Abstract: This study aimed to obtain the gene sequence and expression pattern of bone morphogenetic protein 15 (BMP15) in tissues of New Zealand white rabbits, construct its eukaryotic expression vector, and predict the biological function of BMP15. In this study, 180-day-old healthy New Zealand white rabbits were selected as the study subjects. The CDS region of BMP15 gene was amplified using RT-PCR, the target fragment was ligated to linearized Pmcherry-N1 and pCMV-Myc empty vectors through T4 ligation method, and the eukaryotic expression vector was constructed. The properties and structure of the encoded protein were analyzed by bioinformatics. The overexpression of pCMV-Myc-BMP15 was detected by real-time fluorescence quantitative PCR ( qRT-PCR ) and Western blotting. Subsequently, qRT-PCR was employed to detect the expression level of the BMP15 gene in different tissues. The subcellular localization of BMP15 gene was detected by laser confocal method. The localization of endogenous BMP15 in ovarian tissue of New Zealand white rabbits was determined using immunofluorescence technique. The results revealed that the CDS sequence of BMP15 gene in New Zealand white rabbit was 1 182 bp. PCR and sequencing results confirmed the successful construction of the pCMV-Myc-BMP15 and Pmcherry-N1-BMP15 eukaryotic expression vectors. Bioinformatics analysis indicated that BMP15 gene encoded 393 amino acids. The protein exhibited an instability coefficient of 55.32, with an isoelectric point of 9.69, suggesting stability as a basic protein. BMP15 possessed 26 phosphorylation sites, 15 glycosylation sites, a signal peptides, and lacked transmembrane domains. Phylogenetic analysis revealed that New Zealand white rabbit had a close relationship with Sus Scrofa and a distant relationship with Gallus. Secondary and tertiary structure analysis indicated that BMP15 was a hybrid protein, primarily composed of α-helices, irregular coils, extended strands, and β-turns. Protein interaction prediction suggested that BMP15 protein had interactions with FSHR, FIGLA, BMPR1B, AMHR2, and NOBOX proteins related to ovarian growth and development. Tissue expression analysis demonstrated specific expression in ovarian tissues. Confocal laser scanning revealed cytoplasmic expression of BMP15. Transfection of pCMV-Myc-BMP15 into 293T cells resulted in significant upregulation at both mRNA and protein levels. Immunofluorescence detection of ovarian tissue confirmed cytoplasmic localization of BMP15 in granulosa cells. In this study, the eukaryotic expression vector of BMP15 was successfully constructed, and the physical and chemical properties, as well as biological characteristics of BMP15 gene and its encoded protein, were predicted and analyzed. BMP15 gene overexpression was successfully achieved in HEK293T cells, and information regarding subcellular localization, tissue expression, and distribution in ovarian tissue was obtained. These findings provide a theoretical basis for the subsequent studies on the function and mechanism of BMP15 gene in ovarian growth and development.

Key words: New Zealand white rabbit, BMP15 gene, cloning, tissue expression, cell localization