比格犬β-防御素cBD1基因的原核表达与鉴定  

Prokaryotic expression and identification of Beaglesβ-defensin cBD1 gene

作　　者：冯培祥 潘福星[1] 毕玉敏[1] 郭学金[1] 陈欣[1] 杨莉[1] 徐守振[1] 王建琳[1] 郭妍妍 尹燕博[1,2,3] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]青岛澳兰百特生物工程有限公司,山东青岛266101 [3]青岛博隆实验动物有限公司,山东即墨266225

出　　处：《中国兽医科学》2014年第1期68-72,共5页Chinese Veterinary Science

基　　金："十二五"农村领域国家高技术研究发展计划(863)项目(2012AA101303)

摘　　要：为获得比格犬β-防御素cBD1基因的表达产物,根据GenBank中登录的犬β-防御素基因cBD1(canis familiarisβ-defensin-like peptide 1)的序列设计引物,提取比格犬睾丸组织总RNA,利用PCR扩增cBD1基因,然后将其克隆至pET-32a(+)载体,构建原核表达质粒pET-32a-cBD1,通过PCR、酶切鉴定和测序确认,将重组质粒转化大肠杆菌BL21(DE3)中,并进行IPTG诱导和His-Ni-resin纯化目的蛋白。经SDS-PAGE检测,获得了约28ku的表达产物,与预期大小的β-防御素cBD1的分子质量相一致。Westernblot分析表明,表达产物与抗His标签鼠单克隆抗体发生反应,显示重组蛋白获得了表达。结果表明,成功表达的cBD1为新型抗菌制剂的研制奠定了试验基础。To gain the expression product of Beagles /？-defensin cBD1 gene, the gene was amplified using Beagles testis total RNA by PCR with primers based on the canine/？-defensin gene cBDl（canis famil- iaris ~defensin-like peptide 1） sequence in GenBank. Then it was cloned into the pET-32a（q-） vector. The recombinant plasmid was certified with PCR identification, DNA sequencing and restriction endonuclease digestion, and was then transformed into Escherichia col i BL21 （DE3）. The ^-defensin eBD1 protein was ex- pressed in resultant strains induced with IPTG and purified with His-Ni-resin. The product was confirmed to be about 28 ku in size by SDS-PAGE. The reaction of expression products with mouse monoclonal anti- His tag by Western-blot test suggested that recombinant protein was expressed. The results showed that the successful expression of cBD1 laid a foundation for development of new antibacterial agents.

关 键 词：比格犬 Β-防御素 原核表达 鉴定 

分 类 号：R979.5[医药卫生—药品]