中国野生山葡萄VaERD15基因的原核表达及多克隆抗体制备  

Prokaryotic Expression and Polyclonal Antibody Preparation of the VaERD15 Gene from Chinese Wild V.amurensis

作　　者：张剑侠[1] 翟焕[1] 牛茹萱[1] 李瑞民[1] 

机构地区：[1]西北农林科技大学园艺学院,旱区作物逆境生物学国家重点实验室,农业部西北园艺植物种质资源利用重点开放实验室,陕西杨陵712100

出　　处：《西北林学院学报》2014年第6期100-105,共6页Journal of Northwest Forestry University

基　　金：国家"十二五"科技支撑计划项目(2013BAD02B04)

摘　　要：为探索中国野生山葡萄(Vitis amurensis)抗寒相关基因的抗寒作用机理,以中国野生山葡萄抗寒基因cDNA文库筛选的VaERD15基因(V.amurensis early responsive to dehydration 15)cDNA为模板,采用PCR扩增获得VaERD15基因的开放阅读框为423bp;通过构建pGEX-6p-1/VaERD15原核表达载体并转化大肠杆菌(Escherichia coli)BL21,经IPTG诱导表达出32.2kD的融合蛋白GST-VaERD15。该融合蛋白主要以包涵体表达形式存在,经0.1 mmol·L-1IPTG于37℃下诱导4h,其表达量最大;进一步用电透析法纯化融合蛋白,以纯化产物免疫新西兰大白兔制备多克隆抗体,Western blot检测结果表明其抗原-抗体免疫反应良好,获得的多克隆抗体能够用于VaERD15基因的功能分析。In order to understand the cold resistance mechanism of the related genes from Chinese wild Vitis amurensis,we used the cDNA of VaERD15 as the template,which was gotten from the cDNA library of Chinese wild V.amurensis with resistance to cold-stress,and obtained the open reading frame of 423 bp by PCR amplification.The recombinant plasmids pGEX-6p-1/VaERD15 was built and transformed to Escherichia coli BL21.The specific fusion protein bands of 32.2kD was present after induction by IPTG.Soluble analysis indicated that the GST-VaERD15 fusion protein was mainly expressed in inclusion body.The results of optimizing of the induction system revealed that the best condition GST-VaERD15 fusion protein was being induced by 0.1mmol·L^-1IPTG at 37℃for 4h.Purified GST-VaERD15 fusion protein was obtained by electrodialysis and injected into rabbit to produce polyclonal antibodies.Western blot showed that the antigen-antibody reaction was well.The polyclonal antibodies can be used for the functional analysis of VaERD15 gene.

关 键 词：中国野生葡萄 山葡萄 VaERD15 原核表达 多克隆抗体 

分 类 号：S722.36[农业科学—林木遗传育种]