大鼠Nogo基因片段的克隆及与GST融合蛋白的表达和纯化  

Cloning of adult rat Nogo gene segments and expression and purification of their coding protein fused with GST

作　　者：张萍[1] 程希平[1] 费玲玲[1] 王春婷[1] 杨浩[1] 陈镔复[1] 鞠躬[1] 

机构地区：[1]第四军医大学基础部神经科学研究所,陕西西安710033

出　　处：《第四军医大学学报》2003年第19期1733-1735,共3页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金 (30 1 0 0 1 95)

摘　　要：目的 :获取大鼠Nogo基因片段并高效表达纯化 .方法 :用RT PCR技术 ,从成年SD大鼠脊髓的总RNA中 ,获得编码Nogo基因功能片段的DNA .测序后 ,通过酶切亚克隆至表达载体pGEX 4T 1,构建重组表达载体 ,并导入E .coliDH5α中 ,IPTG诱导表达重组的GST融合蛋白 .对重组融合蛋白过谷胱甘肽琼脂糖柱进行纯化 .结果 :获得成年SD大鼠Nogo(5 70～ 6 91和 10 2 8～ 10 89氨基酸残基 )的基因 ,测序结果与已发表的基因序列相一致 .重组GST融合蛋白经SDS PAGE分析 ,在相对分子质量 (Mr) 390 0 0和 32 0 0 0处 ,有特异的蛋白条带 .重组蛋白经谷胱甘肽琼脂糖柱进行纯化后 ,得到了高纯度的融合蛋白 .结论 :成功克隆成年大鼠No go基因片段 ,并在E .coliDH5α中高效表达 。AIM: To obtain rat Nogo gene segments, express efficiently in E. coli and purify the target proteins. METHODS: Nogo gene segments were amplified by RT PCR from normal adult SD rat spinal cord total RNA. After sequenced, the reconstructed expression vectors were constructed by enzyme digestion and subcloned into expression vector pGEX 4T 1. Then the vectors were transformed into E. coli DH5α. Recombinant GST fusion proteins were expressed via the induction of IPTG and purified through glutathione agarose column. RESULTS: The sequences of cloned adult rat Nogo gene segments (570-691 and 1028- 1089 amine acid residues) were identical with those earlier reported. Two protein bands of M r 39 000 and 32 000 appeared on SDS PAGE gel after the expressed GST fusion proteins were separated by SDS PAGE. Then the expressed fusion proteins were purified to high purity. CONCLUSION : The adult rat Nogo gene segments have been successfully cloned and efficiently expressed in E. coli , and the GST fused target proteins have been purified to high purity.

关 键 词：NOGO基因 克隆 表达 纯化 

分 类 号：R392.13[医药卫生—免疫学]