重组人血清白蛋白在紫花苜蓿中的转基因表达
|
摘要
为获得高产重组人血清白蛋白的转基因紫花苜蓿"游客"(Medicago sativa L. cv.‘Eureka’)株系用于规模化生产rHSA,构建了rHSA植物表达载体。以紫花苜蓿子叶愈伤组织为受体,通过农杆菌介导法进行遗传转化,筛选出潮霉素抗性植株,提取抗性再生紫花苜蓿植株基因组DNA做PCR鉴定,结果表明重组人血清白蛋白基因片段已整合到紫花苜蓿基因组中;提取转基因植株总蛋白,Western blotting检测结果表明rHSA在转基因植株中成功表达。此结果表明转基因紫花苜蓿可稳定表达rHSA。
Human serum albumin(HSA) is the most abundant protein in human plasma, and is widely used in clinical practice and biopharmaceutical industry. Gene-recombinant HSA(rHSA) is an ideal substitute of plasma-derived HSA at present. In order to make high-yielding transgenic Medicago sativa L. cv. ‘Eureka' for industrial production of rHSA, an expression vector was constructed using a modified p CAMBIA1300 plasmid, and the expression vector constructed was transferred into the cotyledon callus of the plant by Agrobacterium tumefaciens-mediated transformation. After selection of hygromycin-resistant plants, the genomic DNA of the regenerated transgenic plant was extracted for PCR, and the results indicated that the rHSA gene was integrated into the genome of M. sativa L. In addition, total proteins of the transgenic plants were extracted and tested by Western blotting, and the results showed that rHSA was successfully expressed in the plants. The study confirmed that rHSA could be expressed steadily in transgenic Medicago sativa L. and it laid a foundation for further study on separation and purification of rHSA from transgenic Medicago sativa L.
Human serum albumin(HSA) is the most abundant protein in human plasma, and is widely used in clinical practice and biopharmaceutical industry. Gene-recombinant HSA(rHSA) is an ideal substitute of plasma-derived HSA at present. In order to make high-yielding transgenic Medicago sativa L. cv. ‘Eureka' for industrial production of rHSA, an expression vector was constructed using a modified p CAMBIA1300 plasmid, and the expression vector constructed was transferred into the cotyledon callus of the plant by Agrobacterium tumefaciens-mediated transformation. After selection of hygromycin-resistant plants, the genomic DNA of the regenerated transgenic plant was extracted for PCR, and the results indicated that the rHSA gene was integrated into the genome of M. sativa L. In addition, total proteins of the transgenic plants were extracted and tested by Western blotting, and the results showed that rHSA was successfully expressed in the plants. The study confirmed that rHSA could be expressed steadily in transgenic Medicago sativa L. and it laid a foundation for further study on separation and purification of rHSA from transgenic Medicago sativa L.
引文
[1]Matejtschuk P,Dash C H,Gascoigne E W.Production of human albumin solution:a continually developing colloid[J].British Journal of Anaesthesia,2000,85(6):887-895.
[2]Hastings G E,Wolf P G.The therapeutic use of albumin[J].Archives of Family Medicine,1992,1(2):281-287.
[3]Sibbald W J.Fluid therapy in sepsis[C]//Reinhard K,Eyrich K,Sprung C.Sepsis:Current perspectives in pathophysiology and therapy.Berlin:Springer,1994:266-273.
[4]Kaminski M V,Williams S D.Review of the rapid normalization of serum albumin with modified total parenteral nutrition solutions[J].Critical Care Medicine,1990,18(3):327-35.
[5]刘丹丹,刘思国,陈建泉,等.人血清白蛋白及其重组表达研究进展[J].生物技术通讯,2016,27(4):572-575.
[6]Chen Z,He Y,Shi B,et al.Human serum albumin from recombinant DNA technology:Challenges and strategies[J].BbaGen Subjects,2013,1830(12):5 515-5 525.
[7]Chuang V T,Otagiri M.Recombinant human serum albumin[J].Drugs of today,2007,43(8):547-561.
[8]Lawn R M,Adelman J,Bock S C,et al.The sequence of human serum albumin c DNA and its expression in E.coli[J].Nucleic acids research,1981,9(22):6 103-6 114.
