老年神经变性疾病机制研究的基础:人α-突触核蛋白基因在原核细胞中的蛋白表达

Basic mechanism of neural degeneration disease in the elder: expression of human α-synuclein gene in prokaryotic cells

目的:为提供人α-突触核蛋白(α-synuclein)抗原,制备抗体以研究其自身聚集在老年神经变性疾病病理机制中所起作用,重组质粒载体、进行DNA测序,以用于α-突触核蛋白基因在原核细胞中的蛋白表达。方法:将质粒pcDNA3NACP(pcDNA3α-synucleincDNA)和质粒载体proEXMTHT1分别进行酶切,取α-突触核蛋白cDNA片段,重组于proEXMTHT1质粒载体上;酶切鉴定后测序;再将重组质粒proEXNACP转染进入DH5α大肠杆菌体内;用IPTG诱导α-突触核蛋白基因产生蛋白;将α突触核蛋白纯化后行Western杂交。结果:质粒proEXMTHT1和pcDNA3NACP经限制性内切酶消化分别得到4750bp和550bpDNA片段。α-突触核蛋白基因在原核细胞中蛋白表达量为10mg/L。表达蛋白经Western杂交证实为a-突触核蛋白。结论:α-突触核蛋白基因在原核细胞的蛋白表达可作为体内外研究老年神经变性疾病病理机制的基础研究。

AIM:To prepare α synuclein antibody so as to study the effect of its auto aggregation on the pathological mechanism of neurodegenerative disease in the elder and to explore the expression of α synuclein gene in the prokaryotic cells by recombination of plasmid vectors and DNA sequencing. METHODS:pcDNAα synuclein cDNA(pcDNA3NACP)and proEXMTHT1 were digested with enzyme to obtain cDNA fragment of α synuclein.The fragment of α synuclein was recombined on the carrier of proEXMTHT1 and identified with enzyme digestion before sequencing.Then the recombinant proEXNACP plasmid was transferred into DN5 a escherichia coli.By inducement of IPTG,α synuclein gene produce protein, which was subjected to western blot analysis after purification. RESULTS:The DNA fragment of 4 750 bp and 550 bp of α synuclein were obtained from proEXNTHT1 and pcDNA3NACP with restriction endonucleases digestion.α synuclein gene was expressed in the prokaryotic cells with the content of 10 mg/L.The expressing proteins of α synuclein were confirmed by Western blot analysis. CONCLUSION:Further investigation on pathological mechamism of neurodegenerative disease in vitro and in vivo can base on the gene expression of a synuclein protein.