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.
出处
《生物医学工程学杂志》
EI
CAS
CSCD
北大核心
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)
关键词
人β防御素4
基因合成
克隆
原核表达
Human β defensin 4（HBD4） Gene synthesis Cloning Prokaryotic expression
