CN101309676A - 生产蜘蛛丝蛋白的纳米胶囊和微胶囊的方法 - Google Patents
生产蜘蛛丝蛋白的纳米胶囊和微胶囊的方法 Download PDFInfo
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Abstract
本发明涉及从蜘蛛丝蛋白质生产纳米胶囊和微胶囊的方法。本发明还涉及通过该方法可以获得的纳米胶囊或微胶囊以及含有所述纳米胶囊或微胶囊的药物、化妆品和食品组合物。
Description
本发明涉及从蜘蛛丝蛋白质生产纳米胶囊和微胶囊的方法。本发明还涉及通过该方法可以获得的纳米胶囊或微胶囊以及含有所述纳米胶囊或微胶囊的药物、化妆品和食品组合物。
小规模的结构作为用于未来设备的运输囊和作为潜在的结构单元具有巨大利益。一项任务是能够以小规模包封反应物或颗粒,并且在将它们放在特定的位置以后容许包封物质的触发释放。这样的问题的一个解决方案是使用化学囊,所谓纳米胶囊。将纳米胶囊设计成在外部触发或刺激时释放所述反应物。由这样的要求引起数个问题:最重要的一个是如何在例如化学或生物学活性的反应物周围以限定的方式制造这样的纳米胶囊。
最近,已经开发了“杂化物”刺激响应性纳米胶囊以达到这样的需要。从例如具有pH-敏感构象的两亲性聚丁二烯(PB)-b-聚(谷氨酸)(PGA)二嵌段共聚物的自装配获得所述结构(囊以及微团)。PH-敏感性可用于卸载所述囊。已经通过组合阴离子的和开环聚合合成了那些含有可交联的疏水性嵌段和亲水性肽嵌段的PB-b-PGA共聚物(Chécot等,2002)。所述共聚物的多分散性是足够小的以获得充分限定的自装配聚集体。例如PB40-b-PGA100共聚物在水中时形成封闭的双层囊,称为聚合物体(polymersomes)(Won等,1999)。所述囊的一个性质是它们通过在尺寸上改变而响应外部pH改变(图1)。这个在pH改变时的转换是可逆的并且对于盐度仅是中度敏感的,因为它不是基于简单的聚电解质溶胀效应,而是基于PGA嵌段的肽属性(图1)。这些囊不仅能包封低分子量化合物(像溶剂分子诸如荧光团(Chécot等,2003)),而且可以稳定较大的纳米颗粒。这样的体系的缺点是与生物体系的部分不相容性,所述生物体系通常对于显著的pH改变是高度敏感的,因为pH改变可以导致包封样品的生物活性损失。
用于说明的图1显示(a)peptosome的流体动力学半径RH的动态光散射作为NaCl浓度和pH的函数。(b)peptosome和作为pH函数的它的尺寸改变的示意图,其原因在于所述肽部分中的卷曲向α-螺旋二级结构的转换。
另一个建立的包封方法是胶态颗粒在油/水界面的自装配。所述自装配方法的驱动力是总表面能量的最小化-由此可以使用各式各样的颗粒和溶剂。众所周知的这样的稳定乳剂是Pickering乳剂。所述颗粒的稳定或交联导致机械稳定的外层,然后可以将其传递到连续相。所谓的微球粒(colloidosomes)的优点是包封的控制和调整外壳的机械和化学稳定性的容易性。所述颗粒的自装配产生几乎结晶的结构并且由此存在所述颗粒之间的孔。这些孔是尺寸选择性的过滤器,其容许穿过所述膜的扩散的控制(Dinsmore等,2000)。所述全部方法可以以生物相容的方式进行。然而,胶态颗粒自身不必是生物相容的。
WO 02/47665描述用于制备自装配的选择性可渗透的弹性显微结构的方法,所述结构称为微球粒,其具有受控的孔尺寸、孔隙率和有利的机械性质。所述方法包括:(a)提供由生物相容材料在第一溶剂中形成的颗粒;(b)通过将第一流体添加到所述第一溶剂而形成乳状液,所述乳状液是由所述第一溶剂围绕的所述第一流体的小滴限定的;(c)用所述颗粒涂布所述小滴的表面;和(d)将所述颗粒稳定在所述小滴的所述表面上以形成具有至少约20帕斯卡的屈服强度的微球粒。WO 02/47665使用生物相容的合成聚合物用于生产这些微球粒。实例是聚苯乙烯、聚甲基丙烯酸甲酯、聚亚烷基、二氧化硅及其组合。例如通过烧结、化学交联等稳定而从其获得微球粒的颗粒。然而,制备那些微球粒的方法是比较困难的并且使用的胶质颗粒对于体内应用可以显示有害的性质,原因在于它们的人工的和非天然的属性。通过使用胶质颗粒也限制所述壳的尺寸范围,因为利用胶体限制具有限定孔的最小尺寸袋。
因此,本发明面临的问题是提供高度生物相容的并且因而适于体内应用的纳米胶囊和微胶囊。本发明的另一个问题是获得能够适应不同类型和多种量的有效试剂或营养物等的纳米胶囊和微胶囊。本发明面临的另外问题是提供可生物降解的纳米胶囊和微胶囊,即所述纳米胶囊和微胶囊能够在体内控制释放所述有效试剂等,例如在局部的或全身性应用中。
这些问题是通过独立权利要求的主题解决的。优选实施方案陈述在从属权利要求中。
在本发明中,出人意料地证明蜘蛛丝蛋白质可以充当形成可以用于多种体内应用的微胶囊和纳米胶囊的基质。特别证实这可以通过生产所述胶囊的改进方法进行,所述方法避开使用通过添加化学品样的交联剂而从其形成胶囊以连接或稳定所述颗粒的步骤的需要,或者其需要对于要包装到所述微胶囊和纳米胶囊中的试剂可以具有有害作用的烧结等。
因为大多数当前使用的包封技术(见例如WO 02/47665)依赖于非生物学颗粒或大分子,本发明人开发了基于自装配蜘蛛丝蛋白质的新的稳定的包封方法。不像其它的包封技术,在本方法中,乳状液表面的疏水性/亲水性属性不仅用于装配胶体颗粒,而且它也用作通过聚结和聚合物网络形成(稳定作用)的胶体固定化的驱动力。该方法不仅代表产生由新类别的生物相容胶体形成的聚合纳米胶囊和微胶囊的方法,它也代表利用蛋白质的聚合物网络形成的新方法。由该方法形成的纳米胶囊和微胶囊的巨大优点是由所述蛋白质给予的微胶囊的生物相容性和功能性。这可以通过数种方式控制释放机理:pH改变、温度改变、或蛋白酶的活性。
例如,纳米胶囊或微胶囊可能被破坏并且它们的组分可以化学地、物理地(例如通过剪切力)或生物学地(通过蛋白水解消化)在体内释放。
蜘蛛丝蛋白质在界面的自装配是通过将所述蛋白质引入到水/油乳状液的水相而实现的(参见图2)。表面能量的最小化将所述蛋白质驱动到界面并且诱导单体聚集成致密的聚合物网络(图3)。
例如,由该方法形成的蜘蛛袋/气囊充满水相的内容物并且可以存在于有机溶剂、醇中以及水中(图3)。因此,它们在强烈不同的环境中显示出乎意料的稳定性。原则上,在反向乳状液表面的蛋白质自装配也是可能的-因而包封油相的内容物(参见下文)。
显著地,所述袋/气囊可以充满蛋白质、化学反应物、纳米和微米规模的颗粒等,这是通过用荧光(FITC)标记的葡聚糖颗粒填充颗粒而例举性地显示的(图4)。
鉴于所述膜的厚度,膜的不渗透性和袋对于渗透压的机械稳定性都相对较高。电子显微图像揭示所述厚度是10~70nm(图5)。
在本方法中,已经采用合成的蜘蛛丝蛋白质,特别是C16的合成序列(Huemmerich等,2004)以产生活性剂的生物学包封。
蜘蛛丝通常是显示特殊物理性质的蛋白质聚合物,但是关于由不同的蜘蛛产生的多种丝的组成仅存在有限的信息(见Scheibel,2004)。在不同类型的蜘蛛丝之中,来自金网蜘蛛(golden orb weaver)(棒络新妇蛛(Nephila clavipes))和十字园蛛(garden cross spider)(十字园蛛(Araneusdiadematus))是研究最深的。拖丝的丝一般由两种主要蛋白质组成,并且还不清楚另外的蛋白质是否在丝装配和最终的丝结构中起显著作用。来自十字园蛛的拖丝的两种主要蛋白质组分是ADF-3和ADF-4(十字园蛛丝心蛋白)。
编码蜘蛛丝样蛋白质的基因是利用克隆策略产生的,所述策略基于合成的DNA组件和PCR扩增的真实基因序列的组合(Huemmerich等,2004)。选择来自园蛛十字园蛛的拖丝丝蛋白质ADF-3和ADF-4作为合成构建体的模板。连续克隆策略容许不同的合成DNA组件以及真实基因片段的受控制的组合。将克隆载体设计成包括具有间隔区的克隆盒,所述间隔区作为合成基因的占位符(placeholder)(Huemmerich等,2004)。
为了模拟ADF-4的重复序列,已经将单一保守重复单元设计成得到一个称为C的共有组件,将其多聚化以获得重复的蛋白质C16,将其以给定方法采用为实例。
对于提供的蜘蛛丝袋/气囊存在许多可能的应用,从功能食品到药物到化妆品应用。例如食品技术中的包封可以保护某些组分诸如维生素不受氧化环境影响。在另一种食品技术应用中,可以掩蔽组分诸如鱼油的味道。在药物应用中,蛋白质壳的扩散屏蔽容许包封材料的缓慢的(控制的)释放过程。蛋白质壳的另外设计可以产生限定的释放容器,其仅在利用某些蛋白酶或其它触发剂的活化以后释放内容物。在化妆品中,在例如由皮肤的蛋白酶缓慢降解蛋白质壳以后,可以通过提供的袋/气囊促进水有效成分到皮肤中的输送。另外,机械剪切可用于在暴露到皮肤时释放内容物。
本发明特别涉及下列方面和实施方案:
根据第一方面,本发明涉及生产纳米胶囊和微胶囊的方法,所述方法包括下列步骤:
a)提供蜘蛛丝蛋白质;
b)在适合的溶剂中形成所述蛋白质的溶液或悬浮液;
c)产生至少两相的乳状液,所述乳状液含有b)中形成的溶液或悬浮液作为第一相以及至少一个基本上与所述第一相不可混合的另外的相;
d)在至少两相的界面处形成蜘蛛丝蛋白质的聚合物网络;
e)从所述乳状液分离(d)中产生的蛋白质聚合物网络。
如上所述,意外地证实,步骤d)中形成聚合物网络不需要添加任何另外的组分(例如交联剂)并且不需要另外的步骤如烧结、交联等。
应当注意,如这里所用的术语“蜘蛛丝蛋白质”不仅包括所有天然的序列而且包括由此衍生的所有人工或合成的序列。
因此,蜘蛛丝序列可以衍生自这里称为“真实的”序列。该术语指的是基本核酸序列是从它们的天然环境分离的而没有在序列本身中进行实质的修改。被接受其存在的唯一修饰是其中真实的核酸序列被修饰,以便适应所述序列在宿主中表达而不改变编码的氨基酸序列。优选的序列是NR3(SEQ ID NO:10;衍生自ADF-3)和NR4(SEQ ID NO:11;衍生自ADF-4)。在两者序列中,为了更有效的翻译,将很少在大肠杆菌中翻译的密码子AGA(Arg)利用PCR诱变突变到CGT(Arg)。
真实的非重复序列优选是从天然存在的蜘蛛丝蛋白质的氨基末端非重复区域(鞭形蛋白质)和/或羧基末端非重复区域(鞭形和拖丝蛋白质)衍生的。那些蛋白质的优选实例将表示在下面。
根据另外的实施方案,所述真实的非重复序列是从天然存在的蜘蛛丝蛋白质的氨基末端非重复区域(鞭形蛋白质)和/或羧基末端非重复区域(鞭形和拖丝蛋白质)衍生的。
鞭形蛋白质的优选真实序列是FlagN-NR(SEQ ID NOs:31和32)和FlagC-NR(SEQ ID NOs:33和34)的氨基酸序列和核酸序列。
本发明的重组蜘蛛丝蛋白质一般可以从来自蜘蛛的主要壶腹状腺的蜘蛛拖丝蛋白质和/或源自鞭形腺的蛋白质所衍生。
根据实施方案,可被用于本发明的重组(合成的/人工的)蜘蛛丝蛋白质一般从来自蜘蛛的主要壶腹状腺的蜘蛛拖丝蛋白质和/或从来源于鞭形腺的蛋白质所衍生。
一般优选从圆网蜘蛛(圆蛛科(Araneidae)和Araneoids)的拖丝或鞭形蛋白质选择拖丝和/或鞭形序列。
更优选地,所述拖丝蛋白质和/或鞭形蛋白质来源于一种或多种下列蜘蛛:希氏尾园蛛(Arachnura higginsi),Araneus circulissparsus,十字园蛛,Argiope picta,条带园蛛(Banded Garden Spider)(三带金蛛(Argiopetrifasciata)),Batik花园网蛛(Batik Golden Web Spider)(星络新妇蛛(Nephila antipodiana)),Beccari′s帐篷蛛(Beccari′s Tent Spider)(Cyrtophorabeccarii),鸟粪蛛(Bird-dropping Spider)(Celaenia excavata),黑白棘蛛(Black-and-White Spiny Spider)(库氏棘腹蛛(Gasteracantha kuhlii)),黑黄园蛛(Black-and-yellow Garden Spider)(Argiope aurantia),流星锤蛛(Bolas Spider)(Ordgarius furcatus),流星锤蛛-巨蜘蛛(Bolas Spiders-Magnificent Spider)(Ordgarius magnificus),棕色水手蛛(Brown SailorSpider)(嗜水新园蛛(Neoscona nautica)),棕腿蛛(Brown-Legged Spider)(Neoscona rufofemorata),带帽黑头蛛(Capped Black-Headed Spider)(帆楚蛛(Zygiella calyptrata)),普通园蛛(Common Garden Spider)(Parawixiadehaani),普通圆蛛(Common Orb Weaver)(Neoscona oxancensis),蟹样棘圆蛛(Crab-like Spiny Orb Weaver)(Gasteracantha cancriformis(elipsoides)),弯曲棘蛛(Curved Spiny Spider)(Gasteracantha arcuata),皿云斑蛛(Cyrtophora moluccensis),Cyrtophora parnasia,Dolophones conifera,Dolophones turrigera,Doria′s棘蛛(Doria′s Spiny Spider)(Gasteracanthadoriae),双点棘蛛(Double-Spotted Spiny Spider)(Gasteracantha mammosa),双尾帐篷蛛(Double-Tailed Tent Spider)(方格云斑蛛(Cyrtophoraexanthematica)),塞若尖腹蛛(Aculeperia ceropegia),Eriophora pustulosa,Flat Anepsion(Anepsion depressium),四棘宝石蛛(Four-spined JewelSpider)(Gasteracantha quadrispinosa),花园圆网蛛(Garden Orb WebSpider)(Eriophora transmarina),巨大地衣网蛛(Giant Lichen Orbweaver)(Araneus bicentenarius),金色网蛛(Golden Web Spider)(Nephila maculata),Hasselt′s棘蛛(Hasselt′s Spiny Spider)(Gasteracantha hasseltii),Tegenariaatrica,Heurodes turrita,Island Cyclosa Spider(岛艾蛛(Cyclosa insulana)),宝石或棘蛛(Jewel or Spiny Spider)(Astracantha minax),肾形园蛛(KidneyGarden Spider)(丽园蛛(Araneus mitificus)),Laglaise′s园蛛(Laglaise′sGarden Spider)(Eriovixia laglaisei),Long-Bellied Cyclosa Spider(Cyclosabifida),Malabar Spider(Nephilengys malabarensis),多色圣安德鲁十字蛛(Multi-Coloured St Andrew′s Cross Spider)(多色金蛛(Argiopeversicolor)),观赏性树干蛛(Ornamental Tree-Trunk Spider)(裂腹蛛(Herennia ornatissima)),卵形圣安德鲁十字蛛(Oval St.Andrew′s CrossSpider)(好胜金蛛(Argiope aemula)),红帐篷蛛(Red Tent Spider)(单色云斑蛛(Cyrtophora unicolor)),俄罗斯帐篷蛛(Russian Tent Spider)(Cyrtophora hirta),圣安德鲁十字蛛(Saint Andrew′s Cross Spider)(凯氏金蛛(Argiope keyserlingi)),猩红阿秋蛛(Scarlet Acusilas)(猩红阿秋蛛(Acusilas coccineus)),银色金蛛(Silver Argiope)(Argiope argentata),棘背圆蛛(Spinybacked Orbweaver)(Gasteracantha cancriformis),斑点圆蛛(Spotted Orbweaver)(Neoscona domiciliorum),圣安德鲁十字(St.Andrews)Cross (Argiope aetheria),圣安德鲁十字蛛(St.Andrew′s CrossSpider)(Argiope Keyserlingi),树干蛛(Tree-Stump Spider)(无鳞波蛛(Poltys illepidus)),三角蛛(Triangular Spider) (Arkys clavatus),三角蛛(Arkys lancearius),双棘蛛(Two-spined Spider)(Poecilopachys australasia),络新妇蛛属(Nephila)物种,例如棒络新妇蛛(Nephila clavipes),Nephilasenegalensis,Nephila madagascariensis和更多(对于另外的蜘蛛物种,还见下文)。最优选地,拖丝蛋白质来源于十字园蛛并且鞭形蛋白质来源于棒络新妇蛛。
在本发明的上下文中,应该清楚,重组蜘蛛丝蛋白质可能不仅包括来自一个物种的蛋白质序列,而且还可以含有来源于不同的蜘蛛物种的序列。作为实例,一种或多种合成的重复蜘蛛丝蛋白质序列可以来源于一个物种,一种或多种真实的非重复蜘蛛丝蛋白质序列来自另一种。作为另外的实例,还可能设计重组蜘蛛丝蛋白质,其含有大于一种类型的重复序列,其中所述不同类型来源于不同的物种。
根据一个优选实施方案,拖丝蛋白质是野生型ADF-3、ADF-4、MaSp I、MaSp II,并且鞭形蛋白质是FLAG。术语ADF-3/-4用于由十字园蛛(十字园蛛丝心蛋白-3/-4)产生的MaSp蛋白质的上下文中。两种蛋白质,ADF-3和-4属于MaSp II蛋白质的类别(主要壶腹状蛛丝蛋白(spidroin)II)。
在另外的实施方案中,提供的核酸序列是ADF-3(SEQ ID NO:1)和/或ADF-4(SEQ ID NO:2),或其变体。
