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

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。

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

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 antibody was determined by ELISA and Western blotting.RESULTS: Recombinant human LC3 protein was expressed and purified successfully.Prepared polyclonal anti-LC3 antibody 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-I and LC3-II in the process of cell autophagy.CONCLUSION: Human recombinant LC3 protein successfully expressed in E.coli and purified.Polyclonal anti-LC3 antibody with high specificity is prepared and applied successfully in detection of cell autophagy.