构建具有突变铜-锌超氧化物歧化酶绿色荧光真核表达载体

Establishing the eukaryon expression vector with mutation Cu/Zn superoxide dismutase and green fluorescence

目的:构建一个具有特殊临床表型的肌萎缩侧索硬化症家系的突变铜-锌超氧化物歧化酶与有增强绿色荧光蛋白基因的pEGFP-N1融合的真核表达载体。方法:实验于2003-09/2005-01在第三军医大学全军免疫学研究所完成。通过RT-聚合酶链扩增突变铜-锌超氧化物歧化酶cDNA,应用基因重组技术,将得到的突变铜-锌超氧化物歧化酶cDNA克隆入荧光真核表达载体pEGFP-N1中,转化DH5α感受态细胞,用含卡那霉素LB培养基平板筛选转化菌,阳性重组子经EcoRⅠ与BamHⅠ双酶切分析、测序鉴定。结果:提取到纯度较高的总RNA,通过RT-聚合酶链和基因重组得到重组质粒,应用EcoRⅠ与BamHⅠ双酶切重组质粒,得到长约130bp和4.6Kb的两条带,前者为插入片段的长度,后者为pEGFP-N1载体本身的长度。测序也最终证实重组载体的方向及序列正确。结论:突变铜-锌超氧化物歧化酶cDNA成功地亚克隆至荧光真核表达载体pEGFP-N1中,获得突变铜-锌超氧化物歧化酶/pEGFP-N1重组质粒,从而为实时监测突变铜-锌超氧化物歧化酶基因在体内的表达和蛋白定位提供一个有用的工具载体。

AIM： To construct eukaryon expression vector, which was the amalgamation of mutation Cu/Zn superoxide dismutase （mSOD1） gene and pEGFP-N1 of enhanced green fluorescent protein gene, with a special clinical phenotyping and familial amyotruphic lateral sclerosis （FAIS）. METHODS： The experiment was done in the Institute of Military Immunology, Third Military Medical University of Chinese PLA from September 2003 to January 2005. The mSOD1 gene was obtained by RT-PCR and was cloned into fluorescent eukaryotic expression vector pEGFP-N1 by genetic recombination technique. The vector DH 5or was transformed with competent cell. The conversion bacteria on LB medium plates with kanamycin were screened and EcoR Ⅰ and BamH Ⅰ restrictive analysis and sequence identified the positive recombinants. RESULTS： High purity RNA was obtained. The recombinant was obtained by RT-PCR and gene recombination. Two bands with the length of 130 bp and 4.6 Kb were obtained by EcoR Ⅰ and BamH Ⅰ restrictive analysis. The former was the length of insertion element. The latter was the length of vector pEGFP-N1. The direction and sequence of the recombination vector were correct after the test of DNA sequence. CONCLUSION： The mSOD1 gene is successfully subcloned to insert into pEGFP-N1 of fluorescent eukaryon expression vector. The recombination plasmid of mSOD1/pEGFP-N1 is obtained. Thus, a useful tool for real time monitoring the express of mSOD1 gene in vivo and localization of protein is provided.