重组人SOD1/3转基因山羊的制备及表达产物的检测
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摘要
设计了以hSOD1、hSOD3为编码序列,以山羊β-酪蛋白/CMV杂合启动增强子构建乳腺特异性表达载体rhSOD1、rhSOD3,共转染母山羊胎儿成纤维细胞,采用PCR和扩增产物序列分析筛选获得SOD1/3克隆细胞株,应用体细胞核移植(SCNT)制备双转基因山羊。出生小羊经PCR和扩增产物序列分析验证是否成功整合外源基因,经Western blotting、ELISA及体外活性检来验证分析表达产物。结果表明:获得SOD1/3转基因山羊胎儿成纤维细胞系6株;原代双转基因体克隆山羊1只(♀);从该转基因羊乳汁中检测到rhSOD1、rhSOD3,浓度分别为:88.81±8.36 mg/L和267.82±12.67 mg/L;转基因羊乳汁中重组人SOD酶活性为1 432±157 U/mL。研究表明,以双载体和单标记基因转染山羊胎儿成纤维细胞可获得双基因整合转基因细胞系,并且以SOD1与SOD3功能基因均可在山羊乳腺中共同表达,表达产物具有较好的生物学活性。
In this study,we employed human SOD1(hSOD1)and SOD3(hSOD3)as encoding sequence along with goat beta-casein/CMV chimeric promoter to construct mammary gland-specific expression vectors rhSOD1 and rhSOD3,which then co-transfected goat fetal fibroblast cells. PCR and sequence analysis of the amplified products were applied to screen and obtain the SOD1/3 cloned lines,and somatic cell nuclear transfer(SCNT)was used to prepare bitransgenic goat. Whether or not successful integration of foreign genes in newborn goats was verified by PCR and sequence analysis of amplification product,and the expressed product was analyzed by Western blotting,ELISA and in vitro activity test. The results were as such:6 SOD1/3 transgenic goat fetal fibroblast cell lines were obtained,one original bitransgenic goat(♀)was prepared,rhSOD1 and rhSOD3 were detected in the milk from the transgenic goats,and their concentrations were 88.81± 8.36 mg/L and 267.82± 12.67 mg/L,respectively. The biological activity of recombinant human SOD in the goat milk was 1 432±1 57 U/mL. This study implies that two expression vectors and single marker gene can simultaneously transfect goat fetal fibroblast cells to obtain the transgenic cell line with double gene integration,the SOD1 and SOD3 functional genes may co-express in the mammary gland,and the expressed products show fine biological activity.
In this study,we employed human SOD1(hSOD1)and SOD3(hSOD3)as encoding sequence along with goat beta-casein/CMV chimeric promoter to construct mammary gland-specific expression vectors rhSOD1 and rhSOD3,which then co-transfected goat fetal fibroblast cells. PCR and sequence analysis of the amplified products were applied to screen and obtain the SOD1/3 cloned lines,and somatic cell nuclear transfer(SCNT)was used to prepare bitransgenic goat. Whether or not successful integration of foreign genes in newborn goats was verified by PCR and sequence analysis of amplification product,and the expressed product was analyzed by Western blotting,ELISA and in vitro activity test. The results were as such:6 SOD1/3 transgenic goat fetal fibroblast cell lines were obtained,one original bitransgenic goat(♀)was prepared,rhSOD1 and rhSOD3 were detected in the milk from the transgenic goats,and their concentrations were 88.81± 8.36 mg/L and 267.82± 12.67 mg/L,respectively. The biological activity of recombinant human SOD in the goat milk was 1 432±1 57 U/mL. This study implies that two expression vectors and single marker gene can simultaneously transfect goat fetal fibroblast cells to obtain the transgenic cell line with double gene integration,the SOD1 and SOD3 functional genes may co-express in the mammary gland,and the expressed products show fine biological activity.
引文
[1]程安阳,范立强,赵健,等.重组人SOD2/3杂合酶的制备及其生物活性初探[J].华东理工大学学报, 2011, 37(6):715-721.
[2]周大海,范立强,赵健,等.重组人SOD2/3杂合酶原核表达及纯化方法的研究[J].食品与药品, 2011, 13(11):385-388.
[3]Wang YL, Zhao SH, Bai L, et al. Expression systems and species used for transgenic animal bioreactors[J]. Biomed Res Int, 2013.doi:10. 1155/2013/580463.
[4]Cheng Y, An LY, Yuan YG, et al. Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice[J]. Molecular Reproduction and Development, 2012, 79(8):573-585.
[5]Yuan YG, An LY, Yu BL, et al. Expression of recombinant human alpha-lactalbumin in the milk of transgenic goats using a hybrid pomoter/enhancer[J]. Journal of Analytical Methods in Chemistry,2014. doi:10. 1155/2014/281031.
[6]Song SZ, Ge X, Cheng YB, et al. High-level expression of a novel recombinant human plasminogen activator(rhPA)in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro[J]. Mol Biol Rep, 2016, 43(8):775-783.
