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荧光标记类鼻疽菌的构建及其与宿主细胞相互作用的应用研究
Construction of fluorescence-labeled
Burkholderia pseudomallei and its interaction with host cells- 陆军军医大学学报 2022年44卷第20期 页码：2105-2112
- 作者机构：
400038 重庆, 陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室
- 作者简介：
[毛旭虎，E-mail: maoxh2012@hotmail.comMAO Xuhu, E-mail: maoxh2012@hotmail.com
- 基金信息：国家自然科学基金面上项目(82272350);陆军军医大学科技创新能力提升专项(2021XJS07)
DOI：10.16016/j.2097-0927.202204009
中图分类号： Q935;R-331;R378.99收稿：2022-04-01，
修回：2022-06-24，
纸质出版：2022-10-30
- 稿件说明：
移动端阅览
陈建高, 饶承龙, 刘文正, 等. 荧光标记类鼻疽菌的构建及其与宿主细胞相互作用的应用研究[J]. 陆军军医大学学报, 2022,44(20):2105-2112.
Jiangao CHEN, Chenglong RAO, Wenzheng LIU, et al. Construction of fluorescence-labeled
Burkholderia pseudomallei and its interaction with host cells[J]. Journal of Army Medical University, 2022, 44(20): 2105-2112.陈建高, 饶承龙, 刘文正, 等. 荧光标记类鼻疽菌的构建及其与宿主细胞相互作用的应用研究[J]. 陆军军医大学学报, 2022,44(20):2105-2112. DOI： 10.16016/j.2097-0927.202204009.
Jiangao CHEN, Chenglong RAO, Wenzheng LIU, et al. Construction of fluorescence-labeled
Burkholderia pseudomallei and its interaction with host cells[J]. Journal of Army Medical University, 2022, 44(20): 2105-2112. DOI： 10.16016/j.2097-0927.202204009.
摘要
目的
2
构建类鼻疽菌(
Burkholderia pseudomallei
，
B. pseudomallei
)的荧光示踪载体，获得自发荧光的类鼻疽菌株，并对其与宿主相互作用的感染过程进行初步应用研究。
方法
2
采用基因重组技术，将绿色、红色和蓝色荧光蛋白(green/red/blue fluorescent protein，GFP/RFP/BFP)基因分别重组到原核表达载体pUCP28T上，构建类鼻疽菌组成型荧光表达载体，并通过体外细菌培养观察外源性荧光蛋白的表达对细菌生长能力的影响，以及荧光标记细菌遗传稳定性的检测。通过炎症因子IL-8的检测评价荧光标记细菌对宿主细胞感染能力的变化。利用活细胞工作站观察类鼻疽菌被宿主细胞自噬体包裹的动态过程。
结果
2
构建了三种荧光标记的类鼻疽菌示踪载体，并且能在体外传代20代内稳定表达，且外源性荧光蛋白的表达不影响类鼻疽菌的生长和对宿主细胞的感染。同时，利用高分辨率活细胞成像系统观察到红色荧光标记的类鼻疽菌与宿主细胞GFP-LC3共定位的动态变化过程。
结论
2
本文成功构建了三种荧光标记的类鼻疽菌，以期为类鼻疽菌致病机制的研究、抗菌药物的筛选提供重要实用工具。
Abstract
Objective
2
To construct the fluorescence-labeled
Burkholderia pseudomallei
(
B. pseudomallei
) to study the pathogenesis of the interaction between the bacteria and host cells.
Methods
2
Gene recombination technique was carried out to recombine the green fluorescent protein (GFP)
red fluorescent protein (RFP) and blue fluorescent protein (BFP) genes into the prokaryotic expression vector pUCP28T
respectively
in order to obtain the fluorescence-labeled
B. pseudomallei
. Furthermore
the biological characteristics of fluorescence-labeled bacteria were detected in bacterial culture
in vitro
and inflammatory factor IL-8 was detected to evaluate the changes in the ability of fluorescence-labeled bacteria infecting host cells. Then
the dynamic process of fluorescent-labeled
B. pseudomallei
being enveloped by host cell autophagosome was observed with aid of living cell workstation.
Results
2
Three fluorescence-labeled
B. pseudomallei
were constructed respectively
and were stably expressed in 20 passages
in vitro
. The expression of exogenous fluorescent proteins did not affect the growth of
B. pseudomallei
and infection of host cells. Meanwhile
the dynamic changes of co-localization between RFP-labeled
B. pseudomallei
and GFP-LC3 were observed by live cell imaging system.
Conclusion
2
Three different fluorescence-labeled
B. pseudomallei
are successfully constructed. And they are important practical tools for the pathogenesis of the bacteria and the screening of antimicrobial agents of melioidosis.
关键词
Keywords
references
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