[9]Ohya T,Ohyama M,Kobayashi K.Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation[J].Biotechnology and bioengineering,2005,90(7):876-887.
[10]Wu X,Lin Y,Xiong F,et al.The extremely high level expression of human serum albumin in the milk of transgenic mice[J].Transgenic research,2012,21(6):1359-1366.
[11]Echelard Y,Williams J L,Destrempes M M,et al.Production of recombinant albumin by a herd of cloned transgenic cattle[J].Transgenic research,2009,18(3):361-376.
[12]Ogawa S,Tomita M,Shimizu K,et al.Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon:production of recombinant human serum albumin[J].Journal of Biotechnology,2007,128(3):531-544.
[13]Sijmons P C,Dekker B M,Schrammeijer B,et al.Production of correctly processed human serum albumin in transgenic plants[J].Biotechnology,1990,8(3):217-221.
[14]Farran I,Sanchez-Serrano J J,Medina J F,et al.Targeted expression of human serum albumin to potato tubers[J].Transgenic research,2002,11(4):337-346.
[15]Huang LF,Liu Y K,Lu C A,et al.Production of human serum albumin by sugar starvation induced promoter and rice cell culture[J].Transgenic research,2005,14(5):569-581.
[16]He Y,Ning T,Xie T,et al.Large-scale production of functional human serum albumin from transgenic rice seeds[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(47):19 078-19 083.
[17]何平,韦正乙,孙卉,等.紫花苜蓿转基因研究进展[J].草业科学,2013,30(4):627-637.
[18]蔡璐,张瑜,李世歌,等.紫花苜蓿转基因的研究进展分析[J].农业与技术,2016,36(10):18.
[19]赵金梅,李芳,周禾,等.紫花苜蓿组织培养体系的建立[J].核农学报,2010,24(3):507-512.
[20]Dong J L,Liang B G,Jin Y S,et al.Oral immunization with p Bs VP6-transgenic alfalfa protects mice against rotavirus infection[J].Virology,2005,339(2):153-163.
[21]Dus Santos M J,Carrillo C,Ardila F,et al.Development of transgenic alfalfa plants containing the foot and mouth disease virus structural polyprotein gene P1 and its utilization as an experimental immunogen[J].Vaccine,2005,23(15):1 838-1 843.
[22]Legocki A B,Miedzinska K,Czaplinska M,et al.Immunoprotective properties of transgenic plants expressing E2 glycoprotein from CSFV and cysteine protease from Fasciola hepatica[J].Vaccine,2005,23(15):1 844-1 846.
[23]Bellucci M,De Marchis F,Arcioni S.Zeolin is a recombinant storage protein that can be used to produce value-added proteins in alfalfa(Medicago sativa L.)[J].Plant cell,Tissue and Organ Culture,2007,90(1):85-91.
[24]Lee R W,Cornelisse M,Ziauddin A,et al.Expression of a modified Mannheimia haemolytica GS60 outer membrane lipoprotein in transgenic alfalfa for the development of an edible vaccine against bovine pneumonic pasteurellosis[J].Journal of Biotechnology,2008,135(2):224-231.
[25]Liu Z H,Zhang H M,Li G L,et al.Enhancement of salt tolerance in alfalfa transformed with the gene encoding for betaine aldehyde dehydrogenase[J].Euphytica,2011,178(3):363-372.
[26]Deak M,Kiss G B,Korkz C,et al.Transformation of Medicago by Agrobacterum mediated gene transfer[J].Plant Cell Reports,1986,5(2):97-100.
[27]Zhang J Y,Broeckling C D,Blancaflor E B,et al.Overexpression of WXP1,a putative Medicago truncatula AP2 domain-containing transcription factor gene,increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa(Medicago sativa)[J].The Plant journal:for cell and molecular biology,2005,42(5):689-707.
[28]Tesfaye M,Temple S J,Allan D L,et al.Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum[J].Plant physiology,2001,127(4):1 836-1 844.
[29]Strizhov N,Keller M,Mathur J,et al.A synthetic cry IC gene,encoding a Bacillus thuringiensis delta-endotoxin,confers Spodoptera resistance in alfalfa and tobacco[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(26):15 012-15 017.
