蜘蛛丝蛋白基因的合成及其串联体的重组表达
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摘要
蜘蛛丝(spider silk)是一类天然蛋白质纤维,具有独特的机械性能(高强度、高弹性和高断裂功等)和卓著的生物学特性(生物可降解性和与生物组织的相容性等)。它在生物医学、材料、纺织和军事等领域都有着很大潜在的应用价值。
但是由于蜘蛛无法驯养和天然蜘蛛丝产量少等原因,大量获取蜘蛛丝惟有通过基因工程手段,才能满足蜘蛛丝潜在的应用需求。
本工作根据已报道的蜘蛛丝蛋白的氨基酸序列,结合蜘蛛丝蛋白的模块结构特性和功能的关系,优化设计了一种新型蜘蛛丝蛋白单元,它理论上集高强度、高弹性等蜘蛛丝主要特性于一体。编码该蛋白单元的基因SPS通过引物重叠延伸法得到了全长合成,为405 bp,并通过了测序验证。
将SPS基因克隆到pET-32a(+)中,与其N端的硫氧还蛋白(TrxA)融合构建成表达载体pET-SPS1;进一步通过同尾酶法构建了蜘蛛丝蛋白基因SPS的多个串联体表达载体pET-SPS(2～8)。然后,所有的含重组蜘蛛丝蛋白基因的表达载体在大肠杆菌BL21(DE3)中进行了诱导表达,并且通过pET-32a(+)自身携带的His标签亲合层析纯化了融合表达的重组蜘蛛丝蛋白。结果表明,所有在大肠杆菌中融合表达的重组蜘蛛丝蛋白几乎全部可溶。其中,1～3串联体蜘蛛丝蛋白基因能够有效表达,但随着串联数的进一步增加,蜘蛛丝蛋白基因融合表达效率下降。
另外,本工作进一步选用植物系统来表达重组蜘蛛丝蛋白。其中构建了含蜘蛛丝蛋白基因的植物表达载体pBI-SPS(5～7),通过冻融法将重组质粒转化到根癌农杆菌LBA 4404感受态细胞中。然后,通过烟草叶盘农杆菌浸染和抗性组织培养,得到了相应的转化再生植株。
总之,本工作成功地获得按照意愿设计的一种新型蜘蛛丝蛋白基因,构建了它的大基因片段,并初步通过大肠杆菌和植物表达系统来表达,为未来这种新型蜘蛛丝的应用研究打下了基础。
Spider silks,being a class of nature protein fibers,can be widely used in the biomedical,material,textile and military areas,because of their unique mechanical features of high strength,elasticity and high energy to break,and eminent biological peculiarities in terms of biodegradability and compatibility to the biological tissues.
Because of inevitable problems in spider cultivation to produce natural spider silks, large amount of spider silks for their applications can only be obtained through genetic engineering.
In this paper,an genetic spider silk protein unit was designed on the basis of amino acid sequences which is previously reported,in which the features of structural modules and their functional correlations were particularly concerned.Theoretically,this new-type spider silk protein is particularly endowed with two main pecaularities of spider silks which are high-intensity and high-elaticsity.The gene SPS(405 bp)encoding this silk protein unit was delicately synthesized by the manners of overlapped primer extension, finally verified by DNA sequencing.
SPS gene was fused behind the tag of thioredoxin(TrxA)in plasmid pET-32a(+)to generate the expression vector pET-SPS1.This vector was further used as the backbone for creation of a set of expression vectors,pET-SPS(2～8)having different multimers of SPS gene by means of isocaudarners,all SPS vectors were expressed in E.coli strain, BL21(DE3)by IPTG induction,and the expression of fusion proteins of spider silk were purified by His-tag affinity chromatography.All SPS fusion proteins expressed in E.coli are almost fully soluble.The vectors with 1-3 multimers of SPS could be efficiently expressed,while more increased multimers could only cause worse expression.
In addition,plant host was used in this work to produce recombinant spider silk proteins.For that,a few of SPS plant expression vectors of pBI-SPS(5～7)were generated,and transformed into Agrobacterium strain LBA 4404 by freeze-thaw method. These engineered strains were subsequently transformed by tobacco plants leaf disc infiltration.Finally,several corresponded lines of regenerated tobacco plants were obtained through tissue culturing with strict antibiotic selection.
