RGD-拖丝蛋白基因的构建及其在毕赤酵母中的分泌表达
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摘要
蜘蛛丝,特别是机械性能优异的拖丝,作为高性能生物材料的研究已引起人们的关注。本室已在大肠杆菌系统中成功表达了含有细胞粘附肽RGD(精氨酸-甘氨酸-天冬氨酸)的重组蜘蛛拖丝蛋白。本文在此基础上,重新设计了RGD-拖丝蛋白单体,以毕赤酵母偏好的密码子合成拖丝蛋白基因单体,同时在单体序列的末端,设计了两组限制性酶切位点,用于基因单体的克隆与多聚化。将基因单体克隆入载体pBluescript SKⅡ,测序鉴定重组质粒。通过“头尾相接”的策略构建拖丝蛋白多聚体基因,依次构建2聚体、4聚体、8聚体、16聚体以及32聚体RGD-重组拖丝蛋白基因工程菌。
选择高聚合度的16聚体与32聚体拖丝蛋白基因,分别克隆到酵母穿梭型表达载体pPIC9K,重组表达载体命名为YNSR16与YNSR32。线性化后电击转化毕赤酵母宿主菌GS115,通过浓度梯度的YPD-G418平板,筛选多拷贝插入的重组菌。对筛选出的重组菌,通过引物(a-factor与3'AOX)进行PCR鉴定,结果显示目的基因已正确整合入GS115基因组中。表明毕赤酵母重组菌的构建在基因水平上达到预期目的。
以16聚体毕赤酵母重组菌的表达产物为分析对象,进行SDS-PAGE分析,通过银染显色,结果显示,16聚体毕赤酵母重组菌分泌表达分子量约47KD的RGD-重组拖丝蛋白,这与理论值相符。在此基础上,摸索建立了毕赤酵母重组菌摇瓶表达的方法,初步确定了诱导表达的条件:培养基pH值为6.0,培养温度为30℃,诱导剂甲醇浓度为1.5%(V/V);诱导时间为96h。
以16聚体与32聚体毕赤酵母重组菌为对象,研究了目的基因性质与整合位点对重组拖丝蛋白表达的影响。16聚体与32聚体拖丝蛋白基因为高度重复性序列,GC含量较高,其mRNA易形成复杂的二级结构,不利于重组拖丝蛋白的高效表达;目的基因可整合于毕赤酵母基因组的不同位点,最终确定目的基因整合于宿主菌基因组的组氨酸脱氢酶基因位点,有利于重组拖丝蛋白的表达。
The study on spider silk,especially dragline,as a high-performance biomaterial has aroused attention.The expression of recombinant dragline protein with RGD motif has been accomplished in E.coli.in our previous work.DNA monomer was redesigned and synthesized with codons bias of Pichia pastoris.Two restriction sites were located in the end of DNA monomer,which was used for the cloning and multimerizing of the monomer,respectively.DNA monomer was cloned to vector- pBluescript SKⅡ.Then,the DNA monomer was used to construct multimers by the "the head to tail" strategy after sequenced.Several genetically engineered strains were obtained,such as 2-multimer strains,4-multimer strains,8-multimer strains,16-multimer strains,32-multimer strains.
Each of 16-multimer and 32-multimer spider dragline gene was cloned to shuttle expression vector- pPIC9K,thus the contructs YNSR-16 and YNSR-32 were obtained.The linized contructs was transformed to Pichia pastoris host strain GS115,then,recombinant strains were acquired, respectively.Stable multicopy recombinant Pichia pastoris were selected by various G418 concentrations.Recombinants of multicopy inserts were identified by PCR(a-factor,3'AOX1 primer).The result indicated that the target gene had been integrated into the genome of GS115. The construction of multicopy engineered strains was successful.
The product of 16-multimer recombinants were analysed by SDS-PAGE.The results demonstrated that recombinant dragline protein was secreted by 16-multimer recombinants,of which the molecular weight was 47KD.The shake flasks methods was established as follows: the medium pH-6.0,the culture temperature-30℃,the concentration of methanol-1.5%,the inducing time-96h.
The obtained 16-multimer and 32-multimer recombinants were used.We investigated the effect of the properties and integrant sites of recombinant dragline protein genes on the expression of recombinant spider dragline silk protein.Because of high repetitive sequence and GC content,recombinant dragline protein genes were transcript into mRNA with complex secondary structure.This led to reduced expression of recombinant dragline protein.The target genes were integrated to different sites in host genome.We concluded that the recombinant dragline protein was secreted as 16-mer gene intergrated to His4 gene in genome.
