Introduction: Helicobacter pylori located in the human stomach is the cause of diseases such as gastritis, peptic ulcers and cancer of lymph nodes and digestive system. However, despite the major problems of H. pylori, a medical vaccine against this bacterium has not yet been developed. The best way to prevent infections is vaccination, and so far research has been done on recombinant subunit vaccines and gene vaccines. The production and purification of recombinant proteins is one of the most expensive stages of antibody production, and therefore, the use of DNA-dependent vaccines due to the ease of mass production of recombinant plasmids compared to the production and purification of recombinant proteins and the increase in the duration of immunity due to the continuous expression of antigens and the occurrence of immune response Humoral and cellular can significantly reduce these processes. The purpose of this research is to collect the fastest research results related to the production of recombinant nucleic acid vaccines based on Helicobacter pylori antigens to stimulate the humoral and cellular immune response.
Methods: In this research, SID, ISI and Google Scholar databases were used and the content was collected using the keywords of recombinant vaccine against Helicobacter pylori. In the articles, methods and results were further investigated and the methods of gene extraction from DNA, gene cloning in plasmid vector, transformation of recombinant vector in prokaryotic expression system and subcloning of the final recombinant construct in eukaryotic systemic expression were investigated. Finally, the methods of analyzing the accuracy of the activities were examined and the best method was chosen.
Results: Diagnostic tests for Helicobacter pylori infection include tests that require endoscopy, such as rapid urease test (RUT) and PCR, and non-endoscopic tests, including antibody tests and urea breath tests. Due to the breakdown of urea molecules and the production of ammonia and carbon dioxide by Helicobacter pylori, which creates a sheath of ammonia around the bacterium that protects it from stomach acid, the production of large amounts of urease enzyme is necessary for the survival and pathogenicity of H. pylori. H. pylori active urease depends on the presence of ureA/B gene constructs to form a 550 kDa holoenzyme. Also, sub-genes ureL/E/F are necessary for the high expression of urease activity and ureB/G/I for the establishment of bacteria in the stomach. ureB is the most effective immunogen of all H. pylori strains that can create a protective immune response in the body against this bacterium. Therefore, ureases, especially ureB, are considered suitable candidates for making a vaccine against H. pylori. Also, recent analyzes indicate active penicillin binding in the HCP protein structure, and more specifically in hcpA, hcpB and hcpD. H. pylori infection can be diagnosed with the immunological assay of this protein, and considering that the hcpD gene in this protein group is able to stimulate the host's immune system, this gene is also considered as a candidate for vaccine production.
In all studies, a DNA extraction kit was used to extract genomic DNA, and then the extraction product was electrophoresed on agarose gel. In one of the studies, the concentration test was also done with a nanodrop device, and then the gene product was amplified by PCR. In the most successful studies, the T/A method was used for cloning, which is a fast method without the requirement for restriction enzymes, and the genes were transformed in the pTZ vector. Different strains of E.coli bacteria were used for transformation, and E.coli Top10F strain was used more than other strains. In all studies, LB-Agar growth medium containing ampicillin was used, and the correctness of cloning was confirmed by enzymatic digestion, PCR, and sequencing. Due to the lack of a promoter in the upstream of the cloned gene in the pTZ vector and the inability to reproduce and express the gene in eukaryotic cells, pcDNA3.1(+) vector was used. The two vectors were cut and the gene was removed from the pTZ vector; Also, the vector was linearized. T4 DNA ligase enzyme was used for binding and 2 studies were similar in using CHO cells. Electroporation was used for this cloning. The most important analyzes used were electrophoresis in different stages, enzymatic digestion, RT-PCR, SDS-PAGE and western blotting.
Conclusion: According to the results of the studies based on the successful cloning of the final recombinant structure in eukaryotic cells and changing part of the behaviors of rats, as well as the possibility of multiplying these structures, Helicobacter pylori genes including ureB and hcpD can be used in vaccine production.