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

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22 kDa，ELISA检测结果显示hGM-CSF在转化细胞系里的表达水平大约为2 814.7 pg/106个细胞。

Abstract:

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.