荧光素酶生物发光法快速评价消毒效果的初步研究

发布时间：2018-04-05 15:14

本文选题：荧光素酶　切入点：生物发光法　出处：《中国人民解放军军事医学科学院》2006年硕士论文

【摘要】：传统的消毒评价方法是将消毒后样本接种到培养基中培养后观察细菌生长情况，从而定性或定量评价消毒效果。此方法费时费力，往往影响后续工作的开展。因此，寻找快速的评价方法一直是研究人员工作的方向。本课题拟通过荧光素酶(luciferase)催化的生物发光反应，达到快速评价消毒效果的目的。荧光素酶在基因工程实验中有广泛应用，特别是作为报告基因，对研究基因活性、基因定位、空间表达等有重要意义。荧光素酶可催化如下生化反应： (?)在此酶促反应中，保证反应物过量，则反应产生的荧光量就与荧光素酶活性成比例，在一定条件下即可认为与荧光素酶的量成比例。本课题首先改构大肠杆菌(8099)，使其表达荧光素酶。通过裂解细菌，加入过量荧光素底物(luciferin)，利用细菌细胞中的ATP，发生上述酶促反应产生荧光。然后利用荧光光度计对反应产生的荧光进行定量，从而定量荧光素酶，进而反映细菌含量，达到评价消毒效果的目的。主要结果总结如下： 1 大肠杆菌(8099)的重组与鉴定 (1) 用CaCl_2制备大肠杆菌(8099)的感受态细胞，用含有荧光素酶基因(luc~+)的pGL3-Control质粒转化感受态细胞，构建表达荧光素酶的重组大肠杆菌。挑选形态和大小典型的菌落，用抗生素阳性培养基培养增殖后低温冷冻保存。 (2) 从重组大肠杆菌中提取质粒，，进行琼脂糖凝胶电泳分析，证明有与pGL3-Control质粒相同大小的DNA条带产生。进一步进行双酶切分析，PCR扩增，测序分析，结果均证明pGL3-Control质粒转化大肠杆菌(8099)成功，所制备的
[Abstract]:The traditional method of disinfection evaluation is to observe the growth of bacteria after inoculating the samples into culture medium, so as to evaluate the disinfection effect qualitatively or quantitatively.This method is time-consuming and laborious, and often affects the development of follow-up work.Therefore, the search for rapid evaluation methods has been the direction of researchers.The aim of this paper is to evaluate the disinfection effect quickly by the bioluminescence reaction catalyzed by luciferase.Luciferase is widely used in gene engineering experiments, especially as a reporter gene, which is of great significance to the study of gene activity, gene location and spatial expression.Luciferase catalyzes the following biochemical reactions:)In this enzymatic reaction, the amount of fluorescence produced by the reaction is proportional to the activity of luciferase, and the amount of luciferase can be considered as proportional to the amount of luciferase.In this study, Escherichia coli 8099 was first constructed to express luciferase.The above enzymatic reaction was produced by pyrolysis of bacteria and addition of excessive fluorescein substrate, luciferin.Then fluorescent photometer was used to quantify the fluorescence produced by the reaction, thus quantifying luciferase, and then reflecting the bacterial content, thus achieving the purpose of evaluating the disinfection effect.The main findings are summarized as follows:Recombination and identification of Escherichia coli 8099)1) Recombinant E. coli cells expressing luciferase were constructed by using pGL3-Control plasmid containing luciferase gene.The typical colonies were selected and cultured in antibiotic positive medium after cryopreservation.2) the plasmids were extracted from recombinant Escherichia coli and analyzed by agarose gel electrophoresis. DNA bands of the same size as pGL3-Control plasmids were produced.The results showed that the pGL3-Control plasmid was successfully transformed into Escherichia coli 8099).
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R187

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