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

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GenbankGSTM 1(GI:1836 6 8)cDNA序列相比较 ,在 6 19位点C→A ,氨基酸由Pro→Thr;在 5 19位点C→G ,编码的氨基酸仍为lys;在 5 2 8位点C→T ,编码的氨基波仍为Asp ,同源性为 99% ,基因登陆号为AY5 32 92 6。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 。

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 pcDNA3 1.The recombined plasmid pcDNA 3 1 hGSTM1 was verified using PCR,restriction analysis and sequencing determination.Results Human GSTM1 gene was recombined correctly with pcDNA3 1,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 AY532926.Conclusion The eukaryotic expression vector pcDNA3 1 hGSTM1 was constructed for research detoxicitic mechanism in eukaryotic cell tranins and animals.