家蝇抗菌肽Defensin基因在COS-7细胞中的瞬时表达  

Expression and Analysis of Its Activity of Anti-bacterial Peptide Defensin in COS-7 Cells

作　　者：金小宝[1] 朱家勇[1] 马艳[1] 刘雷山[1] 

机构地区：[1]广东药学院基础学院寄生虫学教研室,广东广州510224

出　　处：《生物技术》2006年第4期10-11,共2页Biotechnology

基　　金：广州市科技攻关项目资助("家蝇抗菌肽的制备及其生物功能评价";No.2005Z3-E0211)

摘　　要：目的:研究家蝇抗菌肽Defensin cDNA在非洲绿猴肾细胞株COS-7中的表达情况,并对表达产物的抗菌作用进行初步检测。方法:以Defensin基因为模板设计特异性引物,扩增在C端含6×His标签的Defensin开放阅读框序列,将此序列与真核表达载体pcDNA3.1(+)进行重组,构建重组质粒pcDNA3.1(+)/Defensin-His 6。以阳离子脂质体LipofectamineTM2000为载体,对宿主细胞COS-7进行重组质粒pcDNA3.1(+)/Defensin-His 6和空载体pcDNA3.1(+)的转染,72h后收集细胞培养上清液,表达产物经His-Trap HP亲合层析柱分离纯化和Western blotting鉴定后,进行杀菌活性的初步检测。结果:重组质粒pcDNA3.1(+)/Defensin-His 6组细胞培养上清液的纯化物,行Western blotting得到了分子量大小约为10.0kD的单一目的条带,与预期相符;杀菌活性试验中发现:该纯化物对大肠杆菌E.coliK12D31具有一定的杀菌活性。结论:家蝇抗菌肽Defensin基因在宿主细胞COS-7中得到了正确表达。Objective：To investigate the expression and analysis ofits activity of antibacterial peptide defensin in COS- 7 cells.Methods：Tbe Defensin- His_6 was synthesized by the specific primers with the 6 x His tag sequence. The gene was inserted into pcDNA3.1 （ ＋ ）, an eukaryotic vector, after being identified correctly. As a result, the vector pcDNA3.1（ ＋ ）/Defensin- His_6 was constructed. Thereafter, the liposome Lipofectamine^TM 2000 was employed as the vector, and the COS - 7 cells were transfected with liposome pcDNA3.1 （ ＋ ）/Defensin-His_6 and blank vector pcDNA3.1 （ ＋ ）. The normal cells were taken as the control. The supematant was collected for purification by His - Trap HP affnity chromatography column and the detection of its bactericidal activity after 72h ours. Results： Western blotting demonstrated that the protein bands were 10.0 kD or so and were consisent with the expected molecular weight of the fusion proteins of Defensin- His_ 6. The supernatant of the cells transfected by pcDNA3.1 （＋）/Defensin - His _ 6 exhibited bactericidal activity to E. coli K12 D31 when compared with that from normal cells and in cells transfeeted with blank vectors. Conclusion： The expression of Defensin - His _ 6 eDNA was proved to be genuine in COS - 7 cells.

关 键 词：天蚕素 真核表达 转染 杀菌活性 

分 类 号：Q786[生物学—分子生物学]