人β防御素4基因原核表达载体的构建及其表达  

Construction of Prokaryotic Expression Vector and Expression of Human β Defensin 4 Gene

作　　者：曹玉红[1] 张国成[1] 张光运[1] 徐彦鸣[2] 王立锋[2] 

机构地区：[1]第四军医大学西京医院,西安710032 [2]第四军医大学生物化学与分子生物学教研室,西安710032

出　　处：《生物医学工程学杂志》2008年第5期1166-1169,共4页Journal of Biomedical Engineering

基　　金：第四军医大学西京医院科技创新基金资助项目(XJCX06M21)

摘　　要：人β防御素4(Human β defensin 4,HBD4)是一种具有广谱抗微生物活性的抗菌肽。在人体先天免疫尤其上皮及黏膜防御中起重要作用。本研究用重叠延伸PCR扩增合成HBD4cDNA全长,并克隆入pMD18-T载体,用PCR法扩增HBD4成熟肽(mature HBD4,mHBD4)编码序列并克隆入表达载体pGEX-4T-2中,在异丙基-β-D-硫代半乳糖苷(isopropy-β-D-thiogalactoside,IPTG)诱导下,表达HBD4与谷胱甘肽S-转移酶(glutathione S-transferase,GST)的融合多肽。用酶切鉴定、核酸测序、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳等进行鉴定。结果表明:我们成功合成了HBD4全长编码基因,构建了克隆载体pMD18-T/HBD4和原核表达载体pGEX-4T-2/mHBD4,并在大肠杆菌中成功表达融合蛋白GST-HBD4。Human β defensin 4 is a small cationic peptide with a broad range of antimicrobial activity. It plays an important role in innate immunity of human body, especially in mucosal and epithelial defense. In this study, the full-length encoding gene of HBD4 was synthesized by overlap extension polymerase chain reaction and inserted into cloning vector pMD18-T. The gene encoding mature peptide of HBD4 was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-2. Then pGEX-4T-2/mHBIN was transformed into E. coli DH5a, which was induced by isopropy-β-D- thiogalactoside（ IPTG）. The identification was made by means of endonuclease digestion, DNA sequencing, sodium dodecyl sulphate -polyacrylamine gel electrophoresis（SDS-PAGE）. The results showed that the synthesized gene and cloned gene were identical to the HBIN gene sequence registered in GenBank and were successfully cloned into cloning vector pMD18-T and prokaryotic expression vector pGEX-4T-2. After IPTG induction, the GST-HBD4 fusion protein was successfully exoressed in E. coli.

关 键 词：人β防御素4 基因合成 克隆 原核表达 

分 类 号：Q786[生物学—分子生物学]