【作者】 赵越；

【导师】 贡成良；

【作者基本信息】 苏州大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 家蚕丝腺是合成、分泌蚕丝蛋白的重要器官,具有作为生物反应器的基本特点。为了实现人类胰岛素生长因子-I(hIGF-I)在转基因家蚕细胞和丝腺组织中的表达,我们以家蚕丝胶基因启动子(Ser-1)驱动人胰岛素样生长因子(hIGF-I),构建了带有家蚕杆状病毒极早期基因启动子(ie-1)控制的新霉素抗性基因(neo)表达盒的转基因载体pigA3GFP-ser-hIGF-ie-neo,在可表达转座酶的辅助质粒存在下,转染BmN培养细胞,以700~800μg/mL的G418筛选,获得了稳定转化细胞系;ELISA检测检测结果显示,5×105个细胞/ml hIGF-I的表达水平约7.8×103 pg;转基因载体pigA3GFP-Ser-hIGF-ie-neo通过精子介导导入蚕卵,利用neo、gfp基因的双重筛选,经过PCR和点杂交鉴定,获得了转基因家蚕,ELISA检测结果显示,在G1代hIGF-I在每克中部丝腺组织中的表达水平为2.44×103 pg。此外,用家蚕丝素Fhx/P25启动子驱动hIGF-I,构建了带有ie-1启动子控制的neo基因表达盒的转基因载体pigA3GFP- Fhx/P25-hIGF-ie-neo,该载体以精子介导法导入蚕卵,通过筛选并结合分子生物学鉴定,获得了转基因家蚕,ELISA检测结果显示,在G1代hIGF-I在每克后部丝腺组织中的表达水平为152×103 pg。更多还原

【Abstract】 The silk gland of silkworm, as a bioreactor with the basic characteristics, was an important organ for silk protein synthesizing and secreting. To express human insulin-like growth factor-I (hIGF-I) in transformated Bombyx mori cultured cells and silk gland, the transgenic vector pigA3GFP- Ser-hIGF-ie-neo was constructed with a neomycin resistance gene driven by baculovirus ie-1 promoter, hIGF-I gene which under control of silkworm sericin promoter Ser-1. We succeeded to select stable transformation of BmN cells expressing hIGF-I by using antibiotic G418 after BmN cells were transfected with the piggyBac vector and the helper plasmid. The expression level of hIGF-I determined by ELISA was about 7.8×103 pg pg in 5×105 cells. The transgenic vector pigA3GFP-Ser-hIGF-ie-neo was transferred into the eggs by using sperm-mediated gene transfer. Finally, we obtained 2 transgenic silkworms under the screening of neo and gfp genes, and identified with PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2.44×103 pg in each gram of middle silk gland of transgenic silkworm of G1 generation. Besides, another transgenic vector pigA3GFP- Fhx/P25-hIGF-ie-neo was constructed with a neomycin resistance gene driven by ie-1 promoter, hIGF-I gene which under control of silkworm fibroin promoter Fhx/P25. The transgenic vector was also transferred into the eggs by using sperm-mediated gene transfer. We obtained transgenic silkworm aftere being screening and deteced by molecular biology method .Exprssiong lever of hIGF-I was 152×103pg in each gram of posterior silk gland of transgenic silkworm of G1 generation by ELISA.更多还原