一种新杨树菇(Agrocybe aegerita)凝集素的纯化及生化特性  

Purification and Characterization of a Novel Lectin AAVP from Fruiting Bodies of the Edible Fungus, Agrocybe aegerita

作　　者：孙慧[1] 赵辰光[1] 仝鑫[1] 齐义鹏[1] 

机构地区：[1]武汉大学生命科学学院分子病毒室,武汉430072

出　　处：《中国生物化学与分子生物学报》2003年第1期96-102,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助 (No .3 0 10 0 2 3 6)~~

摘　　要：用硫酸铵分级沉淀、离子交换和分子筛等方法 ,从食用菌杨树菇子实体中分离纯化了一种凝集素 ,称作为AAVP(Agrocybeaegeritaantiviralprotein) .经SDS PAGE测定其亚基的相对分子质量为15 8kD ,凝胶过滤分析分子量为 32kD .IEF PAGE计算其等电点为 3 8.AAVP不含糖 ,是一种N端焦谷氨酰环化封闭的蛋白质 ,经N端去封闭后测得N端氨基酸序列为QGVNIYNIVAGA ,用胰蛋白酶消化后得到一大片段 ,测定的氨基酸序列为PDGPWLVEK .AAVP可以凝集供试的 12种动物血和3种血型人血的血红细胞 ,但对各种血红细胞凝集滴度不同 .糖抑制实验表明 ,在供试的 18种单糖和 3种糖蛋白中 ,只有猪胃粘蛋白强烈抑制AAVP的凝血活性 .AAVP具有较好的热稳定性 ,能够忍受极端的酸碱条件 .AAVP的凝血活性不受Ca2 + 、Mg2 + 、Zn2 + 等二价阳离子的影响 .抗肿瘤活性检测表明 ,AAVP对胃癌细胞株SGC 790 1,MGC 80 3,BGC82 3及人急性白血病细胞株HL 6 0有明显的抑制作用 .AAVP对小鼠腹腔注射的半致死剂量为 15 85mg kg .A novel lectin Agrocybe aegerita antiviral protein (AAVP) was purified, from the fruiting bodies of edible fungus Agrocybe aegerita by precipitation of 40%—80% (NH 4) 2SO 4 followed by DEAE SepharoseFast Flow and Sephacryl S 200 chromatography. The relative molecular mass of AAVP was 32 kD tested by gel filtration, the subunit of AAVP was 15 8 kD determined by SDS PAGE. Its pI was about 3 8 by isoelectric focusing. AAVP is probably unglycosylated and blocked by pyroglutamyl at the N terminus. The amino acid sequence of the N terminus was QGVNIYNIVAGA, and the sequence of a segment of AAVP obtained after being digested by trypsin was PDGPWLVEKR. AAVP agglutinated 3 types of human and 12 types of animal erythrocytes regardless of blood group or category. However, its hemagglutinating activity for mammalian erythrocytes is much higher than that for bird and aquatic. In a hemagglutination assay, porcine mucin was the only inhibitor of the lectin among the 18 sugars and 3 glycoconjugates tested. AAVP was thermal stable, acid stable and alkali stable. Its activity was unaffected by demetalization and addition of divalent metals including CaCl 2, MgCl 2 or ZnCl 2. AAVP had inhibiting effect on tumor cell lines SGC 7901, MGC 80 3, BGC 823 and leucocythemia cell line HL 60 in vitro . AAVP is toxic to mouse by celiac injection with an LD 50 of 15.85 mg /kg body weight.

关 键 词：杨树菇凝集素 纯化 理化特性 血凝集活性 抗肿瘤活性 

分 类 号：Q786[生物学—分子生物学]