Preparation and application of a polyclonal antibody against the N structural protein of chilli yellow ringspot orthotospovirus.

Chilli yellow ringspot orthotospovirus (CYRSV), which is a newly discovered virus in Yunnan Province and is a member of the genus Orthotospovirus, family Tospoviridae, order Bunyavirales, causes severe disease in vegetable crops. The nucleocapsid (N) protein is a structural protein that is crucial for viral replication and virulence. To understand the exact role of N from CYRSV in viral replication and the host immune response, a qualified antibody against this protein is needed. In this study, the coding region of the CYRSV N gene was cloned into a bacterial expression vector (pTOPO-D1), and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells. The CYRSV N fusion protein was purified using a His tag and then utilized as an antigen to immunize rabbits and obtain an anti-CYRSV N polyclonal antibody. The polyclonal antibody specificity were confirmed by western blotting, the polyclonal antibody specifically recognized N from CYRSV. Various immunological test methods utilizing polyclonal antibodies were developed, including ELISA and dot immunobinding assay. Field samples exhibited high specificity and sensitivity when tested using these methods. Furthermore, the results obtained from a large number of field samples are consistent with those derived from RT-PCR analysis, further validating the applicability of our approach. This polyclonal antibody will be useful for future studies on N functions and CYRSV identification. In conclusion, our study provides a valuable diagnostic tool for CYRSV rapid detection and epidemiological investigations.