应当注意,两种不同种类的ADF-3和ADF-4编码序列和蛋白质在本发明中是预期的:第一,已经公开的ADF-3和ADF-4序列(在本文:“野生型”序列)和,第二,其变体,由SEQ ID NO:1(ADF-3)和2(ADF-4)编码。野生型序列是已经公开的并且是在登记号U47855和U47856(SEQ IDNO:8和9)之下可以得到的。
可被用于本发明(即单独或与另外的蛋白质组合)的另外的蜘蛛丝蛋白质和它们的数据库登记号是:
蛛丝蛋白2[Araneus bicentenarius]gi|2911272
主要壶腹状腺拖丝丝蛋白-1[大腹园蛛(Araneus ventricosus)]gi|27228957
主要壶腹状腺拖丝丝蛋白-2[大腹园蛛]gi|27228959壶腹状蛛丝蛋白1[Nephila madagascariensis]gi|13562006
主要壶腹状蛛丝蛋白1[Nephila senegalensis]gi|13562010
主要壶腹状蛛丝蛋白1[Latrodectus geometricus]gi|13561998
主要壶腹状蛛丝蛋白1[三带金蛛(Argiope trifasciata)]gi|13561984
主要壶腹状蛛丝蛋白1[Argiope aurantia]gi|13561976
拖丝丝蛋白蛛丝蛋白2[棒络新妇蛛]gi|16974791
主要壶腹状蛛丝蛋白2[Nephila senegalensis]gi|13562012
主要壶腹状蛛丝蛋白2[Nephila madagascariensis]gi|13562008
主要壶腹状蛛丝蛋白2[Latrodectus geometricus]gi|13562002
根据另一个优选实施方案,鞭形蛋白质是SEQ ID NO:6(Flag-N)和/或SEQ ID NO:7(Flag-C)或其变体。
然而,在本文也可以使用已经知道和公开的鞭形序列,特别是下列:
鞭形丝蛋白部分cds[Nephila clavipes]gi|2833646
鞭形丝蛋白部分cds[Nephila clavipes]gi|2833648
在一个优选实施方案中,重组蜘蛛丝蛋白质包括一种或多种合成的重复序列,所述序列含有一种或多种含有共有序列的聚丙氨酸。那些聚丙氨酸序列可以含有6至9个丙氨酸残基。见,例如SEQ ID NO:1,含有6个丙氨酸残基的数个聚丙氨酸基序。
优选地,含有共有序列的聚丙氨酸是从ADF-3衍生的并且具有SEQID NO:3(组件A)的氨基酸序列或其变体。组件A含有具有6个丙氨酸残基的聚丙氨酸。另外优选的从ADF-4衍生的,含有共有序列的聚丙氨酸,是组件C(SEQ ID NO:5),含有8个丙氨酸残基。
根据另外的优选实施方案,在本发明的重组蜘蛛丝蛋白质中,合成的重复序列是从ADF-3衍生的并且包括SEQ ID NO:4(组件Q)的氨基酸序列或其变体的一个或多个重复。
更一般而言,合成的重复序列也可以含有一般基序:GGX或GPGXX,即,富含甘氨酸的区域。如上所述,这些区域将对于蛋白质并且因而对于由含有所述基序的重组蜘蛛丝蛋白质形成的线提供柔性。
应当注意,在本发明中使用的合成重复序列的具体组件还可以相互组合,即,组合A和Q、Q和C等的组件(重复单元)也由本发明所包括。虽然要在蜘蛛丝蛋白质中引入的组件数量不受限制,但是优选对于每个重组蛋白质采用许多合成的重复序列的组件,其数量优选在5-50个组件、更优选10-40并且最优选在15-35个组件之间的范围内。
合成的重复序列优选包括一个或多个作为重复单元的(AQ)和/或(QAQ)。甚至更优选,合成的重复序列是(AQ)12,(AQ)24,(QAQ)8或(QAQ)16。
无论合成的重复序列是何时从ADF-4衍生的,它也可以优选包括一个或多个SEQ ID NO:5(组件C)的氨基酸序列或其变体的一个或多个重复,如上所述,其中总的合成的重复序列是C16或C32。
对于本发明的完全重组蜘蛛丝蛋白质的优选实施方案是(QAQ)8NR3,(QAQ)16NR3,(AQ)12NR3,(AQ)24NR3,C16NR4和C32NR4,即,包含或由所述序列组成的蛋白质。
应当注意,上述合成的重复序列的构型(利用A、Q和C系统)也适用于以上公开的全部其它的重复单元,例如对于A和/或C,可以采用全部含有聚丙氨酸的序列,并且全部富含甘氨酸的序列可以用作Q。
用于从鞭形序列衍生的合成的重复序列的新的组件是组件K(SEQ IDNO:35和36),sp(SEQ ID NO:37和38),X(SEQ ID NO:39和40),和Y(SEQ ID NO:41和42):
合成的重复序列还优选包括或由Y8,Y16,X8,X16,K8,K16组成。
而且,也可以将那些从ADF-3和ADF-4衍生的序列和Flag组合在一个重组序列中。
然而,在本发明中,仍然强烈优选采用步骤a)中的蜘蛛丝蛋白质,其选自或含有ADF-4序列及其衍生物,包括C16、C16NR4、C32、和/或C32NR4的组的序列。
在本发明中,蜘蛛丝蛋白质可以另外改造成在胶囊生产以前含有单一氨基酸取代或直接的化学修饰,或后者也在胶囊生产以后。这可用于将例如特异性结合亲和性引入到所述袋或引入蛋白酶特异性氨基酸序列。这可以产生通过丝膜的蛋白水解消化的包封物的受控制释放。
通过引入例如单一的半胱氨酸,可以实现所述袋的交联或不同的官能团的共价偶联。例如,用编码赖氨酸或半胱氨酸的核酸序列替代编码蜘蛛丝蛋白质中一个或多个氨基酸的核酸,和/或将含有编码赖氨酸和/或半胱氨酸的核酸的核酸序列添加到所述序列,可以实现此。
另外,可以将试剂在纳米胶囊或微胶囊形成前后偶联至所述蜘蛛丝蛋白质,以便将所述胶囊定向到特定的细胞或组织。例如,这可以通过引入或共价偶联特定的RGD序列而实现。因而,可以将RGD肽在纳米胶囊或微胶囊形成前后交联到所述蜘蛛丝蛋白质。环状RGD分子的实例显示在图10中。
而且,可以将细胞或组织特异性抗体和细胞或组织特异性受体偶联到所述蜘蛛丝蛋白质而将所述胶囊定向到特定的靶标。
根据另外的实施方案,b)中的溶剂和/或至少一个另外相的溶剂选自由下列各项组成的组:亲水性溶剂优选地水、醇如乙醇、甘油,或亲脂性溶剂优选地天然油,诸如植物油或动物来源的油,合成的油,诸如miglyol、硅油,有机溶剂,诸如芳香烃,例如甲苯、苯等。
应当注意,一个单一的相也可以含有多于一种溶剂(即混合物),只要所述溶剂基本上相同。“基本上相同”指的是所述溶剂具有类似的溶解度性质因而仅形成一个共同的相。因而,“基本上相同的”溶剂在所述溶剂共混的情况下包括其中不能观察到分离的相的溶剂。作为实例,可以将两种或多种亲脂性溶剂组合成一个相,例如植物油(例如橄榄油和蓖麻油)和miglyol和/或十六烷。或者,作为备选方案,亲水性相可以包括两种或多种亲水性组分,例如水、甘油等。
如上所述,唯一需要的是,用于生产本发明的纳米胶囊和微胶囊的乳状液体系具有至少两个相,其中所述相基本上不可混溶。
全部已知的乳状液类型可以采用在本发明方法的步骤c)中,例如W/O、O/W、O/W/O或W/O/W类型乳状液。这些乳状液类型在本领域中是众所周知的并且对于另外的信息例如参考“雷明顿药物科学(Remington′sPharmaceutical Sciences)”,Mack出版公司,Easton,PA,最新版或另外可以得到的信息。
用于形成本发明的乳状液的优选方法是产生微乳状液。微乳状液是尺寸在50和500nm之间的临界稳定的油滴的分散体,是通过剪切含有油、水、表面活性剂和疏水性试剂的体系而制备的。这样的微乳状液中的聚合,当仔细制备时,产生具有约与初始小滴相同尺寸的颗粒。这指的是,微乳状液的适当制剂抑制小滴的聚结或奥斯特瓦尔德熟化。微乳状液的制备是通过高剪切设备诸如超声和高压匀浆器而进行的。其涉及K.Landfester和同事的多种出版物。
在W/O类型乳状液的情况下,W(亲水性的)相形成乳状液小滴并且在该情况下,蜘蛛丝蛋白质包含在W相中。O相是亲脂性相并且形成连续相。
在O/W类型乳状液的情况下,O(亲脂性的)相形成乳状液小滴并且在该情况下,蜘蛛丝蛋白质包含在O相中。W相是亲水性相并且形成连续相。
上述乳状液中使用的表面活性剂可以选自技术人员将基于药物和相关科学领域中的可以得到的知识所使用的那些化合物。在获得本发明乳状液中使用的表面活性剂的例举性选择是甘油、山梨糖醇及其它多官能醇的脂肪酸酯,优选地,单硬脂酸甘油酯、脱水山梨醇单月桂酸酯、或脱水山梨醇单油酸酯;聚乙二胺(poloxamines);聚氧乙烯醚和聚氧乙烯酯;乙氧基甘油三酸酯;乙氧基酚和乙氧基联苯酚;脂肪酸的金属盐,脂族醇硫酸酯的金属盐,十二烷基硫酸钠;和磺基丁二酸酯的金属盐;聚山梨酯,更优选聚山梨酯20、40、60和80;泊洛沙姆,聚氧乙烯二醇;和所述物质的混合物。
然而,应当明确注意,使用表面活性剂不是本发明的必需特征。技术人员知道不需要表面活性剂的乳状液体系。
在本发明的优选实施方案中,1b)中使用的溶剂还含有一种或多种药剂、化妆品试剂、食品或食品添加剂。换言之,另外的成分通常将存在于所述相中,其也含有蜘蛛丝蛋白质。在该情况下,一种或多种成分/试剂将被包封到在相界面形成的聚合物网络中。
作为备选方案,也可以将上述试剂添加到不含蜘蛛丝蛋白质的连续相。在该情况下,本发明的纳米胶囊和微胶囊将用所述试剂涂布。
作为另外的备选方案,可以将所述试剂在通过本发明方法获得它们以后引入到本发明的纳米胶囊和微胶囊中。
这可以通过用某些溶剂溶胀所述膜并且使包封物(有效试剂)在内部扩散而进行。还可以通过温度、压力或者不仅溶剂而且还有其它化学方法(诸如化学试剂、pH,及其它)而进行溶胀。
也可以将所述包封物结合到所述膜中。该方法可以得到比将所述包封物包封到其中而改良的或改进的释放性质。
另外结合到本发明的纳米胶囊和微胶囊中的试剂类型不以任何方式进行限制。
例如,所述药剂可以选自由下列各项组成的组:镇痛药;催眠药和镇静药;用于治疗精神病病症诸如抑郁和精神分裂症的药物;抗癫痫药和抗惊厥药;用于治疗帕金森病和亨廷顿舞蹈病、衰老和阿尔茨海默病的药物;目的在于治疗CNS损伤或中风的药物;用于治疗成瘾和药物滥用的药物;用于寄生虫感染和由微生物所引起的疾病的化疗剂;免疫抑制剂和抗癌药物;激素和激素拮抗药;用于非金属毒性试剂的拮抗药;用于治疗癌症的细胞抑制剂;供医学用的诊断物质;免疫活性和免疫反应性试剂;抗生素;抗痉挛药;抗组胺剂;止恶心药;松弛剂;刺激物;脑扩张药(dilator);促精神药;脉管扩张药和收缩药;抗高血压药;用于偏头痛治疗的药物;催眠药,高血糖和低血糖药剂;抗哮喘药;抗病毒剂;及其混合物。
食品和食品添加剂可以选自由下列各项组成的组:维生素(抗坏血酸,醋酸维生素E等),无机物(例如,钙、镁、钾、钠),微量元素(硒),天然来源的提取物,天然油(鱼油)等。
例如化妆品试剂可以选自醋酸维生素E,天然或合成来源的油,泛醇,植物提取物,UV吸收剂,消毒剂(desinfectant),抗刺激物试剂,驱除剂。
应当注意,所述试剂可以存在于溶解的、悬浮的或固体形态的溶剂中。在后者情况中,提供由本发明的蜘蛛丝蛋白质涂布的固体核心。
在优选实施方案中,步骤e)中聚合物网络的分离是通过离心或通过破坏步骤c)中形成的乳状液并形成单相溶液而进行的。然而,也可以使用其它方法以便从乳状液体系分离本发明的纳米胶囊和微胶囊。
步骤b)-e)中使用的温度是5-40℃,优选10-30并且更优选室温。步骤b)-e)中使用的pH是3-9,优选5-8,更优选7。
乳状液小滴的尺寸和由此衍生的纳米颗粒和微颗粒优选10nm至40μm,优选在500nm和10μm之间,最优选约5μm。获得的纳米胶囊和微胶囊的壁厚优选是5~100nm,更优选10~70nm(例如见图5)。
在第二方面中,本发明提供由以上公开的方法可获得的纳米胶囊和微胶囊。
本发明的第三方面涉及含有如上所限定的纳米胶囊和微胶囊和一种或多种药用载体的药物组合物。因而,本发明的有效组分优选用于以与可接受的载体或载体材料混合的剂量的这样一种药物组合物,从而可以治疗或至少缓和所述疾病。这样一种组合物可以(除所述活性成分和所述载体之外)包括填充材料、盐、缓冲剂、稳定剂、增溶剂及其它材料,这是已知的本领域现有技术。
术语“药用的”规定为非毒性的材料,其不干扰活性成分的生物活性的效力。所述载体的选择取决于应用。
所述药物组合物可以含有增强活性成分活性或者补充治疗的另外成分。这样的另外的成分和/或因素可以是药物组合物的部分以实现协同效应或使不利的或不希望的效应最小化。
用于本发明的化合物的制剂或制备和应用/药物疗法的技术发表在“雷明顿药物科学(Remington′s Pharmaceutical Sciences)”,Mack出版公司,Easton,PA,最新版(也参见以上)。适当的施用可以包括例如,口服的、皮肤的或跨粘膜的施用和肠胃外的施用,包括肌肉内的、皮下的、髓内(intramedular)的注射以及鞘内的、直接心室内的、静脉内的、腹膜内的或鼻内的注射。
在第四方面中,本发明提供含有如本文以上所公开的纳米胶囊和微胶囊的化妆品或食物产品。
将在这里提到的全部出版物,专利申请,专利,及其它参考文献通过参考全部结合于此。在冲突的情况下,包括定义的本说明书将控制。另外,材料、方法、和实例仅是说明性的并且不意欲是限制性的。
现在还将由附图举例说明本发明,其中:
图1显示(a)作为NaCl浓度和pH的函数的peptosome的流体动力学半径RH的动态光散射。(b)peptosome和作为pH函数的它的尺寸改变的示意图,其原因在于所述肽部分中的卷曲向α-螺旋二级结构的转换。
图2是蜘蛛袋/气囊形成方法的示意图。(A)蛋白质水悬浮液在甲苯中乳化。(B)蛋白质吸附在水-甲苯界面并且变性形成聚合物网络(插入物)。(C)一旦吸附,所述蛋白质网络可以通过离心传递到水中。最终的袋/气囊结构具有内部的水和在外面的水。(D)备选地,一旦吸附,所述蛋白质网络可以通过添加乙醇而传递到单相溶液。
图3显示(A)甲苯/乙醇(50∶50)和(B)在传递到水中以后的蜘蛛袋/气囊的图像。
图4是在传递到连续水相中后的充满FITC-标记的葡聚糖(MW 500kDa)蜘蛛袋/气囊的图像:(A)明亮区域图像。(B)荧光图像。
图5显示由SEM产生的干燥蜘蛛袋/气囊图像。已经确定膜厚度是小于70nm。
图6在微胶囊形成以前()和以后()的D2O中的IR光谱。光谱中的移位显示在微胶囊形成期间蛋白质结构的改变。具体而言,该移位显示β-折叠的形成。微胶囊IR光谱的消卷积和高斯拟合显示四个峰。消卷积的峰是在1621cm-1,1642cm-1,1661cm-1和1683cm-1并且分别指定为β-折叠、无规卷曲结构和两个β-转角的峰。
图7微胶囊渗透性测量。(A)水中微胶囊的样品共焦图像,在所述微胶囊外部添加0.3%的4 kDa的FITC葡聚糖。一部分葡聚糖透过所述膜。(B)如用GPC荧光测量的葡聚糖的分子量分布。(C)51微胶囊的测量截留分子量的直方图。平均截留分子量是2.2kDa。
图8 C16微胶囊的蛋白酶K消化。顶部横行是相对比图像。底部横行是荧光图像。如用荧光损失所表示,在添加蛋白酶K以后立即释放葡聚糖。在13±1分钟以后发生完全的微胶囊消化。
图9对比微胶囊变形所施加的力,如用AFM测量。插入的图是小变形期间的线性力方式(ε<1%)。
图10合成本发明中使用的环状RGD分子的最终步骤。
实施例:
蛋白质制备
形成蜘蛛气囊的蛋白质溶液是用第一溶解的重组蜘蛛拖丝丝蛋白(C16,见Huemmerich等,2004)在10mg/ml在6M硫氰酸胍中制备的。将蛋白质溶液冷却至4℃,并且利用截留分子量为14kDa的来自Carl RothGmbH的透析管,通过将所述蛋白质溶液对10mM Tris缓冲液,pH8.0过夜透析,将硫氰酸胍的浓度减少到1mM以下。通过在100.000×G的力同时将溶液温度保持在4℃而将透析溶液离心30分钟而除去任何不分散的蛋白质。利用UV吸附确定最终的蛋白质浓度,在276nm的波长采用蛋白质消光系数为0.859。
微胶囊形成
通过在300μl甲苯中将5μl的透析蛋白质悬浮液乳化90秒而形成蜘蛛丝的微胶囊(图2A)。在乳化作用期间,丝蛋白质在乳状液小滴的表面吸附并且改变它的结构构象,产生包封乳状液小滴的聚合物网络(图2B)。利用浓度在1至6mg/ml范围内的蛋白质悬浮液并且用短至20秒的乳化时间,形成蜘蛛丝微胶囊。形成的微胶囊的尺寸取决于乳状液小滴的尺寸。
一旦形成,围绕乳状液小滴的蛋白质壳从两相乳状液传递到单相溶液中。在传递所述蛋白质壳中两个不同的方法是有效的。在第一方法中,将300μl的水加入到甲苯形成水次层。将围绕水小滴的蛋白质壳从甲苯层以100×g的力离心4分钟进入到水次层中(图2C)。在第二方法中,通过将300μl乙醇加入到两相乳状液中而形成单相溶液,以便增溶甲苯和水(图2D)。在使用任何一个方法将所述微胶囊传递至单相溶液以后,用光学显微镜研究得到的结构(图3)。
不像可溶的C16,其结构主要是无规卷曲,装配的蛋白质具有β-折叠-丰富的构象。利用IR显微术观察C16构象在装配时的改变。最初,溶解在D2O中的C16在1645cm-1吸收,其是无规卷曲结构的蛋白质的特征(图6)。在微胶囊形成以后,吸附光谱中的两个谷肩(shoulder)出现,表明C16二级结构的改变。光谱的消卷积揭示在1621cm-1、1642cm-1、1661cm-1和1683cm-1的四个高斯峰的贡献。
通过在乳化之前向蛋白质溶液添加0.5重量%,FITC标记的,500kDa葡聚糖(西格玛-奥德里奇(Sigma-Aldrich))而证实离心的微胶囊状蛋白质壳的完整性。在乳化和离心以后,形成的微胶囊状结构继续发荧光,表明这些结构的蛋白质壳在离心期间不分开(图4)。微胶囊膜可以截留大分子诸如高分子量葡聚糖但是对于小分子诸如荧光素是可渗透的。如果将低分子量FITC标记的葡聚糖加入到离心的微胶囊的外部,则一部分葡聚糖透过膜并进入胶囊(图7A)。低分子量葡聚糖的部分许可进入发生是因为葡聚糖具有非限定的多分散性,包括低和高的葡聚糖分子(图7B)。从而所述膜接纳某一截留分子量下的葡聚糖并且排除大于该截留的葡聚糖。通过测量微胶囊内部荧光强度的量并且通过利用葡聚糖的荧光分子量分布(图7B),如通过凝胶渗透层析法测量,确定膜的截留分子量。测量了在13个不同的样品中的52个不同微胶囊的渗透性。这些微胶囊的截留分子量在0.3kDa至6.0kDa范围内,平均截留分子量为2.2kDa(rg~18)(图7C)。
内容物诸如FITC-标记的葡聚糖的酶促触发释放是利用酶蛋白酶K证明的(图8)。如荧光损失所示,在添加蛋白酶K以后不久,微胶囊膜的完整性被破坏并且释放葡聚糖。在葡聚糖释放以后,酶继续消化微胶囊直到在13±1分钟发生完全的消化。