选择高聚合度的16聚体与32聚体拖丝蛋白基因,分别克隆到酵母穿梭型表达载体pPIC9K,重组表达载体命名为YNSR16与YNSR32。线性化后电击转化毕赤酵母宿主菌GS115,通过浓度梯度的YPD-G418平板,筛选多拷贝插入的重组菌。对筛选出的重组菌,通过引物(a-factor与3'AOX)进行PCR鉴定,结果显示目的基因已正确整合入GS115基因组中。表明毕赤酵母重组菌的构建在基因水平上达到预期目的。
以16聚体毕赤酵母重组菌的表达产物为分析对象,进行SDS-PAGE分析,通过银染显色,结果显示,16聚体毕赤酵母重组菌分泌表达分子量约47KD的RGD-重组拖丝蛋白,这与理论值相符。在此基础上,摸索建立了毕赤酵母重组菌摇瓶表达的方法,初步确定了诱导表达的条件:培养基pH值为6.0,培养温度为30℃,诱导剂甲醇浓度为1.5%(V/V);诱导时间为96h。
以16聚体与32聚体毕赤酵母重组菌为对象,研究了目的基因性质与整合位点对重组拖丝蛋白表达的影响。16聚体与32聚体拖丝蛋白基因为高度重复性序列,GC含量较高,其mRNA易形成复杂的二级结构,不利于重组拖丝蛋白的高效表达;目的基因可整合于毕赤酵母基因组的不同位点,最终确定目的基因整合于宿主菌基因组的组氨酸脱氢酶基因位点,有利于重组拖丝蛋白的表达。
The study on spider silk,especially dragline,as a high-performance biomaterial has aroused attention.The expression of recombinant dragline protein with RGD motif has been accomplished in E.coli.in our previous work.DNA monomer was redesigned and synthesized with codons bias of Pichia pastoris.Two restriction sites were located in the end of DNA monomer,which was used for the cloning and multimerizing of the monomer,respectively.DNA monomer was cloned to vector- pBluescript SKⅡ.Then,the DNA monomer was used to construct multimers by the "the head to tail" strategy after sequenced.Several genetically engineered strains were obtained,such as 2-multimer strains,4-multimer strains,8-multimer strains,16-multimer strains,32-multimer strains.
Each of 16-multimer and 32-multimer spider dragline gene was cloned to shuttle expression vector- pPIC9K,thus the contructs YNSR-16 and YNSR-32 were obtained.The linized contructs was transformed to Pichia pastoris host strain GS115,then,recombinant strains were acquired, respectively.Stable multicopy recombinant Pichia pastoris were selected by various G418 concentrations.Recombinants of multicopy inserts were identified by PCR(a-factor,3'AOX1 primer).The result indicated that the target gene had been integrated into the genome of GS115. The construction of multicopy engineered strains was successful.
The product of 16-multimer recombinants were analysed by SDS-PAGE.The results demonstrated that recombinant dragline protein was secreted by 16-multimer recombinants,of which the molecular weight was 47KD.The shake flasks methods was established as follows: the medium pH-6.0,the culture temperature-30℃,the concentration of methanol-1.5%,the inducing time-96h.
The obtained 16-multimer and 32-multimer recombinants were used.We investigated the effect of the properties and integrant sites of recombinant dragline protein genes on the expression of recombinant spider dragline silk protein.Because of high repetitive sequence and GC content,recombinant dragline protein genes were transcript into mRNA with complex secondary structure.This led to reduced expression of recombinant dragline protein.The target genes were integrated to different sites in host genome.We concluded that the recombinant dragline protein was secreted as 16-mer gene intergrated to His4 gene in genome.
引文
[1]Vollrath F,Knight D P.Liquid crystalline spinning of spider silk[J].Nature.2001,410(6828):541-548.
[2]Jelinski L W.Establishing the relationship between structure and mechanical function in silks[J].J Curt Opinion Solid State mater Sci.1998,3(3):237-245.
[3]Vollrath F,Madsen B,Shao Z.Proc R Soc London B Biol.Science.2001,268(1483):2339-2346.
[4]Gosline J m,Guerette P A,Ortlepp C S,Savage K N.The mechanical design of spider silks:from fibroin sequence to mechanical function[J].J Exp Bio1.1999,202(23):3295-32303.