[7]Cui D, Li J, Zhang LL, et al. Generation of bi-transgenic pigs overexpressing human lactoferrin and lysozyme in milk[J].Transgenic Res, 2015, 24(2):365-373.
[8]Simpson RB, Spielmann A, Margossian L, et al. A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes:Frequent co-transformation of two distinct T-DNAs[J].Plant Mol Biol, 1986, 6(6):403-415.
[9]安礼友,袁玉国,于宝利,等.单、双标记基因筛选的转人乳铁蛋白基因供核细胞生产克隆山羊效率的比较[J].生物工程学报, 2012, 28(12):1482-1491.
[10]Eiamphungporn W, Yainoy S, Prachayasittikul V. Enhancement of solubility and specific activity of a Cu/Zn superoxide dismutase by Co-expression with a copper chaperone in Escherichia coli[J].Iranian J Biotechnol, 2016, 14(4):243-249.
[11]Lalrinkima H, Raina OK, Chandra D, et al. Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica[J]. Experimental Parasitology, 2015, 2015(151-152):1-7.
[12]李新鸣,孙冶,肖纯凌.表达人源SOD的毕赤酵母重组子的构建[J].中国医科大学学报, 2017, 46(12):1117-1123.
[13]Garay-Arroyo A, Lledias F, Hansberg W, et al. Cu, Zn-superoxide dismutase of Saccharomyces cerevisiaeis required for resistance to hyperosmosis[J]. FEBS Letters, 2003, 539(1-3):68-72.
[14]时桂芹,韩亚伟,李天笑,等.酿酒酵母Cu/ZnSOD基因的克隆和序列分析[J].基因组学与应用生物学, 2018, 37(7):2836-2842.
[15]He HJ, Yuan QS, Yang GZ, et al. High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells[J]. Protein Expression and Purification, 2002, 24(1):13-17.
[16]Khan AH, Bayat H, Rajabibazl M, et al. Humanizing glycosylation pathways in eukaryotic expression systems[J]. World J Microbiol Biotechnol, 2017, 33(1):1-12.
[17]XuY,DaiXL,LiuBD,etal.Cloning,expression,and characterization of Fe-SOD from Isoetes sinensis[J]. Genet Mol Res, 2016, 15(4):1-10.
[18]Kaouthar F, Ameny FK, Yosra K, et al. Responses of transgenic Arabidopsis plants and recombinant yeast cells expressing a novel durum wheat manganese superoxide dismutase TdMnSOD to various abiotic stresses[J]. J Plant Physiol, 2016, 198(2016):56-68.
[19]Byun SJ, Ji MR, Jang YJ, et al. Human extracellular superoxide dismutase(EC-SOD)expression in transgenic chicken[J].BMB Reports, 2013, 46(8):404-409.
[20]Mamipour M, Yousefi M, Hasanzadeh M. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding[J]. Int J Biol Macromol, 2017, 2017(102):367-375.
[21]Kogelberg H, Tolner B, Sharma SK, et al. Clearance mechanism of a mannosylated antibody-enzyme fusion protein used in experimental cancer therapy[J]. Glycobiology, 2006, 17(1):36-45.
[22]Lima ISS, Garcez BS, Alves AA, et al. Fat protected and profile of fatty acids goat milk:a review[J]. Revista Brasileira de Higiene e Sanidade Animal, 2016, 10(4):830-840.
[23]Gui XF, Yang P, Qiu YX, et al. Studies on the preparation and the stability of SOD modified by lauric acid[J]. Wuhan University Journal of Natural Sciences, 1998, 3(2):247-250.
[24]MilesiMA,LacanD,BrosseH,etal.Effectofanoral supplementation with a proprietary melon juice concentrate(Extramel(R))on stress and fatigue in healthy people:a pilot,double-blind, placebo-controlled clinical trial[J]. Nutrition Journal, 2009, 8(1):1-7.
[25]Viktorinova I, Wimmer EA. Comparative analysis of binary expression systems for directed gene expression in transgenic insects[J]. Insect Biochem Mol Biol, 2007, 37(3):246-254.
[26]Senlin L, Lee T. Genetic mosaic with dual binary transcriptional systems in Drosophila[J]. Nat Neurosci, 2006, 9(5):703-709.
[27]Gonzalez-Nicolini V, Fussenegger M. A novel binary adenovirusbaseddual-regulatedexpressionsystemforindependent transcription control of two different transgenes[J]. J Gene Med,2005, 7(12):1573-1585.
[28]Tatematsu K, Kobayashi I, Uchino K, et al. Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori[J]. Transgenic Res, 2010, 19(3):473-487.
[2]周大海,范立强,赵健,等.重组人SOD2/3杂合酶原核表达及纯化方法的研究[J].食品与药品, 2011, 13(11):385-388.
[3]Wang YL, Zhao SH, Bai L, et al. Expression systems and species used for transgenic animal bioreactors[J]. Biomed Res Int, 2013.doi:10. 1155/2013/580463.
[4]Cheng Y, An LY, Yuan YG, et al. Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice[J]. Molecular Reproduction and Development, 2012, 79(8):573-585.
