人β-防御素-2基因表达及其抗菌活性

Gene expression of human β defensin 2 and its antimicrobial activity

目的获得人β-防御素-2(humanβ-defensin-2,hBD2)基因并构建其真核表达载体,转染小鼠纤维母细胞(L929),检测其表达,观察hBD2的体外抗菌活性。方法采用逆转录(RT)-PCR方法从人宫颈癌组织获取人β-防御素-2基因,然后进行克隆扩增,重组真核表达载体pCAGG-hBD2,通过磷酸钙共沉淀法将pCAGG-hBD2导入L929细胞,并用RT-PCR法检测hBD2的表达,用Kirby-Bauer纸片扩散法检测其抗菌活性。结果RT-PCR凝胶电泳及测序分析的结果显示,所克隆的hBD2基因序列正确,并成功构建了真核表达载体pCAGG-hBD2。Kirby-Bauer纸片法显示,hBD2对金黄色葡萄球菌、铜绿假单胞菌有明显的杀灭效应。结论转染pCAGG-hBD2的L929细胞能高效表达hBD2,其对金黄色葡萄球菌、铜绿假单胞菌有杀灭作用。

Objective To obtain and construct eukaryotic expression vector of hBD2. To examine the expression of hBD2 in L929 and observe the antimicrobial activity of recombinant hBD2. Methods The total RNA was isolated from human cervical carcinoma tissue and hBD2 cDNA fragments were obtained by RT - PCR amplification with specific primers, and inserted into pCAGG vector. The expression of hBD2 in L929 was examined by RT - PCR, the antimicrobial activity of recombinant hBD2 was assessed by Kirby - Bauer disc agar diffusion test. Results It was proved that we successfully cloned the cDNA of hBD2 and constructed its recombinant eukaryotic expression vector pCAGG - hBD2. Recombinant hBD2 in supernate showed antimicrobial activity. Conclusion Recombinant expression vector of hBD2 could be effectively expressed in L929, and the recombinant hBD2 in supernate showed obvious antimicrobial effects toward some standard bacteria lines.