[30]Samac D A,Smigocki A C.Expression of OryzacystatinⅠandⅡin alfalfa increases resistance to the root-lesion nematode[J].Phytopathology,2003,93(7):799-804.
[31]Avraham T,Badani H,Galili S,et al.Enhanced levels of methionine and cysteine in transgenic alfalfa(Medicago sativa L.)plants over-expressing the Arabidopsis cystathionine gammasynthase gene[J].Plant biotechnology journal,2005,3(1):71-79.
[32]Reddy M S,Chen F,Shadle G,et al.Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa(Medicago sativa L.)[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(46):16 573-16 578.
[33]王彦华,李德锋,齐胜利,等.播种量和品种对紫花苜蓿分枝数和株高的影响[J].草业学报,2017,26(3):183-190.
[34]王茜,纪树仁,沈益新.土壤水分和施氮水平对紫花苜蓿苗期生长的互作效应分析[J].草业学报,2017,26(12):48-55.
[35]王坤龙,王千玉,宋彦军,等.留茬高度对紫花苜蓿根系生长及翌年返青的影响[J].中国饲料,2016(1):17-20.
[36]王伟,贾玉山,格根图,等.种植年限对紫花苜蓿生产力和品质的影响[J].草学,2017(4):11-17.
[37]魏小兰,张纯,唐凌,等.紫花苜蓿蛋白的应用研究[J].四川畜牧兽医,2017,44(5):39-41.
[2]Hastings G E,Wolf P G.The therapeutic use of albumin[J].Archives of Family Medicine,1992,1(2):281-287.
[3]Sibbald W J.Fluid therapy in sepsis[C]//Reinhard K,Eyrich K,Sprung C.Sepsis:Current perspectives in pathophysiology and therapy.Berlin:Springer,1994:266-273.
[4]Kaminski M V,Williams S D.Review of the rapid normalization of serum albumin with modified total parenteral nutrition solutions[J].Critical Care Medicine,1990,18(3):327-35.
[5]刘丹丹,刘思国,陈建泉,等.人血清白蛋白及其重组表达研究进展[J].生物技术通讯,2016,27(4):572-575.
[6]Chen Z,He Y,Shi B,et al.Human serum albumin from recombinant DNA technology:Challenges and strategies[J].BbaGen Subjects,2013,1830(12):5 515-5 525.
[7]Chuang V T,Otagiri M.Recombinant human serum albumin[J].Drugs of today,2007,43(8):547-561.
[8]Lawn R M,Adelman J,Bock S C,et al.The sequence of human serum albumin c DNA and its expression in E.coli[J].Nucleic acids research,1981,9(22):6 103-6 114.
[9]Ohya T,Ohyama M,Kobayashi K.Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation[J].Biotechnology and bioengineering,2005,90(7):876-887.
[10]Wu X,Lin Y,Xiong F,et al.The extremely high level expression of human serum albumin in the milk of transgenic mice[J].Transgenic research,2012,21(6):1359-1366.
[11]Echelard Y,Williams J L,Destrempes M M,et al.Production of recombinant albumin by a herd of cloned transgenic cattle[J].Transgenic research,2009,18(3):361-376.
[12]Ogawa S,Tomita M,Shimizu K,et al.Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon:production of recombinant human serum albumin[J].Journal of Biotechnology,2007,128(3):531-544.
[13]Sijmons P C,Dekker B M,Schrammeijer B,et al.Production of correctly processed human serum albumin in transgenic plants[J].Biotechnology,1990,8(3):217-221.
[14]Farran I,Sanchez-Serrano J J,Medina J F,et al.Targeted expression of human serum albumin to potato tubers[J].Transgenic research,2002,11(4):337-346.
[15]Huang LF,Liu Y K,Lu C A,et al.Production of human serum albumin by sugar starvation induced promoter and rice cell culture[J].Transgenic research,2005,14(5):569-581.
[16]He Y,Ning T,Xie T,et al.Large-scale production of functional human serum albumin from transgenic rice seeds[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(47):19 078-19 083.
