【摘要】 建立重组基因酵母筛选雄性内分泌干扰物的关键是雄激素受体(AR)在酵母细胞中的表达。为此,本文用PCR方法从W303-1A酵母基因组扩增3-磷酸甘油醛脱氢酶(GPD)启动子,将其插入到pYestrp2载体的SwaⅠ和BamHⅠ酶切位点,将所构建成的载体命名为pGPD。将5′和3′端含有BamHⅠ和EcoRⅠ黏性末端V5抗原表位DNA,插入到pGPD相应的酶切位点,构建成pGPDV5载体。用PCR方法从pcDNA3.1/AR扩增2 723bp全长的AR开放性阅读框(ORF),将其插入到pGPDV5载体,构建成与V5抗原表位相融合的酵母表达载体pGPDV5/AR,将其转入W303-1A酵母细胞。用Western blot检测该酵母细胞提取液中的AR与V5相融合的表达蛋白,一抗选用小鼠源性V5单克隆抗体(IgG2a),二抗选用偶联有辣根过氧化物酶(HRP)的羊抗小鼠的多克隆抗体(IgG)。结果表明,与V5相融合的AR蛋白在酵母细胞中成功表达。更多还原

【Abstract】 When we try to establish the gene recombinant yeast cell to screen the androgenic endocrine disruptors,the key procedure is the androgen receptor(AR) expression in the yeast cell.For this purpose,we obtained the GPD(glyceraldehyde-3-phosphote dehydrogenase) promoter from the yeast genosome of W303-1A using PCR system and inserting it into SwaⅠand BamH Ⅰsites of pYestrp2.The new constructed vector was named pGPD.The V5 epitope tag DNA with a 5′-BamH Ⅰ and a 3′-EcoR Ⅰ sticky end was cloned into the corresponding site of the pGPD vector to yield the vector of pGPDV5.The 2 723 bp full length AR ORF amplified by PCR from pcDNA3.1/AR was fused to V5 epitope tag DNA in pGPDV5 to give the AR yeast expression vector of pGPDV5/AR.This fused vector was transformed into the yeast cell(W303-1A).Western blot was used to detect the V5 fused protein of AR,in the protocol of which the primary monoclonal antibody(IgG2a) of mouse anti-V5 and the polyclonal secondary antibody of goat anti-mouse(IgG) linked to horseradish peroxidase(HRP) were used to detect the specific protein in the given sample of the transformed yeast extract.The result showed that the fused protein of AR was expressed successfully in the yeast cell.更多还原