猪瘟病毒E2蛋白单克隆抗体的制备与鉴定OA

To prepare monoclonal antibodies against the E2 protein of classical swine fever virus(CSFV),the recombinant plasmid pCAGGS-E2-His was transfected into human embryonic kidney 293T(HEK-293T)cells for eukaryotic expression and purification of the recombinant E2 protein,followed by immuniza-tion of BALB/c mice.Hybridoma cell lines stably secreting specific monoclonal antibodies against the E2 protein were screened using cell fusion technology.The reactivity of the monoclonal antibodies was verified by indirect immunofluorescence assay(IFA)and Western blot,and antibody subclass identifica-tion was performed after purification.The results showed that the recombinant E2 protein was success-fully expressed in HEK-293T cells,with a specific band of approximately 55 kDa detected.In this study,three hybridoma cell lines stably secreting anti-E2 monoclonal antibodies were obtained and designat-ed as 4B11E6,5E9G2 and 6B9F8,respectively.The titers of these monoclonal antibodies were above 1∶64000 with favorable specificity,which could recognize the purified CSFV E2 protein by Western blot and CSFV-infected porcine kidney(PK-15)cells by IFA.Antibody subtype analysis revealed that the heavy chains of 4B11E6 and 6B9F8 belong to the IgG1 subtype,while that of 5E9G2 belongs to the IgG2a subtype,and all light chains are of the κ isotype.In conclusion,the CSFV E2 protein was successfully expressed and high-titer monoclonal antibodies were prepared in this study,which provide important materials for further research on the function of the E2 protein and the establishment of diagnostic methods for classical swine fever.