[5]潘鸿春,宋大祥,周开亚.蜘蛛丝蛋白研究进展[J].蛛形学报,2006,15(1):52-59.
[6]李敏,Kaplan D L.蜘蛛基因组DNA Cosmid文库构建和拖丝蛋白基的克隆[J].动物学报.2001,47(6):713-717.
[7]黄建坤,黄智华,李敏.蜘蛛拖丝蛋白全基因亚克隆及测序对了解其结构与功能的意义[J].中国临床康复.2004,8(8):1462-1463.
[8]李敏,黄建坤,涂桂云,等.RGD一蜘蛛拖丝蛋白聚合物的生物合成与纯化[J].生物医学工程学杂志.2004,21(6):1006-1010.
[9]李敏,涂桂云,黄智华,等.RGD一蛛丝蛋白基因工程茵高密度发酵条件的研究[J].生物医学工程学杂志.2005,22(6):1206-1209.
[10]陈登龙,房乾,李敏,等.重组蛛丝蛋白pNSR-16/聚乙烯醇复合材料的研究[J].功能材料.2007,38(7):1194-1196.
[11]张秋轩.RGD-蛛丝蛋白工程菌高密度发酵条件的优化及支架材料的细胞相容性研究.[福建师范大学硕士学位论文].2007:3-7.
[12]Vollrath F.Biology of spides[J].Biological macromolecules.1999,24:81-88
[13]陈瑶,孟清,卿凤翎.蜘蛛丝纤维的特性与开发生产[J].现代纺织技术,2006,6:53-56.
[14]Knight D P,Vollrath E Liquid crystals and flow elongation in a spiders silk production line[J].ProcR Soc Lonc B.1999,266(1418):519-523.
[15]Knight D P,Vollrath E hexagonal columnar liquid crystal in the cells secreting spider silk[J].Tissue and Cell.1999,31(6):617-620.
[16]吕靖.蚕丝和蜘蛛丝的结构与生物纺丝过程[J].现代纺织技术,2004,1:40-42.
[17]张前军,李敏.蛛丝蛋白的研究进展及应用前景[J].生物工程进展.2001,21(6):19-21.
[18]潘志娟,刘敏,盛家镛.大腹圆蛛丝蛋白的氨基酸组成分析.丝绸.2003,12:22-26
[19]Van Beek J D,Hess S,vollrath F,et al.The molecular structure of spider dragline silk:folding and orientation of the protein backbone.Proc Nata Acad Sci USA.2002,99(16):10266-10271.
[20]邵敬党.蜘蛛丝的性能特征分析[J].棉纺织技术.2005,33(11):653-657.
[21]刘海洋,王伟霞,刘长军,等.纺织新材料-蜘蛛丝[J].纺织导报,2004,22(1):28-30.
[22]Kitagawa M,Yasutomi m,Furukawa K.Microstructure of Spider Dragline.J Soc mat Sci.2001,50(11):1213-1217.
[23]Augsten K et al.Glycoproteins and Skin-core Structure in Nephlia Clavipes Spider Silk Observed by Light and Electron.Scanning.2000,22(1):12-15.
[24]潘志娟,李春萍,刘敏,等.蜘蛛丝的皮芯层及原纤化构造.纺织学报.2003,24(4):298-300.
[25]Hronska m,Van Beek J D,Williamson P T,et al.NmR characterization of native liquid spider dragline silk from Nephila edulis[J].Biomacromolecules.2004,5(3):834-839.
[26]Holland G P,Lewis R V,Yarqer J L.WISE NmR characterization of nanoscale heterogeneity and mobility in supercontracted Nephila clavipes spider dragline silk.J Am Chem Soc.2004,126(18):5867-5872.
[27]Bonev B,Grieve S,Herberstein M E,et al.Orientational order of Australian spider silks as deter mined by solid-state NmR.Biopolymers.2006,82(2):134-143.
[28]Saravanan D.Spider Silk-Structure,Properties and Spinning[J].Journal Of Textile And Apparel Technology And Management.2006,5(1):1-20.
[29]黄君霆.蜘蛛丝的研究动向[J].丝绸.1999,9:47-49.
[30]Cunniff P m,Fossey S A,Auerbachand M A.et,al.In Silk Polymer:materials Science and Biotechnology (D.Kaplan et al.,Eds.)[M].ACS Symposium Series 544,ACS Press,New York,1994,pp.234-251.
[31]袁辉.蜘蛛丝的研究与开发利用[J].丝绸.2003,6:15-17.
