| 摘要: |
| 【目的】构建拟南芥转录因子Dof1基因的原核表达载体，进行原核表达并制备其蛋白抗体。【方法】利用RT-PCR方法从拟南芥中克隆转录因子Dof1基因，将其连接到原核表达载体pET-32a(+)上，构建重组原核表达载体pET32a(+)-Dof1，经酶切鉴定和测序验证后将其转化至大肠杆菌BL21中，经IPTG诱导后，纯化重组蛋白，以分离到的重组蛋白为抗原免疫小鼠制备抗血清，检测植物中Dof1蛋白表达水平。【结果】成功构建了拟南芥转录因子Dof1基因的原核表达载体pET32a(+)-Dof1，在28 ℃、1 mmol/L IPTG诱导6 h的大肠杆菌中可高效表达分子质量约为42 ku的重组蛋白，其分泌到细胞质中，不形成包涵体；Western-blotting检测结果证明，制备的抗血清有较好的抗原-抗体识别反应。【结论】成功实现了Dof1基因的原核表达，制备出的重组蛋白Dof1多克隆抗体可用于植物Dof蛋白表达水平的检测，可为Dof1基因的进一步研究奠定基础。 |
| 关键词: Dof 转录因子 载体构建 原核表达 |
| DOI： |
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| 基金项目:云南省中青年学术与技术后备人才培养基金项目（2006PY01-10） |
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| Construction and application of prokaryotic expression plasmid containing Dof1 gene of Arabidopsis thaliana |
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| Abstract: |
| 【Objective】The prokaryotic expression vector containing Dof1 gene of Arabidopsis thaliana was constructed.The identified construction was transformed into E.coli.Over-expressed protein was purified and polyclonal autiserum was obtained.【Method】The full length cDNA of Dof1 gene was amplified from Arabidopsis by RT-PCR,and cloned into a prokaryote expression vector pET-32a(+).The recombinant prokaryotic expression vector pET32a(+)-Dof1 was used to transform E.coli BL21.The recombinant protein was obtained with induction of IPTG and purification,and was used as an antigen to immunize the healthy rats for production of antiserum.【Result】Prokaryotic expression vector pET32a(+)-Dof1 was successfully constructed,and highly expressed in E.coli BL21 induced with IPTG.A 42 ku fusion protein was secreted into the cytoplasm.Western-blotting analysis indicated that the antiserum could serologically react with Dof1 protein.【Conclusion】Prokaryotic expression vector of Dof1 gene was successfully established.The protein Dof1 polyclonal antibody was used to detect the expression level of Dof1 protein in plant,and provided reliable tools for study in the transgenic plant of Dof1. |
| Key words: Dof transcription factor vector prokaryotic expression |
