非转座子载体介导的稳定转化家蚕BmN细胞表达人粒细胞-巨噬细胞集落刺激因子  

Expression of hGM-CSF in transformed silkworm BmN cells mediated by non-transposon vector

作　　者：陈慧梅[1] 曹广力[1] 薛仁宇[1] 贡成良[1] 

机构地区：[1]苏州大学基础医学与生物科学学院,苏州215123

出　　处：《生物工程学报》2010年第6期830-836,共7页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展计划项目(973计划)(No.2005CB121000);苏州大学重大应用研究培育项目(No.Q3134991)资助~~

摘　　要：为了建立非转座子载体介导的持续表达外源基因的转化家蚕BmN细胞系,将家蚕核型多角体病毒极早期基因(ie-1)启动子控制的人粒细胞-巨噬细胞集落刺激因子(hGM-CSF)基因的表达盒克隆至pIZT/V5-His,获得重组载体pIZT-IE-hGM-CSF,该载体转染家蚕BmN细胞后,通过博莱霉素(Zeocin)筛选获得了稳定转化细胞系IE-hGM-CSF。转基因细胞基因组经PCR鉴定,成功检测到ie-hGM-CSF,Western blotting分析结果显示转化细胞表达的重组hGM-CSF的大小为22kDa,ELISA检测结果显示hGM-CSF在转化细胞系里的表达水平大约为2814.7pg/106个细胞。To develop the stable transformants of the silkworm （Bombyx mori） BmN cells that could continuously express the exogenous gene based on a non-transposon vector,an expression cassette containing human granucyto-macrophage colony-stimulating factor （hGM-CSF） gene driven by ie-1 promoter from B.mori nucleopolyhedrovirus was inserted into pIZT-V5-His to form a recombinant vector pIZT-IE-hGM-CSF,followed by transfecting the constructant into BmN cells,the stable ie-hGM-CSF cell lines were obtained after being selected with Zeocin.PCR result using the genomic DNA of the transformed BmN cells as template illustrated a specific fragment of ie-hGM-CSF,and Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kDa in the transformed cells,meanwhile,the expression level of hGM-CSF determined by ELISA was about 2 814.7 pg in 106 transformed BmN cells.

关 键 词：转化 pIZT/V5-His HGM-CSF BmN细胞 

分 类 号：Q78[生物学—分子生物学]