[5]Yuan YG, An LY, Yu BL, et al. Expression of recombinant human alpha-lactalbumin in the milk of transgenic goats using a hybrid pomoter/enhancer[J]. Journal of Analytical Methods in Chemistry,2014. doi:10. 1155/2014/281031.
[6]Song SZ, Ge X, Cheng YB, et al. High-level expression of a novel recombinant human plasminogen activator(rhPA)in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro[J]. Mol Biol Rep, 2016, 43(8):775-783.
[7]Cui D, Li J, Zhang LL, et al. Generation of bi-transgenic pigs overexpressing human lactoferrin and lysozyme in milk[J].Transgenic Res, 2015, 24(2):365-373.
[8]Simpson RB, Spielmann A, Margossian L, et al. A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes:Frequent co-transformation of two distinct T-DNAs[J].Plant Mol Biol, 1986, 6(6):403-415.
[9]安礼友,袁玉国,于宝利,等.单、双标记基因筛选的转人乳铁蛋白基因供核细胞生产克隆山羊效率的比较[J].生物工程学报, 2012, 28(12):1482-1491.
[10]Eiamphungporn W, Yainoy S, Prachayasittikul V. Enhancement of solubility and specific activity of a Cu/Zn superoxide dismutase by Co-expression with a copper chaperone in Escherichia coli[J].Iranian J Biotechnol, 2016, 14(4):243-249.
[11]Lalrinkima H, Raina OK, Chandra D, et al. Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica[J]. Experimental Parasitology, 2015, 2015(151-152):1-7.
[12]李新鸣,孙冶,肖纯凌.表达人源SOD的毕赤酵母重组子的构建[J].中国医科大学学报, 2017, 46(12):1117-1123.
[13]Garay-Arroyo A, Lledias F, Hansberg W, et al. Cu, Zn-superoxide dismutase of Saccharomyces cerevisiaeis required for resistance to hyperosmosis[J]. FEBS Letters, 2003, 539(1-3):68-72.
[14]时桂芹,韩亚伟,李天笑,等.酿酒酵母Cu/ZnSOD基因的克隆和序列分析[J].基因组学与应用生物学, 2018, 37(7):2836-2842.
[15]He HJ, Yuan QS, Yang GZ, et al. High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells[J]. Protein Expression and Purification, 2002, 24(1):13-17.
[16]Khan AH, Bayat H, Rajabibazl M, et al. Humanizing glycosylation pathways in eukaryotic expression systems[J]. World J Microbiol Biotechnol, 2017, 33(1):1-12.
[17]XuY,DaiXL,LiuBD,etal.Cloning,expression,and characterization of Fe-SOD from Isoetes sinensis[J]. Genet Mol Res, 2016, 15(4):1-10.
[18]Kaouthar F, Ameny FK, Yosra K, et al. Responses of transgenic Arabidopsis plants and recombinant yeast cells expressing a novel durum wheat manganese superoxide dismutase TdMnSOD to various abiotic stresses[J]. J Plant Physiol, 2016, 198(2016):56-68.
[19]Byun SJ, Ji MR, Jang YJ, et al. Human extracellular superoxide dismutase(EC-SOD)expression in transgenic chicken[J].BMB Reports, 2013, 46(8):404-409.
[20]Mamipour M, Yousefi M, Hasanzadeh M. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding[J]. Int J Biol Macromol, 2017, 2017(102):367-375.
[21]Kogelberg H, Tolner B, Sharma SK, et al. Clearance mechanism of a mannosylated antibody-enzyme fusion protein used in experimental cancer therapy[J]. Glycobiology, 2006, 17(1):36-45.
[22]Lima ISS, Garcez BS, Alves AA, et al. Fat protected and profile of fatty acids goat milk:a review[J]. Revista Brasileira de Higiene e Sanidade Animal, 2016, 10(4):830-840.
[23]Gui XF, Yang P, Qiu YX, et al. Studies on the preparation and the stability of SOD modified by lauric acid[J]. Wuhan University Journal of Natural Sciences, 1998, 3(2):247-250.
[24]MilesiMA,LacanD,BrosseH,etal.Effectofanoral supplementation with a proprietary melon juice concentrate(Extramel(R))on stress and fatigue in healthy people:a pilot,double-blind, placebo-controlled clinical trial[J]. Nutrition Journal, 2009, 8(1):1-7.
[25]Viktorinova I, Wimmer EA. Comparative analysis of binary expression systems for directed gene expression in transgenic insects[J]. Insect Biochem Mol Biol, 2007, 37(3):246-254.
[26]Senlin L, Lee T. Genetic mosaic with dual binary transcriptional systems in Drosophila[J]. Nat Neurosci, 2006, 9(5):703-709.
[27]Gonzalez-Nicolini V, Fussenegger M. A novel binary adenovirusbaseddual-regulatedexpressionsystemforindependent transcription control of two different transgenes[J]. J Gene Med,2005, 7(12):1573-1585.
[28]Tatematsu K, Kobayashi I, Uchino K, et al. Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori[J]. Transgenic Res, 2010, 19(3):473-487.