联系我们 加入收藏
 
年期检索 高级检索
33
2025年8月2日 星期六
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
葡萄β型液泡加工酶基因(VvβVPE)的原核表达、纯化与多克隆抗体制备
巩培杰1,张朝红1,魏蓉1,李树秀1,王跃进2
1. 西北农林科技大学
2. 西北农林科技大学园艺学院
Fusion Expression and Purification of β Type Vacuole Processing Enzyme Gene(VvβVPE) in Vitis vinifera and Its Antibody Preparation
1, Wei Rong1, 1
全文: PDF (864 KB)   HTML (1 KB) 
输出: BibTeX | EndNote (RIS)      
摘要 液泡加工酶(VPE)基因是植物内启动和执行细胞程序性死亡的关键基因,β型 VPE 是种子特异表达并且为种子蛋白成熟所必须的基因。本研究以黑比诺葡萄(Vitis vinifera cv. Pinot Noir)花后8个不同时期胚珠的cDNA混合样为模板,通过RT-PCR扩增得到葡萄β型液泡加工酶基因(暂命名VvβVPE , GenBank登录号: KC136352),开放阅读框1 485 bp。进一步将VvβVPE克隆到pGEX-6P-1 原核表达载体并转入大肠杆菌(Escherichia coli)BL21中,经 IPTG 诱导,获得约 81.3 kD的包涵体融合蛋白GST-VvβVPE。将诱导条件优化后,在 37℃、0.05 mmol/L IPTG诱导 5 h 后融合蛋白 GST-VvβVPE的表达量最大,采用电透析与盐酸胍处理后透析复性两种方法获得纯化包涵体蛋白,电透析纯化法获得纯化包涵体蛋白的浓度可达到免疫要求。使用纯化蛋白免疫新西兰大白兔制备多克隆抗血清,经Western blot 检测, 该抗血清(稀释到1∶10 000)与GST-VvβVPE融合蛋白识别反应良好,获得的抗血清可用于以后的VvβVPE的酶活性分析、免疫亚细胞定位分析以及转基因植株蛋白水平的鉴定等,为进一步明确VvβVPE在葡萄胚珠发育中的作用提供基础数据。
服务
把本文推荐给朋友
加入我的书架
加入引用管理器
E-mail Alert
RSS
作者相关文章
巩培杰
张朝红
魏蓉
李树秀
王跃进
关键词 葡萄液泡加工酶原核表达多克隆抗体    
Abstract:Vacuole processing enzyme(VPE) gene family plays an important role in mediating and executing programmed cell death in plants. β type VPE is specifically expressed in seed, which is indispensable in seed development. The ovules were harvested from V. vinifera cv. Pinot Noir in 8 different development stages. RNA was isolated from these ovules. VvβVPE gene was obtained by RT-PCR, and the mixture of cDNA was used as template. The results showed VvβVPE gene contained a complete ORF(open reading frame) sequence (1 485 bp) encoding 494 amino acid(GenBank accession number: KC136352). The full-length sequence was cloned into the prokaryotic expression vector of pGEX-6P-1. An about 81.3 kD fusion protein of inclusion body (GST-VvβVPE) was expressed in the Escherichia coli BL21 through IPTG inducing. After optimizing the inducing condition, two different methods were adopted to purify the protein, containing electro dialysis and dialysis renaturation by GdmCl treating, and then immunized the rabbit to obtain the polyclonal antibody. Western blot displayed a specific immunity-antigen signal, suggesting the obtained antiserum can be used for function analysis and detecting transgenic plants.
Key wordsVitis vinifera    Vacuolar-processing enzyme    Prokaryotic expression    Polyclonal antibody
收稿日期: 2012-11-15     
通讯作者: 张朝红   
引用本文:   
巩培杰1,张朝红1,魏蓉1,李树秀1,王跃进2. 葡萄β型液泡加工酶基因(VvβVPE)的原核表达、纯化与多克隆抗体制备[J]. , 2013, 21(6): 659-667.
1, Wei Rong1, 1. Fusion Expression and Purification of β Type Vacuole Processing Enzyme Gene(VvβVPE) in Vitis vinifera and Its Antibody Preparation. , 2013, 21(6): 659-667.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2013/V21/I6/659
 
版权所有 © 2014 《农业生物技术学报》编辑部   京ICP备11035905号-3
地址:北京市海淀区圆明园西路2号中国农业大学生命科学楼1053室 邮编:100193
电话:010-62733684 传真:010-62731615 E-mail: nsjxb@cau.edu.cn