Application of biotin-streptavidin labeling technology for pathogen tracing, bacteria-containing phagosome purification, and immune evasion mechanisms of intracellular pathogens

Zheng Senquan; Hu Zhiqiang; Tang Zhiheng; Qiao Jianpeng; Mo Shunmin; Wen Yuan; Rao Chenglong; Yan Jingmin; Mao Xuhu; Li Qian

doi:10.16016/j.2097-0927.202605066

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Application of biotin-streptavidin labeling technology for pathogen tracing, bacteria-containing phagosome purification, and immune evasion mechanisms of intracellular pathogens

Basic Medicine | 更新时间：2026-09-16

- Application of biotin-streptavidin labeling technology for pathogen tracing, bacteria-containing phagosome purification, and immune evasion mechanisms of intracellular pathogens
- Journal of Army Medical University Vol. 48, Issue 17, Pages: 2494-2505(2026)
- 作者机构：
  
  1.陆军军医大学（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，重庆
  2.陆军军医大学（第三军医大学）：，创伤与化学中毒全国重点实验室，重庆
- 作者简介：
  
  Li Qian，E-mail： liqianjane@163.com
- 基金信息：
  
  the National Natural Science Foundation for Young Scholars of China(32300161);the Project for New Chongqing Young Innovative Talents(CSTB2024NSCQ-QCXMX0055)
- DOI：10.16016/j.2097-0927.202605066
  CLC： R372;R378;R446.5
- Received：25 May 2026，
  
  Revised：2026-07-03，
  
  Accepted：13 July 2026，
  
  Published：15 September 2026
- 稿件说明：
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郑森泉，胡治强，唐致恒，等.生物素-链霉亲和素标记技术实现病原菌示踪与含菌吞噬体纯化并用于胞内病原菌免疫逃逸机制解析［J］.陆军军医大学学报，2026，48（17）：2494-2505.DOI：10.16016/j.2097-0927.202605066.

Zheng S Q，Hu Z Q，Tang Z H，et al.Application of biotin-streptavidin labeling technology for pathogen tracing， bacteria-containing phagosome purification， and immune evasion mechanisms of intracellular pathogens［J］.J Army Med Univ，2026，48（17）：2494-2505.DOI：10.16016/j.2097-0927.202605066.
郑森泉，胡治强，唐致恒，等.生物素-链霉亲和素标记技术实现病原菌示踪与含菌吞噬体纯化并用于胞内病原菌免疫逃逸机制解析［J］.陆军军医大学学报，2026，48（17）：2494-2505.DOI：10.16016/j.2097-0927.202605066. DOI：

Zheng S Q，Hu Z Q，Tang Z H，et al.Application of biotin-streptavidin labeling technology for pathogen tracing， bacteria-containing phagosome purification， and immune evasion mechanisms of intracellular pathogens［J］.J Army Med Univ，2026，48（17）：2494-2505.DOI：10.16016/j.2097-0927.202605066. DOI：

摘要

目的

基于生物素-链霉亲和素系统（biotin-streptavidin system，BSA）的特异性结合及信号放大效应，假设构建的生物素-链霉亲和素标记技术可在不影响病原菌生物学特性及感染能力前提下，实现病原菌的示踪与含菌吞噬体的分离，并初步用于解析胞内病原菌免疫逃逸的分子机制。本研究旨在验证该假设，为病原菌-宿主互作研究提供技术支撑。

方法

①以类鼻疽伯克霍尔德菌（

Burkholderia pseudomallei

，

B.p

）、鼠伤寒沙门氏菌（

Salmonella typhimurium

，

S.t

）和福氏志贺氏菌（

Shigella flexneri

，

S.f

）为研究对象，以J774A.1巨噬细胞构建细菌-宿主细胞感染互作模型，设置生物素-链霉亲和素标记组与未标记组。通过NHS-生物素与细菌膜氨基共价结合，偶联链霉亲和素-FITC构建示踪体系，或偶联链霉亲和素磁珠用于磁分选分离。②采用免疫荧光实验检测标记效率及胞内分布，并在感染复数（multiplicity of infection，MOI）=10的条件下分别感染1、3、5 h，比较细胞感染率、单细胞胞内菌载量及胞内存活情况。③ELISA检测细胞上清IL-1β、IL-12和TNF-α分泌水平。④进一步以磁珠标记菌感染细胞，于1、2 h或3、5 h磁分选分离含菌吞噬体，Western blotting检测吞噬体标志物Rab5、Rab7、LAMP-2及胞质内参GAPDH表达以鉴定分离纯度。⑤应用该技术对

