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

doi:10.13481/j.1671-587X.20250530

Abstract

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.

Key words: Bacterial outer membrane vesicles, Programmed cell death protein-1, Programmed cell death ligand 1, Nanobody, Immune checkpoint blockade

CLC Number:

R737.95

Zhimin LI,Mingge HUO,Longxue GUAN,Fanlin GU,Dandan LIANG,Zhuorui LIU,Guoqing WANG,Xingang GUAN. Biological properties of bacterial outer membrane vesicles surface-displaying PD-L1 nanobodies and their disrupting effects on PD-1/PD-L1 signaling pathway[J].Journal of Jilin University(Medicine Edition), 2025, 51(5): 1407-1414.

Figures/Tables 6

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Biological properties of bacterial outer membrane vesicles surface-displaying PD-L1 nanobodies and their disrupting effects on PD-1/PD-L1 signaling pathway

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