摘要
对大麦纤维素合成酶(Hordeum vulgare CesA,Hv-CesA)的蛋白质序列进行疏水性分析和跨膜区预测,获得截短的亲水性非跨膜区特征序列,采用PCR扩增截短序列编码区,定向克隆入N端带有His标签的pET-28a(+)表达载体中,并转化大肠杆菌进行诱导表达,利用钴离子螯合层析纯化重组表达蛋白,并制备高效价的多克隆抗体。结果表明,截短的Hv-CesA基因在大肠杆菌Rosetta gami2以包涵体的形式高效表达,western blotting显示制备的多克隆抗体能特异识别其对应的抗原。
A truncated non-transmembrane domain of barley cellulose synthase A（Hv-cesA）was determined by analysis of the hydrophobicity and prediction of transmembrane domains.The coding region of truncated non-transmembrane domains was obtained by PCR,and cloned into prokaryotic expression vector pET-28a（＋） with his-tag at its N terminal.The identified construct was transformed into E.coli and overexpressed protein was purified by cobalt chelating chromatography and polyclonal antiserum was raised against rabbit.The results showed the truncated non-transmembrane domain was expressed in E.coli Rosetta-gami2（DE3）in the form of inclusion bodies,and western blotting analysis showed the raised antibody can specifically react with the antigen.These results laid the detection basis for further research on the expression of Hv-CesA and cellulose synthesis in cell wall.
出处
《西北农业学报》
CAS
CSCD
北大核心
2011年第8期66-70,共5页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家自然科学基金(No.30400282)
西北农林科技大学基本科研业务费青年项目(QN2009064)
关键词
大麦
纤维素合成酶
截短序列
原核表达
多克隆抗体
Barley
Cellulose synthase
Truncated gene
Prokaryotic expression
Polyclonal antibody
