稳定表达荧光素酶的产酸克雷伯菌在评估不同灭菌方法中的应用

doi:10.3877/cma.j.issn.1674-1358.2020.04.002

中华实验和临床感染病杂志(电子版) ›› 2020, Vol. 14 ›› Issue (04) : 272 -277. doi: 10.3877/cma.j.issn.1674-1358.2020.04.002

所属专题：专题评论；文献；

论著

稳定表达荧光素酶的产酸克雷伯菌在评估不同灭菌方法中的应用

滑明溪¹, 袁临天¹, 李昂¹^,^†(

)

^1. 100015　北京，首都医科大学附属北京地坛医院传染病研究所

收稿日期:2019-10-28 出版日期:2020-08-15
通信作者: 李昂
基金资助:
国家自然科学基金(No. 81902025); 北京市医院管理局扬帆计划重点医学专业(No. ZYLX2018-02-感染性疾病重症医学); 北京市博士后科研活动经费(No. ZZ2019-019); 北京市自然科学基金(No. 7184218)

Application of Klebsiella oxytoca with stable expression of luciferase in evaluating different sterilization methods

Mingxi Hua¹, Liutian Yuan¹, Ang Li¹^,^†()

^1. Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China

Received:2019-10-28 Published:2020-08-15
Corresponding author: Ang Li
About author:
Corresponding author: Li Ang, Email: liang@ccmu.edu.cn

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目的

构建稳定表达荧光素酶基因（lux）的产酸克雷伯菌，旨在更直观地观察产酸克雷伯菌（K. oxytoca）在宿主体内的分布以及评估不同杀菌方法的效果。

方法

扩增pBAV1k-T5-Lux质粒的荧光素酶基因簇（Lux A/B/C/D/E），构建含有Lux A/B/C/D/E基因簇的pBBR1质粒（pBBR1-lux），并获得能够表达荧光素基因基团的大肠埃希菌（E. coli-pBBR1-lux）。将pBBR1-lux质粒电转化至产酸克雷伯菌感受态细胞，经荧光强度鉴定及菌株的3次传代，筛选能够稳定表达lux基因的产酸克雷伯菌（K. oxytoca-pBBR1-lux）。

结果

连接成功的环状质粒产物命名为pBBR1-lux。与大肠埃希菌对照株相比，含E. coli-pBBR1-lux的Luminesence荧光信号值显著升高[15 345（14 676，18 654） vs. 63（60，82）]，差异具有统计学意义（t = 21.14、P = 0.035）。K. oxytoca-pBBR1-lux的Luminesence荧光信号值[399 303（265 245，617 192）]显著高于产酸克雷伯菌对照菌株[83（63.5，86.75）]，差异具有统计学意义（t = 7.07、P = 0.014）。E. coli-pBBR1-lux和K. oxytoca-pBBR1-lux均能够在Veritas微孔板光度计和小动物成像仪上检测到Luminesence荧光信号。将所得菌株按照1︰10稀释6个梯度，荧光值检测结果显示，Luminesence值随菌落浓度降低而下降。多次传代后pBBR1-lux能够在产酸克雷伯菌中稳定表达荧光素酶，不同理化杀菌方法对产酸克雷伯菌杀菌效果不同，紫外线和84消毒液（10%）是最有效的杀灭产酸克雷伯菌方法。

结论

本研究获得了可被微孔板光度计和小动物成像仪检测到的稳定表达荧光素酶的K. oxytoca-pBBR1-lux。

关键词: 产酸克雷伯菌, 生物发光, 荧光素酶基因

Objective

To construct a Klebsiella oxytoca (K. oxytoca)-pBBR1-lux strain that stably express luciferase (lux) gene, and to investigate the distribution of K. oxytoca in vivo and to evaluate the effect of different methods of sterilization.

Methods

The luciferase gene cluster (Lux A/B/C/D/E) of pBAV1k-T5-Lux plasmid was amplified, and the pBBR1 plasmid (pBBR1-lux) containing Lux A/B/C/D/E gene cluster was constructed; while Escherichia coli which expressed fluorescein gene group (E. coli-pBBR1-lux) was obtained. The expression of luciferase in E. coli-pBBR1-lux and K. oxytoca-pBBR1-lux were tested using luminescence microplate spectrophotometer. The pBBR1-lux plasmid was electroconverted to the receptive cells of K. oxytoca; after fluorescence intensity identification and three passages of the strain, K. oxytoca-pBBR1-lux which stably express lux genes were screened. Conjugated circular plasmid product was named pBBR1-lux. The value of Luminesence fluorescence signal of E. coli-pBBR1-lux increased significantly compared with E. coli control strain [15 345 (14 676, 18 654) vs. 63 (60, 82)], with significant difference (t = 21.14, P = 0.035). The value of Luminesence fluorescence signal of K. oxytoca-pBBR1-lux [399 303 (265 245, 617 192)] was significantly higher than that of the control strain K. oxytoca [83 (63.5, 86.75)], with significant difference (t = 7.07, P = 0.014). E. coli-pBBR1-lux and K. oxytoca-pBBR1-lux were both able to detect fluorescence signals on Veritas microplate photometer and small animal imager. According to 1︰10 dilution of 6 gradients, the fluorescence detection results showed that the Luminesence value decreased with the decreasing of colony concentration. After repeated passage pBBR1-lux luciferase could be stably expressed in K. oxytoca. Different physicochemical methods had different bactericidal effect on K. oxytoca-producing bacteria. Ultraviolet ray and 84 disinfectant (10%) were the most effective methods to inactivate K. oxytoca-producing bacteria.

Key words: Klebsiella oxytocca, Bioluminescence, Luciferase gene cassette

图1 PBBR1-LUX A/B/C/D/E质粒构建过程示意图

图2 E. coli-pBBR1-lux能够稳定表达荧光素酶

图3 K. oxytoca-pBBR1-lux稳定表达荧光素酶

图4 传代至第十代后K. oxytoca-pBBR1-lux稳定表达荧光素酶

图5 不同浓度K. oxytoca-pBBR1-lux的Luminescence荧光强度

图6 不同理化杀菌方法对K. oxytoca-pBBR1-lux的杀菌效果

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