一种新型的通过免疫富集纯化溶酶体方法  

A Novel Purification Method for Lysosomes by Lmmunoprecipitation

作　　者：张磊[1] 阳宇[1] 吴传芳[1] 

机构地区：[1]四川大学生命科学院,成都610064

出　　处：《中国生物工程杂志》2013年第11期51-55,共5页China Biotechnology

摘　　要：用传统的密度浓度梯度法分离溶酶体具有耗时长,样品需求量大,而且无法得到纯度较高的溶酶体等不足,因此有必要开发一种简便分离溶酶体的方法。依据溶酶体膜蛋白LAMP2的特性,开创性地尝试用免疫富集溶酶体膜蛋白的方式纯化溶酶体:首先构建了带Flag标签的LAMP2真核表达载体,并转染Hela细胞,用免疫荧光鉴定其亚细胞定位情况,发现重组蛋白LAMP2-Flag特异性定位于溶酶体。在Hela细胞中筛选得到了稳定表达LAMP2-Flag的细胞系。大规模培养该细胞系,在非变性条件下分离细胞质提取物,利用anti-Flag抗体做免疫沉淀,用蛋白质免疫印迹检测,发现沉淀产物中特异性富集了溶酶体标识蛋白LAMP1。用糖苷酶活性检测,发现沉淀产物中酸化蛋白酶活性很高,表明富集到较纯的溶酶体。这种改良的新型方法步骤简捷,样品需求量少,获得的溶酶体纯度高,为从少量细胞中分离溶酶体提供了一个种优化方案。A novel purification method for lysosomes by IP lysosomal membrane protein was explored in this paper. LAMP2-Flag was ligated into the eukaryotic expression vector pcDNA3.1, and was transfected into Hela cell line. The lysosomal location of LAMP2-Flag was conformed by Immunofluorescence labeling. Hela cell lines that can stably express LAMP2-Flag were cultured in large numbers. Highly pure lysosomes were isolated from cell extractions using anti-Flag immunopreci highlyenriched lysosomal marker proteins an indicate the efficiency of this method in the Hela cell lines. pitation under native d enzymems, and no isolation of lysosomes condition. The purifiedlysosomes contained mitochondrial contamination. Our rResults of high purity from LAMP2-Flag expressing

关 键 词：分子生物学 细胞器分离 免疫共沉淀溶酶体 

分 类 号：Q813[生物学—生物工程]