In one word,this work was completed with the success of gaining the gene encoding a new- type of artificial spider silk protein with distinct features of high intensity and elasticity.Prelimilarily expression of this gene and its mutimers were investigated by using the systems of E.coli and plants,which could Laying the foundation of applications of this new-type silk protein in the future.
但是由于蜘蛛无法驯养和天然蜘蛛丝产量少等原因,大量获取蜘蛛丝惟有通过基因工程手段,才能满足蜘蛛丝潜在的应用需求。
本工作根据已报道的蜘蛛丝蛋白的氨基酸序列,结合蜘蛛丝蛋白的模块结构特性和功能的关系,优化设计了一种新型蜘蛛丝蛋白单元,它理论上集高强度、高弹性等蜘蛛丝主要特性于一体。编码该蛋白单元的基因SPS通过引物重叠延伸法得到了全长合成,为405 bp,并通过了测序验证。
将SPS基因克隆到pET-32a(+)中,与其N端的硫氧还蛋白(TrxA)融合构建成表达载体pET-SPS1;进一步通过同尾酶法构建了蜘蛛丝蛋白基因SPS的多个串联体表达载体pET-SPS(2～8)。然后,所有的含重组蜘蛛丝蛋白基因的表达载体在大肠杆菌BL21(DE3)中进行了诱导表达,并且通过pET-32a(+)自身携带的His标签亲合层析纯化了融合表达的重组蜘蛛丝蛋白。结果表明,所有在大肠杆菌中融合表达的重组蜘蛛丝蛋白几乎全部可溶。其中,1～3串联体蜘蛛丝蛋白基因能够有效表达,但随着串联数的进一步增加,蜘蛛丝蛋白基因融合表达效率下降。
另外,本工作进一步选用植物系统来表达重组蜘蛛丝蛋白。其中构建了含蜘蛛丝蛋白基因的植物表达载体pBI-SPS(5～7),通过冻融法将重组质粒转化到根癌农杆菌LBA 4404感受态细胞中。然后,通过烟草叶盘农杆菌浸染和抗性组织培养,得到了相应的转化再生植株。
总之,本工作成功地获得按照意愿设计的一种新型蜘蛛丝蛋白基因,构建了它的大基因片段,并初步通过大肠杆菌和植物表达系统来表达,为未来这种新型蜘蛛丝的应用研究打下了基础。
Spider silks,being a class of nature protein fibers,can be widely used in the biomedical,material,textile and military areas,because of their unique mechanical features of high strength,elasticity and high energy to break,and eminent biological peculiarities in terms of biodegradability and compatibility to the biological tissues.
Because of inevitable problems in spider cultivation to produce natural spider silks, large amount of spider silks for their applications can only be obtained through genetic engineering.
In this paper,an genetic spider silk protein unit was designed on the basis of amino acid sequences which is previously reported,in which the features of structural modules and their functional correlations were particularly concerned.Theoretically,this new-type spider silk protein is particularly endowed with two main pecaularities of spider silks which are high-intensity and high-elaticsity.The gene SPS(405 bp)encoding this silk protein unit was delicately synthesized by the manners of overlapped primer extension, finally verified by DNA sequencing.
SPS gene was fused behind the tag of thioredoxin(TrxA)in plasmid pET-32a(+)to generate the expression vector pET-SPS1.This vector was further used as the backbone for creation of a set of expression vectors,pET-SPS(2～8)having different multimers of SPS gene by means of isocaudarners,all SPS vectors were expressed in E.coli strain, BL21(DE3)by IPTG induction,and the expression of fusion proteins of spider silk were purified by His-tag affinity chromatography.All SPS fusion proteins expressed in E.coli are almost fully soluble.The vectors with 1-3 multimers of SPS could be efficiently expressed,while more increased multimers could only cause worse expression.
In addition,plant host was used in this work to produce recombinant spider silk proteins.For that,a few of SPS plant expression vectors of pBI-SPS(5～7)were generated,and transformed into Agrobacterium strain LBA 4404 by freeze-thaw method. These engineered strains were subsequently transformed by tobacco plants leaf disc infiltration.Finally,several corresponded lines of regenerated tobacco plants were obtained through tissue culturing with strict antibiotic selection.