B.p

野生型与

bopA

基因敲除株（Δ

bopA

株）的含菌吞噬体进行蛋白质谱分析。

结果

该标记体系对

B.p

、

S.t

、

S.f

的标记效率均达90%以上，且荧光信号均一、无非特异性吸附；标记组与未标记组在感染1、3、5 h后的细胞感染率、单细胞胞内菌载量、胞内存活趋势及胞内分布模式等差异无统计学意义（

0.05）；ELISA结果显示，标记组与未标记组IL-1β、IL-12和TNF-α分泌水平差异无统计学意义（

0.05）。磁分选获得的含菌吞噬体中，Rab5、Rab7和LAMP-2蛋白表达显著高于胞质组分（

0.05），胞质GAPDH在吞噬体中污染极低。蛋白质组学分析显示，与

B.p

野生型相比，Δ

bopA

株含菌吞噬体中VAMP7、EEA1、RABEP1和RAB3GAP2等囊泡转运

Abstract

Objective

Based on the specific binding and signal amplification effects of the biotin-streptavidin system

a hypothesis is proposed that the constructed biotin-streptavidin labeling technology can achieve tracing of pathogenic bacteria and isolation of bacteria-containing phagosomes without affecting the biological characteristics and infectivity of pathogenic bacteria

and can be preliminarily applied to elucidate the molecular mechanisms of immune evasion by intracellular pathogenic bacteria. This study aims to verify this hypothesis and provide technical support for research on pathogen-host interactions.

Methods

①

Burkholderia pseudomallei

(

B.p

)

Salmonella typhimurium

(

S.t

) and

Shigella flexneri

(

S.f

) were used as research objects

and J774A.1 macrophages were employed to construct a bacteria-host cell infection interaction model. Biotin-streptavidin-labeled and unlabeled groups were established. Through covalent conjugation of NHS-biotin to amino groups on the bacterial membrane

a tracing system was constructed by coupling with streptavidin-FITC

or with streptavidin magnetic beads for magnetic sorting and isolation. ② Immunofluorescence assay was utilized to detect labeling efficiency and intracellular distribution. After the cells were infected at a multiplicity of infection (MOI) of 10 for 1

and 5 h respectively

cell infection rates

intracellular bacterial load per cell

and intracellular survival were compared. ③ ELISA was applied to detect the secretion levels of IL-1β

IL-12 and TNF-α in cell supernatants. ④ Furthermore

the cells were infected with magnetic bead-labeled bacteria

and bacteria-containing

phagosomes were isolated by magnetic separation at 1 and 2 h or 3 and 5 h. Western blotting was performed to detect the expression of phagosome markers Rab5

Rab7

and LAMP-2

as well as the cytosolic internal reference GAPDH

to identify separation purity. ⑤ This technology was also applied to perform proteomic analysis of bacteria-containing phagosomes from wild-type

B.p

and

bopA

knockout strain (

ΔbopA

Results

The labeling efficiency of this labeling system for

B.p

S.t

and

S.f

all reached above 90%

with uniform fluorescence signals and no non-specific adsorption. There were no significant differences between the labeled group and the unlabeled group in terms of cell infection rate

intracellular bacterial load per cell

intracellular survival trend and intracellular distribution patterns at 1

3 and 5 h after infection (

0.05). ELISA showed no statistically significant differences in the secretion levels of IL-1β

IL-12 and TNF-α between the 2 groups (

0.05). In bacteria-containing phagosomes obtained by magnetic separation

the protein levels of Rab5

Rab7

and LAMP-2 were significantly higher than those in the cytosolic fractions (

0.05)

the level of cytoplasmic GAPDH contamination in phagosomes was extremely low. Proteomic analysis revealed that host proteins related to vesicle transport

including VAMP7

EEA1

RABEP1

and RAB3GAP2

were significantly upregulated in bacteria-containing phagosomes of the Δ

bopA

strain compared with the wild-type strain.

Conclusion

The biotin-streptavidin labeling and magnetic sorting system established in this study does not change the biological characteristics of pathogens

host cell infectivity

or the secretion of inflammatory factors. This system can accomplish fluorescent tracing of pathogenic bacteria and isolate high-purity bacteria-containing phagosomes

and be applied to preliminary elucidation of the potential mechanisms by which pathogen-related effector proteins mediate host defense evasion research.

关键词

Keywords

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