家蝇抗菌肽基因Cecropin在COS-7细胞中的表达及产物活性初步研究

The expression of anti-bacterial peptide cecropin gene in COS-7 cells and the preliminary study on the activities of its gene product

目的初步研究家蝇抗菌肽Cecropin cDNA在非洲绿猴肾细胞株COS-7中的表达及其产物的抗菌作用。方法以Cecropin基因为模板设计2条特异引物,扩增在C端含6×His标签的Cecropin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(+)/Cecropin-His_6。以脂质体LipofectamineTM2000为载体,对COS-7细胞进行重组质粒pcDNA3.1(+)/Cecropin-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-TrapHP亲合层析柱分离纯化和Tricine-SDS-PAGE电泳鉴定后,进行杀菌活性的初步检测。结果转染了重组质粒pcDNA3.1(+)/Cecropin-His_6细胞培养上清液的纯化物,行Tricine-SDS-PAGE电泳得到与预期分子量大小相符的单一目的条带,该纯化物对大肠杆菌E.coli K12D31具有一定的杀菌活性。结论家蝇抗菌肽Cecropin cDNA在COS-7中得到了正确表达。

To investigate the expression of anti-bacterial peptide cecrop;n in COS-7 cells, the open reading frame （ORF） sequence of cecropin was amplified at the C-terminus containing 6 x His-marker with two specific primer designed with cecropin gene as template. The gene sequences were recombinant with eukaryotie expression vector pcDNA 3.1（＋ ） after being identified correctly, to construct the recombinant plasmid pcDNA 3.1 （＋）/cecropin-His-6. Thereafter, the liposome lipofectaminer^TM2 000 was used as vector, and COS-7 cells were transfeeted with recombinant plasmid peDNA 3.1 （＋）/eecropin-His-6 and blank vector pcDNA3, 1 （＋）. The normal cells were used as controls. Meanwhile, the supernatants were collected for purification by His-Trap HP affinity chromatograohy column and its bacteriocidal activity was detected 72 hours later. It was demonstrated that the purified product of the cell culture supernatants transfected with the recombinant plasmid pcDNA 3.1 （＋）/ eecropin-His-6 showed only a single target band as demonstrated in the trieine-SDS-PAGE electrophoresis,and it was consistent with the expected molecular weight of the cecropin-His-6 fusion protein. The supernatants of cell cultures transfected with this recombinant plasmid exhibited bacteriocidal activity to E. coli K12 D3l in comparison with that from the normal cells and cells transfected with blank vector, It is concluded that the eDNA of cecropin-Hia-6 can be successfully in COS-7 cells,thus providing the basis for further study of its eukaryotic expression.