In one word,this work was completed with the success of gaining the gene encoding a new- type of artificial spider silk protein with distinct features of high intensity and elasticity.Prelimilarily expression of this gene and its mutimers were investigated by using the systems of E.coli and plants,which could Laying the foundation of applications of this new-type silk protein in the future.
引文
[1]Yang Z,Grubb DT,Jelinsky LW.Small Angle X-ray Scattering of Spider Dragline Silk.Macromolecules,1997,30:8254-8261
[2]David H.C NMR of Nephlia Clavipes Major Ampullate Silk Gland.Biophysical Journal,1996,71:3442-3447
[3]Winkler S,Szela S,Avtges P,et al.Designing recombinant spider silk proteins to control assembly.International Journal of Biological Macromolecules,1999,24:265-270
[4]Xiaoyi Hu,Kristin K,Arnold M,et al.Egg Case Protein-1.The Journal of Biological Chemistry,2005,280(22):21220-21230
[5]Beckwitt R,Arcidiacono S.Sequence Conservation In the C-terminal Region of Spider Silk Proteins(Spidroin)from Nephlia Clavipes(Tetragnathidae)and Araneus bicentenarius(Araneidae).Biol.Chem,1994,269:6661-6663
[6]Gatesy J,Hayashi CY,Motriuk D,et al.Extreme Diversity,Conservation,and Convergence of Spider Silk Fibroin Sequences.Science,2001,291:2603-2605
[7]Hayashi CY,Lewis RV.Spider Flagelliform Silkaessons in Protein Design,Gene Structure,and Molecular Evolution.Bio.Essays,2001,23:750-756
[8]Becker N,Oroudjev E,Mutz S,et al.Molecular Nanosprings in Spider Capture-silk Threads.Nat.Materials,2003,2:278-283
[9]Hu X,Kristin K,Arnold M,et al.Egg Case Protein-1.The Journal of Biological Chemistry,2005,280(22):21220-21230
[10]Fahnestock RS,Bedzyk LA.Production of synthetic Spider Dragline silk Protein in Pichapastoris.Appl.Microbiol Biotechnol,1997,47:33-39
[11]Kummerlin J,Beek J,Vollrath F,et al.Local Structure in Spider Dragline Silk Investigated by 2-dimensionalspin-diffusion NMR.Macromolecules,1996,29:2920-2928
[12]Foelix RF.Biology of spider.Harvard University Press.Cambridge MA,1992
[13]黄君霆.蜘蛛丝研究的动向.丝绸,1999,(9):47
[14]Lewis R.Spider silk:The unraveling of a mystery.Acc.Chem.Res.1992,25(9):392
[15]Tso IM,Wu HC,Hwang IR.Giant wood spider Nephila pilipes alters silk protein in response to prey variation.The Journal of Experimental Biology,2005,208:1053-1061.
[16]盛家镛,潘志娟.蜘蛛丝的化学组成与结构初探.丝绸,2000,(4):8
[17]Fritz V,David P.Liquid crystalline spinning of spider silk.Nature,2001,410:541
[18]Dicko C,Vollrath F,Kenney JM.Spider Silk Protein refolding is controlled by changing pH.Biomacromolecules.2004,5:704-710
[19]Knight DP,Vollrath F.Changes in element composition along the spinning duct in a Nephlia spider.Naturwissenschaflen,2001,88:179-182
[20]Vollrath F,Knight DP.Structure and function of the silk production pathway in the spider Nephila edulis.Biological Macromolecules,1999,24:243-249
[21]Knight DP,Vollrath F.Liquid crystals and flow elongation in a spider's silk production line.Proc.R.Soc.Lond.B 1999,266:519-523
[22]潘志娟等.蜘蛛丝的皮芯层及原纤化结构.纺织学报,2002,4(4):298-300
[23]Vollrath F.Biology of spider silk,Biological Macromolecules,1999,24:81-88
[24]Shao Z,Vollrath F,Sirichaisit J,et al.Analysis of spider silk in native and supercontracted states using Raman spectroscopy.Polymer.40:2493-2500
[25]李春萍,潘志娟,刘敏.大腹圆蛛丝的拉伸机械性能.丝绸,2002,9(9):46-48
[26]潘志娟,邱芯薇.蜘蛛丝的物理性能研究.苏州大学学报(工科版),2003,2(1):18-21
[27]陈瑶.大腹园蛛拖丝蛋白基因的克隆和序列分析.东华大学,硕士论文,2007
[28]蒋平.三种生态类型蜘蛛的生物学观察及三种蜘蛛丝的结构与功能研究,硕士论文,2003
[29]潘志娟,盛家镛,陈宇岳.大腹圆珠牵引丝的结构与性能分析.中国纺织大学学报,2000,26(5):82-84
[30]黄君霆.蜘蛛丝研究的动向.丝绸,1999,(9):47-49
[31]李栋高,蒋赢钩.丝绸材料学冲国纺织出版社,1994
[32]Xu M,Lewis RV.Structure of a protein superfiber:Spider dragline silk.Biochemistry,1990,87:7120-7124.
