Abstract
Background and aims: The role of Helicobacter pylori in the development of gastric ulcer and gastrointestinal cancer was identified in this study. More precisely, the study focused on the creation of a DNA vaccine based on the cagT gene of this bacterium and the investigation of its immunogenicity against H. pylori in infused BALB/c mice. Materials and Methods: To this end, the pcDNA3.1(+)-cagT was prepared and transformed into Escherichia coli. Then, animals were injected with recombinant pcDNA3.1(+)-cagT plasmid, pcDNA3.1(+)-cagT + nanoparticles, and pcDNA3.1(+). After the plasmid purification and confirmation of the transformation by digestion and polymerase chain reaction (PCR), chitosan nanoparticles were synthesized using the ionic gelation method. Next, the animals were classified into three groups each including 21 mice. The injectable solutions including pcDNA3.1(+)-cagT, pcDNA3.1(+)-cagT + nanoparticles, or empty pCDNA3.1 (as a control group) were injected into the quadriceps muscle of mice, separately. Finally, the blood and tissue samples of each mouse were collected 15, 30, and 45 days after the last injection, and the expression levels of transforming growth factor-beta (TGF-β1), interleukin-4 (IL-4), and interferon-gamma (IFNγ) were evaluated by real-time PCR. Results: The IFNγ and TGF-β1 expression increased in the infused mice (P<0.01) while the IL4 expression represented a significant decrease (P<0.01). Moreover, the IFNγ and IL4 expression level in pcDNA3.1(+)-cagT + nanoparticle significantly altered (P<0.01) compared to the pcDNA3.1(+)-cagT group although the TGF-β1 expression was not significantly different (P=0.075). Contrarily, the cagT gene expression in the tissue samples of both groups was significantly different 15, 30, and 45 days after the last injection (P<0.01). Eventually, the expression of the cagT gene in the infused mice by pcDNA3.1(+)-cagT and in the nanoparticle group was not significantly different 45 days after the last injection (P=0.105). Conclusion: In general, the decrease of IL-4 expression was observed in the injected mice by pcDNA3.1(+)-cagT and indicated that the immune system work by a Th1 pattern. The findings showed that a pcDNA3.1(+)-cagT construct combined with chitosan nanoparticles can increase the stimulation of the immune system in an animal model and thus it can be used as an appropriate method for controlling H. pylori infection.
Highlights
Helicobacter pylori is a spiral-shaped microaerophilic Gram-negative bacterium that colonizes the luminal surface of the gastric mucosa [1,2]
Verifying the Inserted Gene in the Recombinant Vector The recombinant pcDNA3.1(+)-cagT vector was successfully transformed into competent E. coli, and the accuracy of the transformation on the extracted plasmids and formation of the final construct were determined by enzymatic digestion and polymerase chain reaction (PCR)
The PCR showed the presence of the inserted gene in the vector, and the digestion of the pcDNA3.1(+)-cagT recombinant vector by XhoI and XbaI restriction endonucleases on 2% agarose gel electrophoresis revealed two fragments including 5428 and 855 bp as pcDNA3.1(+) and the cagT gene, respectively (Figure 2)
Summary
Helicobacter pylori is a spiral-shaped microaerophilic Gram-negative bacterium that colonizes the luminal surface of the gastric mucosa [1,2] The infection with this human pathogen causes chronic inflammation, duodenal and gastric ulcers, and gastric cancer [3]. Several effectors and toxins released by H. pylori in the host play an important role in its pathogenicity This human pathogen can escape from stomach acid and colonize the gastric epithelium by the possession of polar flagella, encoding cag pathogenicity island (cag PAI), vacuolating cytotoxin (Vac), and the urease enzyme causing tissue damage [8,9,10]. The expression of the cagT gene in the infused mice by pcDNA3.1(+)-cagT and in the nanoparticle group was not significantly different 45 days after the last injection (P = 0.105).
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