可以通过光启动的用过氧化硫酸铵(APS)和Tris(2,2′-联吡啶)二氯钌(II)(Rubpy)的氧化用化学交联C16而防止微胶囊的酶促消化。为了化学交联C16,将10mM的APS和100mM的Rubpy加入到离心的溶液,并且通过将所述混合物暴露于来自钨灯的光达5分钟而光启动所述反应。该交联致使C16微胶囊对于用蛋白酶K的处理是稳定的。在交联以后,即使在37℃温育一小时以后,添加100μm蛋白酶K至交联的微胶囊对于胶囊完整性也没有影响。该行为显著不同于在相同条件之下几乎立即释放包封的葡聚糖的非交联微胶囊。
观察到形成的微胶囊是高度弹性的。用AFM通过压缩测量微胶囊的弹性。对于压缩测量,对于尺寸在1至4微米范围内的微胶囊,获得了用弹簧常数为10pN/nm的附着于AFM悬臂的35微米的玻璃球和对比变形曲线的力(图9)。在小的变形,施加的力,f,和得到的变形,ε,之间的关系由下式描述
其中h是膜厚度,E是杨氏模量,σ是泊松比,并且前因子是一级常数。利用由使用的丝单体初始浓度计算的最大胶囊壁厚并且通过假定泊松比为0.5,确定所述微胶囊具有杨氏模量在E=0.7-3.6GPa之间。所述胶囊也表明优异的化学稳定性。蛋白质变性剂诸如2%十二烷基硫酸钠(SDS)和8M脲的添加对于胶囊完整性没有影响。观察所述微胶囊在这些条件之下稳定数周。
参考文献:
Chécot F,Lecommandoux S,Gnanou Y,Klok HA(2002)应用化学国际版(Angew.Chem.Int.Ed.)41,1339
Chécot F,Lecommandoux S,Klok HA,Gnanou Y(2003)欧洲物理杂志(Euro.Phys.J.)E 10,25
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Scheibel T(2004)蜘蛛丝:合成蛋白质的重组合成,装配,纺丝,和工程化(Spider silks:recombinant synthesis,assembly spinning,andengineering of synthetic proteins),微生物细胞工厂(Microbial Cell Factories)3,14
序列表
<110>慕尼黑技术大学
<120>生产纳米胶囊和微胶囊的方法
<130>P21347
<140>PCT/EP
<141>2006-08-01
<150>EP 05016712.1
<151>2005 08 01
<160>55
<170>专利版本3.1
<210>1
<211>653
<212>PRT
<213>十字园蛛(Araneus diadematus)
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Ala Arg Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
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Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala
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Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly
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Pro Ser Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro
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Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly
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Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro
100 105 110
Gly Ser Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly Pro Gly Ser
115 120 125
Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
130 135 140
Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly
145 150 155 160
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly
165 170 175
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly
180 185 190
Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro
195 200 205
Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly
210 215 220
Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln
225 230 235 240
Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala
245 250 255
Ala Ala Ala Gly Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly Pro Gly
260 265 270
Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala
275 280 285
Ala Ser Ala Ala Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly
290 295 300
Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala
305 310 315 320
Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln
325 330 335
Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln
340 345 350
Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala
355 360 365
Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly
370 375 380
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln
385 390 395 400
Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln
405 410 415
Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly Pro Gly
420 425 430
Ala Ser Ala Ala Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly
435 440 445
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln
450 455 460
Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln
465 470 475 480
Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala
485 490 495
Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly
500 505 510
Gln Gln Gly Pro Gly Gln Gln Gly Pro Val Gly Gln Gly Pro Tyr Gly
515 520 525
Pro Gly Ala Ala Ser Ala Ala Val Ser Val Gly Gly Tyr Gly Pro Gln
530 535 540
Ser Ser Ser Ala Pro Val Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser
545 550 555 560
Pro Ala Ala Ser Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser
565 570 575
Ser Gly Pro Thr Asn Gln Ala Ala Leu Ser Asn Thr Ile Ser Ser Val
580 585 590
Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val
595 600 605
Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu
610 615 620
Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Gly Ala Ser Ala Gln Tyr
625 630 635 640
Thr Gln Met Val Gly Gln Ser Val Ala Gln Ala Leu Ala
645 650
<210>2
<211>671
<212>PRT
<213>十字园蛛
<400>2
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Ala Ala Arg Ala
1 5 10 15
Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ser Gly Ser Gly Gly Tyr
20 25 30
Gly Pro Glu Asn Gln Gly Pro Ser Gly Pro Val Ala Tyr Gly Pro Gly
35 40 45
Gly Pro Val Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Gly Pro
50 55 60
Gly Gly Tyr Gly Pro Glu Asn Gln Gly Pro Ser Gly Pro Gly Gly Tyr
65 70 75 80
Gly Pro Gly Gly Ser Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala
85 90 95
Ser Gly Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro
100 105 110
Gly Gly Ser Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro
115 120 125
Gly Ala Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly
130 135 140
Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Ala Tyr Gly
145 150 155 160
Pro Gly Gly Pro Gly Ser Ser Ala Ala Ala Ser Gly Pro Gly Gly Tyr
165 170 175
Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Gly Ser Gly Gly Tyr
180 185 190
Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Gly Pro Gly Ala Ser
195 200 205
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly Tyr Gly
210 215 220
Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Ala Tyr Gly Pro Gly Gly
225 230 235 240
Pro Gly Ser Ser Ala Ala Ala Ser Gly Pro Gly Gly Tyr Gly Pro Gly
245 250 255
Ser Gln Gly Pro Ser Gly Pro Gly Ala Tyr Gly Pro Gly Gly Pro Gly
260 265 270
Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Gly Pro Gly Gly Tyr
275 280 285
Gly Pro Gly Asn Gln Gly Pro Ser Gly Pro Gly Gly Tyr Gly Pro Gly
290 295 300
Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly
305 310 315 320
Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Val Tyr Gly
325 330 335
Pro Gly Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser
340 345 350
Gly Pro Gly Gly Tyr Gly Pro Gly Asn Gln Gly Pro Ser Gly Pro Gly
355 360 365
Gly Tyr Gly Pro Gly Gly Ser Gly Ser Ser Ala Ala Ala Ala Ala Ala
370 375 380
Ala Ala Ser Gly Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser
385 390 395 400
Gly Pro Gly Gly Ser Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser
405 410 415
Gly Pro Gly Ala Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly
420 425 430
Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Ala
435 440 445
Tyr Gly Pro Gly Gly Pro Gly Ser Ser Ala Ala Ala Ser Gly Pro Gly
450 455 460
Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Ala Tyr Gly
465 470 475 480
Pro Gly Gly Pro Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ser Gly
485 490 495
Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Gly
500 505 510
Ser Arg Gly Tyr Gly Pro Gly Ser Gln Gly Pro Gly Gly Pro Gly Ala
515 520 525
Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly Tyr
530 535 540
Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Tyr Gln Gly Pro Ser
545 550 555 560
Gly Pro Gly Ala Tyr Gly Pro Ser Pro Ser Ala Ser Ala Ser Val Ala
565 570 575
Ala Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser Ala
580 585 590
Val Ser Ser Leu Val Ser Ser Gly Pro Thr Asn Gly Ala Ala Val Ser
595 600 605
Gly Ala Leu Asn Ser Leu Val Ser Gln Ile Ser Ala Ser Asn Pro Gly
610 615 620
Leu Ser Gly Cys Asp Ala Leu Val Gln Ala Leu Leu Glu Leu Val Ser
625 630 635 640
Ala Leu Val Ala Ile Leu Ser Ser Ala Ser Ile Gly Gln Val Asn Val
645 650 655
Ser Ser Val Ser Gln Ser Thr Gln Met Ile Ser Gln Ala Leu Ser
660 665 670
<210>3
<211>24
<212>PRT
<213>人工序列
<220>
<223>组件A(ADF-3)
<400>3
Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly
1 5 10 15
Tyr Gly Pro Gly Ser Gly Gln Gln
20
<210>4
<211>20
<212>PRT
<213>人工序列
<220>
<223>组件Q(ADF-3)
<400>4
Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
1 5 10 15
Pro Gly Gln Gln
20
<210>5
<211>35