[32]Winkler S,Kaplan D L.Molecular biology of spider silk[J].J Biotechnol.2000,74(2):85-93.
[33]Savage K M,Guerette P A,Gosline J m.Supercontraction stress in spider webs[J].Biomacromolecules.2004,5(3):675-679.
[34]Fossey S A,Tripathy S.Atomistic modeling of interphases in spider silk firbers[J].Int J Biol macromol.1999,24(2):119-125.
[35]Liu Y,Sponner A,Vollrath F.Proline and processing of spider silks[J].Biomacromolecules.2008,9(1):116-121.
[36]Hakimi O,Knight P D,Vollrath F,et al.Spider and mulberry silkworm silks as compatible biomaterials[J].Composites PartB:Engineering.2007,38(3):324-337.
[37]Allmeling C,Jokuszies A,Voqt P m,et al.Use of spider silk fibres as an innovative material in a biocompatible artificial nerve conduit[J].J Cell Mol Med.2006,10(3):770-777.
[38]许箐,潘志娟.蜘蛛丝蛋白人工合成及人造蜘蛛丝[J].苏州大学学报(工科版).2005,25(1):47-51.
[39]Xu M,Lewis R V.Structure of a protein superfiber:spider dragline silk.Proc.Natl.Acad.Sci.USA.1990,87:7120-7124.
[40]Hinman MB,Lewis R V.Isolation of a clone encoding a second dragline silk fibroin Nephila clavipes dragline silk is a two protein fiber[J].J Biol Chem.1992,267(27):19320-19324.
[41]Beckwitt R,Arcidiacono S.Sequence conservation in the C-ter minal region of spider silk proteins (Spidorin)from Nephilia clavipes(Tetragnathidae)and Araneus bicentenarius(Araneidae)[J].J Biol Chem.1994,269:6661-6663.
[42]Guerrete P,Ginzinger D,Gosline J.Silk properties deter mined by gland-specific expression of a spider fibroin gene family[J].Science.1996,272(5258):112-115.
[43]Avoub N A,Garb J E,Hayashi C Y,et al.Blueprint for a high-performance biomaterial:full-length spider dragline silk genes[J].Plos ONE.2007,2(6):502-514.
[44]Colgin m A,Lewis R V.Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved nonsilk-like 'spacer regions[J].Protein Sci.1998,7(3):667-672.
[45]Hayashi C Y,Lewis R V.Evidence from flagelliform silk cDNA for the structural basis of elasticity and modular nature of spider silks[J].J Mol Biol.1998,275(5):773-784.
[46]Fahnestock S R,Irwin S L.Synthetic spider silk dragline proteins and their production in Escherichia coli[J].Appl microbiol Biotechnol.1997,47(1):23-32.
[47]Fahnestock S R,Bedzyk L A.Production of synthetic spider dragline silk protein in Pichia pastoris[J].Appl microbiol Biotechnol.1997,47(1):33-39.
[48]Fahnestock S R,Yao Z,Bedzyk L A.Microbial production of spider silk proteins[J],tool.Biotechnol.2000,74(2):105-119.
[49]Wong Po Foo C,Patwardhan S V,Kaplan D L,et al.Novel nanocomposites from spider silk-silica fusion (chimeric)proteins[J].PNAS,2006,103(25):9428-9433.
[50]Jia huang,Cheryl Wong,Kaplan D L,et al.The effect of genetically engineered spider silk-dentin matrix proteinl chimeric protein on hydroxyapatite nucleation[J].Biomaterials.2007,28(14):2358-2367.
[51]阮超然,黄晶星,李敏,等.高分子量RGD-蛛丝蛋白重组体的构建、高密度发酵及纯化p].生物工程学报.2007,23(5):858-861.
[52]Lazaris A,Arcidiacono S,Huang Y,et al.Spider silk fibers spun from soluble recombinant silk produced in mammalian cells[J].Science.2002,295:472-476.
[53]王晓玉,柳增善,任洪林.“生物钢性及弹性蛋白'一蜘蛛丝研究进展[J].第四军医大学学报.2002,23:31-35.
[54]Yamao M,Katayma N,Nakazawa h,et al.Gene targeting in the silkworm by use of a Baculovirus[J].Gene Development.1999,13(5):511-516.
[55]刘辉芬,李维,王宇,等.蜘蛛拖牵丝蛋白基因转家蚕表达质粒的构建[J].湖南大学学报(自然科学版).2006,33(5):105-109.
