本文選題：熒光素酶　切入點(diǎn)：生物發(fā)光法　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2006年碩士論文

【摘要】：傳統(tǒng)的消毒評(píng)價(jià)方法是將消毒后樣本接種到培養(yǎng)基中培養(yǎng)后觀察細(xì)菌生長(zhǎng)情況，從而定性或定量評(píng)價(jià)消毒效果。此方法費(fèi)時(shí)費(fèi)力，往往影響后續(xù)工作的開(kāi)展。因此，尋找快速的評(píng)價(jià)方法一直是研究人員工作的方向。本課題擬通過(guò)熒光素酶(luciferase)催化的生物發(fā)光反應(yīng)，達(dá)到快速評(píng)價(jià)消毒效果的目的。 熒光素酶在基因工程實(shí)驗(yàn)中有廣泛應(yīng)用，特別是作為報(bào)告基因，對(duì)研究基因活性、基因定位、空間表達(dá)等有重要意義。熒光素酶可催化如下生化反應(yīng)： (?)在此酶促反應(yīng)中，保證反應(yīng)物過(guò)量，則反應(yīng)產(chǎn)生的熒光量就與熒光素酶活性成比例，在一定條件下即可認(rèn)為與熒光素酶的量成比例。 本課題首先改構(gòu)大腸桿菌(8099)，使其表達(dá)熒光素酶。通過(guò)裂解細(xì)菌，加入過(guò)量熒光素底物(luciferin)，利用細(xì)菌細(xì)胞中的ATP，發(fā)生上述酶促反應(yīng)產(chǎn)生熒光。然后利用熒光光度計(jì)對(duì)反應(yīng)產(chǎn)生的熒光進(jìn)行定量，從而定量熒光素酶，進(jìn)而反映細(xì)菌含量，達(dá)到評(píng)價(jià)消毒效果的目的。主要結(jié)果總結(jié)如下： 1 大腸桿菌(8099)的重組與鑒定 (1) 用CaCl_2制備大腸桿菌(8099)的感受態(tài)細(xì)胞，用含有熒光素酶基因(luc~+)的pGL3-Control質(zhì)粒轉(zhuǎn)化感受態(tài)細(xì)胞，構(gòu)建表達(dá)熒光素酶的重組大腸桿菌。挑選形態(tài)和大小典型的菌落，用抗生素陽(yáng)性培養(yǎng)基培養(yǎng)增殖后低溫冷凍保存。 (2) 從重組大腸桿菌中提取質(zhì)粒，，進(jìn)行瓊脂糖凝膠電泳分析，證明有與pGL3-Control質(zhì)粒相同大小的DNA條帶產(chǎn)生。進(jìn)一步進(jìn)行雙酶切分析，PCR擴(kuò)增，測(cè)序分析，結(jié)果均證明pGL3-Control質(zhì)粒轉(zhuǎn)化大腸桿菌(8099)成功，所制備的
[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).
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R187

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本文編號(hào)：1715316