| 摘要: |
| 用PCR扩增出绿色荧光蛋白(GFP)基因,插入到pGEX-KG表达载体中,并将构建出的重组质粒命名为pKG-GFP. 将重组载体导入大肠杆菌DH10β中,经IPTG诱导产生GST-GFP融合蛋白,同时以可溶蛋白和包涵体两种形式存在.GST-GFP分子量大约为53kDa,与其理论值大小一致,用亲和层析以及凝血酶处理纯化GFP.纯化的产物经证实具有很好的均一性.以GFP免疫新西兰家兔,制备多克隆抗体,Western blotting测定抗血清效价. |
| 关键词: GFP 原核表达 GST融合蛋白 |
| DOI： |
| 分类号:Q78 |
| 基金项目: |
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| Prokaryotic Expression and Purification of Green Fluorescent Protein and Its Antibody Preparation |
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| Abstract: |
| PCR amplified green fluorescent protein (GFP) gene was inserted into pGEX-KG expression vector, and resulting plasmid was designated as pKG-GFP. A recombinant fusion protein GST-GFP was expressed in DH10β after IPTG induction, in both soluble and insoluble forms. The molecular weight of GST-GFP was 53kDa in homonymous to its theoretical size. GFP was purified by using affinity binding and thrombin treatment. Purified protein demonstrated good homogeneity. Polyclonal antibody against purified GFP raised in rabbit recognized eukaryotic expressed GFP specifically. Titer of this antibody was measured by Western blotting. |
| Key words: GFP prokaryotic expression GST fusion protein |
