番鸭MAPK1蛋白多克隆抗体制备及其在卵泡中表达定位分析OA

[Objective]This study aimed to prepare a polyclonal antibody against mitogen‑activated protein kinase 1(MAPK1)protein,and determine its specificity and applicability.Furthermore,the expression and localization of MAPK1 protein in follicular granulosa cells and follicles were further investigated,thereby providing an effective tool for investigating its role in follicular development and reproductive regulation of Muscovy duck.[Method]Using the cDNA of duck ovary as the template,MAPK1 gene was amplified by PCR and then sequenced.The physicochemical properties and main antigenic epitopes of MAPK1 protein were analyzed using bioinformatics tools,and the immunogenic regions were screened.The immunogen sequence was optimized for Escherichia coli codon usage,synthesized,and cloned into the prokaryotic expression vector pET-30a(+)through homologous recombination.The recombinant expression vector pET30a-MAPK1 was constructed and transformed into Escherichia coli BL21(DE3)competent cells.Recombinant protein MAPK1 expresed in the HB-PET self-induction medium and purifid.The purified protein was mixed with QuickAntibody-Rabbit8W adjuvant and used to immunize New Zealand White rabbits to prepare polyclonal antibodies.The titers,specificities and applicability of the polyclonal antibodies were detected by ELISA,Western blotting,cell immunofluorescence(CIF),tissue immunofluorescence(TIF),and immunohistochemistry(IHC).And using the granulosa cell marker follicle-stimulating hormone receptor(FSHR)as a reference,the expression and localization of MAPK1 were analyzed.[Result]The MAPK1 gene was successfully cloned,with a size of 1 107 bp,encoding 368 amino acids.The theoretical molecular weight of MAPK1 protein was approximately 42 ku.Bioinformatics prediction indicate that MAPK1 protein lacked signal peptide or transmembrane structure.Strong hydrophilicity was observed in 50-100,200-300,and 250-350 amino acid segments,high antigenicity was found in 30-90 and 200-300 amino acid segments,and the amino acid surface exposure probabilities of 30-70,120-150,200-260,and 320-360 amino acid segments were relatively high.The secondary structure of MAPK1 protein was mainly composed of alpha-helices,with typical conserved kinase motifs and a disordered activation loop,and was mainly localized in the cytoplasm and nucleus.Based on these characteristics,the 4-364 amino acids were finally selected as the candidate immunogenic region.The prokaryotic recombinant expression vector pET30-MAPK1 was successfully constructed.The recombinant MAPK1 protein was expressed in a soluble form,with a molecular weight of 48 ku.The polyclonal antibody against duck MAPK1 protein from rabbits was successfully prepared.ELISA results showed that the titer of antibody reached 1∶102 400.The results of Western blotting,CIF,TIF,and IHC showed that the prepared polyclonal antibody could specifically recognize the recombinant protein MAPK1,as well as the endogenous MAPK1 protein in duck follicular granulosa cells and follicles.In granulosa cells,MAPK1 was mainly located in cytoplasm and nucleus of follicular granulosa cells.In follicles,MAPK1 was mainly distributed in granulosa cell layer of follicles,and its expression localization was basically consistent with that of FSHR.[Conclusion]This experiment successfully prepared a polyclonal antibody for MAPK1 of Muscovy duck.This antibody had good specificity and was suitable for the immunological detection of MAPK1 in follicular-related cells and tissues of Muscovy duck.This results provided technical support for the study of the function of MAPK1 in follicle development and reproductive regulation.