猪源核糖体RNA加工蛋白1B的多克隆抗体制备及与核糖体和核仁共定位研究OA

This study aimed to obtain polyclonal antibodies against porcine ribosome RNA process-ing protein 1B(RRP1B)and investigate its co-localization with ribosomes and nucleoli.The full-length RRP1B gene was amplified from IPEC-J2 using RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1.The recombinant plasmid pcDNA3.1(+)-RRP1B-His was constructed.After sequencing verifica-tion,the plasmid was transfected into HEK-293T and IPEC-J2.Expression of RRP1B was detected by indirect immunofluorescence assay(IFA)and Western-blot.The recombinant His-RRP1B was purified by nickel affinity chromatography and used as an immunogen to generate polyclonal antibodies in BALB/c mice via subcutaneous immunization.Antibody titers were determined by Western-blot and IFA to evaluate immunogenicity.Meanwhile,the prepared polyclonal antibody was applied to investigate the co-localization of RRP1B with ribosomes and nucleoli.The results showed that the recombinant plasmid pcDNA3.1(+)-RRP1B-His was successfully expressed in both HEK-293T and IPEC-J2.Immunization with the recombinant RRP1B induced high-level antibody production in mice,with titers reaching above 1∶2 000.Immunofluorescence co-localization assays revealed that RRP1B was localized in the nucleoli,with no significant co-loca-lization observed with ribosomes.This study lays a foundation for further investigation on the biological functions of RRP1B.