HBD-2转基因小鼠的建立及鉴定

人β防御素2（Human beta defensin 2，HBD-2）是人体抗菌肽的重要分子，体外试验研究证明具有广谱抗微生物活性，为开展其在整体水平上的功能研究，建立HBD-2转基因小鼠模型。用分子克隆方法构建了带CMV启动子的HBD-2全长cDNA真核重组表达质粒pCDNA3．1-HBD2，以此为摸板，PCR扩增出带CMV启动子和BGH polyA尾的HBD-2基因片段，用显微注射技术将此微基因导入小鼠雄原核中，于M16培养液培养后经输卵管移植到受体鼠体内让其怀孕产生子代小鼠。PCR法检测外源基因在小鼠基因组中的整合，RT—PCR和免疫组化法检测HBD-2在小鼠组织的表达。PCR检测结果显示17只F1代转基因鼠中4只检测到阳性信号，RT—PCR和免疫组化检测显示HBD-2在其F1代转基因小鼠生殖道、呼吸道、泌尿道以及血管内皮等组织部位广泛表达。实验表明HBIN2基因已整合到小鼠基因组且广泛表达，为进一步研究HBD-2的生物学功能及基因调控提供了有用的整体动物模型。

Construction and Identification of HBD-2 Transgenic Mice

Human beta defensin 2 (HBD-2) may play an important role in human defense against infection. Its antimicrobial capacity has been fully documented in in vitro study. In order to evaluae its in vivo effects, we developed an HBD-2 transgenic mouse model. The HBD-2 minigene containing CMV promoter, full length of HBD-2 cDNA and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57 X ICR hybridized mouse by microinjection, and offspring were produced. DNA was isolated from the tails of the mouse pups, and the HBD-2 minigene incorporation was analyzed by PCR using HBD-2 specific primers. The HBD-2 gene expression in the multi-tissues of transgenic mice was determined at mRNA level by RT-PCR and at peptide level by immunohistological staining with the use of HBD-2 monoclonal antibody. The results showed that among 17 F0 transgenic mice, HBD-2 positive signal was determined by PCR in 4 mice, suggesting that HBD-2 minigene has been incorporated into the offspring mice. Meanwhile, a widespread expression of HBD-2 mRNA and peptide was detected in the F1 transgenic mice's multi-tissues such as trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium and brain.