表达PRRSV结构蛋白的saRNA制备及免疫原性初步分析OA

Getah virus(GETV)can serve as a candidate vector for novel vaccines,and the structural proteins of porcine reproductive and respiratory syndrome virus(PRRSV)can induce specific immune responses.This study aimed to construct self-amplifying RNA(saRNA)expressing PRRSV structural proteins using GETV and to preliminarily evaluate the humoral immune response induced by the saRNA in mice.The genes encoding GP2 to M proteins of the PRRSV JXwn06 strain were linked using the P2A peptide sequence derived from porcine teschovirus and subjected to mouse codon optimization.The synthesized sequence was designated"2A".Using an infectious cDNA clone of GETV,the GETV nonstructural proteins,5' untranslated region(UTR),and 3' UTR were retained,while the structural protein subgenomic region was replaced with"2A"to successfully construct the recombinant plasmid.Following in vitro transcription and purification,GETV-based saRNA was obtained.Indirect immunofluorescence assay(IFA)and Western blotting(WB)showed that PRRSV structural proteins were expressed in baby hamster kidney fibroblast BHK-21 cells transfected with the saRNA.The prepared saRNA was encapsulated in lipid nanoparticle(LNP)to generate LNP-saRNA,which was used to transfect human embryonic kidney HEK-293T cells and immunize 6-8-week-old BALB/c mice.IFA results showed that the transfected HEK-293T cells expressed PRRSV structural proteins and that sera from the immunized mice were positive for PRRSV-specific antibodies.These results provide technical support for the development of a PRRSV saRNA vaccine using GETV as a vector.