Recombinant Production and Immunological Evaluation of Tobamovirus fructirugosum (ToBRFV) Capsid and Movement Proteins

Tomato brown rugose fruit virus ( Tobamovirus fructirugosum , ToBRFV) is an emerging tobamovirus threatening tomato and pepper production worldwide. Accurate and scalable detection methods are essential to limit its agricultural impact. In this study, we report the recombinant production and characterization of ToBRFV capsid (CP ToBRFV ) and movement (MP ToBRFV ) proteins as candidate antigens for serological detection. Both coding sequences were amplified from infected tomato tissue and heterologously expressed in Escherichia coli BL21(DE3). CP ToBRFV exhibited partial solubility under low-temperature induction, allowing purification under native conditions, whereas MP ToBRFV accumulated in inclusion bodies and was purified under denaturing conditions. Purified proteins were validated by SDS-PAGE and immunoblotting. In silico analyses indicated the presence of multiple B- and T-cell epitopes in both proteins. Immunization of mice with purified proteins elicited IgG responses. Interestingly, MP ToBRFV induced a higher humoral response, but antisera raised against CP ToBRFV showed superior ELISA reactivity with crude extracts from ToBRFV-infected plants. Together, these results support CP ToBRFV as a robust antigen for serological detection and provide a reproducible framework for the development of recombinant protein-based diagnostics for ToBRFV.