<212>PRT
<213>人工序列
<220>
<223>组件C(ADF-4)
<400>5
Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly
1 5 10 15
Tyr Gly Pro Glu Asn Gln Gly Pro Ser Gly Pro Gly Gly Tyr Gly Pro
20 25 30
Gly Gly Pro
35
<210>6
<211>942
<212>PRT
<213>棒络新妇蛛(Nephila clavipes)
<400>6
Ala Cys Phe Thr Ser Ala Val Ile Phe Leu Phe Leu Ala Gln Cys Ala
1 5 10 15
Ser Thr Tyr Gly Arg Gly Ile Ile Ala Asn Ser Pro Phe Ser Asn Pro
20 25 30
Asn Thr Ala Glu Ala Phe Ala Arg Ser Phe Val Ser Asn Ile Val Ser
35 40 45
Ser Gly Glu Phe Gly Ala Gln Gly Ala Glu Asp Phe Asp Asp Ile Ile
50 55 60
Gln Ser Leu Ile Gln Ala Gln Ser Met Gly Lys Gly Arg His Asp Thr
65 70 75 80
Lys Ala Lys Ala Lys Ala Met Gln Val Ala Leu Ala Ser Ser Ile Ala
85 90 95
Glu Leu Val Ile Ala Glu Ser Ser Gly Gly Asp Val Gln Arg Lys Thr
100 105 110
Asn Val Ile Ser Asn Ala Leu Arg Asn Ala Leu Met Ser Thr Thr Gly
115 120 125
Ser Pro Asn Glu Glu Phe Val His Glu Val Gln Asp Leu Ile Gln Met
130 135 140
Leu Ser Gln Glu Gln Ile Asn Glu Val Asp Thr Ser Gly Pro Gly Gln
145 150 155 160
Tyr Tyr Arg Ser Ser Ser Ser Gly Gly Gly Gly Gly Gly Gln Gly Gly
165 170 175
Pro Val Val Thr Glu Thr Leu Thr Val Thr Val Gly Gly Ser Gly Gly
180 185 190
Gly Gln Pro Ser Gly Ala Gly Pro Ser Gly Thr Gly Gly Tyr Ala Pro
195 200 205
Thr Gly Tyr Ala Pro Ser Gly Ser Gly Ala Gly Gly Val Arg Pro Ser
210 215 220
Ala Ser Gly Pro Ser Gly Ser Gly Pro Ser Gly Gly Ser Arg Pro Ser
225 230 235 240
Ser Ser Gly Pro Ser Gly Thr Arg Pro Ser Pro Asn Gly Ala Ser Gly
245 250 255
Ser Ser Pro Gly Gly Ile Ala Pro Gly Gly Ser Asn Ser Gly Gly Ala
260 265 270
Gly Val Ser Gly Ala Thr Gly Gly Pro Ala Ser Ser Gly Ser Tyr Gly
275 280 285
Pro Gly Ser Thr Gly Gly Thr Tyr Gly Pro Ser Gly Gly Ser Glu Pro
290 295 300
Phe Gly Pro Gly Val Ala Gly Gly Pro Tyr Ser Pro Gly Gly Ala Gly
305 310 315 320
Pro Gly Gly Ala Gly Gly Ala Tyr Gly Pro Gly Gly Val Gly Thr Gly
325 330 335
Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly
340 345 350
Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala
355 360 365
Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly
370 375 380
Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro
385 390 395 400
Gly Gly Tyr Gly Pro Gly Gly Thr Gly Pro Gly Gly Tyr Gly Pro Gly
405 410 415
Gly Thr Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Pro Gly Gly
420 425 430
Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala
435 440 445
Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly
450 455 460
Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Pro
465 470 475 480
Ser Gly Ala Gly Leu Gly Gly Ala Gly Pro Gly Gly Ala Gly Leu Gly
485 490 495
Gly Ala Gly Pro Gly Gly Ala Gly Thr Ser Gly Ala Gly Pro Gly Gly
500 505 510
Ala Gly Pro Gly Gly Ala Gly Gln Gly Asp Ala Gly Pro Gly Gly Ala
515 520 525
Gly Arg Gly Gly Ala Gly Arg Gly Gly Val Gly Arg Gly Gly Ala Gly
530 535 540
Arg Gly Gly Ala Gly Arg Gly Gly Ala Arg Gly Ala Gly Gly Ala Gly
545 550 555 560
Gly Ala Gly Gly Ala Gly Gly Ser Gly Gly Thr Thr Ile Val Glu Asp
565 570 575
Leu Asp Ile Thr Ile Asp Gly Ala Asp Gly Pro Ile Thr Ile Ser Glu
580 585 590
Glu Leu Thr Ile Gly Gly Ala Gly Ala Gly Gly Ser Gly Pro Gly Gly
595 600 605
Ala Gly Pro Gly Asn Val Gly Pro Gly Arg Ser Gly Pro Gly Gly Val
610 615 620
Gly Pro Gly Gly Ser Gly Pro Gly Gly Val Gly Pro Gly Ser Phe Gly
625 630 635 640
Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Val Gly Ser
645 650 655
Gly Gly Ser Gly Gln Gly Gly Val Arg Pro Ser Gly Ser Gly Pro Gly
660 665 670
Gly Val Gly Thr Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Pro Tyr
675 680 685
Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Ser Ala Gly Gly Thr
690 695 700
Tyr Gly Pro Gly Gly Phe Gly Gly Pro Gly Gly Phe Gly Gly Pro Gly
705 710 715 720
Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly
725 730 735
Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly Pro
740 745 750
Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly
755 760 765
Gly Ser Tyr Gly Leu Gly Gly Ala Gly Gly Ser Gly Gly Val Gly Pro
770 775 780
Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly
785 790 795 800
Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly
805 810 815
Ser Gly Ser Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Ser
820 825 830
Gly Pro Gly Gly Tyr Gly Pro Gly Gly Thr Gly Pro Gly Gly Ser Glu
835 840 845
Ser Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Ser Gly Pro
850 855 860
Gly Gly Ser Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly
865 870 875 880
Gly Ser Gly Pro Ser Ser Phe Val Pro Gly Gly Ser Gly Pro Gly Gly
885 890 895
Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Val
900 905 910
Gly Leu Gly Gly Ala Gly Arg Gly Gly Ala Gly Arg Gly Gly Ala Gly
915 920 925
Ser Val Gly Ala Gly Arg Gly Gly Ala Gly Arg Gly Gly Thr
930 935 940
<210>7
<211>907
<212>PRT
<213>棒络新妇蛛
<400>7
Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly
1 5 10 15
Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro
20 25 30
Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly
35 40 45
Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly
50 55 60
Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Tyr
65 70 75 80
Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Thr Gly
85 90 95
Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro
100 105 110
Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly
115 120 125
Gly Phe Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly
130 135 140
Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Phe
145 150 155 160
Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly
165 170 175
Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro
180 185 190
Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly
195 200 205
Gly Ala Gly Pro Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala
210 215 220
Gly Gly Ser Gly Gly Ala Gly Gly Ser Gly Gly Thr Thr Ile Ile Glu
225 230 235 240
Asp Leu Asp Ile Thr Ile Asp Gly Ala AsP Gly Pro Ile Thr Ile Ser
245 250 255
Glu Glu Leu Thr Ile Ser Gly Ala Gly Gly Ser Gly Pro Gly Gly Ala
260 265 270
Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Val Gly
275 280 285
Pro Gly Gly Ser Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro
290 295 300
Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly
305 310 315 320
Ser Gly Pro Gly Gly Ala Gly Gly Ala Gly Gly Pro Gly Gly Ala Tyr
325 330 335
Gly Pro Gly Gly Ser Tyr Gly Pro Gly Gly Ser Gly Gly Pro Gly Gly
340 345 350
Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro Gly Gly Ala Gly
355 360 365
Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly
370 375 380
Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Gly Pro Tyr Gly Pro
385 390 395 400
Gly Gly Ser Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly
405 410 415
Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro Gly Gly Ala Gly Gly Pro
420 425 430
Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr
435 440 445
Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala Gly
450 455 460
Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro
465 470 475 480
Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly
485 490 495
Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Ser Gly Gly
500 505 510
Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr
515 520 525
Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Thr Gly
530 535 540
Pro Gly Gly Thr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro
545 550 555 560
Gly Gly Ser Gly Pro Gly Gly Ser Gly Pro Gly Gly Ser Gly Pro Gly
565 570 575
Gly Tyr Gly Pro Ser Gly Ser Gly Pro Gly Gly Tyr Gly Pro Ser Gly
580 585 590
Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr
595 600 605
Gly Pro Gly Gly Ser Gly Ala Gly Gly Thr Gly Pro Gly Gly Ala Gly
610 615 620
Gly Ala Gly Gly Ala Gly Gly Ser Gly Gly Ala Gly Gly Ser Gly Gly
625 630 635 640
Ala Gly Gly Ser Gly Gly Ala Gly Gly Ser Gly Gly Val Gly Gly Ser
645 650 655
Gly Gly Thr Thr Ile Thr Glu Asp Leu Asp Ile Thr Ile Asp Gly Ala
660 665 670
Asp Gly Pro Ile Thr Ile Ser Glu Glu Leu Thr Ile Ser Gly Ala Gly
675 680 685
Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly
690 695 700
Ser Gly Pro Gly Gly Val Gly Pro Gly Val Ser Gly Pro Gly Gly Val
705 710 715 720
Gly Pro Gly Gly Ser Gly Pro Gly Gly Val Gly Ser Gly Gly Ser Gly
725 730 735
Pro Gly Gly Val Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Ser
740 745 750
Gly Gly Val Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Gly Phe
755 760 765
Tyr Gly Pro Gly Gly Ser Glu Gly Pro Tyr Gly Pro Ser Gly Thr Tyr
770 775 780
Gly Ser Gly Gly Gly Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly
785 790 795 800
Pro Gly Ser Pro Gly Gly Ala Tyr Gly Pro Gly Ser Pro Gly Gly Ala
805 810 815
Tyr Tyr Pro Ser Ser Arg Val Pro Asp Met Val Asn Gly Ile Met Ser
820 825 830
Ala Met Gln Gly Ser Gly Phe Asn Tyr Gln Met Phe Gly Asn Met Leu
835 840 845
Ser Gln Tyr Ser Ser Gly Ser Gly Thr Cys Asn Pro Asn Asn Val Asn
850 855 860
Val Leu Met Asp Ala Leu Leu Ala Ala Leu His Cys Leu Ser Asn His
865 870 875 880
Gly Ser Ser Ser Phe Ala Pro Ser Pro Thr Pro Ala Ala Met Ser Ala
885 890 895
Tyr Ser Asn Ser Val Gly Arg Met Phe Ala Tyr
900 905
<210>8
<211>636
<212>PRT
<213>十字园蛛
<400>8
Ala Arg Ala Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
1 5 10 15
Gln Gln Gly Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala
20 25 30
Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly
35 40 45
Pro Ser Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro
50 55 60
Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly
65 70 75 80
Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro
85 90 95
Gly Ser Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly Pro Gly Ser
100 105 110
Gly Gln Gln Gly Ala Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly
115 120 125
Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly
130 135 140
Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly
145 150 155 160
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly
165 170 175
Ser Gly Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr
180 185 190
Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro
195 200 205
Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
210 215 220
Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala
225 230 235 240
Ala Ala Gly Gly Tyr Gly Pro Gly Tyr Gly Gln Gln Gly Pro Gly Gln
245 250 255
Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala
260 265 270
Ser Ala Ala Ser Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro
275 280 285
Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser
290 295 300
Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln
305 310 315 320
Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
325 330 335
Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala
340 345 350
Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln
355 360 365
Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln
370 375 380
Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
385 390 395 400
Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Ala Tyr Gly Pro Gly Ala
405 410 415
Ser Ala Ala Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln
420 425 430
Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln
435 440 445
Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
450 455 460
Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala
465 470 475 480
Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gln
485 490 495
Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro
500 505 510
Gly Ala Ala Ser Ala Ala Val Ser Val Gly Gly Tyr Gly Pro Gln Ser
515 520 525
Ser Ser Val Pro Val Ala Ser Ala Val Ala Ser Arg Leu Ser Ser Pro
530 535 540
Ala Ala Ser Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser Ser
545 550 555 560
Gly Pro Thr Lys His Ala Ala Leu Ser Asn Thr Ile Ser Ser Val Val
565 570 575
Ser Gln Val Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu
580 585 590
Val Gln Ala Leu Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu Gly
595 600 605
Ser Ser Ser Ile Gly Gln Ile Asn Tyr Gly Ala Ser Ala Gln Tyr Thr
610 615 620
Gln Met Val Gly Gln Ser Val Ala Gln Ala Leu Ala
625 630 635
<210>9
<211>410
<212>PRT
<213>十字园蛛
<400>9
Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ser Gly Ser Gly Gly
1 5 10 15
Tyr Gly Pro Glu Asn Gln Gly Pro Ser Gly Pro Val Ala Tyr Gly Pro
20 25 30
Gly Gly Pro Val Ser Ser Ala Ala Ala Ala Ala Ala Ala Gly Ser Gly
35 40 45
Pro Gly Gly Tyr Gly Pro Glu Asn Gln Gly Pro Ser Gly Pro Gly Gly
50 55 60
Tyr Gly Pro Gly Gly Ser Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala
65 70 75 80
Ala Ser Gly Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly
85 90 95
Pro Gly Gly Ser Gly Gly Tyr Gly Pro Gly Ser Gln Gly Ala Ser Gly
100 105 110
Pro Gly Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala
115 120 125
Ala Ser Gly Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly
130 135 140
Pro Gly Ala Tyr Gly Pro Gly Gly Pro Gly Ser Ser Ala Ala Ala Ala
145 150 155 160
Ala Ala Ala Ala Ser Gly Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly
165 170 175
Pro Ser Gly Pro Gly Val Tyr Gly Pro Gly Gly Pro Gly Ser Ser Ala
180 185 190
Ala Ala Ala Ala Ala Ala Gly Ser Gly Pro Gly Gly Tyr Gly Pro Glu
195 200 205
Asn Gln Gly Pro Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly
210 215 220
Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly Tyr
225 230 235 240
Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Gly Ser Gly Gly Tyr
245 250 255
Gly Pro Gly Ser Gln Gly Gly Ser Gly Pro Gly Ala Ser Ala Ala Ala
260 265 270
Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly Tyr Gly Pro Gly Ser Gln
275 280 285
Gly Pro Ser Gly Pro Gly Tyr Gln Gly Pro Ser Gly Pro Gly Ala Tyr
290 295 300
Gly Pro Ser Pro Ser Ala Ser Ala Ser Val Ala Ala Ser Val Tyr Leu
305 310 315 320
Arg Leu Gln Pro Arg Leu Glu Val Ser Ser Ala Val Ser Ser Leu Val
325 330 335
Ser Ser Gly Pro Thr Asn Gly Ala Ala Val Ser Gly Ala Leu Asn Ser
340 345 350
Leu Val Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp
355 360 365
Ala Leu Val Gln Ala Leu Leu Glu Leu Val Ser Ala Leu Val Ala Ile
370 375 380
Leu Ser Ser Ala Ser Ile Gly Gln Val Asn Val Ser Ser Val Ser Gln
385 390 395 400
Ser Thr Gln Met Ile Ser Gln Ala Leu Ser
405 410
<210>10
<211>140
<212>PRT
<213>人工序列
<220>
<223>NR3(ADF-3)
<400>10
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Gly
1 5 10 15
Ala Ala Ser Ala Ala Val Ser Val Gly Gly Tyr Gly Pro Gln Ser Ser
20 25 30
Ser Ala Pro Val Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro Ala
35 40 45
Ala Ser Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser Ser Gly
50 55 60
Pro Thr Asn Gln Ala Ala Leu Ser Asn Thr Ile Ser Ser Val Val Ser
65 70 75 80
Gln Val Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val
85 90 95
Gln Ala Leu Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu Gly Ser
100 105 110
Ser Ser Ile Gly Gln Ile Asn Tyr Gly Ala Ser Ala Gln Tyr Thr Gln
115 120 125
Met Val Gly Gln Ser Val Ala Gln Ala Leu Ala Gly
130 135 140
<210>11
<211>125
<212>PRT
<213>人工序列
<220>
<223>NR4(ADF-4)
<400>11
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Gly
1 5 10 15
Ala Tyr Gly Pro Ser Pro Ser Ala Ser Ala Ser Val Ala Ala Ser Arg
20 25 30
Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser Ala Val Ser Ser
35 40 45
Leu Val Ser Ser Gly Pro Thr Asn Gly Ala Ala Val Ser Gly Ala Leu
50 55 60
Asn Ser Leu Val Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu Ser Gly
65 70 75 80
Cys Asp Ala Leu Val Gln Ala Leu Leu Glu Leu Val Ser Ala Leu Val
85 90 95
Ala Ile Leu Ser Ser Ala Ser Ile Gly Gln Val Asn Val Ser Ser Val
100 105 110
Ser Gln Ser Thr Gln Met Ile Ser Gln Ala Leu Ser Gly
115 120 125
<210>12
<211>1959
<212>DNA
<213>十字园蛛
<400>12
atggctagca tgactggtgg acagcaaatg ggtcgggatc cgaattcggc acgagccgga 60
tctggacaac aaggacccgg acaacaagga cccggacaac aaggacccgg acaacaagga 120
ccatatggac ccggtgcatc cgccgcagca gcagccgctg gaggttatgg acccggatct 180
ggacaacaag gacccagcca acaaggacct ggccaacaag gacccggtgg tcaaggacca 240
tatggacccg gtgcatccgc cgccgcagca gccgctggtg gatatggacc cggttccgga 300
caacaaggac caggaggtca aggaccatat ggacctggtt catccgctgc cgcagcagcc 360
