人NR1靶片段的原核表达

Expression of the cDNA encoding a target fragment of human NR1 protein in E.coli

目的 :克隆人N_甲基_D_门冬氨酸受体 (NMDAR ,NR)靶片段cDNA ,构建其原核表达载体并建立相应的表达体系。方法 :用常规RT_PCR法从脑肿瘤切除组织中克隆人NR1靶片段cDNA ,测序鉴定后构建其原核表达载体 ,并在大肠杆菌DH5α中升温诱导表达 ,表达产物通过SDS_PAGE分析及免疫印迹分析进行鉴定。结果 :成功克隆了人NR1靶片段 4 89bp长cDNA ,测序结果与文献报道一致 ,原核表达载体在宿主菌中诱导表达后得到了NR1蛋白靶片段的高效表达产物 ,占全菌体 15 %～ 30 %。SDS_PAGE及免疫印迹分析与预计相同。结论

Objective:To obtain the cDNA fragment encoding a polypeptide of human NR1 protein and to construct its prokaryotic expression vector.Methods:The cDNA fragment encoding a polypeptide of human NR1 protein was isolated by RT_PCR from total RNA of fresh human brain tumor tissue. After sequencing, the cDNA fragment was inserted into the expression plasmid pBV220. Then the recombinant plasmid was introduced into E.coli DH5α and induced by raising temperature. The expressed product was identified by SDS_PAGE and Western blot. Results and Conclasions:A 489?bp cDNA fragment encoding a polypeptide of human NR1 protein was obtained. DNA sequencing indicated that the sequence of isolated cDNA was same as that reported in the literature. After induction, the human NR1 was highly expressed, accounting for 15-30% of total bacterial protein. The results of SDS_PAGE and Western blot analysis were same as those anticipated.