表达DEC-205结合肽重组乳杆菌的构建及其对树突状细胞成熟的影响

doi:10.16431/j.cnki.1671-7236.2025.12.036

摘要/Abstract

摘要： 【目的】构建表达免疫原融合鸡DEC-205结合肽的重组神话猪联合乳杆菌(LigiLactobacillus saerimneri,L.sae)，探究其对鸡外周血树突状细胞(PB-MoDC)的影响。【方法】构建含组成型启动子HCE、增强子T7 g10、信号肽ssUSP及细胞壁锚定蛋白LPXTDG的乳酸菌表达质粒p612。插入Flag标签、鸡DEC-205短肽(DL(结合肽)/PS(对照肽))融合鸡传染性法氏囊病病毒(IBDV)VP2基因序列，构建重组质粒p612-VP2-PS、p612-VP2-DL。重组质粒经双酶切鉴定后，电转入神话猪联合乳杆菌M11，构建重组乳杆菌p612-VP2-PS/M11、p612-VP2-DL/M11。利用Western blotting及免疫电镜鉴定蛋白表达和分布情况；通过荧光显微镜观察未成熟PB-MoDC细胞对重组乳杆菌的吞噬效果；重组菌作用PB-MoDC细胞4 h后，利用实时荧光定量PCR检测细胞表面标志物(MHC-Ⅱ、CD40、CD80、CD83、DEC-205)及炎性因子(IFN-γ、IL-1β、IL-12、IL-6、TNF-α、CXCLi1)的转录水平。【结果】试验成功获得了携带空载体p612、重组质粒p612-VP2-DL(表达VP2和结合肽)和p612-VP2-PS(表达VP2和对照肽)的神话猪联合乳杆菌M11。Western blotting结果显示，重组乳杆菌p612-VP2-PS/M11、p612-VP2-DL/M11菌体沉淀中均可检测到目的蛋白表达；免疫电镜观察显示，重组蛋白主要分布在胞质及细胞壁内壁；重组菌可被未成熟的PB-MoDC细胞吞噬。与p612-VP2-PS/M11组相比，p612-VP2-DL/M11组PB-MoDC细胞中的MHC-Ⅱ、CD80、CD40、CD83、IL-1β、IL-12、IFN-γ、CXCLi1基因转录水平显著或极显著上调(P＜0.05；P＜0.01)；两组间PB-MoDC细胞中DEC-205、IL-6、TNF-α基因转录水平无显著差异(P＞0.05)。【结论】本研究成功构建了表面表达免疫原融合鸡DEC-205结合肽的重组鸡肠道乳杆菌，其能有效刺激PB-MoDC细胞成熟。试验结果为研发畜禽疫苗的新型靶向递送策略提供了理论依据。

关键词: DEC-205结合肽; 神话猪联合乳杆菌; 鸡外周血树突状细胞; 表面标志物; 炎性因子

Abstract: 【Objective】 The purpose of this experiment was to construct a recombinant LigiLactobacillus saerimneri (L.sae) expressing an immunogen fused with chicken DEC-205 binding peptide,and investigate its effects on peripheral blood monocyte-derived dendritic cells (PB-MoDCs) in chickens. 【Method】 The lactic acid bacteria expression plasmid p612 containing the HCE constitutive promoter,T7 g10 enhancer,ssUSP signal peptide,and the cell wall anchoring protein LPXTDG was constructed.The Flag tag and the chicken DEC-205 short peptide (DL (binding peptide)/PS (control peptide)) column fusion was inserted into the chicken Infectious bursal disease virus (IBDV) VP2 gene sequence,and the recombinant plasmids p612-VP2-PS and p612-VP2-DL were constructed.After the recombinant plasmid was identified by double enzyme digestion,they were electroporated into the LigiLactobacillus saerimneri M11 to construct recombinant Lactobacillus plasmids p612-VP2-PS/M11 and p612-VP2-DL/M11.The expression and distribution of proteins were identified by Western blotting and immunoelectron microscopy.The phagocytic effect of immature PB-MoDC cells on the recombinant Lactobacillus was observed through fluorescence microscopy.After the recombinant bacteria acted on PB-MoDC cells for 4 h,the transcriptional levels of cell surface markers (MHC-Ⅱ,CD40,CD80,CD83 and DEC-205) and inflammatory factors (IFN-γ,IL-1β,IL-12,IL-6,TNF-α and CXCLi1) were detected using Real-time quantitative PCR. 【Result】 The experiment successfully obtained LigiLactobacillus saerimneri M11 carrying the empty vector p612,the recombinant plasmid p612-VP2-DL (expressing VP2 and binding peptide) and p612-VP2-PS (expressing VP2 and control peptide).Western blotting results showed that the target protein could be detected in the bacterial precipitates of both recombinant Lactobacillus p612-VP2-PS/M11 and p612-VP2-DL/M11.Immunoelectron microscopy observations revealed that the recombinant protein was mainly distributed in the cytoplasm and the inner wall，and the recombinant bacteria could be phagocytosed by immature PB-MoDC cells.Compared with p612-VP2-PS/M11 group,the transcriptional levels of MHC-Ⅱ,CD80, CD40,CD83,IL-1β,IL-12,IFN-γ and CXCLi1 genes in PB-MoDC cells of p612-VP2-DL/M11 group were significantly or extremely significantly upregulated (P＜0.05 or P＜0.01).There were no significant difference in the transcriptional levels of DEC-205,IL-6 and TNF-α genes in PB-MoDC cells between the two groups (P＞0.05). 【Conclusion】 This study successfully constructed a recombinant chicken intestinal Lactobacillus that expresses immunogenic fusion peptides of chicken DEC-205 on its surface.This strain could effectively stimulate the maturation of PB-MoDC cells.This experimental results provided a theoretical basis for developing a new targeted delivery strategy for livestock and poultry vaccines.

Key words: DEC-205 binding peptide; LigiLactobacillus saerimneri; PB-MoDCs; surface markers; inflammatory factors

中图分类号:

S852.4

郭双霖, 马孙婷, 谭飞, 张磊, 秦浩然, 陈蓉, 欧阳伟, 徐彬, 张纹纹, 杨蕾蕾, 袁厅, 朱枰潞, 冯志新, 熊祺琰, 郝飞, 谢星. 表达DEC-205结合肽重组乳杆菌的构建及其对树突状细胞成熟的影响[J]. 中国畜牧兽医, 2025, 52(12): 5923-5932.

GUO Shuanglin, MA Sunting, TAN Fei, ZHANG Lei, QIN Haoran, CHEN Rong, OUYANG Wei, XU Bin, ZHANG Wenwen, YANG Leilei, YUAN Ting, ZHU Pinglu, FENG Zhixin, XIONG Qiyan, HAO Fei, XIE Xing. Construction of Recombinant Lactobacillus Expressing DEC-205 Binding Peptide and Its Effect on Dendritic Cell Maturation[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5923-5932.

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