بررسی بیان ژن lnT هلیکوباکتر پیلوری در سلول‌های HDF انسان به روش RT-PCR

Background and Objectives: Helicobacter pylori is a gram-negative and spiral bacterium that causes stomach and duodenal disease in humans. Because of the presence of disadvantages in antibiotic therapies, increasing efforts  have been made to produce effective vaccine for this infection. The aim of this study was to generate a construct   carrying the lnT gene and to survey its expression in human cells with RT-PCR method. Materials and Methods: In this laboratory study, lnT gene from the genome of Helicobacter pylori bacterial was isolated via polymerase chain reaction (PCR). Cloning of the PCR products was done by T/A cloning method in the appropriate T vector. Then, the lnT gene was subcloned into a pEGFP-C2 eukaryotic expression vector. To study the lnT gene expression, the final pEGFP-C2-lnT construct was transformed into human dermal fibroblasts (HDF) cells by electroporation and its expression was analyzed by RT-PCR. Results: The performance of the PCR resulted  in amplification of 1290 bp segment as to lnT gene. This gene was successfully cloned in pTZ vector and enzyme digestion and sequencing results showed lnT gene was subcloned in the expression vector and final construction of the pEGFP-C2-lnT was created. Gene expression analysis by RT-PCR showed the relevant band. Conclusion: Based on the obtained results, lnT gene cloned in the pEGFP-C2 eukaryotic expression vector has the ability to produce  the specific product of this gene in eukaryotic cells. Therefore, this gene construction has the required potential to evaluate the immunogenicity in an animal model as a gene vaccine against Helicobacter pylori. Key words: Helicobacter pylori, Cloning, lnT gene, RT-PCR   Funding: This research was funded by Shahrekord Branch, Islamic Azad University. Conflict of interest: None declared. Ethical approval: The Ethics Committee of Department of Biology, Shahrekord Branch, Islamic Azad University approved the study (IR.IAUSHK.1395.5213).   How to cite this article: Rahmani-Piani R, Doosti A. Survey of Helicobacter Pylori lnT Gene Expression in HDF Cells by RT-PCR Method. J Rafsanjan Univ Med Sci 2017; 16(9): 807-18. [Farsi]