[33]Hinman MB,Lewis RV.Isolation of a clone encoding a second dragline silk fibroin:Nephila clavipes dragline silk is a two protein fiber.J Biol Chem,1992,267(27):1932-1934.
[34]Beckwitt R,Arcidiacono S.Sequence conservation in the C-terminal region of spider silk proteins(spidroin)from Nephila clavipes(Tetragnathidae)and Araneus bicentenarius(Araneidae).J Biol.Chem,1994,269:6661-6663.
[35]Colgin MA,Lewis RV.Spiderminor ampullate silk proteins contain new repetitive sequences and highly conserved nonsilklike "spacer regions".Protein Sci.,1998,7(3):667-672
[36]Hayashi CY,Lewis RV.Evidence from flagelliform silk eDNA for the structural basis of elasticity and modular nature of spider silks.J Mol Biol,1998,275:773-784.
[37]Cheryl Y,Hayashi,Shipley NH,et al.Hypotheses that correlate the sequence,structure,and mechanical properties of spider silk proteins.International Journal of Biological Macromolecules,1999,24:271-275.
[38]Urry D,Luan CH,Peng S.Molecular biophysics of elastin structure,function and pathology.Ciba Found Symp,1995,19:24-30.
[39]Chang DK,Venkatachalam CM,Urry DW.Nuclear Overhauser effect and computational characterization of the beta-spiral of the polypentapeptide of elastin.J Biomol Struct Dyn,1989,6:851-858.
[40]Jelinski LW.Establishing the relationship between structure and mechanical function in silks.Current Opinion in Solid State & Materials Science,1998,3:237-245.
[41]Yang Z,Grubb DT,Jelinsky LW.Small angle X-ray scattering of spider dragline silk.Macro molecules,1997,30:8254-8261.
[42]潘志娟,盛家镛.蜘蛛丝的结构与力学性能.南通工学院学报,1999,15(2):6
[43]Thiel BL.β-sheet and spider silk.Science,1996,27(3):12
[44]Dong Z,Lewis RV,Middaugh CR.Molecular mechanisms of spider silk elasticity.Arch Biochem Biophys,1991,284:53-57.
[45]Rising A,Nimmervoll H,Grip S,et al.Spider silk proteins-mechanical property and gene sequence.Zoological science,2005,22:273-281.
[46]Fahnestock RS,Yao Z J,Bedzyk LA,et al.Microbial production of spider silk proteins.Molecular Biotechnology,2000,74:105-119.
[47]Arcidiacono S,Mello C,Kaplan D,et al.Purification and characterization of recombinant spider silk expressed in Escherichia colil.Microbiol Bioteclmology,1998,49:31-38.
