Efficient expression of UreB by Helicobacter pylori in rapeseed (Brassica napus) using a plant viral vector.

Abstract

Helicobacter pylori is recognized as the main cause of chronic gastritis, peptic ulcers, and stomach cancer. The urease subunit B (UreB) has been identified as the most effective antigen for vaccination and prevention of bacterial infection. Transient expression in plants using virus-based vectors allows for high levels of gene expression within a short time. In this study, we investigated the transient expression of UreB in rapeseed (Brassica napus) using A vector derived from turnip mosaic virus (TuMV). The efficiency of the vector for inoculation and foreign gene expression in rapeseed was first confirmed using the infectious p35STuMVGFPHis construct, validated through fluorescence microscopy, RT-PCR (amplifying a 127-bp fragment), dot blotting, and ELISA. Leaves were inoculated using surface abrasion with carborundum and harvested 14 days post-inoculation for analysis. Following this validation, the construct was engineered by replacing the GFP coding sequence with the UreB gene through NcoI/NheI double digestion and ligation. Successful translation and accumulation of the UreB protein were verified by SDS-PAGE, western blotting, and ELISA. Analyses revealed a distinct protein band corresponding to the predicted molecular weight of 66 kDa and significantly higher expression levels in inoculated plants compared to wild-type controls (OD 450: 0.45 ± 0.06). Our results represent the first report of UreB antigen expression in rapeseed using viral-based vector system and reveal its potential as a rapid and low-cost strategy for plant-based production of vaccine antigens.