肾上腺脑白质营养不良蛋白ATP结合区的原核表达与鉴定

Prokaryotic Expression and Characterization of ATP-binding Domain of X-linked Adrenoleukodystrophy Protein

目的 构建人肾上腺脑白质营养不良 ( adrenoleukodystrophy,ALD)蛋白 ( ALD protein,ALDP) ATP结合区的原核重组载体并进行表达和鉴定。方法 自行设计引物 ,通过 PCR技术 ,以人全血 RNA为模板扩增ALDP ATP结合区编码序列 ,PCR产物经 Eco R /Xho 双酶切后 ,将其克隆至 p GEX-4T-2载体中 ,在大肠杆菌 BL2 1中诱导 GST融合蛋白的表达。结果 在 IPTG的诱导下 ,重组菌高效表达出一相对分子质量约为42 0 0 0的产物 ,与预期大小相符。对重组质粒的序列分析表明 ,插入片段的序列与 Genbank登录的编码序列完全一致。Western blot结果显示 ,目的条带可被鼠抗人 ALDP单克隆抗体识别。结论 人 ALDP ATP结合区的编码序列已被克隆至 GST融合表达载体 p GEX-4T-2 ,并在大肠杆菌 BL2 1中获得高表达。

Objective To construct a prokaryotic recombinant vector of ATP-binding domain of human X-linked adrenoleukodystrophy(ALD) protein and to detect its expression in E. coli BL 21. Methods The coding sequence of ATP-binding domain of ALD protein was amplified from human blood RNA with primers designed in our laboratory, and cloned into pGEX-4T-2 after the restrictive digestion of EcoRⅠ and XhoⅠ. GST fusion protein was expressed after induction with IPTG. Results Sequencing and restrictive digestion of the recombinant plasmid revealed the existence of coding sequence for ATP-binding domain of ALD protein. A protein band of about 42 000 could be induced by IPTG in the recombinant plasmid. And the band could be identified by mouse anti-human ALD protein monoclonal antibody with Western blotting technique. Conclusions The coding sequence of ATP-binding domain of ALD protein was introduced into the pGEX-4T-2 plasmid and a GST-fused protein could be induced at a high level.