[48]马鹤雯,张立树,郑伟等.外源丙氨酸提高蜘蛛牵引丝蛋白天然基因在原核系统中的表达.中国生物工程杂志,2007,27(1):47-51
[49]田保中,汪牛鹏,王建南等.类蜘蛛丝丝素蛋白SPF198在毕赤酵母中的分泌表达.蚕业科学,2006,32(2):276-279
[50]Piruzian ES,Bogush VG,Sidoruk KV,et al.Construction of Synthetic Genes for Analogs of Spider Silk Spidroin 1 and Their Expression in Tobacco Plants.Molecular Biology,2003,37:554-560
[51]Scheller J,Guhrs KH,Grosse F.Production of spider silk proteins in tobacco and potato.Nat Biotechnol,2001,19(6):573-577
[52]Yamao M,Katayama N,Nakazawa H,et al.Gene targeting in the silkworm by use of a baculovirus.Genes & Dev,1999,13:511-516
[53]Lazafis A,Arcidiacono S,Huang Y,et al.Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells.Science,2002,295:472-476
[54]王晓玉,柳增善,任洪林.牛物钢性及弹性蛋白——蜘蛛丝研究进展.第四军医大学学报,2002,23:31-34
[55]Andreas Seidel,Oskar Liivak.Artificial spinning of spider silk [.Macromolecules,1998(31):6733-6736
[56]Oskar Liivak,Amy Blye,et al.A microfabricated wet—spinning apparatus to spin fibers of silk proteins.Structure-property correlations.Macromolecules,1998(31):2947-2951
[57]许箐,潘志娟,钱丹娜等.不同工艺条件下再生蜘蛛丝蛋白的分子结构.苏州大学学报,2005,25(2):11-14
[58]Arcidiacono S,Mello CM,Butler M,et al.Aqueous processing and fiber spinning of recombinant spider silks.Macromolecules,2002,35:1262-1266
[59]Andreas Seidel,Oskar Liivak,et al.Regenerated spider silk:processing,properties and structure.Macromolecules,2000,33:775-780
[60]张长胜,潘志娟,于洁松.蜘蛛丝多肽链段结构的优化设计.科学技术与工程,2002,2(6):35-37
[61]Fukushima Y.Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk.Biopolymers.1998,45(4):269-279
[62]张立树,马鹤雯,陆一鸣,等.蜘蛛牵引丝蛋白cDNA的扩增、克隆与序列分析.2002,18(5):641-643
[63].韩志勇,沈革志,潘建伟.一种改良的质粒DNA小量提取法.生物技术通报,2000,4:45.
[64].Sambrook J,et al.分子克隆实验指南(第三版).科学出版社,2002.
[65]Jiang Y F,Lin C T,Yin B,et al.Effects of the configuration ofa muti—epitope chimeric malaria DNA vaccine on its antigenicity to mice.J Chinese Medical,1999,112(8):686-690
[66]Terpe K.Overview of bacterial expression systems for heterologous protein production:from molecular and biochemical fundamentals to ommercial systems.Appl.Microbiol.Biotechnol 2006,72:211-222.
[67]Cabrita LD,Bottomley SP.Protein expression and refolding-a practical guide to getting the most out of inclusion bodies.Biotechnol Annu Rev2004,10:31-50.
[68]Hammarstrom M,Hellgren N,Berg S,et al.Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli.Protein Sci.2002,11:313-321.
[69]DummlerA,Lawrence AM,Marco A,et al.Simplified screening for the detection of soluble fusion constructs expressed in E.coli using a modular set of vectors.Microb Cell Fact,2005,4:34.
[70]Marblestone JG,Edavettal SC,Lira Y,et al.Comparison of SUMO fusion technology with traditional gene fusion systems:enhanced expression and solubility with SUMO.Protein Sci.2006,15:182-189.
[71]多聚组氨酸标签融合蛋白纯化操作手册,Novagen.
[72]Bollag DM,Rozycki MD,Edelstein SJ,et al.Protein Methods 2nd ed.NewYork:Wiley-Liss,1996,12:108-149.
