摘要
目的人β-防御素4(humanβ-defensin 4,HBD 4)对多种病原菌有杀伤作用。HBD4的自然表达水平极低,体外获得难度较大且价格昂贵。利用基因工程技术生产重组的HBD4成为研究的焦点。文中旨在构建HBD4真核表达载体,探讨真核细胞表达HBD4的可行性。方法用PCR法从已构建的重组克隆载体pMD18-T/HBD4中扩增出HBD4全长编码基因,克隆入真核表达载体pEGFP-N2,构建pEGFP-N2/HBD4重组真核表达载体;通过脂质体转染法将pEGFP-N2/HBD4导入HEK293细胞,采用RT-PCR、荧光显微镜、Western blot检测HBD4的mRNA和蛋白表达情况,并对表达的HBD4进行抗菌活性的初步研究。结果构建的重组真核表达载体pEGFP-N2/HBD4,经酶切鉴定、测序,证实插入的序列与Genebank中的HBD4基因序列完全相同。将pEGFP-N2/HBD4转染HEK293细胞,在转染细胞检测到HBD4mRNA表达。荧光显微镜下可在细胞膜及细胞质中观察到绿色荧光。Western blot分析显示:在pEGFP-N2/HBD4转染的HEK293细胞及细胞培养液中检测到了HBD4蛋白的表达。Kirby—Bauer纸片扩散法证实表达的HBD4蛋白对铜绿假单胞菌具有较强的杀伤作用。结论成功构建了HBD4基因的真核表达载体,并在HEK293细胞中表达出具有抗菌活性的HBD4多肽。
Objective Human β-defensin 4 （HBIM） has lethal effects on many kinds of pathogens. It is difficult and expen- sive to obtain HBD4 in vitro because of its low natural expression. Therefore it has become a focus of research to produce recombinant HBD4 using genetic engineering technology. This study was to construct a eukaryotic expression vector of the HBD4 gene, and explore the possibility of the expression of HBD4 in eukaryotic cells. Methods The full-length gene of HBD4 was amplified by PCR from the re- combinant cloning vector pMD18-T/HBIM and cloned into the eukaryotic expression vector pEGFP-N2 to construct pEGFP-N2/HBIM. Then the pEGFP-N2/HBD4 was transfected into HEK293 cells with a liposome infection protocol. RT-PCR, fluorescence microscopy and Western blot were used to identify the expressions of HBD4 mRNA and HBD4 peptide and the antimicrobial activity of the secreted HBD4 was evaluated. Results Endonuclease digestion and sequence analysis demonstrated that the inserted sequences of the recombinant ex- pression vector pEGFP-N2/HBD4 was identical to that of the full-length HBD4 gene in Gene Bank. The expression of HBD4 mRNA was detected by RT-PCR in the transfected cells. Green fluorescence was located mainly in the cellular membrane and cytoplasm of the trans- fected cells. HBIM was expressed both in the transfected cells and cell culture fluid, and HBIM proteins secreted into the culture medium showed strong antimicrobial activity against P. acruginosa ATCC 27853. Conclusion The eukaryotic expression vector pEGFP- N2/HBIM was successfully constructed. HEK293 ceils transfected with pEGFP-N2/HBIM can express bioactive HBIM.
出处
《医学研究生学报》
CAS
北大核心
2012年第10期1020-1023,共4页
Journal of Medical Postgraduates
基金
陕西省科技攻关项目(2009K12-01)
关键词
人β-防御素4
基因克隆
真核表达载体
基因转染
Human β-defensin 4
Gene cloning
Eukaryotic expression vector
Gene transfection
