Cloning and Sequencing of ompf Salmonella typhimurium Salmonella ompf Gene in Escherichia coli Origami

Salmonella Typhimurium belongs to the family Enterobacteriaceae, gram-negative bacilli and causes gastrointestinal diseases such as typhoid. This bacterium has a special structure and various genes, including the ompf gene (outer membrane protein). Recent studies have shown the possibility of using ompf in the development of a diagnostic tuberculosis vaccine. Therefore, the aim of this study was to clone and sequence the ompf gene of Salmonella typhimurium, the causative agent of typhoid in Escherichia coli Oragami, in order to obtain a vaccine.
Purines are the outer membrane proteins of gram-negative bacteria, which act as receptors for bacteriophages and are effective in a variety of functions such as solution transport, pathogenicity, and immunity. Salmonella typhi purines show heterologous epitopes on their loops, giving them the potential for diagnosis and vaccination. The outer membrane proteins of Salmonella typhi allow ions to pass through. Omp (outer membrane protein) is a member of the purine family, which is highly expressed in gram-negative bacteria.OmpC, D Omp, and F Omp purines of Escherichia coli and Salmonella typhi are ternary proteins that create pores on both outer membrane surfaces. The presence of protein pores makes this membrane permeable to low molecular weight solutes. Large molecules of antibiotics slowly perforate the membrane, which is one of the reasons that gram-negative bacteria are resistant to many antibiotics.

Sampling of people with salmonellosis was performed during 4 months from the infectious ward of medical centers in Tehran province. After identifying and performing specific biochemical tests, Salmonella typhi was isolated and DNA was extracted. Salmonella typhi strains with ompf gene were then extracted by PCR. The ompf gene of the positive strains was transfected into the Escherichia coli bacterium by vector and cloned by TA technique. Finally, the expression of genes in Escherichia coli Oregami was measured by Real time PCR technique. ClustalX and Mega5 software were used to draw the phylogenetic tree. After visiting the following site and studying and searching in various articles, suitable primers for Ompf gene were selected. The primers were compared and blasted at the site (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and ordered from Sinagen. The primers were diluted with distilled water to a concentration of 100 picomoles and then to a suitable concentration of 10 picomoles. According to studies, 3 suitable pairs of primers were selected and ordered from Bioneer.
In order to clone the PCR product faster and more efficiently, the TA-Cloning method is used. The TA-Cloning PCR kit was prepared by Sina Gene Company. As stated in the kit protocol, this method uses a linear vector called PTG19-T with the thymine base at the end ´3. The use of PTG19-T linear vector, which has a thymine base at the end of 3, leads to direct, fast and easy binding of the PCR product to the cloning vector. As a result, a cyclic molecule containing the gene we want is formed, which has the ability to reproduce spontaneously in a suitable host such as E. coli. In this method, no enzymatic digestion step is required and this step has been eliminated

A total of 12 Salmonella typhi isolates were isolated from the screening of clinical specimens sent to the laboratory, which were identified based on morphological characteristics, microscopy and biochemical tests. Of these, only one wasolate had the ompf gene. After cloning, ompf genes, cloned selection colony strains (blue / white) were isolated. In order to confirm the results of DNA cloning, it was extracted from suspicious colonies and analyzed by Real time PCR. Sequence, m13 and proliferation curves confirmed gene expression in Escherichia coli origami.
In order to determine the molecular identity of Salmonella typhi, 16s general primers were used (PCR result in Figure 7) and finally the PCR product was sent to Bioneer for sequencing and BLAST.

In this study, by genomic evaluation of Salmonella typhimurium isolated from patients with tuberculosis, ompf gene with immunization potential was extracted from this bacterium in order to make a vaccine and cloning was successful. Finally, by examining the phylogenetic tree drawn in this study, the degree of similarity and kinship of Salmonella typhi with other species was shown.Typhoid is an infectious disease caused by Salmonella typhi and is considered an important protein in immunological research due to the ability of the Ompf gene. This gene has the potential to stimulate immune responses by isolating and cloning the ompf gene separately. Salmonella typhimurium in Escherichia coli can be used to meet treatment needs and achieve an effective vaccine. Out of 12 Salmonella typhi isolates, only one strain carried the ompf gene. The gene was transferred to the host bacterium via the ptg19 plasmid and cloning was successful. This recombinant protein has the potential to be used in immunization and vaccine development. The phylogenetic tree drawn in this study showed the similarity and kinship of Salmonella typhi with other species.In addition, S. typhi induces the expression of excitatory molecules on antigen-containing cells through conventional signaling pathways. However, the main potential of S. Typhi for use as vaccine compounds is unknown. Here, the characteristics of S. typhi against a range of related laboratory and clinically antigens were investigated. Co-immunization of S. Typhi with ovalbumin protein (OVA), in addition, immunization of S. Typhi protein, generates anti-influenza IgG elements, changes antibody class and matures. In general, OmpF proteins are versatile vaccine compounds, which can be used to enhance cellular immune responses and improve antibody responses.