gctggaggta atggacccgg atctggacaa caagggcccg gtcaacaagg tcctggacaa 420
caaggacccg gtgcatccgc cgccgcagca gccgctggag gatacggacc cggatctgga 480
caacaaggac ccggacaaca aggaccagga ggtcaaggac catatggacc tggtgcatcc 540
gccgctgcag cagccgctgg aggatacgga cccggatctg gacaacaagg acccggacaa 600
caaggaccag gaggtcaagg accatatgga cccggtgcat ccgctgcagc agcagccgct 660
ggaggttatg gacccggatc tggacaacaa ggacccggac aacaaggacc tggacaacaa 720
ggacccggtg gtcaaggacc atatggaccc ggtgcatccg ccgccgcagc agccgctgga 780
ggatacggac ccggttatgg acagcaagga ccaggacaac aaggaccagg aggtcaagga 840
ccatatggac ctggtgcatc cgccgcctca gcagcctctg gaggatacgg acccggatct 900
ggacaacaag gacccggaca acaaggacct ggaggtcaag gaccatatgg acctggtgca 960
tccgccgcag cagcagccgc tggaggttat ggacccggat ctggacaaca aggaccaggc 1020
caacaaggac ccggtcaaca aggacctgga caacaaggac ccggtggtca aggaccatat 1080
ggacctggtg catccgccgc agcagcagcc gctggaggtt atggacccgg atctggacaa 1140
caaggacccg gtcaacaagg acccggtcaa caaggacccg gtcaacaagg acccggtcaa 1200
caaggacccg gccaacaagg acccggtcaa caaggacccg gccaacaagg acctggtcaa 1260
caaggtcccg gtggtcaagg ggcatatgga cctggtgcat ccgccgcagc aggagccgct 1320
ggaggttatg gacccggatc tggacaacaa ggacccggac aacaaggacc cggacaacaa 1380
ggacccggac aacaaggacc cggacaacaa ggacccggac aacaaggacc cggacaacaa 1440
ggacccggac aacaaggacc atatggacct ggtgcatccg ccgcagcagc agccgctgga 1500
ggttatggac ccggatctgg acaacaagga cccggccaac aaggacctgg acaacaagga 1560
cccgttggtc aaggaccata tggacctggt gcggcttctg cagctgtatc tgttggagga 1620
tatggaccac aaagctcctc ggctcctgtt gcatcagcag ccgcttctcg cctttcttct 1680
ccagcggcca gttctagagt ttcatcggct gtatcatctt tggtatctag tggacctact 1740
aatcaagctg cactttctaa tactatcagt agcgttgtat cgcaagttag tgcaagtaat 1800
cctggtcttt ctggttgcga tgtacttgtg caagcattgc tcgaagttgt atcggccctg 1860
gtatctatcc ttggatcttc tagtatcggg caaattaact atggtgcctc tgctcagtac 1920
acccaaatgg taggtcaatc tgtagctcaa gcccttgct 1959
<210>13
<211>2013
<212>DNA
<213>十字园蛛
<400>13
atggctagca tgactggtgg acagcaaatg ggtcgcgcgg cacgagcagg atcttcagca 60
gcagcggccg cggcagcaag tggatctgga ggatacggac ctgaaaacca aggaccatct 120
ggacctgtag catatggacc tggtggaccc gtatcttcag ctgcagcagc agccgctgca 180
ggaagtggac ctggtggata cggacctgaa aaccaaggac catctggacc cggaggatat 240
ggacctggtg gttccggatc ttcagcagca gcagcagccg ctgcagcaag tggacctgga 300
ggatatggac ctggaagcca aggaccatct ggacctggtg gatccggagg atatggtccc 360
ggaagccaag ggccatctgg acctggtgca tcttcggcag cagcagcagc cgctgcagca 420
agtggacctg gaggatatgg acctggaagc caaggaccat ctggacctgg agcatatgga 480
cctggtggac ccggatcttc agctgcagca agtggacctg gaggatatgg acctggaagc 540
caaggaccat ctggacctgg tggatccgga ggatatggtc ccggaagcca agggccatct 600
ggacctggtg ggcctggtgc atctgcggca gcagcagcag ccgctgcagc aagtggacct 660
ggaggatatg gacctggaag ccaaggacca tctggacctg gagcatatgg acctggtgga 720
cccggatctt cagctgcagc aagtggacct ggaggatatg gacctggaag ccaaggacca 780
tctggacctg gagcatatgg acctggtgga cccggatctt cagctgcagc agcagccgct 840
gcaggaagtg gacctggtgg atacggacct ggaaaccaag gaccatctgg acccggagga 900
tatggacctg gtggtcccgg atcttcagca gcagcagccg ctgcagcaag tggacctgga 960
ggatatggac ctggaagcca aggaccatct ggacctggag tatatggacc tggtggaccc 1020
ggatcttcag ctgcagcagc agccgctgca ggaagtggac ctggtggata cggacctgga 1080
aaccaaggac catctggacc cggaggatat ggacctggtg gttccggatc ttcagcagca 1140
gcagcagccg ctgcagcaag tggacctgga ggatatggac ctggaagcca aggaccatct 1200
ggacctggtg gatccggagg atatggtccc ggaagccaag ggccatctgg acctggtgca 1260
tcttcggcag cagcagcagc cgctgcagca agtggacctg gaggatatgg acctggaagc 1320
caaggaccat ctggacctgg agcatatgga cctggtggac ccggatcttc agctgcagca 1380
agtggacctg gaggatatgg acctggaagc caaggaccat ctggtcctgg agcatatgga 1440
cctggtggac ccggatcttc agctgcagca gccgctgcag caagtggacc tggaggatat 1500
ggacctggaa gccaaggacc atctggacct ggtggatccc gaggatatgg tcccggaagc 1560
caaggacctg gtgggcctgg agcatctgcg gcagcagcag cagccgctgc agcaagtgga 1620
cctggaggat atggacctgg aagccaagga ccatctggac ctggatatca aggccctagt 1680
ggtcctggag catatggccc atctccttct gcttccgcat ccgttgcagc ctctcgttta 1740
tcttcgcctg cagcctcgtc tagagtgtct tccgctgtat cgtctttagt gtctagcgga 1800
cctacgaatg gtgctgctgt ttctggagct ttgaatagtt tagtatctca gattagtgca 1860
agtaatccag gtttatcggg atgtgatgct cttgtgcagg cattattgga attagtgtct 1920
gctcttgtgg caattctttc atctgcaagt attggccaag tcaacgtcag ctctgttagt 1980
cagtcaactc aaatgattag ccaagctctt tca 2013
<210>14
<211>420
<212>DNA
<213>人工序列
<220>
<223>NR3(ADF-3)
<400>14
atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgggtgc ggcttctgca 60
gctgtatctg ttggaggata tggaccacaa agctcctcgg ctcctgttgc atcagcagcc 120
gcttctcgcc tttcttctcc agcggccagt tctcgtgttt catcggctgt atcatctttg 180
gtatctagtg gacctactaa tcaagctgca ctttctaata ctatcagtag cgttgtatcg 240
caagttagtg caagtaatcc tggtctttct ggttgcgatg tacttgtgca agcattgctc 300
gaagttgtat cggccctggt atctatcctt ggatcttcta gtatcgggca aattaactat 360
ggtgcctctg ctcagtacac ccaaatggta ggtcaatctg tagctcaagc ccttgctggc 420
<210>15
<211>375
<212>DNA
<213>人工序列
<220>
<223>NR4(ADF-4)
<400>15
atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgggagc atatggccca 60
tctccttctg cttccgcatc cgttgcagcc tctcgtttat cttcgcctgc agcctcgtct 120
cgtgtgtctt ccgctgtatc gtctttagtg tctagcggac ctacgaatgg tgctgctgtt 180
tctggagctt tgaatagttt agtatctcag attagtgcaa gtaatccagg tttatcggga 240
tgtgatgctc ttgtgcaggc attattggaa ttagtgtctg ctcttgtggc aattctttca 300
tctgcaagta ttggccaagt caacgtcagc tctgttagtc agtcaactca aatgattagc 360
caagctcttt caggc 375
<210>16
<211>2828
<212>DNA
<213>棒络新妇蛛
<400>16
gcttgcttta cctcggcagt gatatttctt ttcttagcgc agtgtgcgtc gacgtacgga 60
agggggatta tagccaactc ccctttctca aaccctaaca cagcggaagc ttttgcacga 120
tctttcgtga gcaatattgt ttctagtgga gaatttggag cccaaggagc cgaagacttc 180
gatgacataa ttcagagtct catacaggcc cagagcatgg gcaaagggcg gcatgatacg 240
aaggccaagg cgaaagcgat gcaggtagcc cttgcttctt ctatagccga attggttatt 300
gcagaaagca gcggaggcga tgtgcaacgc aaaaccaacg ttatctccaa cgctttgaga 360
aacgccttga tgtctacaac aggcagccca aacgaagagt tcgtccatga agttcaagac 420
ctcatccaga tgttatctca agaacagatc aacgaggtag atacttcagg accagggcag 480
tactacaggt cgtcttcttc cggtggagga ggtggaggac aaggaggtcc tgtagttact 540
gaaacactga ccgttacagt tggcggatcc ggtggagggc aaccttcagg tgcaggtcct 600
agtggtacag gtggatatgc accaactgga tacgccccaa gcggctcagg tgcaggtggc 660
gttcgaccta gtgcctccgg tccaagtggt agtggaccta gtggtggatc tcgtcctagt 720
agtagtggac ctagtggaac tcgtcccagc cctaatggtg caagtggatc tagccctggt 780
ggtatcgcac ctggtggatc caattctggt ggtgctggag tatccggcgc aactggagga 840
cctgcatcca gcggctccta cggaccagga agtacaggtg gaacatatgg acctagtgga 900
ggaagtgaac ctttcggacc aggagtggct ggaggaccat acagcccagg tggagctgga 960
cctggtggtg caggtggagc ctatggacca ggaggtgtag gaactggtgg agccggacca 1020
ggaggttacg gacctggtgg agccggacca ggaggttatg gacctggtgg agccggacca 1080
ggaggttacg gacctggtgg agctggacca ggaggttacg gacctggtgg agctgggcct 1140
ggaggttacg gacctggtgg agctggacct ggaggttacg gacctggtgg agctggacct 1200
ggaggttacg gacctggtgg aactggacct ggtggatacg gacctggtgg aactggacct 1260
ggaggagttg gacctggagg agctggacca ggaggatatg gacctggtgg tgctggacct 1320
ggtggtgctg gacctggtgg tgctggacct ggtggtgctg gacctggtgg tgctggacct 1380
ggtggtgctg gacctggtgg atacggccct ggtggatctg gacctggtgg tgctggacct 1440
agtggtgccg gacttggtgg tgctggacct ggaggtgcgg gacttggtgg agcaggacct 1500
ggaggagcag gaaccagtgg tgccggaccc ggtggagcag gacccggtgg agcaggacaa 1560
ggtgatgctg gacccggtgg tgcaggacgt ggaggagcag gtcgtggtgg tgtaggtcgt 1620
ggtggtgcag gtcgtggagg tgcaggacgt ggtggagcta gaggtgctgg tggagcagga 1680
ggtgctggtg gagcaggagg atccggcggc acaacaatcg tagaggactt ggatattaca 1740
attgatggtg cagatggccc gataacaata tcagaagaat taacaatcgg tggagcaggc 1800
gctggaggtt ccggacccgg tggtgctgga ccaggaaacg ttggacctgg tcgctctgga 1860
ccaggaggag taggacctgg tggctctgga ccaggaggcg taggacctgg tagctttgga 1920
ccaggaggcg taggacctgg tggctccgga ccaggaggcg taggatctgg tggctccgga 1980
caaggaggag taagacctag tggctccgga ccaggtggcg taggaactgg aggcgtagga 2040
cccggtggtg ctggaggacc ttacggtcct ggtggttccg gacccggagg tgcaggaagc 2100
gctggaggaa cttatggacc tggtggtttc ggaggacccg gtggtttcgg aggacccggt 2160