[56]Jiirgen Scheller,Udo Conrad.Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation[J].Transgenic Research..2004,13(1):51-57.
[57]刘庆生,段亚峰.蜘蛛丝的结构性能与研究现状[J].四川丝绸.2005,18(2):16-18.
[58]余祖华,王红宁.利用巴斯德毕赤酵母表达外源蛋白的研究进展[J].生物技术通讯.2004,15(6):614-616.
[59]Daly R,Heam M T W.Expression of heterologous proteins in Pichia pastoris:a useful experimental tool in protein engineering and production[J].J mol Recognit.2005,18(2):119-138.
[60]Romanos M A.Advances in the use of Pichia pastoris for high-level gene expression[J].Curr Opin Biotechnol.1995,6(5):527-533.
[61]Inan M,Meagher M M.Non-repressing carbon sources for alcohol oxidase(AOX1)promoter of Pichia pastoris[J].J Biosci Bioengng.2001,92(6):585-589.
[62]Shen S,Sulter G,Jeffries T W,et al.A strong nitrogen source regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris[J].Gene.1998,216(1):93-102.
[63]Waterham hR,Digan m E,Koutz P J,et al.Isolation of the Pichia pastoris glyceraldehyde-3- phosphate dehydrogenase gene and regulation of its promoter[J].Gene.1997,186(1):37-44.
[64]李健仔.巴斯德毕赤酵母外源基因表达系统[J].生物学通报.2005,40(3):21-23.
[65]Vassileva A,Chugh D A,Khanna N.Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris.Protein Express Purif.2001,21(1):71-80.
[66]Mansur M,Cabello C,Hernandez L,et al.multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris[J].Biotechnol Lett.2005,27(5):339-345.
[67]Chen.Y S,Jin M,Egborge T,et al.Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase a produced in Pichia pastoris[J].Protein Express.Purif.2000,20(3):472-484.
[68]Trimble R B,Lubowski C,Kumar S A,et al.Characterization of N-and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris[J].Glycobiology.2004,14(3):265-274.
[69]Boraston A B,Sandercock L E,Kilburn D G,et al.O-glycosylation of a recombinant carbohydrate -binding module mutant secreted by Pichia pastoris[J].J mol microbiol Biotechnol.2003,5(1):29-36.
[70]罗竞红,游自立.巴斯德毕赤酵母表达系统在外源基因表达中的研究进展.生物技术通报.2007,3:75-79.
[71]Gustafsson C,Govindarajan S,Minshull J.Condon bias and heterologous protein expression.Trends in Biotechnology[J].2004,22(7):346-353.
[72]赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析.生物工程学报[J].2000,16(3):308-311.
[73]陈惠,赵海霞,王红宁,等.植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量[J].中国生物化学与分子生物学报.2005,21(2):171-175.
[74]Sreekrishna K,Brankamp RG,Kropp KE,et al.Strategies for optimal synthesis and secretion of heterologous protein in the methylotrophics yeast Pichia pastoris.Gene.1997,190(1):55-62.
[75]谢涛,王红宁.外源基因在巴斯德毕赤酵母中多拷贝整合的研究进展[J].生物技术通讯.2006,17(3):415-417.
[76]White C E,Kempi NM,Komives E A.Expression of highly disulfide-bonded proteins in Pichia pastoris.Structure.1994,2(11):1003-1005
[77]方曙光,储炬,张嗣良,等.温度对巴斯德毕赤酵母表达瑞替普酶(reteplase)的影响.工业微生物.2007,37(4):11-15.
[78]黄建坤.RGD-蛛丝蛋白生物合成与丝膜制备.[福建师范大学硕士学位论文].2004:39.
[79]Sreekrishna K.Strategies for optimal synthesis and secretion of heterologous proteins in the methylctrophic yeast Pichia pastoris.Gene.1997,190:55-62.
[80]Bini E,Huang J,Kaplan D L,et al.RGD-Functionalized bioengineered spider dragline silk biomaterial.Biomacromolecules.2006,7(11):3139-3145.
[81]Pakkanen.O,Pirskanen.A,Myllyharju.J.Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris[J].Journal of Biotechnology.2006,123(2):248-256.
[2]Jelinski L W.Establishing the relationship between structure and mechanical function in silks[J].J Curt Opinion Solid State mater Sci.1998,3(3):237-245.
[3]Vollrath F,Madsen B,Shao Z.Proc R Soc London B Biol.Science.2001,268(1483):2339-2346.
