水稻Qb-SNARE蛋白OsNPSN11多克隆抗体制备、鉴定与应用

doi:10.3724/SP.J.1005.2010.00961

遗传 ›› 2010, Vol. 32 ›› Issue (9): 961-965.doi: 10.3724/SP.J.1005.2010.00961

水稻Qb-SNARE蛋白OsNPSN11多克隆抗体制备、鉴定与应用

鲍永美, 刘永惠, 许冬清, 黄骥, 王州飞, 王建飞, 张红生

南京农业大学作物遗传与种质创新国家重点实验室, 南京210095

收稿日期:2009-12-30 修回日期:2010-04-19 出版日期:2010-09-20 发布日期:2010-09-20
通讯作者: 张红生 E-mail:hszhang@njau.edu.cn
基金资助:
国家自然科学基金项目(编号：30900888), 教育部博士点新教师基金(编号：200903070015)和南京农业大学青年创新基金 (编号：KJ08033)资助

Preparation, characterization and application of rice Qb-SNARE protein OsNPSN11 polyclonal antibody

BAO Yong-Mei, LIU Yong-Hui, XU Dong-Qing, HUANG Ji, WANG Zhou-Fei, WANG Jian-Fei, ZHANG Hong-Sheng

State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China

Received:2009-12-30 Revised:2010-04-19 Online:2010-09-20 Published:2010-09-20
Contact: ZHANG Hong-Sheng E-mail:hszhang@njau.edu.cn

摘要/Abstract

摘要： 在真核生物细胞囊泡运输过程中的膜融合主要是由SNARE蛋白介导的, OsNPSN11是从水稻中克隆的Qb-SNARE家族基因, 文章将OsNPSN11构建到原核表达载体pET-30a中与6个His标签融合, 重组质粒pET-OsNPSN11转化BL21(DE3)0.5 mmol/L IPTG诱导4 h后获得了高效表达。用镍离子亲和树脂(Ni²⁺-NTA His Bind Resin) 纯化融合蛋白, 以纯化后的蛋白为抗原免疫新西兰家兔制备多克隆抗体, Western blotting结果显示, 该抗体能特异识别在原核系统表达的抗原, 以及水稻不同组织质膜组分中的OsNPSN11, 可用于转基因水稻中目标蛋白的表达分析。

关键词: 水稻, Qb-SNARE蛋白, OsNPSN11, 多克隆抗体, 转基因表达分析

Abstract: Membrane fusion in vesicle trafficking in the cells of eukaryotic organisms is mediated by soluble-N-ethyl- maleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins. OsNPSN11 is a member of Qb-SNARE gene family isolate from rice. The cDNA of OsNPSN11 was subcloned into pET-30a and fusion to the 6 × His tag. Induced by 0.5 mmol/L IPTG for four hours, the recombinant protein was highly expressed in Escherichia coli, which was purified by Ni²⁺-NTA His-bind resin affinity chromatography column to be used as an antigen to raise the antibody in New Zealand rabbits. Western blotting analysis showed that the antibody can specifically recognize the expressed antigen and the OsNPSN11 in plasma membrane protein from various rice tissues. This indicated that the antibody can be used for expres-sion analysis in transgenic rice.

Key words: rice, Qb-SNARE protein, OsNPSN11, polyclonal antibody, expression analysis in transgenic plants

鲍永美，刘永惠，许冬清，黄骥，王州飞，王建飞，张红生. 水稻Qb-SNARE蛋白OsNPSN11多克隆抗体制备、鉴定与应用[J]. 遗传, 2010, 32(9): 961-965.

BAO Yong-Mei, LIU Yong-Hui, HU Dong-Qing, HUANG Ji, WANG Zhou-Fei, WANG Jian-Fei, ZHANG Gong-Sheng. Preparation, characterization and application of rice Qb-SNARE protein OsNPSN11 polyclonal antibody[J]. HEREDITAS, 2010, 32(9): 961-965.

参考文献

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水稻Qb-SNARE蛋白OsNPSN11多克隆抗体制备、鉴定与应用

Preparation, characterization and application of rice Qb-SNARE protein OsNPSN11 polyclonal antibody

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