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

吉林大学学报(医学版) ›› 2025, Vol. 51 ›› Issue (5): 1407-1414.doi: 10.13481/j.1671-587X.20250530

• 方法学 • 上一篇

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

李智敏^1,²,霍明歌¹,管龙雪¹,顾凡林¹,梁丹丹¹,刘倬睿¹,王国庆¹(),关新刚²()

^1.北华大学医学技术学院临床检验系，吉林吉林 132013
^2.台州学院医学院基础医学系，浙江台州 318000

收稿日期:2024-09-30 接受日期:2024-12-02 出版日期:2025-09-28 发布日期:2025-11-05
通讯作者: 王国庆,关新刚 E-mail:wgq@beihua.edu.cn;guanxg@ciac.ac.cn
作者简介:李智敏（1998-），男，浙江省台州市人，在读硕士研究生，主要从事工程化细胞膜功能方面的研究。
基金资助:
吉林省教育厅科研项目(JJKH20180370KJ);浙江省科技厅自然科学基金探索一般项目(LY23C100001);浙江省台州市科技局科技发展计划项目(22gya03)

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

Zhimin LI^1,²,Mingge HUO¹,Longxue GUAN¹,Fanlin GU¹,Dandan LIANG¹,Zhuorui LIU¹,Guoqing WANG¹(),Xingang GUAN²()

^1.Department of Clinical Laboratory Diagnostics，School of Medical Technology，Beihua University，Jilin 132013，China
^2.Department of Basic Medicine，School of Medical Science，Taizhou University，Taizhou 318000，China

Received:2024-09-30 Accepted:2024-12-02 Online:2025-09-28 Published:2025-11-05
Contact: Guoqing WANG,Xingang GUAN E-mail:wgq@beihua.edu.cn;guanxg@ciac.ac.cn

摘要/Abstract

摘要：

目的制备表面表达程序性细胞死亡配体1（PD-L1）纳米抗体的细菌外膜囊泡（OMV），探讨其结构特性、细胞相容性、细胞内分布及其对程序性细胞死亡受体1（PD-1）/PD-L1信号轴的阻断效果。方法构建pET28a-ClyA-PD-L1nb原核表达载体，转化至大肠杆菌BL21（DE3）感受态细胞，通过超速离心法从转化PD-L1nb表达载体的BL21（DE3）单克隆菌落中分离OMV，利用组氨酸（His）标签进行蛋白纯化。采用透射电子显微镜和纳米粒径分析仪对OMV进行分析鉴定。将转化PD-L1nb表达载体的BL21（DE3）单克隆菌落所分离的OMV作为实验组，以未转化的BL21（DE3）单克隆菌落所分离的OMV作为对照组，采用Western blotting 法检测2组OMV中ClyA-PD-L1nb融合蛋白表达情况，采用细胞计数试剂盒8（CCK-8）法检测OMV处理后小鼠巨噬细胞RAW264.7、小鼠三阴性乳腺癌细胞4T1和人胚胎肾细胞HEK293T活性，应用荧光成像技术观察OMV的肿瘤细胞内吞情况，采用流式细胞术检测PBS组、OMV-PD-L1nb组和aPD-L1＋OMV-PD-L1nb组OMV与肿瘤细胞表面PD-L1的结合效果。结果十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），大肠杆菌诱导后在预计相对分子质量（约49 000）附近出现明显加粗的蛋白条带，ClyA-PD-L1nb蛋白成功表达，纯化后泳道中无明显杂蛋白存在。利用超速离心法分离得到尺寸约120 nm的OMV-PD-L1nb，透射电子显微镜下呈现为尺寸均一的圆球形结构。Western blotting法，实验组OMV中检测到ClyA-PD-L1nb特异性条带。CCK-8法，经不同浓度OMV处理后，RAW 264.7细胞、4T1细胞和HEK293T细胞活性均接近100%。荧光成像，OMV-PD-L1nb被4T1细胞内吞后分散在细胞质中。与OMV-PD-L1nb组比较，aPD-L1＋OMV-PD-L1nb组细胞中平均荧光强度明显降低（P<0.001）。结论成功制备并分离了表面表达PD-L1nb的OMV即OMV-PD-L1nb，其在小鼠巨噬细胞、肿瘤细胞和人胚胎肾细胞上显示出良好的相容性，可被肿瘤细胞内吞并成功阻断PD-1/PD-L1信号通路。

关键词: 细菌外膜囊泡, 程序性细胞死亡受体1, 程序性细胞死亡配体1, 纳米抗体, 免疫检查点阻断

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

中图分类号:

R737.95

李智敏,霍明歌,管龙雪,顾凡林,梁丹丹,刘倬睿,王国庆,关新刚. 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[J]. 吉林大学学报(医学版), 2025, 51(5): 1407-1414.

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.

