Hsp60受体靶向重组乳酸菌的构建及特性OA

To systematically evaluate the protective effects of engineered probiotics on the intestinal mucosal barrier by regulating inflammatory responses,this research amplified the adhesion-related antigen genes aha(adhesin-encoding gene),Inp(invasin-encoding gene),CsgA and CsgG(fimbrial subunit-encoding genes)from Aeromonas veronii strain TH0426,which are responsible for the bacterial adhesive capacity.Using Lactobacillus plantarum NC8 as the delivery vector,four recombinant lactobacilli strains,namely LP-pPG-aha,LP-pPG-Inp,LP-pPG-CsgA and LP-pPG-CsgG,were successfully constructed.The expression of target proteins was verified by Western blot and indirect immunofluorescence assay.Furthermore,the zebrafish fibroblast epithelial cell line AB.9 was employed as the cell model to evaluate the adhesion and invasion abilities of the four recombinant strains in vitro.In addition,RT-qPCR was performed to detect the transcriptional levels of Hsp60,pro-inflammatory cytokine genes and intestinal tight junction protein genes in AB.9 cells co-incubated with A.veronii and recombinant lactobacilli.The results demonstrated that clear protein bands of the four recombinant lactobacilli were observed at molecular weights of 44,15,55 and 30 kDa,respectively,and distinct green fluorescent signals were observed on the surface of all recombinant bacterial cells.All four recombinant lactobacilli exhibited strong adhesion and invasion capacities to AB.9 cells,and they could attenuate the infectivity of A.veronii through competitive adhesion and invasion.Moreover,the recombinant lactobacilli promoted the expression of Hsp60 in AB.9 cells,down-regulated the transcriptional levels of pro-inflammatory cytokines including IL-6,IL-1β and TNF-α,and up-regulated the mRNA expression of tight junction protein genes such as Occludin,CLDN1 and ZO-1.The findings provide a solid foundation for investigating the mechanism underlying mucosal immune responses mediated by engineered probiotics against A.veronii infection.