[4]Gosline J m,Guerette P A,Ortlepp C S,Savage K N.The mechanical design of spider silks:from fibroin sequence to mechanical function[J].J Exp Bio1.1999,202(23):3295-32303.
[5]潘鸿春,宋大祥,周开亚.蜘蛛丝蛋白研究进展[J].蛛形学报,2006,15(1):52-59.
[6]李敏,Kaplan D L.蜘蛛基因组DNA Cosmid文库构建和拖丝蛋白基的克隆[J].动物学报.2001,47(6):713-717.
[7]黄建坤,黄智华,李敏.蜘蛛拖丝蛋白全基因亚克隆及测序对了解其结构与功能的意义[J].中国临床康复.2004,8(8):1462-1463.
[8]李敏,黄建坤,涂桂云,等.RGD一蜘蛛拖丝蛋白聚合物的生物合成与纯化[J].生物医学工程学杂志.2004,21(6):1006-1010.
[9]李敏,涂桂云,黄智华,等.RGD一蛛丝蛋白基因工程茵高密度发酵条件的研究[J].生物医学工程学杂志.2005,22(6):1206-1209.
[10]陈登龙,房乾,李敏,等.重组蛛丝蛋白pNSR-16/聚乙烯醇复合材料的研究[J].功能材料.2007,38(7):1194-1196.
[11]张秋轩.RGD-蛛丝蛋白工程菌高密度发酵条件的优化及支架材料的细胞相容性研究.[福建师范大学硕士学位论文].2007:3-7.
[12]Vollrath F.Biology of spides[J].Biological macromolecules.1999,24:81-88
[13]陈瑶,孟清,卿凤翎.蜘蛛丝纤维的特性与开发生产[J].现代纺织技术,2006,6:53-56.
[14]Knight D P,Vollrath E Liquid crystals and flow elongation in a spiders silk production line[J].ProcR Soc Lonc B.1999,266(1418):519-523.
[15]Knight D P,Vollrath E hexagonal columnar liquid crystal in the cells secreting spider silk[J].Tissue and Cell.1999,31(6):617-620.
[16]吕靖.蚕丝和蜘蛛丝的结构与生物纺丝过程[J].现代纺织技术,2004,1:40-42.
[17]张前军,李敏.蛛丝蛋白的研究进展及应用前景[J].生物工程进展.2001,21(6):19-21.
[18]潘志娟,刘敏,盛家镛.大腹圆蛛丝蛋白的氨基酸组成分析.丝绸.2003,12:22-26
[19]Van Beek J D,Hess S,vollrath F,et al.The molecular structure of spider dragline silk:folding and orientation of the protein backbone.Proc Nata Acad Sci USA.2002,99(16):10266-10271.
[20]邵敬党.蜘蛛丝的性能特征分析[J].棉纺织技术.2005,33(11):653-657.
[21]刘海洋,王伟霞,刘长军,等.纺织新材料-蜘蛛丝[J].纺织导报,2004,22(1):28-30.
[22]Kitagawa M,Yasutomi m,Furukawa K.Microstructure of Spider Dragline.J Soc mat Sci.2001,50(11):1213-1217.
[23]Augsten K et al.Glycoproteins and Skin-core Structure in Nephlia Clavipes Spider Silk Observed by Light and Electron.Scanning.2000,22(1):12-15.
[24]潘志娟,李春萍,刘敏,等.蜘蛛丝的皮芯层及原纤化构造.纺织学报.2003,24(4):298-300.
[25]Hronska m,Van Beek J D,Williamson P T,et al.NmR characterization of native liquid spider dragline silk from Nephila edulis[J].Biomacromolecules.2004,5(3):834-839.
[26]Holland G P,Lewis R V,Yarqer J L.WISE NmR characterization of nanoscale heterogeneity and mobility in supercontracted Nephila clavipes spider dragline silk.J Am Chem Soc.2004,126(18):5867-5872.
[27]Bonev B,Grieve S,Herberstein M E,et al.Orientational order of Australian spider silks as deter mined by solid-state NmR.Biopolymers.2006,82(2):134-143.
[28]Saravanan D.Spider Silk-Structure,Properties and Spinning[J].Journal Of Textile And Apparel Technology And Management.2006,5(1):1-20.
[29]黄君霆.蜘蛛丝的研究动向[J].丝绸.1999,9:47-49.
