| 摘要: |
| 【目的】制备拟南芥PRL1蛋白的多克隆抗体，并检测PRL1在拟南芥真叶中的积累情况。【方法】扩增拟南芥PRL1基因的CDS序列，将其克隆至原核表达载体pET-28a中，转化到BL21（DE3）表达菌，经IPTG诱导后，对诱导产生的PRL1重组蛋白进行镍柱亲和纯化。将制备的PRL1重组蛋白通过皮下注射免疫家兔，从抗血清中分离、纯化PRL1抗体。扩增拟南芥PRL1基因的启动子序列及其基因组DNA序列，将其克隆至植物表达载体pBI111L中，再转化到农杆菌GV3101中，利用农杆菌侵染法构建prl1回复株系。利用纯化的抗体检测PRL1在野生型、prl1点突变体、PRL1 T-DNA插入突变体和prl1转基因回复系中蛋白积累量的差异。【结果】克隆了PRL1基因的CDS序列，成功构建了pET-28a-PRL1原核表达载体，实现了PRL1重组蛋白在大肠杆菌中的表达（包涵体）。克隆了PRL1基因的启动子序列及其基因组DNA序列，成功构建了pBI111L-PPRL1-Strep-gPRL1植物表达载体及prl1回复株系。制备获得了PRL1重组蛋白的多克隆抗体，用该抗体能够有效地在拟南芥样品中检测出一条分子质量约为54 ku的蛋白，该蛋白在prl1点突变体中表达量下降，在PRL1 T-DNA插入突变体中缺失，而在prl1回复株系中表达量升高，并且在分裂旺盛的幼嫩组织中PRL1积累量较高。【结论】成功制备了PRL1蛋白的多克隆抗体，可用于拟南芥中PRL1蛋白含量的检测。 |
| 关键词: 拟南芥 PRL1蛋白 原核表达 蛋白纯化 多克隆抗体制备 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目（31300988，31741010）；教育部博士点基金新教师项目（20120204120036） |
|
| Preparation and application of polyclonal antibody against Arabidopsis PRL1 |
|
FAN Ying,LIANG Shuang,LIU Siyu,et al
|
| Abstract: |
| 【Objective】A polyclonal antibody against Arabidopsis PRL1 was prepared,and the accumulation of PRL1 in Arabidopsis true leaf was detected.【Method】The Arabidopsis PRL1 CDS sequence was amplified and cloned into prokaryotic expression vector pET-28a,followed by transformation into BL21(DE3) E.coli.After IPTG induction,recombinant PRL1 was purified via Nickel affinity chromatography.Recombinant PRL1 was used to immunize rabbits through subcutaneous injection.Specific PRL1 antibody was separated and purified from the PRL1 anti serum.The Arabidopsis PRL1 promoter and genomic DNA sequence were amplified and cloned into the plant expression vector pBI111L,followed by transformation into GV3101 Agrobacterium.Then prl1 complement lines were constructed by Agrobacterium infection.Purified PRL1 antibody was used to test the accumulation of PRL1 in the wild type,prl1 mutants,PRL1 T-DNA insertion mutants and prl1 complement lines.【Result】The Arabidopsis PRL1 CDS sequence was cloned,and prokaryotic expression vector pET-28a-PRL1 was constructed successfully.The PRL1 recombinant protein was expressed in E.coli as inclusion body.The PRL1 promoter and genomic DNA sequence were cloned,and plant expression vector pBI111L-PPRL1-Strep-gPRL1 and prl1 complement lines were constructed successfully.Obtained polyclonal antibody against PRL1 recombinant protein efficiently detected a specific protein with molecular weight of 54 ku in Arabidopsis.The protein was recognized as PRL1 protein,as it was expressed low in the prl1 mutant,not found in PRL1 T-DNA insertion mutants,while expressed high in prl1 complement lines.Accumulation of PRL1 was high in tissues with active cell division.【Conclusion】The specific antibody against PRL1 protein was prepared successfullyfor detection of PRL1 in Arabidopsis. |
| Key words: Arabidopsis PRL1 prokaryotic expression protein purification polyclonal antibody preparation |
