转基因家蚕丝腺生物反应器表达hIGF-Ⅰ的研究
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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。
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.
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.
引文
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「3」刘春,徐汉福,陈玉琳,等.家蚕转基因研究及肌动蛋白A3启动子的表达分析[J].蚕学通讯.2007.27(3):1～6
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「7」周文林,王崇龙,刘波,曹广力,薛仁宇,沈卫德,贡成良.piggyBac转座子介导的家蚕细胞转基因研究初探,蚕业科学,2007,33(1):30-35
「8」诸戌娴,曹广力,薛仁宇,周文林,刘炜彬,贡成良.家蚕丝胶蛋白基因1(ser-1)启动子的克隆及其活性分析.科技通报2007,23(6):828-834
「9」郭秀洋,周泽扬,冯丽春等.利用精子介导法向蚕卵导入外源基因的研究[J].生物化学与生物物理进展, 2001, 28(3): 423-425
「10」曹广力,薛仁宇,沈卫德,贡成良.基于piggyBac转座子转hGM-CSF基因的家蚕研究.蚕业科学,2006,32(3):324-327
「11」徐岩.人胰岛素样生长因子Ⅰ型在大肠杆菌和家蚕中的表达[D].硕士学位论文,苏州:苏州大学,2006
[1] Tomita M, Munetsuna H, Sato T, et al. Transgenic silkworms produce recombinant human type III procollagen in cocoons [J]. Nat Biotechnol, 2003,21:52-56
[2] Takahiro A, Masahiro T, Katsuhiko S, et al. Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylaseα-subunits and human collagens in posterior silk glands: Production of cocoons that contained collagens with hydroxylated praline residues [J]. J Biotech, 2006,126:205-219
[3] Hino R, Tomita M, Yoshizato K. The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor [J]. Biomaterials, 2006,27:5 715-5 724
[4] Ogawa S, Tomita M, Shimizu K, et al. Generation of transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: Production of recombinant human serum albumin [J]. J Biotechmol, 2007,128:531-54
[5] Kurihara H, Sezutsu H, Tamura T, et al. Production of an active felineinterferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system [J]. Biochem Biophy Research Communicat, 2007,355:976-980
[6]刘炜彬.家蚕丝蛋白Fhx/P25基因启动子的克隆与活性分析及转基因研究[D].[硕士学位论文],苏州:苏州大学,2007
[7]郭秀洋,周泽扬,冯丽春等.利用精子介导法向蚕卵导入外源基因的研究[J].生物化学与生物物理进展, 2001, 28(3): 423-425
[8]曹广力,薛仁宇,何泽等.基于piggyBac转座子转hGM-CSF基因家蚕的研究[J].蚕业科学, 2006, 32(3): 324-327
[9] Xianzong Shi, Robert L Harrison, Jason R Hollister, Ahmed Mohammed, Malcolm J Fraser Jr and Donald L Jarvis. Construction and characterization of new piggyBac vectors for constitutive or inducible expression of heterologous gene pairs and the identification of a previously unrecognized activator sequence in piggybac.BMC Biotechnology 2007, 7:5 doi:10.1186/1472-6750-7-5
[10] Tamura T, Thibert C, Royer C, et al. A piggyBac-derived vector efficiently promotes germ-line transformation in the silkworm Bombyx mori L. [J]. Nat Biotechnol, 2000,18:81-84;
「2」柯为.生物反应器研究新进展(J).生物工程学报,2005 21.No 5
「3」Handler AM, McCombs SD, Fraser MJ, Saul SH: The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly. Proc Natl Acad Sci U S A .1998, 95(13):7520-7525
「4」徐岩.人胰岛素样生长因子Ⅰ型在大肠杆菌和家蚕中的表达[D].硕士学位论文,苏州:苏州大学,2006
「5」何泽.家蚕生物反应器的研究[D].[硕士学位论文],苏州:苏州大学,2006
「6」陈淼.HChEGF在原核和真核中的高效表达及应用研究[D].[硕士学位论文],苏州:苏州大学,2006
「7」Polayes D, Harris R, Anderson D, et al. Focus. 1996,18:10-13.
「8」李保同,邓小昭,乔仁良等.中国公共卫生学报.1998,17(4):219-220.
「1」Gordon J W, Scangos G A, Plotkin D J, et al. Genetic transformation of mouse embryo by microinjection of purified DNA [J]. Proc Natl Acad Sci USA, 1980, 77: 7280-7384.
「2」Palmiter R D, Brinster R E, Hammer M E, et al. Dramatic growth of mice that developed from eggs microinjected with metallothionein-grow thhormone fusion genes [J]. Nater, 1982, 300: 611-615.
「3」Nagueh SF, Chen S, Patel R, et al. Evolution of expression of cardiac phenotypes over a 4-year period in the beta-myosin heavy chain-Q403 transgenic rabbit model of human hypertrophic cardiomyopathy [J]. J Mol Cell Cardiol, 2004, 36(5):663-673.
「4」田村俊樹.トランスジェニックカイコの作出と有用物質の生産[M]. 日本,高分子学会編.バイオロジクス.2004: 044-068
「5」Tamura T, Kanda T, Takiya S, et al. Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm Bombyx mori [J]. Jpn J Genet, 1990, 65:401-410;
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