猪肺炎支原体P97R1的多克隆抗体制备及亚细胞定位OA

To explore the biological function of the Mycoplasma hyopneumoniae adhesion factor P97 pro-tein,the P97R1 target gene was synthesized based on the GenBank database,and the prokaryotic expression vector pET28a-P97R1 was constructed.The plasmid was transformed into BL21(DE3)competent cells for protein expression verification.The verified P97R1 protein was mixed with adjuvant to immunize animals and prepare rabbit polyclonal antibodies.The antibody titer and immunogenicity were detected by indirect ELISA.Meanwhile,the physicochemical properties,hydrophobicity,signal peptide,transmembrane region,subcellular localization and secondary structure of the P97R1 protein were analyzed and predicted by bioin-formatics software.In addition,the eukaryotic expression vector pEGFP-N1-P97R1 was constructed.After double enzyme digestion and sequencing verification,it was transiently transfected into HEK293T cells,and the subcellular localization of the P97R1 protein was analyzed by indirect immunofluorescence assay(IFA).The results showed that the molecular weight of the prokaryotic expressed pET28a-P97R1 recombinant protein was approximately 33 ku;indirect ELISA confirmed the successful preparation of rabbit polyclonal antiserum specifically recognizing the P97R1 protein.Bioinformatics analysis predicted that the P97R1 pro-tein was composed of 190 amino acids,with a molecular weight of approximately 17.98 ku,and had no transmembrane region or signal peptide.After the successfully constructed pEGFP-N1-P97R1 eukaryotic vector was transfected into HEK293T cells,IFA confirmed that the P97R1 protein was mainly located in the nucleus.The results indicated that the prokaryotic and eukaryotic expression systems of P97R1 were successfully constructed in this study,and specific polyclonal antibodies were prepared.The nuclear localiza-tion characteristics of the P97R1 protein in eukaryotic cells were also clarified,laying a foundation for in-depth analysis of the biological function of the P97R1 protein and the development of new vaccine strate-gies based on this antigen for porcine mycoplasma pneumonia.