Survey of Helicobacter Pylori lnT Gene Expression in HDF Cells by RT-PCR Method

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.
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.
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.
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.