[73]pET表达系统操作指南,Novagen
[74]Fahnestock SR,Irwin SL.Synthetic spider dragline silk proteins and their production in Escherichia coli.Appl Microbiol Biotechnol,1997,47:23-32
[75]王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社.1998
[76]Zupan JR,Zambryski PC.Transfer of T-DNA from Agrobacterium to the plant cell.Plant Physiol,1995,107:1041-1047
[78]顾红雅 瞿礼佳.植物分子生物学实验手册.高等教育出版社1998
[79]郭丽.盐生杜氏藻MAPK基因的克隆及对烟草的转化.甘肃农业大学,硕士论文,2006
[80]李兴宝,秦明辉.芥菜型油菜抗虫转基因植株及其后代株系的研究.生物工程学报,1999,15(4):482-488
[81]李兴宝,郑世学.甘蓝型油菜抗虫转基因植株及其抗性分析.遗传学报, 1999;26(3):262-268
[82]蓝海燕,王长海.导入β-1,3-葡聚糖及几丁质酶基因的转基因可育油菜及其抗菌核病的研究.生物工程学报,2000,16(2):142-145
[2]David H.C NMR of Nephlia Clavipes Major Ampullate Silk Gland.Biophysical Journal,1996,71:3442-3447
[3]Winkler S,Szela S,Avtges P,et al.Designing recombinant spider silk proteins to control assembly.International Journal of Biological Macromolecules,1999,24:265-270
[4]Xiaoyi Hu,Kristin K,Arnold M,et al.Egg Case Protein-1.The Journal of Biological Chemistry,2005,280(22):21220-21230
[5]Beckwitt R,Arcidiacono S.Sequence Conservation In the C-terminal Region of Spider Silk Proteins(Spidroin)from Nephlia Clavipes(Tetragnathidae)and Araneus bicentenarius(Araneidae).Biol.Chem,1994,269:6661-6663
[6]Gatesy J,Hayashi CY,Motriuk D,et al.Extreme Diversity,Conservation,and Convergence of Spider Silk Fibroin Sequences.Science,2001,291:2603-2605
[7]Hayashi CY,Lewis RV.Spider Flagelliform Silkaessons in Protein Design,Gene Structure,and Molecular Evolution.Bio.Essays,2001,23:750-756
[8]Becker N,Oroudjev E,Mutz S,et al.Molecular Nanosprings in Spider Capture-silk Threads.Nat.Materials,2003,2:278-283
[9]Hu X,Kristin K,Arnold M,et al.Egg Case Protein-1.The Journal of Biological Chemistry,2005,280(22):21220-21230
[10]Fahnestock RS,Bedzyk LA.Production of synthetic Spider Dragline silk Protein in Pichapastoris.Appl.Microbiol Biotechnol,1997,47:33-39
[11]Kummerlin J,Beek J,Vollrath F,et al.Local Structure in Spider Dragline Silk Investigated by 2-dimensionalspin-diffusion NMR.Macromolecules,1996,29:2920-2928
[12]Foelix RF.Biology of spider.Harvard University Press.Cambridge MA,1992
[13]黄君霆.蜘蛛丝研究的动向.丝绸,1999,(9):47
[14]Lewis R.Spider silk:The unraveling of a mystery.Acc.Chem.Res.1992,25(9):392
[15]Tso IM,Wu HC,Hwang IR.Giant wood spider Nephila pilipes alters silk protein in response to prey variation.The Journal of Experimental Biology,2005,208:1053-1061.
[16]盛家镛,潘志娟.蜘蛛丝的化学组成与结构初探.丝绸,2000,(4):8
[17]Fritz V,David P.Liquid crystalline spinning of spider silk.Nature,2001,410:541
[18]Dicko C,Vollrath F,Kenney JM.Spider Silk Protein refolding is controlled by changing pH.Biomacromolecules.2004,5:704-710
[19]Knight DP,Vollrath F.Changes in element composition along the spinning duct in a Nephlia spider.Naturwissenschaflen,2001,88:179-182
[20]Vollrath F,Knight DP.Structure and function of the silk production pathway in the spider Nephila edulis.Biological Macromolecules,1999,24:243-249
[21]Knight DP,Vollrath F.Liquid crystals and flow elongation in a spider's silk production line.Proc.R.Soc.Lond.B 1999,266:519-523
[22]潘志娟等.蜘蛛丝的皮芯层及原纤化结构.纺织学报,2002,4(4):298-300
[23]Vollrath F.Biology of spider silk,Biological Macromolecules,1999,24:81-88
[24]Shao Z,Vollrath F,Sirichaisit J,et al.Analysis of spider silk in native and supercontracted states using Raman spectroscopy.Polymer.40:2493-2500
[25]李春萍,潘志娟,刘敏.大腹圆蛛丝的拉伸机械性能.丝绸,2002,9(9):46-48
[26]潘志娟,邱芯薇.蜘蛛丝的物理性能研究.苏州大学学报(工科版),2003,2(1):18-21
[27]陈瑶.大腹园蛛拖丝蛋白基因的克隆和序列分析.东华大学,硕士论文,2007
[28]蒋平.三种生态类型蜘蛛的生物学观察及三种蜘蛛丝的结构与功能研究,硕士论文,2003
[29]潘志娟,盛家镛,陈宇岳.大腹圆珠牵引丝的结构与性能分析.中国纺织大学学报,2000,26(5):82-84
[30]黄君霆.蜘蛛丝研究的动向.丝绸,1999,(9):47-49
[31]李栋高,蒋赢钩.丝绸材料学冲国纺织出版社,1994
[32]Xu M,Lewis RV.Structure of a protein superfiber:Spider dragline silk.Biochemistry,1990,87:7120-7124.
