| 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 |
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| 引用本文： | 刘缙,王亚红,王强,刘曾甄,郭蔼光. 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[J]. 农业生物技术学报, 2010, 18(2): 246-253. DOI: 10.3969/j.issn.1674-7968.2010.02.007 |
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| 作者姓名： | 刘缙 王亚红 王强 刘曾甄 郭蔼光 |
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| 作者单位： | 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 |
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| 基金项目： | 国家转基因植物研究与产业化开发项目 |
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| 摘 要： | 通过40%及80%(NH4)2>SO4>分级盐析,CM Sephorose F.F.离子交换层析和Superdex 75凝胶过滤层析;从银杏(Ginkgo biloba)种仁中分离纯化到一种表观分子量约为12 KD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Western blot分析表明,融合蛋白在大肠杆菌中能够高效表达.
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| 关 键 词： | 银杏 抗菌蛋白 克隆 原核表达 |
Purification and Gene Cloning of an Antimicrobial Protein from Seeds of Ginkgo biloba L.and its Prokaryotic Expression |
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| Liu Jin,Wang Yahong,Wang Qiang,Liu Zengzeng,Guo Aiguang. Purification and Gene Cloning of an Antimicrobial Protein from Seeds of Ginkgo biloba L.and its Prokaryotic Expression[J]. Journal of Agricultural Biotechnology, 2010, 18(2): 246-253. DOI: 10.3969/j.issn.1674-7968.2010.02.007 |
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| Authors: | Liu Jin Wang Yahong Wang Qiang Liu Zengzeng Guo Aiguang |
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| Affiliation: | Liu Jin Wang Yahong Wang Qiang Liu Zengzeng Guo Aiguang College of Life Science,Northwest Agriculture , Forestry University,Key Laboratory for Agriculture Molecule Biotechnology,Yangling,712100 |
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| Abstract: | 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 seq... |
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| Keywords: | Ginkgo biloba Antimicrobial protein Cloning Prokaryotic expression |
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