短尾蝮蛇毒金属蛋白酶和磷脂酶A2的分离纯化及磷酸化修饰检测  

Purification and Phosphorylation Modification Detection of Metalloproteinase and PLA2 from the Snake Venom in Gloydius brevicaudus

作　　者：罗亚军 林楚霞 文琳 汪妍 徐宁 高建芳[1] LUO Yajun;LIN Chuxia;WEN Lin;WANG Yan;XU Ning;GAO Jianfang(College of Life and Environmental Sciences,Hangzhou Normal University,Hangzhou 311121,China)

机构地区：[1]杭州师范大学生命与环境科学学院,浙江杭州311121

出　　处：《杭州师范大学学报（自然科学版）》2020年第3期290-296,共7页Journal of Hangzhou Normal University(Natural Science Edition)

基　　金：国家大学生创新训练项目(2017103461008);浙江省自然科学基金项目(LY14C030007).

摘　　要：利用阴离子层析、凝胶排阻色谱和反相高效液相色谱从短尾蝮蛇毒中分离金属蛋白酶和磷脂酶A2,用蛋白印迹法检测两种组分的磷酸化修饰信号,结合LC-MS质谱鉴定并预测磷酸化修饰位点.结果表明,利用3种色谱法串联组合,最终分离获得两种条带单一且纯度高的短尾蝮蛇毒蛋白,经LC-MS质谱鉴定分别为蛇毒金属蛋白酶和磷脂酶A2.免疫印迹检测发现这两种蛇毒蛋白受到明显的磷酸化修饰,解析质谱图发现蛇毒金属蛋白酶有4个氨基酸位点受到潜在的磷酸化修饰(含1个丝氨酸和3个苏氨酸),磷脂酶A2仅有1个酪氨酸位点受到潜在的修饰.所得结果是磷酸化位点修饰抗体在蛇毒蛋白磷酸化修饰信号检测中的一次尝试,基于修饰位点的预测可为蛇毒蛋白功能受磷酸化修饰影响的研究提供基础.Using anion chromatography,gel exclusion chromatography and RP-HPLC,the metalloproteinase and PLA2 were separated from the snake venom of Gloydius brevicaudus,the phosphorylation modification signals in these two components were detected by western blot,and the modification sites were also predicted by LC-MS mass spectrometry.The results showed the snake venom metalloproteinase and PLA2 with high purity were successfully isolated using a three-step chromatography protocol,and confirmed by LC-MS.Western blot analysis found both snake venom metalloproteinase and PLA2 were modified by phosphorylation.It was found four phosphorylated sites(one serine and three threonine)in snake venom metalloproteinase,and only one phosphorylated site(tyrosine)in PLA2.This study was an attempt at detecting the phosphorylated modification signals in snake venom proteins by anti-phosphorylation antibody,and the prediction based on modified sites could provide information for revealing the effects of phosphorylation modification on the function of snake venom proteins.

关 键 词：短尾蝮 纯化 蛇毒 金属蛋白酶 磷脂酶A2 磷酸化 

分 类 号：Q956[生物学—动物学]