| 基于硒蛋白生物合成机制的硒生物检测器 |
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| 引用本文： | 邱洋松,孙婧,孙爱,邬丹妮,张冰倩,马建辉,徐丽珊,孙梅好.基于硒蛋白生物合成机制的硒生物检测器[J].安徽农业科学,2013,41(11):4729-4734. |
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| 作者姓名： | 邱洋松 孙婧 孙爱 邬丹妮 张冰倩 马建辉 徐丽珊 孙梅好 |
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| 作者单位： | 浙江师范大学化学与生命科学学院,浙江金华,321004;浙江师范大学化学与生命科学学院,浙江金华,321004;浙江师范大学化学与生命科学学院,浙江金华,321004;浙江师范大学化学与生命科学学院,浙江金华,321004;浙江师范大学化学与生命科学学院,浙江金华,321004;浙江师范大学化学与生命科学学院,浙江金华,321004;浙江师范大学化学与生命科学学院,浙江金华,321004;浙江师范大学化学与生命科学学院,浙江金华,321004 |
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| 基金项目： | 国家自然科学基金项目，浙江省自然科学基金项目，教育部留学归国科研启动基金 |
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| 摘 要： | 硒是许多生物的必需微量元素,与人类健康关系密切。硒在自然界中分布的不平衡导致不同地区的硒含量差异较大,易引起硒缺乏或者中毒。原核生物的硒代半胱氨酸(Selenocysteine,Sec)及硒蛋白的生物合成途径已较为清楚,此为构建硒酸盐生物检测器,进而补充目前常用硒含量测定方法提供了可能。该研究利用原核载体构建硒代半胱氨酸插入所需茎环结构(Selenocysteine insertion se-quence,SECIS)及其突变载体,通过氨苄青霉素筛选获得高效、高准确率的SECIS,并以此构建GFP超表达载体pSEGFP。通过pSEGFP和pSelABC共转化大肠杆菌BL21(DE3),并进行IPTG诱导表达,测定分析发现胞质蛋白的荧光强度与培养基硒酸盐浓度在一定范围内具有较好的线性关系。此结果表明,试验已初步构建了大肠杆菌硒生物检测器,为利用此方法进一步分析测定环境及生物样品中的硒含量奠定了基础。
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| 关 键 词： | 硒蛋白 硒代半胱氨酸 生物检测器 茎环结构 |
Biological Selenium Detector Based on Mechanism of Selenoprotein Biosynthesis in Prokaryotes |
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| Institution: | QIU Yang-song et al(School of Chemistry and Life Science,Zhejiang Normal University,Jinhua,Zhejiang 321004) |
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| Abstract: | Selenium,one trace essential elements for many organisms,is crucial to human health.Imbalance of selenium distribution in nature would result the marked variation in local environmental selenium levels which would lead to the selenosis or selenium deficiency.The elucidation of pathways for biosynthesis of selenocysteine（Sec） and selenoproteins in prokaryotes made it possible for the construction of biological selenium detector which would supplement the current selenium measuring methods.Selenocysteine insertion sequence（SECIS） is necessary for the Sec insertion,and its preceding nucleotides had been found influenced the efficiency for Sec insertion.By varying the preceding nucleotides,high accurate and efficient SECIS were screened based on the effects on translation of β-lactmase which activity was easily detected by bacterial anti-ampicillin activity.The best SECIS would be further used to direct translation of GFP reporter gene together with SelABC to quantitatively report selenate concentration.After induction with IPTG and incubation with selenate,fluorescence intensity of the cell lysate was found linearly correlated with selenate concentrations very well in our testing selenate concentrations.This linear correlation indicated that the constructed biological detector is successful so far and laid foundation for measuring selenate contents of environmental and biological samples with this detector in the future. |
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| Keywords: | Selenoprotein Selenocysteine Biological detector Selenocysteine insertion sequence |
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