丝瓜籽核糖体失活蛋白luffin-a的克隆表达及其cDNA序列分析  

Cloning expression of a novel luffin-a and analysis of its cDNA sequence

作　　者：王怡[1] 孙萍[1] 詹林盛[1] 宋丽洁[1] 李晖[1] 孙红琰[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《军事医学科学院院刊》2006年第4期333-336,356,共5页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的:从丝瓜籽中克隆核糖体失活蛋白luffin-a基因,并在原核系统进行表达。方法:根据已报道的cDNA序列设计引物,用RT-PCR的方法从成熟的丝瓜籽中克隆luffin-a基因,克隆后鉴定核苷酸序列。构建表达载体pET-28 a(+)-luffin-a,转化大肠杆菌BL21(DE3),并经IPTG诱导表达。利用金属离子亲和层析的方法纯化所得目的蛋白。结果:克隆的luffin-a基因及其所编码的氨基酸序列与国外报道的核苷酸序列及氨基酸序列同源性分别为95%和92%,因而所获得的luffin-a基因是丝瓜籽核糖体失活蛋白luffin-a一个新的cDNA序列。经SDS-PAGE分析:luffin-a主要存在于包涵体中。金属离子亲和层析可纯化目的蛋白。结论:本研究成功地克隆了luf-fin-a一个新的序列,并建立了该蛋白的原核表达系统,同时利用亲和层析可一步法纯化获得目的蛋白。Objective： To isolate ribosome inactivating protein luffin-a gene from the seed of Luffa cylindrica and express it in E. coli. nethods：Luffin-a gene was obtained by RT-PCR using the primers designed according to known luffin-a cDNA sequence and cloned in pGEM-T vector. The luffin-a gene obtained was sequenced and analyzed, Expression vector pET-28a（ ＋ ） -luffin-a was constructed and expressed in E, coli BL21 （ DE3 ）. The interest protein was purified using immobilized metal ion affinity chromatography （IMAC）. Results：The cDNA and amino acid sequence of the novel luffin-a obtained showed 95% and 92% homology with those reported by others. SDS-PAGE showed that the protein luffin-a was located in the inclusion body. The luffin-a protein could be purified by IMAC. Conclusion： A novel luffin-a gene is isolated, expressed in E. coli and purified by one-step IMAC.

关 键 词：核糖体 luffin-a 序列分析 DNA 金属离子亲和层析 基因表达 

分 类 号：Q943.2[生物学—植物学]