[17]何平,韦正乙,孙卉,等.紫花苜蓿转基因研究进展[J].草业科学,2013,30(4):627-637.
[18]蔡璐,张瑜,李世歌,等.紫花苜蓿转基因的研究进展分析[J].农业与技术,2016,36(10):18.
[19]赵金梅,李芳,周禾,等.紫花苜蓿组织培养体系的建立[J].核农学报,2010,24(3):507-512.
[20]Dong J L,Liang B G,Jin Y S,et al.Oral immunization with p Bs VP6-transgenic alfalfa protects mice against rotavirus infection[J].Virology,2005,339(2):153-163.
[21]Dus Santos M J,Carrillo C,Ardila F,et al.Development of transgenic alfalfa plants containing the foot and mouth disease virus structural polyprotein gene P1 and its utilization as an experimental immunogen[J].Vaccine,2005,23(15):1 838-1 843.
[22]Legocki A B,Miedzinska K,Czaplinska M,et al.Immunoprotective properties of transgenic plants expressing E2 glycoprotein from CSFV and cysteine protease from Fasciola hepatica[J].Vaccine,2005,23(15):1 844-1 846.
[23]Bellucci M,De Marchis F,Arcioni S.Zeolin is a recombinant storage protein that can be used to produce value-added proteins in alfalfa(Medicago sativa L.)[J].Plant cell,Tissue and Organ Culture,2007,90(1):85-91.
[24]Lee R W,Cornelisse M,Ziauddin A,et al.Expression of a modified Mannheimia haemolytica GS60 outer membrane lipoprotein in transgenic alfalfa for the development of an edible vaccine against bovine pneumonic pasteurellosis[J].Journal of Biotechnology,2008,135(2):224-231.
[25]Liu Z H,Zhang H M,Li G L,et al.Enhancement of salt tolerance in alfalfa transformed with the gene encoding for betaine aldehyde dehydrogenase[J].Euphytica,2011,178(3):363-372.
[26]Deak M,Kiss G B,Korkz C,et al.Transformation of Medicago by Agrobacterum mediated gene transfer[J].Plant Cell Reports,1986,5(2):97-100.
[27]Zhang J Y,Broeckling C D,Blancaflor E B,et al.Overexpression of WXP1,a putative Medicago truncatula AP2 domain-containing transcription factor gene,increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa(Medicago sativa)[J].The Plant journal:for cell and molecular biology,2005,42(5):689-707.
[28]Tesfaye M,Temple S J,Allan D L,et al.Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum[J].Plant physiology,2001,127(4):1 836-1 844.
[29]Strizhov N,Keller M,Mathur J,et al.A synthetic cry IC gene,encoding a Bacillus thuringiensis delta-endotoxin,confers Spodoptera resistance in alfalfa and tobacco[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(26):15 012-15 017.
[30]Samac D A,Smigocki A C.Expression of OryzacystatinⅠandⅡin alfalfa increases resistance to the root-lesion nematode[J].Phytopathology,2003,93(7):799-804.
[31]Avraham T,Badani H,Galili S,et al.Enhanced levels of methionine and cysteine in transgenic alfalfa(Medicago sativa L.)plants over-expressing the Arabidopsis cystathionine gammasynthase gene[J].Plant biotechnology journal,2005,3(1):71-79.
[32]Reddy M S,Chen F,Shadle G,et al.Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa(Medicago sativa L.)[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(46):16 573-16 578.
[33]王彦华,李德锋,齐胜利,等.播种量和品种对紫花苜蓿分枝数和株高的影响[J].草业学报,2017,26(3):183-190.
[34]王茜,纪树仁,沈益新.土壤水分和施氮水平对紫花苜蓿苗期生长的互作效应分析[J].草业学报,2017,26(12):48-55.
[35]王坤龙,王千玉,宋彦军,等.留茬高度对紫花苜蓿根系生长及翌年返青的影响[J].中国饲料,2016(1):17-20.
[36]王伟,贾玉山,格根图,等.种植年限对紫花苜蓿生产力和品质的影响[J].草学,2017(4):11-17.
[37]魏小兰,张纯,唐凌,等.紫花苜蓿蛋白的应用研究[J].四川畜牧兽医,2017,44(5):39-41.