人NMDA受体主亚基M3-M4环基因片段的高效表达、纯化与鉴定

用基因工程方法获得人N 甲基 D 天冬氨酸 (N methyl D aspartate ,NMDA)受体主亚基M3 M4环靶片段 ,以此为免疫原 ,用于进一步免疫原性及相关应用研究 .自人脑胶质瘤组织中提取总RNA ,采用RT PCR扩增出人NMDA受体主亚基M3 M4环的基因片段 ,并按照计算机辅助原核表达载体pBV2 2 0中外源基因高效表达的数学模型预测方法 ,将其进行优化改构 .将目的基因克隆到pBV2 2 0中 ,转化大肠杆菌DH5α ,升温诱导表达 ,从蛋白质水平检测重组体在大肠杆菌中的表达情况 ,通过制备性SDS PAGE进行纯化 ,从相对分子质量、免疫反应性、肽质谱指纹分析等方面进行鉴定 .结果表明 ,成功构建了人NMDA受体主亚基M3 M4环的原核表达载体 (命名为pBV NR1L3) ,通过基因优化 ,实现了高效表达 .凝胶扫描分析表达量约占菌体总蛋白 2 9% ,重组肽纯度达 95 %以上

High Expression,Purification and Characterization of cDNA Encoding M3-M4 Loop in the Essential Subuint of hNMDA Receptor

A cDNA fragment encoding M3 M4 loop in the essential subuint of human N \|methyl\| D \|aspartate(NMDA) receptor was obtained by RT PCR using the total RNA extracted from a human brain glioma as template. The cDNA fragment was structurally optimized by means of CAD and then inserted into the high expression vector pBV220. The resultant recombinant plasmid which was confirmed by DNA sequencing was introduced into the competent cell of E.coli DH5α and inducibly expressed by means of shifting the culture temperature from 30℃ to 42℃. The recombinant protein obtained was assayed and purified by the preparative PAGE, and then characterized by a series method of identification, such as apparent molecular weight, immunoreactivity, and peptide fingerprinting. As a result, a prokaryotic expression vector for M3 M4 loop in the essential subuint of human NMDA receptor was constructed and named as pBV NR1L3, the high expression of which was achieved by structure optimization. The recombinant protein accounted for 29% of the total bacterial protein, and was purified to a purity of 95%.