人LC3蛋白在大肠杆菌中的表达纯化及其抗体的制备与鉴定  

Expression and purification of human recombinant LC3 in E.coli and preparation of its polyclonal antibodies

作　　者：李骅 胡嘉[1,3] 李艳君[1,3] 潘欢[3] 陈英玉[1,3] 

机构地区：[1]卫生部医学免疫学重点实验室,北京大学基础医学院,北京100191 [2]北京诺和诺德医药科技有限公司,北京102206 [3]北京大学人类疾病基因研究中心,北京100191

出　　处：《细胞与分子免疫学杂志》2012年第5期517-519,523,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展计划(973)(2011CB910103)

摘　　要：目的:制备兔抗人LC3的多克隆抗体,为细胞自噬的研究提供有用的检测工具。方法:构建pET32a(+)LC3的原核细胞表达载体并进行序列鉴定,大肠杆菌BL21(DE3)中诱导表达LC3蛋白,SP-Sepharose XL柱纯化;纯化的人LC3蛋白免疫兔制备抗血清,通过LC3蛋白偶联的Sepharose 4B(LC3-Sepharose 4B)亲和层析纯化兔抗人LC3的多克隆抗体,以间接ELISA和Western blot鉴定其特异性。结果:成功实现了LC3蛋白在大肠杆菌中的高效表达和纯化,并用质谱分析证明表达蛋白相对分子质量(Mr)为15 500。制备的兔抗人LC3多克隆抗体具有很高的特异性,与大肠杆菌成分无交叉反应。Western blot能够特异检测自噬过程中LC3Ⅰ型和Ⅱ型蛋白,观察到自噬发生时LC3Ⅰ向LC3Ⅱ的转化。结论:获得了重组人LC3蛋白,成功地制备了该分子的特异性多克隆抗体,为细胞自噬的检测提供了有用的探针。AIM： To prepare the polyclonal antibodies against human LC3 protein and to provide a useful tool for monitoring cell autophagy. METHODS： The plasmid of pET32a（ ＋ ）LC3 was successfully constructed, human LC3 protein was purified from E. coli through one-step purification with SP-Sepharose XL. The purified LC3 protein was used to immunize rabbits for preparing the specific antibody. Simultaneously, it was also coupled with CNBr-activated Sepharose 4B. The specificity of the polyclonal anti-LC3 anti- body was determined by ELISA and Western blotting. RESULTS： Recombinant human LC3 protein was expressed and purified successfully. Prepared polyclonal anti-LC3 anti- body showed high specificity against LC3 without cross- reaction with E. coli components in Western blotting. It was also indicated that the antibody could recognize both LC3-1 and LC3-11 in the process of cell autophagy. CONCLU- SION： Human recombinant LC3 protein successfully expressed in E. coli and purified. Polyclonal anti-LC3 anti- body with high specificity is prepared and applied success- fully in detection of cell autophagy.

关 键 词：细胞自噬 LC3 抗体 原核表达 

分 类 号：Q78[生物学—分子生物学] R392-33[医药卫生—免疫学]