[30]Cunniff P m,Fossey S A,Auerbachand M A.et,al.In Silk Polymer:materials Science and Biotechnology (D.Kaplan et al.,Eds.)[M].ACS Symposium Series 544,ACS Press,New York,1994,pp.234-251.
[31]袁辉.蜘蛛丝的研究与开发利用[J].丝绸.2003,6:15-17.
[32]Winkler S,Kaplan D L.Molecular biology of spider silk[J].J Biotechnol.2000,74(2):85-93.
[33]Savage K M,Guerette P A,Gosline J m.Supercontraction stress in spider webs[J].Biomacromolecules.2004,5(3):675-679.
[34]Fossey S A,Tripathy S.Atomistic modeling of interphases in spider silk firbers[J].Int J Biol macromol.1999,24(2):119-125.
[35]Liu Y,Sponner A,Vollrath F.Proline and processing of spider silks[J].Biomacromolecules.2008,9(1):116-121.
[36]Hakimi O,Knight P D,Vollrath F,et al.Spider and mulberry silkworm silks as compatible biomaterials[J].Composites PartB:Engineering.2007,38(3):324-337.
[37]Allmeling C,Jokuszies A,Voqt P m,et al.Use of spider silk fibres as an innovative material in a biocompatible artificial nerve conduit[J].J Cell Mol Med.2006,10(3):770-777.
[38]许箐,潘志娟.蜘蛛丝蛋白人工合成及人造蜘蛛丝[J].苏州大学学报(工科版).2005,25(1):47-51.
[39]Xu M,Lewis R V.Structure of a protein superfiber:spider dragline silk.Proc.Natl.Acad.Sci.USA.1990,87:7120-7124.
[40]Hinman MB,Lewis R V.Isolation of a clone encoding a second dragline silk fibroin Nephila clavipes dragline silk is a two protein fiber[J].J Biol Chem.1992,267(27):19320-19324.
[41]Beckwitt R,Arcidiacono S.Sequence conservation in the C-ter minal region of spider silk proteins (Spidorin)from Nephilia clavipes(Tetragnathidae)and Araneus bicentenarius(Araneidae)[J].J Biol Chem.1994,269:6661-6663.
[42]Guerrete P,Ginzinger D,Gosline J.Silk properties deter mined by gland-specific expression of a spider fibroin gene family[J].Science.1996,272(5258):112-115.
[43]Avoub N A,Garb J E,Hayashi C Y,et al.Blueprint for a high-performance biomaterial:full-length spider dragline silk genes[J].Plos ONE.2007,2(6):502-514.
[44]Colgin m A,Lewis R V.Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved nonsilk-like 'spacer regions[J].Protein Sci.1998,7(3):667-672.
[45]Hayashi C Y,Lewis R V.Evidence from flagelliform silk cDNA for the structural basis of elasticity and modular nature of spider silks[J].J Mol Biol.1998,275(5):773-784.
[46]Fahnestock S R,Irwin S L.Synthetic spider silk dragline proteins and their production in Escherichia coli[J].Appl microbiol Biotechnol.1997,47(1):23-32.
[47]Fahnestock S R,Bedzyk L A.Production of synthetic spider dragline silk protein in Pichia pastoris[J].Appl microbiol Biotechnol.1997,47(1):33-39.
[48]Fahnestock S R,Yao Z,Bedzyk L A.Microbial production of spider silk proteins[J],tool.Biotechnol.2000,74(2):105-119.
[49]Wong Po Foo C,Patwardhan S V,Kaplan D L,et al.Novel nanocomposites from spider silk-silica fusion (chimeric)proteins[J].PNAS,2006,103(25):9428-9433.
[50]Jia huang,Cheryl Wong,Kaplan D L,et al.The effect of genetically engineered spider silk-dentin matrix proteinl chimeric protein on hydroxyapatite nucleation[J].Biomaterials.2007,28(14):2358-2367.
[51]阮超然,黄晶星,李敏,等.高分子量RGD-蛛丝蛋白重组体的构建、高密度发酵及纯化p].生物工程学报.2007,23(5):858-861.
[52]Lazaris A,Arcidiacono S,Huang Y,et al.Spider silk fibers spun from soluble recombinant silk produced in mammalian cells[J].Science.2002,295:472-476.
[53]王晓玉,柳增善,任洪林.“生物钢性及弹性蛋白'一蜘蛛丝研究进展[J].第四军医大学学报.2002,23:31-35.
[54]Yamao M,Katayma N,Nakazawa h,et al.Gene targeting in the silkworm by use of a Baculovirus[J].Gene Development.1999,13(5):511-516.