ggtgctggtg gaccctacgg tccaggtggt gctggtggac cctacggacc aggtggtgct 2220
ggtggaccct acggaccagg tggtgctggt ggaccctacg ggccgggtgg tgctggtgga 2280
ccctacgggc cgggaggtgc tggtggatcc tacgggctgg gtggtgctgg tggatcagga 2340
ggtgtaggac ctggtggaag tggacctgga ggttatggac ccggtggagc gggacctgga 2400
ggttacggac ccggtggttc tggtccaggt ggatacggac ctggcggttc tggatctggt 2460
ggatacggac ctggaggttc tggacctggt ggttctggac ctggtggata cggacctggt 2520
ggtactggac ctggtggttc tgaatctggt ggatacggac ctggtggatc tggacctggc 2580
ggttctggac ctggtggatc tggacctggc ggttctggac ctggtggata cggacctggt 2640
ggttctggac ctagcagttt tgtacctggc ggttctggac ctggtggctc tggacccggt 2700
ggcgctggac ccggtggcgc tggacccggt ggtgttggac ttggaggtgc tggacgtggt 2760
ggagctggac gtggtggagc tggaagtgtt ggagctggac gtggtggagc tggacgtggt 2820
ggaactgg 2828
<210>17
<211>2724
<212>DNA
<213>棒络新妇蛛
<400>17
ggaccaggag gtgtaggacc tggtggaagt ggacctggag gttatggacc cggtggagct 60
ggacctggag gttacggacc tggtggttct ggtccaggtg gatacggacc cggtggttcg 120
ggaccaggag gatacggacc tggcggttct ggacctggtg gatacggacc aggcggttct 180
ggacctggtg gatacggacc aggcggttct ggacctggtg gatacggacc tggtggatat 240
ggacctggtg gttctggacc tggtggatat ggacctggtg gtactggacc tggtggttct 300
ggacccggcg gatacggacc tggtggttct ggacctggcg gttctggacc tggtggatac 360
ggacctggtg gttctggacc tggcggtttt ggacctggcg gttctggacc tggtggatac 420
ggacctggtg gctctggacc cggtggtgct ggtcccggtg gtgttggacc cggtggtttt 480
ggacctggtg gtgctggacc cggtggagct ggacctggtg gtgctggacc tggtggtgct 540
ggacctggtg gtgctggacc tggtggagct ggacctggtg gtgctggacc tggtggagct 600
ggacctggtg gtgctggacc tggtggagct ggacctggtg gtgctggtgg cgctggagga 660
gcaggcggag caggaggttc aggtggagca ggaggatccg gcggtacaac aatcatagaa 720
gacttggata ttacaattga tggcgctgat ggcccgataa cgatttcaga agaattaaca 780
attagtggtg ctggaggttc cggacccggt ggtgctggac caggaggtgt agggcctggt 840
ggctccggac caggaggtgt aggacctgga ggctctggac caggaggtgt aggacctggt 900
ggttctggtc caggaggcgt aggacctggt ggtgctggtg gaccttacgg acctggcggt 960
tctggacctg gaggtgcagg cggagctgga ggacctggtg gagcatacgg acctggtgga 1020
tcatatggac ctggtggttc cggaggaccc ggtggtgctg gcggaccata cggacctgga 1080
ggtgaaggac ccggtggtgc tggcggaccc tacggacctg gtggtgcagg tggaccttac 1140
ggcccaggtg gtgcaggtgg accctacgga ccaggtggtg aaggtggacc ctacggacca 1200
ggtggatcat acggaccggg tggtgctggt ggaccatacg gaccaggtgg accctacgga 1260
cctggaggtg aaggaccagg tggtgctggc ggaccctatg gaccaggagg tgtaggacct 1320
ggtggaagtg gacctggagg ttatggacct ggtggaagtg gacctggagg ttatggacct 1380
ggtggagctg gacctggagg ttacggacct ggtggttctg gtccaggtgg atacggaccc 1440
ggtggttctg gtccaggtgg atacggaccc ggtggttccg gaccaggagg atacggacct 1500
ggcggttctg gacctggtgg atacggatct ggcggtgctg gacctggtgg atacggacct 1560
ggcggttctg gacctggtgg atacggtcct ggaggttctg gacctggtgg ttatggacct 1620
ggtggtactg gacctggtgg tactggacct ggtggttctg gacctggcgg atacggacct 1680
ggtggttctg gacctggcgg ttctggacct ggcggttctg gacctggtgg atacggacct 1740
agtggttcgg gacctggtgg atacggacct agtggttctg gacctggcgg atacggtcct 1800
ggcggttctg gacctggtgg atacggaccg ggtggctctg gagccggtgg tactggacct 1860
ggtggcgctg gaggagcagg cggagcagga ggttcaggtg gagcaggagg ttcaggtggt 1920
gcaggaggtt caggtggagc aggaggttca ggtggagtag gaggatccgg cggtacaaca 1980
atcaccgaag acttggatat tacaattgat ggcgcagatg gcccgataac gatttcagaa 2040
gaattaacaa ttagtggtgc tggaggttct ggacccggtg gtgctggacc aggtggtgta 2100
gggcctggtg gctctggacc aggaggtgta ggacctggag tctctggacc aggaggcgta 2160
ggacctggtg gttctggacc aggaggcgta ggttctggtg gttctggacc aggaggcgta 2220
ggacctggtg gttacggacc tggaggttct ggatcaggag gcgtaggacc tggtggttac 2280
ggacctggag gttcaggagg attttacgga cctggaggtt cagaaggacc ttatggacct 2340
agtggaactt atggttctgg aggaggatat ggtcctggtg gtgctggagg accatatgga 2400
cctggaagtc ctggaggagc ttatggacct ggaagccctg gaggagctta ttatcctagc 2460
tcgcgtgttc ccgatatggt gaatggtata atgagtgcta tgcaaggatc tggttttaac 2520
taccaaatgt ttggtaatat gctatcacaa tattcgtctg gttcaggaac atgcaatcca 2580
aataatgtta atgttttgat ggatgctttg ttagctgctt tgcactgtct aagtaaccac 2640
ggatcatcat cttttgcacc ttctccaact ccggctgcta tgagtgcgta ttctaattct 2700
gtaggaagaa tgttcgctta ttaa 2724
<210>18
<211>52
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>18
gatcgaggag gatccatggg acgaattcac ggctaatgaa agcttactgc ac 52
<210>19
<211>52
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>19
agctgtgcag taagctttca ttagccgtga attcgtccca tggatcctcc tc 52
<210>20
<211>72
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>20
tccgtacggc ccaggtgcta gcgccgcagc ggcagcggct ggtggctacg gtccgggctc 60
tggccagcag gg 72
<210>21
<211>72
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>21
ctgctggcca gagcccggac cgtagccacc agccgctgcc gctgcggcgc tagcacctgg 60
gccgtacgga cc 72
<210>22
<211>60
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>22
tccgggccag cagggcccgg gtcaacaggg tcctggccag caaggtccgg gccagcaggg 60
<210>23
<211>60
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>23
ctgctggccc ggaccttgct ggccaggacc ctgttgaccc gggccctgct ggcccggacc 60
<210>24
<211>105
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>24
ttctagcgcg gctgcagccg cggcagctgc gtccggcccg ggtggctacg gtccggaaaa 60
ccagggtcca tctggcccgg gtggctacgg tcctggcggt ccggg 105
<210>25
<211>105
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>25
cggaccgcca ggaccgtagc cacccgggcc agatggaccc tggttttccg gaccgtagcc 60
acccgggccg gacgcagctg ccgcggctgc agccgcgcta gaacc 105
<210>26
<211>36
<212>DNA
<213>人工序列
<220>
<223>引物
<400>26
gaaaaaccat gggtgcggct tctgcagctg tatctg 36
<210>27
<211>45
<212>DNA
<213>人工序列
<220>
<223>引物
<400>27
gaaaagaagc tttcattagc cagcaagggc ttgagctaca gattg 45
<210>28
<211>34
<212>DNA
<213>人工序列
<220>
<223>引物
<400>28
gaaaaaccat gggagcatat ggcccatctc cttc 34
<210>29
<211>45
<212>DNA
<213>人工序列
<220>
<223>引物
<400>29
gaaaagaagc tttcattagc ctgaaagagc ttggctaatc atttg 45
<210>30
<211>12
<212>PRT
<213>人工序列
<220>
<223>T7标记
<400>30
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
1 5 10
<210>31
<211>216
<212>PRT
<213>人工序列
<220>
<223>FlagN-NR
<400>31
Gly Glu Ser Ser Gly Gly Asp Val Gln Arg Lys Thr Asn Val Ile Ser
1 5 10 15
Asn Ala Leu Arg Asn Ala Leu Met Ser Thr Thr Gly Ser Pro Asn Glu
20 25 30
Glu Phe Val His Glu Val Gln Asp Leu Ile Gln Met Leu Ser Gln Glu
35 40 45
Gln Ile Asn Glu Val Asp Thr Ser Gly Pro Gly Gln Tyr Tyr Arg Ser
50 55 60
Ser Ser Ser Gly Gly Gly Gly Gly Gly Gln Gly Gly Pro Val Val Thr
65 70 75 80
Glu Thr Leu Thr Val Thr Val Gly Gly Ser Gly Gly Gly Gln Pro Ser
85 90 95
Gly Ala Gly Pro Ser Gly Thr Gly Gly Tyr Ala Pro Thr Gly Tyr Ala
100 105 110
Pro Ser Gly Ser Gly Ala Gly Gly Val Arg Pro Ser Ala Ser Gly Pro
115 120 125
Ser Gly Ser Gly Pro Ser Gly Gly Ser Arg Pro Ser Ser Ser Gly Pro
130 135 140
Ser Gly Thr Arg Pro Ser Pro Asn Gly Ala Ser Gly Ser Ser Pro Gly
145 150 155 160
Gly Ile Ala Pro Gly Gly Ser Asn Ser Gly Gly Ala Gly Val Ser Gly
165 170 175
Ala Thr Gly Gly Pro Ala Ser Ser Gly Ser Tyr Gly Pro Gly Ser Thr
180 185 190
Gly Gly Thr Tyr Gly Pro Ser Gly Gly Ser Glu Pro Phe Gly Pro Gly
195 200 205
Val Ala Gly Gly Pro Tyr Ser Pro
210 215
<210>32
<211>648
<212>DNA
<213>人工序列
<220>
<223>FlagN-NR
<400>32
ggcgaaagca gcggaggcga tgtgcaacgc aaaaccaacg ttatctccaa cgctttgaga 60
aacgccttga tgtctacaac aggcagccca aacgaagagt tcgtccatga agttcaagac 120
ctcatccaga tgttatctca agaacagatc aacgaggtag atacttcagg accagggcag 180
tactacaggt cgtcttcttc cggtggagga ggtggaggac aaggaggtcc tgtagttact 240
gaaacactga ccgttacagt tggcggatcc ggtggagggc aaccttcagg tgcaggtcct 300
agtggtacag gtggatatgc accaactgga tacgccccaa gcggctcagg tgcaggtggc 360
gttcgaccta gtgcctccgg tccaagtggt agtggaccta gtggtggatc tcgtcctagt 420
agtagtggac ctagtggaac tcgtcccagc cctaatggtg caagtggatc tagccctggt 480
ggtatcgcac ctggtggatc caattctggt ggtgctggag tatccggcgc aactggagga 540
cctgcatcca gcggctccta cggaccagga agtacaggtg gaacatatgg acctagtgga 600
ggaagtgaac ctttcggacc aggagtggct ggaggaccat acagccca 648
<210>33
<211>93
<212>PRT
<213>人工序列
<220>
<223>FlagC-NR
<400>33
Gly Ala Tyr Tyr Pro Ser Ser Arg Val Pro Asp Met Val Asn Gly Ile
1 5 10 15
