cotton lectin [J]. Acta Bot Sin,1995,37(3):204-211. [曾仲奎,吴洽庆,鲍锦库. 棉花凝集素的纯化及性质研究,植物学报,1995,37(3):204-211][9] Zeng Z K, Wu Q Q. The physiological role of plantlectin in plant [J]. Natural product. research and devlopment. 1993,5(3):69-76. [曾仲奎,吴洽庆. 植物凝集素在植物体内的生理作用. 天然产物研究与开发,1993,5(3):69-76][10] Suen C, Zhu Z, Mo HQ. Lectin [M]. Beijing: Science Press, 1986,65-66. [孙册,朱政,莫汉庆. 凝集素. 北京:科学出版社,1986,65-66][11] Hori K, Miyazawa K, Ito K. Some common properties of lectins from marine algae [J]. Hydrobiologia. 1990, 203/205:561-566[12] Hou X W, Wu B L, Zeng Z K. Purification and characterization of a lectin from Alocasia eucullata [J].1998,19(2):106-111. [侯学文,吴伯良,曾仲奎,等. 尖尾芋凝集素的纯化及性质研究[J]. 华南农业大学学报,1998,19(2):106-111][13] Kakita H, Gukuoka S, Obika G, et al. Purification and properties of a high molecular weight hemagglutinin from the red alga, Gracilaria verrucosa [J]. Bot mar. 1997, 40:241-247[14] Zheng Y, Yu P, Liu Y R. Partial properties and cell agglutination of Gracilaria bursa-pastoris lectin [J]. 2002,8(1):66-70. [郑怡,余萍,刘燕如. 脆江蓠凝集素的部分性质及细胞凝集作用. 应用与环境生物学报,2002,8(1):66-70]
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