Biological properties of bacterial outer membrane vesicles surface-displaying PD-L1 nanobodies and their disrupting effects on PD-1/PD-L1 signaling pathway
10.13481/j.1671-587X.20250530
- VernacularTitle:表面表达PD-L1纳米抗体的细菌外膜囊泡的生物学特性及其对PD-1/PD-L1信号通路的阻断作用
- Author:
Zhimin LI
1
;
Mingge HUO
;
Longxue GUAN
;
Fanlin GU
;
Dandan LIANG
;
Zhuorui LIU
;
Guoqing WANG
;
Xingang GUAN
Author Information
1. 北华大学医学技术学院临床检验系,吉林吉林 132013;台州学院医学院基础医学系,浙江台州 318000
- Keywords:
Bacterial outer membrane vesicles;
Programmed cell death protein-1;
Programmed cell death ligand 1;
Nanobody;
Immune checkpoint blockade
- From:
Journal of Jilin University(Medicine Edition)
2025;51(5):1407-1414
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To prepare the bacterial outer membrane vesicles(OMV)that can express programmed death ligand 1(PD-L1)nanobody on surface,and to discuss its structural characteristics,cell compatibility,intracellular distribution,and its blocking effect on the programmed cell death protein-1(PD-1)/PD-L1 signaling axis.Methods:The pET28a-ClyA-PD-L1nb prokaryotic expression vector was constructed and transformed into Escherichia coli BL21(DE3)competent cells;the OMV was isolated from the BL21(DE3)monoclonal colonies transformed with the PD-L1nb expression vector by ultracentrifugation;the protein purification was performed using the histidine(His)tag;transmission electron microscope and nanoparticle size analyzer were used to analyze and identify the OMV;the OMV isolated from the BL21(DE3)monoclonal colonies transformed with the PD-L1nb expression vector was used as experimental group;the OMV isolated from the untransformed BL21(DE3)monoclonal colonies was used as control group;Western blotting method was used to detect the expression levels of ClyA-PD-L1nb fusion protein in the OMV in two groups;cell counting kit-8(CCK-8)assay was used to detect the activities of mouse macrophage RAW 264.7 cells,mouse triple-negative breast cancer 4T1 cells,and human embryonic kidney HEK293T cells after treated with OMV;fluorescence imaging technology was used to observe the tumor cell endocytosis of OMV;flow cytometry was used to detect the binding effect of OMV to the PD-L1 on surface of the tumor cells in PBS group,OMV-PD-L1nb group,and aPD-L1+OMV-PD-L1nb group.Results:The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)results showed that after induction of Escherichia coli,significantly thickened protein bands appeared near the predicted relative molecular mass(about 49 000),and after purification,no obvious impurity proteins existed in the lanes;the OMV-PD-L1nb with a size of about 120 nm was isolated by ultracentrifugation,and it presented a uniform spherical structure under transmission electron microscope;the Western blotting results showed that the specific band of ClyA-PD-L1nb was detected in the OMV in experimental group;the CCK-8 assay results showed that after treated with different concentrations of OMV,the viabilities of the RAW 264.7 cells,4T1 cells,and HEK293T cells were all close to 100％;the fluorescence imaging results showed that OMV-PD-L1nb was endocytosed by 4T1 cells and dispersed in the cytoplasm;compared with OMV-PD-L1nb group,the average fluorescence intensity in the cells in aPD-L1+OMV-PD-L1nb group was significantly decreased(P＜0.001).Conclusion:The OMV surface-displaying PD-L1nb,OMV-PD-L1nb,is successfully prepared and isolated;OMV-PD-L1nb shows good compatibility on mouse macrophage cells,tumor cells,and human embryonic kidney cells,can be endocytosed by tumor cells,and successfully blocks the PD-1/PD-L1 signaling pathway.
