人类5,10-亚甲基四氢叶酸还原酶N端部分的克隆和表达 |
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引用本文: | 万琪,孙文萍,苏明权,俞英欣,李力,张瑞国,郭以川. 人类5,10-亚甲基四氢叶酸还原酶N端部分的克隆和表达[J]. 医学争鸣, 2003, 24(7): 600-602 |
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作者姓名: | 万琪 孙文萍 苏明权 俞英欣 李力 张瑞国 郭以川 |
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作者单位: | 1. 第四军医大学西京医院神经内科,陕西,西安,710033 2. 第四军医大学西京医院检验科,陕西,西安,710033 |
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摘 要: | 目的 :克隆人 5 ,10 亚甲基四氢叶酸还原酶 (MTH FR)N端部分编码基因 ,构建重组表达载体并对其诱导表达 .方法 :通过RT PCR方法扩增出人胎肝组织MTHFR的N端部分 1116bp的核苷酸序列 ,克隆于载体pGEM Teasy ,测序分析后亚克隆于表达载体pGEX 4T 2 ,构建重组表达载体pGEX MTHFR(N) .结果 :经过转化E .coliJM10 9和BL2 1后 ,诱导表达出Mr 为 66× 10 3的蛋白 .SDS PAGE分析显示 ,表达量占全菌总蛋白质的 3 1%.结论 :获得了人MTHFR(N)编码基因及其原核表达菌株 ,对研究人MTHFR的生物学功能具有重要的意义 .
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关 键 词: | 甲基四氢叶酸还原酶 克隆 原核表达 |
文章编号: | 1000-2790(2003)07-0600-03 |
修稿时间: | 2002-10-09 |
Cloning and expression of N terminal human 5, 10-methylenetetrahydrofolate reductase code gene |
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Abstract: | AIM: To clone human N terminal human 5, 10 methylenetetrahydrofolate reductase code gene, construct the recombinant vector and express its product. METHODS: Human MTHFR(N) code gene was amplified by RT PCR from fetal liver tissue and cloned into vector pGEM T easy, which was sequenced and subcloned into prokaryotic expressive vector pGEX 4T 2. After a 4 hour induction by IPTG, the recombinant Mr 66ku fusion protein GST MTHFR(N) was expressed and confirmed by SDS PAGE. RESULTS: Human MTHFR(N) gene was cloned. Recombinant expression plasmid pGEX 4T 2/ MTHFR(N) was constructed. The expressed fusion protein was about 31% of the total bacterial protein by gel thin layer chromatography scanning. CONCLUSION: The human MTHFR(N) gene and prokaryotic expression products have been obtained, which is of great importance for further study on the function of human MTHFR. |
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Keywords: | MTHFR cloning prokaryotic expression |
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