fib_H启动子驱动hGM_CSF在转基因家蚕丝腺中的表达研究_李曦.pdf

fib-H 启 子驱 hGM-CSF 在转基因家蚕丝腺中的表达研究 李曦 赵越 曹广力 薛仁宇 贡成良 (苏州大学基础医学与生物科学学院，江苏苏州 215123) 摘 要 为了建立家蚕表达hGM-CSF 的转基因技术体系，我们构建了基于piggyBac 转座子的带有人粒细胞-巨噬 细胞集落刺激因子基因（hGM- CSF ）和新霉素抗性基因(neo)表达元件的转基因载体pigA GFP-FH-hGM-CSF-IE-neo。 3 将转基因载体导入家蚕进行转基因丝腺工厂表达hGM-CSF 探索，本研究对几种传 的导入方法做了比较，并对新 的更简便的导入方法进行了探索，转基因处理后的家蚕通过G418 和荧光双重筛选，结合PCR 和southern blot 进行 分子生物学鉴定，获得了转基因家蚕，ELISA 检测表明G3 代转基因家蚕丝腺 hGM-CSF 的表达水平为5.149ng/g 冻干组织。 关键词 转基因家蚕、hGM-CSF、p iggyBac、GFP、丝腺生物工厂 Expression of hGM-CSF in Silkworm Silk Gland Bioreactor LI Xi ZHAO Yue ZHOU Wen-Lin CAO Guang-Li XUE Ren-Yu GONG Cheng-Liang (College of Life Sciences, Soochow University, Suzhou Jiangsu 215123, China) Abstract To generate stable transformation of Bombyx mori silkworm expressing hGM-CSF, The transgenic vector pigA GFP-FH-hGM-CSF-IE-neo contains a hGM- CSF gene controlled by fibroin H-chain promoter and a neomycin 3 resistance gene controlled by baculovirus early promoter. In order to introduce the transposon vector into Bombyx mori, several transgenic methods such as sperm-mediated gene transfer, particle delivery system transfer, egg pricking and gonadal inj ection were used. And screened with GFP and antibiotics G418, combined identification of PCR and southern blot, transgenic silkworms were obtained. The result of western blot displayed that the molecular weight of hGM-CSF expressed by transgenic silkworm silk gland was 22 kDa. The result of ELISA showed that the expression level of hGM-CSF in transgenic silkworm silk gland was 5.149 ng per gram of freeze-dried silk gland. Keywords: Bombyx mori, p iggyBac transposonm, hGM-CSF, Transgenic silkworm 家蚕（Bombyx mori ）是我国传 的经济昆虫，其五龄幼虫丝腺可分泌大量的丝蛋白，并具 有表达高纯度外源蛋白的能力。目前，利用家蚕作为生物反应器，生产医药、保健用途的蛋白以 成为养蚕业新的发