摘要
【目的】制备乙烯应答基因HLS1多克隆抗体,在过表达植物中检测HLS1的积累,为深入研究HLS1响应乙烯信号通路的分子机制奠定基础。【方法】构建pET28a-HLS1c原核表达载体,转化到大肠杆菌BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导获得重组蛋白HLS1c-His;镍柱亲和纯化重组蛋白后免疫家兔获得抗HLS1血清,利用抗原亲和纯化抗血清获得高纯度的HLS1多克隆抗体;用HLS1抗体和GFP抗体,检测原生质体瞬时转化体系中GFP-HLS1c融合蛋白的表达;用农杆菌蘸花法获得过表达GFP-HLS1g的转基因植物,用HLS1抗体和GFP抗体检测GFP-HLS1g融合蛋白的表达。【结果】成功构建了原核表达载体pET28a-HLS1c,并在大肠杆菌系统中诱导获得重组蛋白HLS1c-His,且多以包涵体形式存在。纯化的HLS1c-His多克隆抗体,能清晰检测到约80 ng原核表达的HLS1抗原。纯化的HLS1抗体不仅能检测到原生质体中瞬时表达的GFP-HLS1c融合蛋白,也能检测到过表达转基因植物株系中的GFP-HLS1g融合蛋白,并且与标签蛋白GFP对应抗体所检测到的特异性条带大小一致。【结论】成功制备了拟南芥HLS1蛋白多克隆抗体,为HLS1蛋白在植物发育中的功能研究奠定了基础。
【Objective】The HLS1 polyclonal antibodies were prepared and the accumulation of HLS1 in HLS1 overexpression plants was detected to understand the molecular mechanism of HLS1 in response to ethylene signaling pathway.【Method】The prokaryotic expression vector pET28a-HLS1c was constructed,followed by transformation into BL21(DE3)E.coli.After IPTG induction,HLS1c-His was purified via Nickel affinity chromatography.HLS1c-His antiserum was obtained by immunization of rabbits.After affinity purification of antibody,the accumulation of HLS1 was identified by HLS1 antibody in transgenic plants.The expression of fusion protein GFP-HLS1c was detected using HLS1 antibody and GFP antibody through protoplast transient transformation system.【Result】The prokaryotic expression vector pET28a-HLS1c was constructed successfully and the induced recombinant protein HLS1c-His was obtained in E.coli system mostly as inclusion body.Purified HLS1 antibody clearly detected about 80 ng prokaryotic expressed HLS1 antigen.Purified HLS1 antibody did not only detect the expression of GFP-HLS1c in protoplast system,but detected GFP-HLS1g in transgenic lines,and the same specific bands were detected by GFP antibody.【Conclusion】The specific antibody against HLS1 protein was prepared successfully in Arabidopsis.
作者
郭佳
郁品泽
贾敏
郁飞
GUO Jia;YU Pinze;JIA Min;YU Fei(State Key Laboratory of Crop Stress Biology for Arid Areas,College of Life Sciences,Northwest A&F University,Yangling,Shaanxi 712100,China)
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2020年第7期141-147,共7页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(31300988)。
关键词
乙烯应答基因
拟南芥
原核表达
蛋白纯化
多克隆抗体
gene responding to ethylene
Arabidopsis
prokaryotic expression
protein purification
polyclonal antibody
