人谷胱甘肽硫转移酶M1真核表达系统序列分析

陈萍萍; 张朝武; 谢东; 韩良峰; 吴逸明; 陈玲玲

人谷胱甘肽硫转移酶M1真核表达系统序列分析

Construction of an eukaryotic expression vector pcDNA3.1-glutathione-S-transferase M1 and sequence determination

摘要

摘要:
目的对人谷胱甘肽硫转移酶M1真核表达系统的构建及序列分析。
方法采用RT-PCR方法将肺组织中提取总RNA扩增人谷胱甘肽硫转移酶M1基因的cDNA序列, 将其插入到真核表达载体pcDNA3.1多克隆位点中, 构建重组表达质粒, 并用PCR扩增、酶切分析及序列测定等方法对重组质粒进行鉴定。
结果人谷胱甘肽硫转移酶M1克隆到真核表达质粒pcDNA3.1中, 测序结果同Genbank GSTM1(GI: 183668)cDNA序列相比较, 在619位点C→A, 氨基酸由Pro→Thr; 在519位点C→G, 编码的氨基酸仍为lys; 在528位点C→T, 编码的氨基波仍为Asp, 同源性为99%, 基因登陆号为AY532926。
结论人谷胱甘肽硫转移酶M1真核表达系统的构建, 为进一步进行真核细胞和动物的解毒机制研究, 提供应用材料。

Abstract:
Objective To construct an eukaryotic expression vector carrying human glutathione-S-transferase(GST)M1 gene and to determine partial sequence analysis.
Methods The GSTM1 cDNA was amplified and extracted from human lung total RNAs by RT-PCR approach and recombined with eukaryotic expression vector pcDNA311.The recombined plasmid pcDNA 3.1-hGSTM1 was verified using PCR, restriction analysis and sequencing determination.
Results Human GSTM1 gene was recombined correctly with pcDNA311, compared with genbank, in code 619 C→A, amino acid Pro→Thr, in code 519, C→G, in code 528, C→T, animo acids were not changed.GenBank accession was A Y532926.
Conclusion The eukaryotic expression vector pcDNA3.1-hGSTM1 was constructed for research detoxicitic mechanism in eukaryotic cell tranins and animals.

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