华东葡萄转录因子VpSBP3基因原核表达、纯化及多克隆抗血清制备  

Prokaryotic expression,purification and polyclonal antiserum preparation of the transcription factor VpSBP3 gene from Vitis pseudoreticulata

作　　者：王浩[1] 侯鸿敏[1] 殷向静[1] 宋军阳[1] 王西平[1] 

机构地区：[1]西北农林科技大学园艺学院·农业部西北地区园艺作物生物学与种质创制重点开放实验室·旱区作物逆境生物学国家重点实验室,西北农林科技大学,陕西杨凌712100

出　　处：《果树学报》2013年第5期737-743,共7页Journal of Fruit Science

基　　金：国家自然科学基金(31071782);基本科研业务费科技创新专项(QN2011056)

摘　　要：【目的】制备华东葡萄(Vitis pseudoreticulata)转录因子VpSBP3基因抗血清,为研究VpSBP3基因功能奠定基础。【方法】构建华东葡萄转录因子VpSBP3基因重组原核表达载体pGEX-VpSBP3,转化大肠杆菌(E.coli)Rosetta(DE3),以IPTG为诱导剂诱导目的融合蛋白的表达。切胶电透析回收诱导表达的VpSBP3融合蛋白免疫新西兰兔,制备VpSBP3抗血清。【结果】重组原核表达载体pGEX-VpSBP3在DE3中高效表达出了分子质量为68 kD的GSTVpSBP3融合蛋白。回收的目的融合蛋白免疫新西兰兔后得到了抗血清,经Western-Blot检测,抗血清的免疫-抗原反应良好。【结论】在27℃、0.7 mmol·L-1IPTG过夜培养的诱导条件下,融合蛋白GST-VpSBP3过量表达。免疫大白兔获得的血清特异性强,效价高,可用于VpSBP3基因功能分析。[Objective] The objective of this study is to obtain polyclonal antiserum of the transcription factor gene VpSBP3 from Vitis pseudoreticulata for the sake of supporting the study of VpSBP3 function. [Method] The recombinant prokaryotic expression vector pGEX-VpSBP3 of the VpSBP3 gene was con- structed and translated into cells of E. coli Rosetta （DE3）. The induced fusion protein with IPTG was purified by the electrodialysis, and the rabbit was immunized to obtain the polyclonal antiserum. [Result] The recombinant vectors pGEX-VpSBP3 was constructed in E. coli DE3 and the 68 kD fusion protein of GST- VpSBP3 was efficiently expressed. Then the polyclonal antiserum was obtained by immunizing the rabbit with the purified protein. The result of Western blot indicated that the polyclonal antiserum had a specific antigen-antibody recognition feature. [Conclusion] The results suggested that the fusion protein GST- VpSBP3 was expressed at high levels overnight under the induction condition of 0.7 mmol·L^-1 IPTG, 27 ℃. The polyclonal antiserum obtained by immunizing a rabbit with the purified fusion protein had high specificity and titer, and could be used for the functional analysis of VpSBP3 gene.

关 键 词：华东葡萄 VpSBP3 转录因子 原核表达 多克隆抗血清 

分 类 号：S663.1[农业科学—果树学]