Abstract：Abstract: Dictyostelium discoideum is one of the models for studying neurodegenerative diseases and can be used to explore the roles and pathogenesis of DJ-1 in Parkinson’s disease (PD). Herein, the specific primers were designed to amplify the D. discoideum DJ-1 fragment DAM, the homologue of human DJ-1 gene. The endonuclease recognition sites of BamH Ⅰ and Hind Ⅲ were used to insert DAM into vector pQE-30 for generation of the construct pPROF696. After gene sequencing, bioinformatics analysis of D. discoideum DJ-1 was conducted. The construct pPROF696 was electroporated into E. coli M15 to induce expression of DJ-1 protein using IPTG. After SDS-PAGE analysis and Western-blot, DJ-1 protein was separated and trans-ferred to PVDF membrane, and further purified by His-tagged purification resin. The purified DJ-1 protein was used for polyclonal antibody production. After affinity purification, the antibody was verified by detecting the DJ-1 protein from E. coli M15 and D. discoideum strains. The results showed that the prokaryotic con-struct pPROF696 containing D. discoideum DJ-1 was successfully built and the produced polyclonal anti-body could detect the DJ-1 protein from E. coli M15 and D. discoideum strains. This study plays an impor-tant role in detecting the expression levels of D. discoideum DJ-1, building the relativity between the DJ-1 expression level and the phenotypes of D. discoideum, labelling the subcellular localization of DJ-1 and fur-ther studying the mechanisms of DJ-1 in PD in future.
引用本文:
侯碧巍, 陈苏维. 盘基网柄菌DJ-1蛋白的原核表达及抗体制备[J]. 生命科学研究, 2021, 25(2): 102-108. HOU Bi-wei, CHEN Su-wei. Prokaryotic Expression and Antibody Preparation of Dictyostelium discoideum DJ-1 Protein. Life Science Research, 2021, 25(2): 102-108.
