经济动物学报

维氏气单胞菌（Aeromonas veronii）广泛分布于各种淡水、污水等水环境中，可感染鱼、虾、贝类等水生生物及人类等哺乳动物，临床症状严重。乳酸菌可定植并存活于动物及人类胃肠道黏膜表面，作为肠道常生菌，其调节免疫应答、异源蛋白表达能力和维持肠道菌群平衡的特点成为新一代黏膜疫苗载体的强有力候选者。本研究扩增了A.veronii TH0426株具有黏附能力的抗原黏附蛋白aha、侵袭蛋白Inp、菌毛亚基CsgA及CsgG基因，以植物乳杆菌（Lactobacillus plantarum）NC8为递呈载体，构建4株重组乳酸菌LP-pPG-aha、LP-pPG-Inp、LP-pPG-CsgA及LP-pPG-CsgG。通过Western blot和间接免疫荧光验证靶标蛋白表达情况，并以斑马鱼成纤维上皮细胞AB.9为体外细胞模型进一步检测4株重组乳酸菌对细胞的黏附与侵袭能力。此外，通过荧光定量PCR检测A. veronii与重组乳酸菌共孵育后Hsp60基因、促炎因子及肠道紧密连接蛋白因子的表达水平。结果显示，4株重组乳酸菌蛋白分子量分别在43 kDa、15 kDa、55 kDa和30 kDa处见清晰条带，且均可在菌体表面检测到绿色荧光信号。4株重组乳酸菌对AB.9细胞均有较强的黏附能力与侵袭能力，并通过与致病菌竞争黏附和侵袭的方式来降低A.veronii的感染能力。同时，可促进AB.9细胞内Hsp60表达，下调促炎因子IL-6、IL-10、IL-1β及TNF-α的转录水平，并促进紧密连接蛋白Occludin、CLDN1和ZO-1基因的表达。本研究旨在评估肠上皮细胞被致病菌侵袭感染过程中调节炎症反应的保护作用，为探究工程益生菌抗A. veronii感染介导的黏膜免疫应答机制研究提供基础。

Aeromonas veronii is a ubiquitous pathogen thriving in diverse aquatic habitats， including freshwater and sewage. It can infect aquatic organisms such as fish， shrimp， shellfish， and mammals such as humans， with severe clinical symptoms. Lactic acid bacteria can colonize and survive on the surface of the gastrointestinal mucosa in animals and humans. As a common gut bacterium， they have become a strong candidate for the new generation of mucosal vaccine carriers due to their ability to regulate immune response， heterologous protein expression， and maintain gut microbiota balance. This study amplified the antigen adhesion protein aha， invasion protein Inp， bacterial pili subunit CsgA， and CsgG genes of strain A.veronii TH0426 with adhesion ability. Four recombinant lactic acid bacteria LP-pPG-aha， LP-pPG-Inp， LP-pPG-CsgA， and LP-pPG-CsgG were constructed using Lactobacillus plantarum NC8 as the presentation vector. Western blot and Indirect Immunofluorescence were used to verify the expression of target proteins， and the adhesion and invasion ability of four recombinant lactic acid bacteria on zebrafish fibroblast epithelial cells AB.9 was further tested using an in vitro cell model. In addition， the expression levels of Hsp60 gene， pro-inflammatory factors and intestinal tight junction proteins were detected by real-time PCR after A.veronii was incubated with recombinant lactic acid bacteria. The results showed that the molecular weights of the four engineered strains were 43 kDa， 15 kDa， 55 kDa and 30 kDa， respectively， and the green fluorescence signals could be detected on the surface of the strains. 4 strains recombinant lactic acid bacteria on AB. 9 cells have strong ability of adhesion and invasion ability， and through the way of competition with pathogenic bacteria adhesion and invasion to reduce A veronii infection ability. Simultaneously， they can promote Hsp60 expression in AB.9 cells， cut proinflammatory factor IL-6， IL-10 and TNF-α， IL-1β transcription level， and promote the tight junction protein Occludin， CLDN1 and ZO-1 gene expression. The purpose of this study was to evaluate the protective effect of intestinal epithelial cells on regulating inflammatory response during invasion and infection by pathogenic bacteria， in order to explore the anti-A.veronii. The study of the mucosal immune response mechanism mediated by infection provides a basis.