[33]Hinman MB,Lewis RV.Isolation of a clone encoding a second dragline silk fibroin:Nephila clavipes dragline silk is a two protein fiber.J Biol Chem,1992,267(27):1932-1934.
[34]Beckwitt R,Arcidiacono S.Sequence conservation in the C-terminal region of spider silk proteins(spidroin)from Nephila clavipes(Tetragnathidae)and Araneus bicentenarius(Araneidae).J Biol.Chem,1994,269:6661-6663.
[35]Colgin MA,Lewis RV.Spiderminor ampullate silk proteins contain new repetitive sequences and highly conserved nonsilklike "spacer regions".Protein Sci.,1998,7(3):667-672
[36]Hayashi CY,Lewis RV.Evidence from flagelliform silk eDNA for the structural basis of elasticity and modular nature of spider silks.J Mol Biol,1998,275:773-784.
[37]Cheryl Y,Hayashi,Shipley NH,et al.Hypotheses that correlate the sequence,structure,and mechanical properties of spider silk proteins.International Journal of Biological Macromolecules,1999,24:271-275.
[38]Urry D,Luan CH,Peng S.Molecular biophysics of elastin structure,function and pathology.Ciba Found Symp,1995,19:24-30.
[39]Chang DK,Venkatachalam CM,Urry DW.Nuclear Overhauser effect and computational characterization of the beta-spiral of the polypentapeptide of elastin.J Biomol Struct Dyn,1989,6:851-858.
[40]Jelinski LW.Establishing the relationship between structure and mechanical function in silks.Current Opinion in Solid State & Materials Science,1998,3:237-245.
[41]Yang Z,Grubb DT,Jelinsky LW.Small angle X-ray scattering of spider dragline silk.Macro molecules,1997,30:8254-8261.
[42]潘志娟,盛家镛.蜘蛛丝的结构与力学性能.南通工学院学报,1999,15(2):6
[43]Thiel BL.β-sheet and spider silk.Science,1996,27(3):12
[44]Dong Z,Lewis RV,Middaugh CR.Molecular mechanisms of spider silk elasticity.Arch Biochem Biophys,1991,284:53-57.
[45]Rising A,Nimmervoll H,Grip S,et al.Spider silk proteins-mechanical property and gene sequence.Zoological science,2005,22:273-281.
[46]Fahnestock RS,Yao Z J,Bedzyk LA,et al.Microbial production of spider silk proteins.Molecular Biotechnology,2000,74:105-119.
[47]Arcidiacono S,Mello C,Kaplan D,et al.Purification and characterization of recombinant spider silk expressed in Escherichia colil.Microbiol Bioteclmology,1998,49:31-38.