图/表 6

图1

图2

图3

图4

图5

图6

参考文献 24

[1]	PATWEKAR M， SEHAR N， PATWEKAR F， et al. Novel immune checkpoint targets： a promising therapy for cancer treatments［J］. Int Immunopharmacol， 2024， 126： 111186.
[2]	SHARMA P， GOSWAMI S， RAYCHAUDHURI D， et al. Immune checkpoint therapy-current perspectives and future directions［J］. Cell， 2023， 186（8）： 1652-1669.
[3]	GE W F， SONG S Y， QI X C， et al. Review and prospect of immune checkpoint blockade therapy represented by PD-1/PD-L1 in the treatment of clear cell renal cell carcinoma［J］. Oncol Res， 2023， 31（3）： 255-270.
[4]	ZHANG Q， YANG C Y， GAO X S， et al. Phytochemicals in regulating PD-1/PD-L1 and immune checkpoint blockade therapy［J］. Phytother Res， 2024， 38（2）： 776-796.
[5]	张蓝方，张明娟，许进秀，等. PD-1/PD-L1通路、Th17和Tregs细胞在肿瘤免疫中的作用［J］. 现代肿瘤医学， 2023， 31（18）： 3519-3523.
[6]	罗丽波，任胜祥. KRAS和TP53共突变对肺腺癌生物学行为的影响［J］. 同济大学学报（医学版）， 2024， 45（03）： 345-353.
[7]	THOMMEN D S， PEEPER D S. Rational combination of cancer therapies with PD1 axis blockade［J］. Nat Rev Cancer， 2024. DOI： 10.1038/s41568-024-00727-1 . doi: 10.1038/s41568-024-00727-1
[8]	ZHANG H， LIU L， LIU J B， et al. Roles of tumor-associated macrophages in anti-PD-1/PD-L1 immunotherapy for solid cancers［J］. Mol Cancer， 2023， 22（1）： 58.
[9]	ZHOU Y J， LI G L， WANG J Y， et al. PD-L1： expression regulation［J］. Blood Sci， 2023， 5（2）： 77-91.
[10]	LI W， WU F L， ZHAO S L， et al. Correlation between PD-1/PD-L1 expression and polarization in tumor-associated macrophages： a key player in tumor immunotherapy［J］. Cytokine Growth Factor Rev， 2022， 67： 49-57.
[11]	YIN S N， CHEN Z J， CHEN D G， et al. Strategies targeting PD-L1 expression and associated opportunities for cancer combination therapy［J］. Theranostics， 2023， 13（5）： 1520-1544.
[12]	AGOSTINI M， TRALDI P， HAMDAN M. Proteomic investigation of immune checkpoints and some of their inhibitors［J］. Int J Mol Sci， 2024， 25（17）： 9276.
[13]	XUE F X， REN X T， KONG C Y， et al. Polymeric PD1/PDL1 bispecific antibody enhances immune checkpoint blockade therapy［J］. Mater Today Bio， 2024， 28： 101239.
[14]	FODA B M， MISEK S A， GALLO K A， et al. Inhibition of the Rho/MRTF pathway improves the response of BRAF-resistant melanoma to PD1/PDL1 blockade［J］. Int J Cancer， 2024， 155（7）： 1303-1315.
[15]	钟丰羽，王婷，古文姝，等. Pembrolizumab联合曲妥珠单抗治疗HER2阳性胃癌研究进展［J］. 现代医药卫生， 2023， 39（21）： 3730-3735.
[16]	曹佳丽，熊枝繁，靳泽，等. 免疫检查点抑制剂在肝细胞癌治疗中的研究进展［J］. 肿瘤防治研究， 2023， 50（5）： 525-530.
[17]	NASEER F， AHMAD T， KOUSAR K， et al. Formulation for the targeted delivery of a vaccine strain of oncolytic measles virus （OMV） in hyaluronic acid coated thiolated chitosan as a green nanoformulation for the treatment of prostate cancer： a viro-immunotherapeutic approach［J］. Int J Nanomedicine， 2023， 18： 185-205.
[18]	JI P P， WU P Y， WANG L T， et al. Lysosome-targeting bacterial outer membrane vesicles for tumor specific degradation of PD-L1［J］. Small， 2024， 20（43）： e2400770.
[19]	LI Y C， ZHU T C， CHEN J， et al. Dual-targeted engineered bacterial outer membrane vesicles for hepatocellular carcinoma immunotherapy［J］. Adv Funct Mater， 2024， 34（41）： 2401355.
[20]	JIN M L， HUO D， SUN J J， et al. Enhancing immune responses of ESC-based TAA cancer vaccines with a novel OMV delivery system［J］. J Nanobiotechnology， 2024， 22（1）： 15.
[21]	CUI C Y， HE Q， WANG J J， et al. Targeted miR-34a delivery with PD1 displayed bacterial outer membrane vesicles-coated zeolitic imidazolate framework nanoparticles for enhanced tumor therapy［J］. Int J Biol Macromol， 2023， 247： 125692.
[22]	SU L Y， TIAN Y， ZHENG Q， et al. Anti-tumor immunotherapy using engineered bacterial outer membrane vesicles fused to lysosome-targeting chimeras mediated by transferrin receptor［J］. Cell Chem Biol， 2024， 31（6）： 1219-1230.e5.
[23]	CHEN X， LI P Z， LUO B， et al. Surface mineralization of engineered bacterial outer membrane vesicles to enhance tumor photothermal/immunotherapy［J］. ACS Nano， 2024， 18（2）： 1357-1370.
[24]	ZHENG K S， FENG Y P， LI L， et al. Engineered bacterial outer membrane vesicles： a versatile bacteria-based weapon against gastrointestinal tumors［J］. Theranostics， 2024， 14（2）： 761-787.

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

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

RichHTML

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 24

相关文章 2

Metrics

本文评价

推荐阅读 0

[1]	曾洁,俞雪燕,罗婷,徐江. PD-L1对人口腔鳞状细胞癌细胞增殖、迁移和侵袭的影响[J]. 吉林大学学报(医学版), 2024, 50(1): 18-24.
[2]	任爱华,刘婉,王大伟. 基于CRISPR/Cas9技术敲除PD-L1对非小细胞肺癌 PC-9/T790M细胞吉非替尼耐药敏感性的影响[J]. 吉林大学学报(医学版), 2021, 47(2): 292-298.