摘要
目的克隆编码大鼠α-突触核蛋白的cDNA,探讨α-突触核蛋白基因在原核细胞中的表达过程并制备基因重组型α-突触核蛋白。方法设计合成含适当酶切位点的DNA片段作为引物,采用RT-PCR法从Wistar大鼠脑总RNA中扩增编码α-突触核蛋白的cDNA并亚克隆至原核表达质粒pGEX-4T-1,重组质粒转化大肠杆菌BL21(DE3),使其表达以谷胱甘肽S转移酶(glutathione-S-transferase,GST)为标签的融合蛋白质。用凝血酶定点分解融合蛋白,再用谷胱甘肽-琼脂糖4B和Superdex S200纯化基因重组型α-突触核蛋白。结果核酸序列测定表明克隆的cDNA含420 bp编码140个氨基酸,重组质粒pGEX-raSYN在原核细胞中表达为可溶性组分并受IPTG诱导,纯化的目的蛋白质在SDS-PAGE上表现为单一条带,推测其相对分子质量约为18 000。每升细菌培养液可纯化目的蛋白质3 mg。Western blot分析结果表明纯化的目的蛋白质可以被抗α-突触核蛋白识别。结论大鼠α-突触核蛋白基因在原核细胞高效表达,可制备高纯度基因重组型大鼠α-突触核蛋白。
Objective To investigate bacteria expression of rat α-Synuclein cDNA, and prepare the recombinant protein. Methods Total RNA were isolated from Wistar Rat brain. A cDNA fragment encoding the α-Synuclein was amplified by RT-PCR, and was subcloned into plasmid pGEX-4T-1. The recombinant plasmid DNA pGEX-raSYN was expressed with bacteria BL21 （ DE3 ）, and Glutathione S transferase （GST） tagged protein was purified by column chromatography. Recombinant rat α-Synuclein was freed by thrombin cleavage, and confirmed with SDS-PAGE and western blot analysis. Results Amplified cDNA fragment was composed of 420 bp and encodes 140 amino acids. The fusion protein GST-raSYN was expressed in the bacteria, with IPTG present in the condition medium. The recombinant rat α-Synuclein was purified to homogeneity from soluble fraction of the bacteria. The molecular weight of the purified protein was calculated to be approximately 524 000 on superdex S-200HR and to be 18 000 on SDS-PAGE in the presence of bmercaptoethanol. Western blot analysis demonstrates that the purified recombinant protein was reactive to the anti α-Synuclein monoclonal or polyclonal antibodes. Conclusion . Rat α-Synuclein cDNA was effectively expressed with bacteria, and pure recombinant rat α- Synuclein was prepared, which may be samples to studies physiological function of the protein.
出处
《首都医科大学学报》
CAS
2007年第3期333-336,共4页
Journal of Capital Medical University
基金
国家自然科学基金重点项目(30430280)
国家自然科学基金(30271437
30270482)资助项目~~
关键词
Α-突触核蛋白
纯化
Parkinson disease
α-Synuclein
purification
rat
