盘基网柄菌DJ-1蛋白的原核表达及抗体制备

Prokaryotic Expression and Antibody Preparation of Dictyostelium discoideum DJ-1 Protein

盘基网柄菌(Dictyostelium discoideum)是研究神经退行性病变的模式生物,可用以研究帕金森病相关基因DJ-1的作用和致病机理。本研究设计特异性引物扩增了人类DJ-1的盘基网柄菌同源基因片段DAM,并利用酶切位点BamHⅠ和HindⅢ将DAM插入质粒载体pQE-30产生重组载体pPROF696,序列测定后对DJ-1进行了生物信息学分析。同时,通过电穿孔法将pPROF696转入大肠杆菌M15,并利用IPTG诱导DJ-1蛋白表达。经SDS-PAGE分析、Western-blot印迹和His标签树脂对DJ-1粗蛋白纯化后进行抗体制备,并在大肠杆菌M15转化株和盘基网柄菌转化株与野生型内对抗体进行检验。结果表明:本研究成功构建了盘基网柄菌DJ-1的原核表达载体pPROF696,所制备的盘基网柄菌DJ-1多克隆抗体能成功检测大肠杆菌M15和盘基网柄菌野生型与转化株中的DJ-1蛋白。这为今后检测盘基网柄菌细胞内DJ-1的表达水平,建立其与盘基网柄菌表现型相关性,标记DJ-1的亚细胞定位和进一步研究DJ-1在帕金森病中的作用机理奠定了基础。

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 Bam H 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 construct pPROF696 containing D. discoideum DJ-1 was successfully built and the produced polyclonal antibody could detect the DJ-1 protein from E. coli M15 and D. discoideum strains. This study plays an important 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 further studying the mechanisms of DJ-1 in PD in future.