犬β防御素-1真核表达载体的构建及其在HEK293T细胞中的表达

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

哺乳动物的β-防御素是一类具有广谱抗微生物活性的阳离子小肽。为了克隆和分析犬β防御素-1基因,并建立一套在HEK293T细胞中高效表达犬β防御素-1的方法,本研究采用RT-PCR方法从犬睾丸组织中扩增出犬β防御素-1(cBD-1)的cDNA基因,并将其克隆到pcDNA3.1A载体中,构建了犬β防御素-1基因的真核表达载体pcDNA3.1A-cBD-1,经磷酸钙介导转染HEK293T细胞进行表达。结果表明:克隆的犬β防御素-1基因序列与已发表序列(GenBank编号:NM-001024641)的同源性为99.7%。表达犬β防御素-1经Western-blot检测,证明构建的犬β防御素-1基因表达载体能够在真核细胞中表达,表达产物能够分泌到细胞外。为进一步研究犬β防御素的功能奠定了基础。

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-lgene 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