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目的 :克隆并表达人类抗砷基因 human arsenite resistance related gene(h ARRG)。 方法 :从人胎脑中获得 m RNA,建立 c DNA文库 ,通过大规模测序克隆出一人类新基因。 结果 :该基因全长 1170 bp,读框为 (ORF)72 3bp,经序列分析 ,与中国仓鼠抗砷基因 arsenite resistance gene (asr2 )同源性达 88%。根据 ORF设计引物 ,PCR扩增 ,扩增产物酶切后装入 PQE30质粒 ,诱导后获得 >40 %的高效表达。用镍亲和层析法纯化后纯蛋白回收率 >80 %。 结论 :首次在国内成功克隆并表达了人类抗砷相关基因 h ARRG。
Objective: To clone and express human arsenite resistance related gene (h ARRG). Methods: Obtaining m RNA from human fetal brain, establishing c DNA library and cloning a new human gene by large-scale sequencing. Results: The gene was 1170 bp in length and 72 bp in reading frame (ORF). The sequence of the gene was 88% homologous to the arsenite resistance gene (asr2) in Chinese hamster. Primers were designed according to ORF. PCR amplification and amplification products were digested and inserted into PQE30 plasmid to obtain high expression of> 40% after induction. After purification by nickel affinity chromatography pure protein recovery> 80%. Conclusion: The human anti-arsenic related gene h ARRG was successfully cloned and expressed for the first time in China.