银杏果仁抗菌蛋白的分离纯化及其基因克隆和原核表达初步研究  

Purification and Gene Cloning of an Antimicrobial Protein from Seeds of Ginkgo biloba L.and its Prokaryotic Expression

作　　者：刘缙[1] 王亚红[1] 王强[1] 刘曾甄[1] 郭蔼光[1] 

机构地区：[1]西北农林科技大学生命学院,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2010年第2期246-253,共8页Journal of Agricultural Biotechnology

基　　金：国家转基因植物研究与开发专项(JY03-A-11-01)资助

摘　　要：通过40%及80%(NH4)2SO4分级盐析,CM SephoroseF.F.离子交换层析和Superdex75凝胶过滤层析;从银杏(Ginkgobiloba)种仁中分离纯化到一种表观分子量约为12kD的抗菌蛋白。经过MALDI-TOF-MSPMF鉴定表明,该蛋白与一种新型抗菌蛋白Gnk2(ginkbilobin-2)具较高匹配率(P<0.05);根据ESI-MS/MS内肽氨基酸测序结果设计简并引物,扩增该蛋白基因一段,经比对发现与Gnk2基因序列相似性也达99%。按照Gnk2基因序列设计同源引物,利用RT-PCR克隆该抗菌蛋白全长基因,提交GeneBank(Accession No.FJ865399),并以此构建原核表达载体pGEX-GK2-1转化大肠杆菌(Escherichia coli)BL21(DE3),SDS-PAGE和Westernblot分析表明,融合蛋白在大肠杆菌中能够高效表达。An antimicrobial protein, with an apparent molecular mass of 12 kD, was isolated from the seeds of Ginkgo biloba.The isolation procedure entailed extraction, ammonium sulfate precipitation, cation exchange chromatography on CM Sephorose F.F, gel filtration chromatography on Superdex 75.MALDI-TOF-MS PMF identification showed higher homology with a novel antifungal protein Gnk2（Ginkbilobin-2）.Degenerate primers were designed to clone a fragment of the antimicrobial protein gene, according to the ESI-MS/MS sequence results of internal peptide.The amplified fragment also showed 99% similarity with the Gnk2 gene.Further, the homological primers were designed for cloning the full length cDNA of the antimicrobial protein by RT-PCR.The sequence has been deposited in GenBank（Accession No.FJ865399）.In addition, the prokaryotic expression vector pGEX-GK2-1 was constructed to express the antimicrobial protein in vitro.Results of SDS-PAGE and Western blot showed that the recombinant protein with the calculated molecular mass of 38 kD was highly expressed in Escherichia coli BL21（DE3）.

关 键 词：银杏 抗菌蛋白 克隆 原核表达 

分 类 号：Q78[生物学—分子生物学]