| 手掌参中赤霉酸诱导的富含半胱氨酸蛋白基因的表达、纯化及鉴定 |
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| 引用本文： | 刘渊,蒙国权,周建平,张腾,冯娟,任正隆.手掌参中赤霉酸诱导的富含半胱氨酸蛋白基因的表达、纯化及鉴定[J].农业生物技术学报,2009,17(3):510-515. |
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| 作者姓名： | 刘渊 蒙国权 周建平 张腾 冯娟 任正隆 |
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| 作者单位： | 1. 电子科技大学生命科学与技术学院,成都,610054
2. 电子科技大学生命科学与技术学院,成都610054;四川农业大学植物遗传育种省级重点实验室,雅安625014 |
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| 基金项目： | 国家自然科学基金重点项目 |
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| 摘 要： | 目的: 构建含赤霉酸诱导的富含半胱氨酸蛋白基因(gcgasa)的原核表达载体，并在E.coli BL21(DE3)中表达。材料：手掌参。方法：设计带有EcoR I和Hind III酶切位点的引物，分别对gcgasa基因编码区全长及信号肽缺失的片段进行PCR扩增，将目标片段克隆入原核表达载体pET-32(a)，构建重组质粒。经测序鉴定后，转化大肠杆菌BL21(DE3)，IPTG诱导表达融合蛋白，并采用SDS-PAGE、Western-blotting、电镜超薄切片技术检测外源蛋白的表达及定位情况。对于水溶性蛋白，采用Ni2+-NTA亲和层析及凝胶过滤手段纯化。结果: 含有信号肽的外源基因在大肠杆菌周质空间中以包含体形式存在，而信号肽缺失的片段主要以可溶形式表达，经Ni2+-NTA纯化可获得目的蛋白。结论：首次在原核表达系统中高效表达了水溶性的gcgasa蛋白，为其结构和功能的研究奠定了基础。
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| 关 键 词： | 手掌参 赤霉酸诱导的富含半胱氨酸蛋白 原核表达 |
| 收稿时间： | 2008-9-10 |
| 修稿时间： | 2008-11-7 |
Expression,Purlfication and Identification of Gibberellin-induced Cysteine-rich Protein of Gymanadenia conopsea |
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| LIU Yuan,MENG Guo-quan,ZHOU Jian-ping,ZHANG Teng,FENG Juan,REN Zheng-long.Expression,Purlfication and Identification of Gibberellin-induced Cysteine-rich Protein of Gymanadenia conopsea[J].Journal of Agricultural Biotechnology,2009,17(3):510-515. |
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| Authors: | LIU Yuan MENG Guo-quan ZHOU Jian-ping ZHANG Teng FENG Juan REN Zheng-long |
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| Abstract: | Objective: To construct the prokaryotic expression vector for gcgasa gene encoding Gibberellin-induced cysteine-rich protein, and to express the gene in E.coli BL21(DE3). Material: Gymanadenia Conopsea R.Br. Methods: The primers bearing restriction enzyme site of EcoR I and Hind III were designed and were ultilized to amplify the full-length of ORF and the signal peptide-truncated fragment. The target fragments were then cloned into the pET-32(a) vector to produce the recombinant plasmids pET-32(a)/gcgasa and pET-32(a)/gcgasa. After they were identified by DNA sequencing, the recombinants were transformed into E.coli BL21(DE3) to express the fusion protein with the induction of IPTG. SDS-PAGE, Western blotting, as well as Transmission Electron Micrograph of ultrathin section were done to identify the expression and location of heterogenous protein. Following the solubility analysis, the expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods. Results: DNA sequencing showed that the recombinant plasmids have been successfully constructed. SDS-PAGE and Western blotting analysis revealed that the presence of signal peptide gave rise to the formation of inclusion body, which was located in periplasmic space, however, the absence of signal peptide greatly enhanced the solubility of the target protein. In the latter case, further purification was achieved using Ni2+-NTA and Sephadex G-75 gel filtration column. Conculsion: It was for the first time that the soluble gcgasa protein was successfully obtained from E. coli system with high efficiency. This study will be very fundamental and essential for further investigation on the stucture and function of gcgasa. |
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