表达红色荧光蛋白鸡白痢沙门菌的构建与应用

doi:10.16431/j.cnki.1671-7236.2025.08.040

摘要/Abstract

摘要： 【目的】构建表达红色荧光蛋白(mCherry)的鸡白痢沙门菌，并探究其在巨噬细胞中的定位示踪作用。【方法】将外源基因mCherry插入原核表达载体pUC19获得pUC19-mCherry质粒，以鸡白痢沙门菌基因组为模板，通过PCR扩增其内源性毒力基因virK启动子，并将其插入pUC19-mCherry质粒以启动mCherry，构建重组质粒PvirK-mCherry并转化鸡白痢沙门菌CVCC 533菌株，挑取单克隆后通过荧光正置显微镜检测荧光表达。将感染复数(MOI)为200的重组荧光菌株与小鼠单核巨噬细胞(RAW264.7)和禽巨噬细胞(HD11)共培养，经DAPI染色及流式细胞术检测两种巨噬细胞对荧光菌株的吞噬效果。【结果】 pUC19-mCherry质粒双酶切结果可见720 bp mCherry基因片段和2 600 bp的载体片段，转化大肠杆菌DH5α感受态细胞后可见红色荧光。重组质粒PvirK-mCherry测序结果显示，启动子成功插入pUC19-mCherry质粒，双酶切可见990 bp virK基因启动子片段和3 300 bp载体片段；荧光镜检转化PvirK-mCherry质粒的鸡白痢沙门菌可见红色荧光。巨噬细胞吞噬试验结果显示，经DAPI染色后荧光镜检可见红色荧光鸡白痢沙门菌菌株聚集在蓝色细胞核周围；流式细胞术检测结果显示，RAW264.7细胞吞噬效率为88.06%，HD11细胞吞噬效率为34.18%。【结论】本研究成功构建重组质粒PvirK-mCherry,转染鸡白痢沙门菌后表达红色荧光，且红色荧光菌株能被巨噬细胞吞噬。试验结果不仅有助于精确观察细菌在宿主细胞内的定位，还为研究沙门菌与宿主细胞的互作提供了经济有效的工具，对禽类健康和养殖业发展具有重要意义。

关键词: 鸡白痢沙门菌; 荧光菌株; 巨噬细胞; 吞噬

Abstract: 【Objective】 The purpose of this experiment was to construct Salmonella Pullorum expressing red fluorescent protein (mCherry) to explore its localization and tracing effect in macrophages. 【Method】 The exogenous gene mCherry was inserted into the prokaryotic expression vector pUC19 to obtain the pUC19-mCherry plasmid.Using the genome of Salmonella Pullorum as a template,the endogenous virulence gene virK promoter was amplified by PCR and inserted into the pUC19-mCherry plasmid to drive the expression of mCherry.The recombinant plasmid PvirK-mCherry was constructed and transformed into the Salmonella Pullorum CVCC 533.Single colonies were selected,and fluorescence expression was confirmed using upright fluorescence microscopy.The recombinant fluorescent strain with an multiplicity of infection (MOI) of 200 was co-cultured with RAW264.7 and HD11 cells.The phagocytic effects of the two types of macrophages on the fluorescent strain were detected by DAPI staining and flow cytometry. 【Result】 The double enzyme digestion results of the pUC19-mCherry plasmid showed a 720 bp mCherry gene fragment and a 2 600 bp vector fragment.Red fluorescence was observed after transforming the Escherichia coli DH5α competent cells.The sequencing results of the recombinant plasmid PvirK-mCherry showed that the promoter was successfully inserted into the pUC19-mCherry plasmid.Double enzyme digestion revealed a 990 bp virK gene promoter fragment and a 3 300 bp vector fragment.Fluorescence microscopy of Salmonella Pullorum transformed with PvirK-mCherry plasmid showed red fluorescence.The results of the macrophage phagocytosis test showed that after DAPI staining,fluorescence microscopy revealed that the red fluorescent Salmonella Pullorum strains were aggregated around the blue nuclei.Flow cytometry results showed that the phagocytic efficiency of RAW264.7 cells was 88.06%,and that of HD11 cells was 34.18%. 【Conclusion】 This study successfully constructed the plasmid PvirK-mCherry，after transfection with Salmonella Pullorum,it expressed red fluorescence,and the red fluorescent strain could be phagocytosed by macrophages.This results not only helped to precisely observe the localization of bacteria within host cells,but also provided an economical and effective tool for studying the interaction between Salmonella and host cells,which was of great significance to the health of poultry and the development of breeding industry.

Key words: Salmonella Pullorum; fluorescent bacteria; macrophage; phagocytosis

中图分类号:

S852.61

刘子敬, 孙佳, 张丽, 罗成龙. 表达红色荧光蛋白鸡白痢沙门菌的构建与应用[J]. 中国畜牧兽医, 2025, 52(8): 3918-3926.

LIU Zijing, SUN Jia, ZHANG Li, LUO Chenglong. Construction and Application of Salmonella Pullorum Expressing Red Fluorescent Protein[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3918-3926.

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