[55]刘辉芬,李维,王宇,等.蜘蛛拖牵丝蛋白基因转家蚕表达质粒的构建[J].湖南大学学报(自然科学版).2006,33(5):105-109.
[56]Jiirgen Scheller,Udo Conrad.Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation[J].Transgenic Research..2004,13(1):51-57.
[57]刘庆生,段亚峰.蜘蛛丝的结构性能与研究现状[J].四川丝绸.2005,18(2):16-18.
[58]余祖华,王红宁.利用巴斯德毕赤酵母表达外源蛋白的研究进展[J].生物技术通讯.2004,15(6):614-616.
[59]Daly R,Heam M T W.Expression of heterologous proteins in Pichia pastoris:a useful experimental tool in protein engineering and production[J].J mol Recognit.2005,18(2):119-138.
[60]Romanos M A.Advances in the use of Pichia pastoris for high-level gene expression[J].Curr Opin Biotechnol.1995,6(5):527-533.
[61]Inan M,Meagher M M.Non-repressing carbon sources for alcohol oxidase(AOX1)promoter of Pichia pastoris[J].J Biosci Bioengng.2001,92(6):585-589.
[62]Shen S,Sulter G,Jeffries T W,et al.A strong nitrogen source regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris[J].Gene.1998,216(1):93-102.
[63]Waterham hR,Digan m E,Koutz P J,et al.Isolation of the Pichia pastoris glyceraldehyde-3- phosphate dehydrogenase gene and regulation of its promoter[J].Gene.1997,186(1):37-44.
[64]李健仔.巴斯德毕赤酵母外源基因表达系统[J].生物学通报.2005,40(3):21-23.
[65]Vassileva A,Chugh D A,Khanna N.Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris.Protein Express Purif.2001,21(1):71-80.
[66]Mansur M,Cabello C,Hernandez L,et al.multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris[J].Biotechnol Lett.2005,27(5):339-345.
[67]Chen.Y S,Jin M,Egborge T,et al.Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase a produced in Pichia pastoris[J].Protein Express.Purif.2000,20(3):472-484.
[68]Trimble R B,Lubowski C,Kumar S A,et al.Characterization of N-and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris[J].Glycobiology.2004,14(3):265-274.
[69]Boraston A B,Sandercock L E,Kilburn D G,et al.O-glycosylation of a recombinant carbohydrate -binding module mutant secreted by Pichia pastoris[J].J mol microbiol Biotechnol.2003,5(1):29-36.
[70]罗竞红,游自立.巴斯德毕赤酵母表达系统在外源基因表达中的研究进展.生物技术通报.2007,3:75-79.
[71]Gustafsson C,Govindarajan S,Minshull J.Condon bias and heterologous protein expression.Trends in Biotechnology[J].2004,22(7):346-353.
[72]赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析.生物工程学报[J].2000,16(3):308-311.
[73]陈惠,赵海霞,王红宁,等.植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量[J].中国生物化学与分子生物学报.2005,21(2):171-175.
[74]Sreekrishna K,Brankamp RG,Kropp KE,et al.Strategies for optimal synthesis and secretion of heterologous protein in the methylotrophics yeast Pichia pastoris.Gene.1997,190(1):55-62.
[75]谢涛,王红宁.外源基因在巴斯德毕赤酵母中多拷贝整合的研究进展[J].生物技术通讯.2006,17(3):415-417.
[76]White C E,Kempi NM,Komives E A.Expression of highly disulfide-bonded proteins in Pichia pastoris.Structure.1994,2(11):1003-1005
[77]方曙光,储炬,张嗣良,等.温度对巴斯德毕赤酵母表达瑞替普酶(reteplase)的影响.工业微生物.2007,37(4):11-15.
[78]黄建坤.RGD-蛛丝蛋白生物合成与丝膜制备.[福建师范大学硕士学位论文].2004:39.
[79]Sreekrishna K.Strategies for optimal synthesis and secretion of heterologous proteins in the methylctrophic yeast Pichia pastoris.Gene.1997,190:55-62.
[80]Bini E,Huang J,Kaplan D L,et al.RGD-Functionalized bioengineered spider dragline silk biomaterial.Biomacromolecules.2006,7(11):3139-3145.
[81]Pakkanen.O,Pirskanen.A,Myllyharju.J.Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris[J].Journal of Biotechnology.2006,123(2):248-256.