جداسازي باسيلوس سوبتيليس با توان بالقوه پروبيوتيك از فرآورده¬هاي غذايي، مطالعه بيان cagL در سطح اسپور باسيلوس سوبتيليس و ارزيابي ايمني¬زايي آن در تجويز خوراكي به موش BALB/c

Isolation of Bacillus subtilis with probiotic potential from food products, study of cagL expression on surface of Bacillus subtilis spore and eva‎luation of its immunogenicity by oral administration in BALB/c mice

زيست شناسي سلولي مولكولي و ميكروبيولوژي

Helicobacter pylori is a pathogenic bacterium which is located in the gastric mucosa of about half of the worldʹs population. The cagPAI locus encoding type IV secretion system is one of the important pathogenic factors of bacteria, that increases the risk of gastric cancer. The conserved protein CagL is a part of this secretion system, which plays an important role in the transfer of virulence factors to the target cell by binding and activating the 51 and v5 integrin receptors on the surface of gastric epithelial cells. Therefore, its application as a vaccine for immunization against Helicobacter pylori has been considered. Moreover, bacterial spores, with the ability to survive in harsh environmental conditions have attracted the attention of researchers as a useful tool for surface display technique. Therefore, investigating the putative role of CagL protein in induction of immune responses using Bacillus subtilis spore display system was the main aim of this study. In this study, CagL protein from H. pylori was selected as a target protein, and CotE and CotG proteins, which are spore coat proteins, were selected as anchoring proteins. pCotE-CagL and pCotG-CagL vectors were made from pSDJH100 vector for spore engineering. Then the vectors were transferred to Bacillus subtilis DB104. This vector contains homologous sequences with B. subtilis chromosome, and due to recombination, CotG-CagL and CotE-CagL gene fragments are inserted into the chromosome. Therefore, the target protein is expressed on the spore surface with the expression of spore coat proteins concurrently. The expression of the gene encoding CagL protein in two vectors with CotG and CotE coat proteins on the surface of B. subtilis spores was eva‎luated using specific antibodies labeled with HRP and FITC in immunoblotting methods and immunofluorescence microscopy. 200 l of suspension equivalent to 1010 of recombinant bacterial spores containing pCotE-CagL and pCotG-CagL vectors as well as non-recombinant B. subtilis DB104 were fed orally to BALB/c mice at specific time intervals, and the immune response was eva‎luated by Western blotting analysis. In this test, the serum of mice orally gavaged with recombinant and non-recombinant spores was used as the primary antibody. The observation of band formation for recombinant bacteria and the absence of the band in non-recombinant bacteria indicates the production of specific antibody against CagL protein in the serum of mice gavaged with recombinant spores, which is a qualitative verification of the immunogenicity of mice. Therefore, the results indicate that CagL of H. pylori was successfully expressed on the surface of B. subtilis spores, and the recombinant bacteria can be introduced as oral vaccine candidates against H. pylori infection.

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