鹅VDR基因克隆及其多克隆抗体制备与鉴定

doi:10.16431/j.cnki.1671-7236.2024.03.031

Abstract

Abstract: 【Objective】 Vitamin D₃ (VD₃) is an important steroid hormone involved in the regulation of various physiological activities in animals.Its bioactive metabolite 1,25 (OH)₂D₃ performs the biological function by binding to vitamin D receptor (VDR).Cloning of goose VDR gene CDS and preparation of its polyclonal antibody would provide the basic support for further research on the molecular mechanism of VDR-mediated VD₃ regulation of various physiological activities in geese.【Method】 The CDS sequence of VDR gene in geese was obtained by the gene cloning method,and the structure and function of the encoded protein were predicted by bioinformatic methods.The recombinant plasmid of VDR gene in geese was constructed by gene synthesis and sub-cloning methods,and then was transformed into Escherichia coli BL21(DE3) competent cells.The recombinant VDR protein in geese was obtained by induction with IPTG and purification with a nickel column.The rabbit anti-goose VDR polyclonal antibody was prepared with the purified recombinant VDR protein as an immunogen.The antibody titer was detected by indirect ELISA,and the antibody specificity was detected by Western blotting.【Result】 The CDS sequence of VDR gene in geese was successfully cloned with a length of 1 356 bp and was predicted to encode 451 amino acid residues.The coding protein shared a high identity with chicken VDR protein (91.4%),and no signal peptide and transmembrane domain were observed.The coding protein contained an N-terminal nuclear localization sequence and two conserved domains of DNA binding domain and ligand binding domain,suggesting that it belonged to the steroid/thyroid hormone receptor superfamily.The recombinant plasmid pET-30a(+)-VDR was successfully constructed and the recombinant VDR protein in geese was expressed.The purity of the purified recombinant protein was more than 90% and its size was 52 ku as expected.The rabbit anti-goose VDR polyclonal antibody was successfully prepared,and the titer was greater than 1∶1 093 500.Except for the liver tissue,the polyclonal antibody was able to recognize two isoforms of native VDR protein in kidney,duodenum,jejunum and ileum in laying geese.【Conclusion】 The CDS region of VDR gene in geese was successfully cloned,and the rabbit anti-goose VDR polyclonal antibody was prepared.The polyclonal antibody had a high specificity and could be used for further study of VDR-mediated biological function in geese.

Key words: goose; VDR gene; cloning; polyclonal antibody

CLC Number:

S835

QIN Yifei, CHEN Rong, SHI Zhendan. Cloning of VDR Gene and Preparation and Identification of Its Polyclonal Antibody in Geese[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 1194-1202.

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Cloning of VDR Gene and Preparation and Identification of Its Polyclonal Antibody in Geese

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