Construction Of Spv Gene Compensation Strains In Salmonella Typhimurium And Its Effects On Intestinal Mucosal Epithelial Cell Apoptosis

Objective:To construct the compensating Salmonella strain implemented with typhimurium spv gene and study its stability on the basis of the construction of good Salmonella expression vector. We establish Henle-407 cell model of Salmonella typhimurium infection in vitro and observe the effect of Salmonella plasmid virulence genes spvC on intestinal epithelial cell apoptosis and its molecular mechanisms. Besides, to provide theoretical and experimental basis and subsequent use of the animal model for studying on the function of the gene, we explore the pathogenic mechanisms of Salmonella typhimurium virulence plasmid gene spvC. Methods:1.The construction of the fluorecent compensating Salmonella typhimurium inserting spv gene strain and investigation of its stability.The low copy cloning vector pACYC-184 as the foundation, according to the eGFP gene sequence published in GenBank database, Trc Promoter promoter in the upstream and downstream rrnB ribosomal terminator downstream sequence ends site design appropriate restriction sites, using the method of multiple PCR synthesis of pACYC184-eGFP carrier, and in turn the transfer and cloning of aphA-hok-parDE gene and spv gene, construct pACYC184-eGFP-HOK vector and pACYC184-spvB-eGFP-HOK vector and pACYC184-spvC-eGFP-HOK vector were transformed into corresponding Salmonella typhimurium spv mutant was obtained covering strains, and the expression of the plasmid stability and protein fluorescence was detected.2.Study on the effect of Salmonella plasmid virulence gene spvC on intestinal epithelial cell apoptosis and its molecular mechanism.Wild type Salmonella typhimurium 211 carrying spv gene, knockout spvC defects variation strains(ST211-?-e-spvC) and compensating strain(ST211-c-spvC) and Henle-407 cells were co cultured, intervention at different time points of cell experiments were performed. Fluorescence microscope was used to observe cell morphological changes and intracellular bacteria; Promega luciferase luminescence assay Caspase3/7 activity; Western blot was used to detect ERK and p-ERK expression; plate colony count assays cells in colony forming units(colony forming unit, CFU). Results:1. The construction of the fluorecent compensating Salmonella typhimurium inserting spv gene strain and investigation of its stability.1)By PCR and gene sequencing confirmed successful construction of Salmonella typhimurium spvB, spvC gene fluorescence complemented strain.2)There were no loss of the 12 generation or 144 H in the continuous culture of the strain containing low copy plasmid, which was observed by fluorescence microscopy in the bacterial infection cell model.2.Study on the effect of Salmonella plasmid virulence gene spvC on intestinal epithelial cell apoptosis and its molecular mechanism.1)The fluorescent microscopy under ST211 infection group and ST211-c-spvC infection cell group in 4H appears to be a large number of nuclei a corrugated or crease sample, chromatin condensation, early apoptosis morphological changes, and ST211-?-e-spvC infection group and blank control group without obvious apoptosis morphological changes. 24 hours after the infection, ST211 infection group and ST211-c-spvC infection group cell chromatin condensed highly marginalized, produce apoptotic bodies and the emergence of a lot to change the necrosis of the morphological characteristics, ST211-?-e-spvC only part of the cells are apoptotic and necrotic morphology change.2)The detection of Caspase-3/7 activity results showed that each group of Caspase-3/7 activity increased with time prolonged infection, 0h, 4H three bacterial infection between groups of fluorescence values with no significant difference(P> 0.05), 4 h after infection, bacterial infection group compared with the blank control group have significant difference(P<0.01). Infection of 8h, 12 h and 24 h, ST211-]-e-spvC infection group than in the ST211-eGFP infection ST211-c-spvC infection group, the fluorescence value difference(P<0.05).3)The ratio of p-ERK protein(24h; blank control group, 0.89 0.03; ST211-eGFP infection group, 0.12 + 0.02; ST211-?-e-spvC infection group, 0.74+0.05; ST211-cspvC infection group, 0.11+0.04) respectively. ST211-eGFP infection group and ST211-c-spvC infection group, p-ERK protein ratio is ST211-?-e-spvC infection group was significantly lower(P<0.01).4)Before 4h, there was no significant difference in the number of viable bacteria(P>0.05) between the groups. In the intervention after 8h, 12 h and 24 h, ST211-eGFP infection group and ST211-c-spvC cellular infection living bacteria number higher than infection ST211-?-e-spvC group(P < 0. 05). Conclusions:1.Highly stable and containing green fluorescent protein tagged Salmonella expression vector and spv gene back to fill the strain was successfully constructed and laid the foundation for the in-depth study of Salmonella typhimurium virulence gene function.2.Salmonella plasmid virulence genes spvC can promote the bacteria within the host cell survival and proliferation through inducing cell apoptosis, and aggravate the injury of the cells. This effect may be related to its effect on ERK phosphorylation.