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Construction of eukaryotic expression vector carrying canine β-defensin-1 gene and its expression in HEK293T cells

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Author(s): JIN Hui-Jun | ZHONG Fei | WU Ling-Juan | XIE Min | WANG Wei | HAN Dong-Mei

Journal: Acta Zoologica Sinica
ISSN 0001-7302

Volume: 54;
Issue: 5;
Start page: 897;
Date: 2008;
Original page

Keywords: Canine β-defensin-1 | Vector construction | HEK 293T cells | Expression

ABSTRACT
Mammalian β-defensins are small cationic peptides that possess broad antimicrobial and physiological activities. To obtain the gene construct of canine β-defensin-1 (cBD-1), we cloned cBD-1gene and established a method to express cBD-1 in HEK293T cells. The cDNA encoding cBD-1 was amplified from canine testicular tissues by RT-PCR, and the cBD-1 gene was inserted into eukaryotic expression vector pcDNA3.1A. Then, the recombinant pcDNA3.1A-cBD-1 plasmids were transfected into HEK293T cells using calcium phosphate. The results showed that the sequence of cBD-1 gene amplified in this research was almost identical to the published sequence (GenBank accession number: NM_001024641) since the homology between them was 99.7%. The expressed cBD-1 protein isolated from cell culture medium was detected by Western-blot suggesting that cBD-1 gene could be secretively expressed in eukaryotic cells. These results also provide a basis for further investigation of canine β-defensin function [Acta Zoologica Sinica 54(5): 897- 902, 2008].
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