[48]马鹤雯,张立树,郑伟等.外源丙氨酸提高蜘蛛牵引丝蛋白天然基因在原核系统中的表达.中国生物工程杂志,2007,27(1):47-51
[49]田保中,汪牛鹏,王建南等.类蜘蛛丝丝素蛋白SPF198在毕赤酵母中的分泌表达.蚕业科学,2006,32(2):276-279
[50]Piruzian ES,Bogush VG,Sidoruk KV,et al.Construction of Synthetic Genes for Analogs of Spider Silk Spidroin 1 and Their Expression in Tobacco Plants.Molecular Biology,2003,37:554-560
[51]Scheller J,Guhrs KH,Grosse F.Production of spider silk proteins in tobacco and potato.Nat Biotechnol,2001,19(6):573-577
[52]Yamao M,Katayama N,Nakazawa H,et al.Gene targeting in the silkworm by use of a baculovirus.Genes & Dev,1999,13:511-516
[53]Lazafis A,Arcidiacono S,Huang Y,et al.Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells.Science,2002,295:472-476
[54]王晓玉,柳增善,任洪林.牛物钢性及弹性蛋白——蜘蛛丝研究进展.第四军医大学学报,2002,23:31-34
[55]Andreas Seidel,Oskar Liivak.Artificial spinning of spider silk [.Macromolecules,1998(31):6733-6736
[56]Oskar Liivak,Amy Blye,et al.A microfabricated wet—spinning apparatus to spin fibers of silk proteins.Structure-property correlations.Macromolecules,1998(31):2947-2951
[57]许箐,潘志娟,钱丹娜等.不同工艺条件下再生蜘蛛丝蛋白的分子结构.苏州大学学报,2005,25(2):11-14
[58]Arcidiacono S,Mello CM,Butler M,et al.Aqueous processing and fiber spinning of recombinant spider silks.Macromolecules,2002,35:1262-1266
[59]Andreas Seidel,Oskar Liivak,et al.Regenerated spider silk:processing,properties and structure.Macromolecules,2000,33:775-780
[60]张长胜,潘志娟,于洁松.蜘蛛丝多肽链段结构的优化设计.科学技术与工程,2002,2(6):35-37
[61]Fukushima Y.Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk.Biopolymers.1998,45(4):269-279
[62]张立树,马鹤雯,陆一鸣,等.蜘蛛牵引丝蛋白cDNA的扩增、克隆与序列分析.2002,18(5):641-643
[63].韩志勇,沈革志,潘建伟.一种改良的质粒DNA小量提取法.生物技术通报,2000,4:45.
[64].Sambrook J,et al.分子克隆实验指南(第三版).科学出版社,2002.
[65]Jiang Y F,Lin C T,Yin B,et al.Effects of the configuration ofa muti—epitope chimeric malaria DNA vaccine on its antigenicity to mice.J Chinese Medical,1999,112(8):686-690
[66]Terpe K.Overview of bacterial expression systems for heterologous protein production:from molecular and biochemical fundamentals to ommercial systems.Appl.Microbiol.Biotechnol 2006,72:211-222.
[67]Cabrita LD,Bottomley SP.Protein expression and refolding-a practical guide to getting the most out of inclusion bodies.Biotechnol Annu Rev2004,10:31-50.
[68]Hammarstrom M,Hellgren N,Berg S,et al.Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli.Protein Sci.2002,11:313-321.
[69]DummlerA,Lawrence AM,Marco A,et al.Simplified screening for the detection of soluble fusion constructs expressed in E.coli using a modular set of vectors.Microb Cell Fact,2005,4:34.
[70]Marblestone JG,Edavettal SC,Lira Y,et al.Comparison of SUMO fusion technology with traditional gene fusion systems:enhanced expression and solubility with SUMO.Protein Sci.2006,15:182-189.
[71]多聚组氨酸标签融合蛋白纯化操作手册,Novagen.
[72]Bollag DM,Rozycki MD,Edelstein SJ,et al.Protein Methods 2nd ed.NewYork:Wiley-Liss,1996,12:108-149.
[73]pET表达系统操作指南,Novagen
[74]Fahnestock SR,Irwin SL.Synthetic spider dragline silk proteins and their production in Escherichia coli.Appl Microbiol Biotechnol,1997,47:23-32
[75]王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社.1998
[76]Zupan JR,Zambryski PC.Transfer of T-DNA from Agrobacterium to the plant cell.Plant Physiol,1995,107:1041-1047
[78]顾红雅 瞿礼佳.植物分子生物学实验手册.高等教育出版社1998
[79]郭丽.盐生杜氏藻MAPK基因的克隆及对烟草的转化.甘肃农业大学,硕士论文,2006
[80]李兴宝,秦明辉.芥菜型油菜抗虫转基因植株及其后代株系的研究.生物工程学报,1999,15(4):482-488
[81]李兴宝,郑世学.甘蓝型油菜抗虫转基因植株及其抗性分析.遗传学报, 1999;26(3):262-268
[82]蓝海燕,王长海.导入β-1,3-葡聚糖及几丁质酶基因的转基因可育油菜及其抗菌核病的研究.生物工程学报,2000,16(2):142-145