非转座子载体介导的稳定转化家蚕 BmN 细胞表达人粒.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2010, June 25; 26(6): 830−836 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ ©2010 CJB, All rights reserved. 生物技术与方法 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 陈慧梅，曹广力，薛仁宇，贡成良 苏州大学基础医学与生物科学学院，苏州 215123 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。 关键词: 转化，pIZT/V5-His ，hGM-CSF ，BmN 细胞 Expression of hGM-CSF in transformed silkworm BmN cells mediated by non-transposon vector Huimei Chen, Guangli Cao, Renyu Xue, and Chengliang Gong College of Pre-clinical Medical and Biological Science, Soochow University, Suzhou 215123, China 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 demonstra