中文摘要：

目的：通过克隆LC3．I基因，体外原核表达LC3-I蛋白后制备抗LC3单克隆抗体，作为自噬研究中的标记分子检测自噬的发生和发展过程。方法：RT．PCR方法从RAW264．7细胞基因组中克隆LC3基因，亚克隆至pQE80L原核表达载体后转化E．cobDH5a进行诱导表达，SDS—PAGE电泳及Westemblot鉴定表达蛋白。蛋白纯化后免疫BALB／c小鼠。采用淋巴细胞杂交瘤技术，制备分泌抗LC3．I杂交瘤细胞株，体内诱生腹水制备mAb，间接ELISA法测定其效价，辛酸一硫酸铵沉淀法及亲和层析法纯化mAb。结果：成功克隆了LC3一I基因，并对其在E．coilDH5a进行诱导表达，SDS-PAGE分析表明在相对分子量Mr为20×10^3有特异条带，Westernblot验证表达产物具有一定的生物学活性。建立了3株稳定分泌特异性抗LC3-ImAb的杂交瘤细胞株，诱导产生的腹水获得的抗体效价在10^5-10^7之间，结论：在E．coli中对LC3-I进行表达，并制备特异性较强抗LC3-I蛋白的单克隆抗体。为自噬研究提供了良好的标记分子，可对自噬形成和发展进行有效的检测。

英文摘要：

Objective： To clone the Microtubule associated protein I light chain 3 （LC3-I） gene, and prokaryotic expression LC3-I protein in vitro to prepare LC3 monoclonal antibody resistance for autophytic research provides the experimental material. Methods： In order to obtain the related experimental materials for study of the autophagy, LC3-I gene was cloned and constructed a recombinant plas- mid. This recombinant plasmid containing LC3-I gene was expressed in E.coli. The expressed recombinant protein was identified by SDS-PAGE and Western blot. BALB/c mice was immunized by purified LC-1 protein to produce monoclonal antibodies against LC3-I. Spleen cell suspension from immunized mice was fused with SP2/0 cells and positive clones were identified by selective culture medium. The positive cell clones were then incubated BALB/c mice by intrapulmonary inoculation to produce ascitic fluid. Results： In this study, we obtained 5 high titer strains hybridoma cell lines which could recognize specifically LC3-I protein. Conclusion： This provided a good reagent for study autophagy.