Met Ser Ala Met Gln Gly Ser Gly Phe Asn Tyr Gln Met Phe Gly Asn
20 25 30
Met Leu Ser Gln Tyr Ser Ser Gly Ser Gly Thr Cys Asn Pro Asn Asn
35 40 45
Val Asn Val Leu Met Asp Ala Leu Leu Ala Ala Leu His Cys Leu Ser
50 55 60
Asn His Gly Ser Ser Ser Phe Ala Pro Ser Pro Thr Pro Ala Ala Met
65 70 75 80
Ser Ala Tyr Ser Asn Ser Val Gly Arg Met Phe Ala Tyr
85 90
<210>34
<211>279
<212>DNA
<213>人工序列
<220>
<223>FlagC-NR
<400>34
ggtgcttatt atcctagctc gcgtgttccc gatatggtga atggtataat gagtgctatg 60
caaggatctg gttttaacta ccaaatgttt ggtaatatgc tatcacaata ttcgtctggt 120
tcaggaacat gcaatccaaa taatgttaat gttttgatgg atgctttgtt agctgctttg 180
cactgtctaa gtaaccacgg atcatcatct tttgcacctt ctccaactcc ggctgctatg 240
agtgcgtatt ctaattctgt aggaagaatg ttcgcttat 279
<210>35
<211>27
<212>PRT
<213>人工序列
<220>
<223>组件K
<400>35
Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly
1 5 10 15
Pro Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr
20 25
<210>36
<211>81
<212>DNA
<213>人工序列
<220>
<223>组件K
<400>36
ggtccgggcg gtgctggcgg tccgtacggc cctggtggcg caggtgggcc atatggtccg 60
ggcggtgcgg gcggtccgta c 81
<210>37
<211>28
<212>PRT
<213>人工序列
<220>
<223>组件sp
<400>37
Gly Gly Thr Thr Ile Ile Glu Asp Leu Asp Ile Thr Ile Asp Gly Ala
1 5 10 15
Asp Gly Pro Ile Thr Ile Ser Glu Glu Leu Thr Ile
20 25
<210>38
<211>84
<212>DNA
<213>人工序列
<220>
<223>组件sp
<400>38
ggtggcacca ccatcattga agatctggac atcactattg atggtgcgga cggcccgatc 60
acgatctctg aagagctgac catc 84
<210>39
<211>18
<212>PRT
<213>人工序列
<220>
<223>组件x
<400>39
Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ser Gly Gly Ala Gly
1 5 10 15
Gly Ser
<210>40
<211>54
<212>DNA
<213>人工序列
<220>
<223>组件x
<400>40
ggtggcgctg gtggcgccgg tggcgcaggt ggctctggcg gtgcgggcgg ttcc 54
<210>41
<211>30
<212>PRT
<213>人工序列
<220>
<223>组件Y
<400>41
Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly
1 5 10 15
Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr
20 25 30
<210>42
<211>90
<212>DNA
<213>人工序列
<220>
<223>组件Y
<400>42
ggtccgggcg gtgcgggccc aggtggctat ggtccgggcg gttctgggcc gggtggctac 60
ggtcctggcg gttccggccc gggtggctac 90
<210>43
<211>32
<212>DNA
<213>人工序列
<220>
<223>PCR引物
<400>43
gaaaaaccat gggcgaaagc agcggaggcg at 32
<210>44
<211>36
<212>DNA
<213>人工序列
<220>
<223>PCR引物
<400>44
gaaaagaagc tttcattagc ctgggctgta tggtcc 36
<210>45
<211>33
<212>DNA
<213>人工序列
<220>
<223>PCR引物
<400>45
gaaaaaccat gggtgcttat tatcctagct cgc 33
<210>46
<211>42
<212>DNA
<213>人工序列
<220>
<223>PCR引物
<400>46
gaaaagaagc tttcattagc cataagcgaa cattcttcct ac 42
<210>47
<211>90
<212> DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>47
tccgggcggt gcgggcccag gtggctatgg tccgggcggt tctgggccgg gtggctacgg 60
tcctggcggt tccggcccgg gtggctacgg 90
<210>48
<211>90
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>48
gtagccaccc gggccggaac cgccaggacc gtagccaccc ggcccagaac cgcccggacc 60
atagccacct gggcccgcac cgcccggacc 90
<210>49
<211>84
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>49
tggcaccacc atcattgaag atctggacat cactattgat ggtgcggacg gcccgatcac 60
gatctctgaa gagctgacca tcgg 84
<210>50
<211>84
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>50
gatggtcagc tcttcagaga tcgtgatcgg gccgtccgca ccatcaatag tgatgtccag 60
atcttcaatg atggtggtgc cacc 84
<210>51
<211>81
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>51
tccgggcggt gctggcggtc cgtacggccc tggtggcgca ggtgggccat atggtccggg 60
cggtgcgggc ggtccgtacg g 81
<210>52
<211>81
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>52
gtacggaccg cccgcaccgc ccggaccata tggcccacct gcgccaccag ggccgtacgg 60
accgccagca ccgcccggac c 81
<210>53
<211>54
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>53
tggcgctggt ggcgccggtg gcgcaggtgg ctctggcggt gcgggcggtt ccgg 54
<210>54
<211>54
<212>DNA
<213>人工序列
<220>
<223>合成寡核苷酸
<400>54
ggaaccgccc gcaccgccag agccacctgc gccaccggcg ccaccagcgc cacc 54
<210>55
<211>3238
<212>DNA
<213>人工序列
<220>
<223>克隆载体pAZL
<400>55
tgtcgagaag tactagagga tcataatcag ccataccaca tttgtagagg ttttacttgc 60
tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 120
tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 180
cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 240
atcttatcat gtctggatct gatcactgct tgagcctagg agatccgaac cagataagtg 300
aaatctagtt ccaaactatt ttgtcatttt taattttcgt attagcttac gacgctacac 360
ccagttccca tctattttgt cactcttccc taaataatcc ttaaaaactc catttccacc 420
cctcccagtt cccaactatt ttgtccgccc acagcggggc atttttcttc ctgttatgtt 480
tttaatcaaa catcctgcca actccatgtg acaaaccgtc atcttcggct actttttctc 540
tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt aatgtttgta attgactgaa 600
tatcaacgct tatttgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa 660
gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 720
ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 780
ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 840
aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 900
gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 960
cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 1020
attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 1080
cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 1140
ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 1200
aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 1260
tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 1320
atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 1380
agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 1440
tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 1500
tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 1560
atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 1620
ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 1680
tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 1740
acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 1800
ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 1860
aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 1920
ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 1980
cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 2040
gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 2100
actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 2160
agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 2220
cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 2280
tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 2340
agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 2400
tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 2460
acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 2520
ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 2580
gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 2640
gtgagcattg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 2700
gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 2760
tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 2820
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 2880
tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 2940
gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 3000
agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt 3060
gcggtatttc acaccgcaga ccagccgcgt aacctggcaa aatcggttac ggttgagtaa 3120
taaatggatg ccctgcgtaa gcgggtgtgg gcggacaata aagtcttaaa ctgaacaaaa 3180
tagatcgagg aggatccatg ggacgaattc acggctaatg aaagcttact gcacagct 3238
Claims (16)
1.生产纳米胶囊和微胶囊的方法,所述方法包括下列步骤:
a)提供蜘蛛丝蛋白质;
b)在适合的溶剂中形成所述蛋白质的溶液或悬浮液;
c)产生至少两相的乳状液,所述乳状液含有b)中形成的溶液或悬浮液作为第一相以及至少一个基本上与所述第一相不可混溶的另外的相;
d)在至少两相的界面处形成蜘蛛丝蛋白质的聚合物网络;
e)从所述乳状液分离(d)中产生的蛋白质聚合物网络。
2.权利要求1的方法,其中a)中提供的蜘蛛丝蛋白质选自ADF-4及其衍生物,包括C16、C16NR4、C32、C32NR4的组。
3.权利要求1或2的方法,其中b)中的溶剂和/或所述至少一个另外的相的溶剂选自由亲水性溶剂优选地水和醇,或亲脂性溶剂优选天然油或合成油组成的组。
4.上述权利要求的一项或多项的方法,其中c)中形成的乳状液是W/O、O/W、O/W/O或W/O/W类型的。
5.上述权利要求的一项或多项的方法,其中1b)中使用的溶剂还含有一种或多种药剂、化妆品试剂、食品或食品添加剂。
6.权利要求5的方法,其中所述药剂以溶解的、悬浮的或固体形态存在于所述溶剂中。
7.上述权利要求的一项或多项的方法,其中步骤e)中聚合物网络的分离是通过离心或通过破坏步骤c)中形成的乳状液并形成单相溶液而进行的。
8.上述权利要求的一项或多项的方法,其中步骤b)-e)中使用的温度是5-40℃,优选10-30并且更优选室温,并且其中步骤b)-e)中使用的pH是3-9,优选5-8,更优选7。
9.上述权利要求的一项或多项的方法,其中所述乳状液小滴和由此衍生的纳米颗粒和微颗粒的尺寸是10nm-40μm,优选500nm-10μm,最优选约5μm。
10.上述权利要求的一项或多项的方法,其中获得的纳米胶囊和微胶囊的壁厚是5-100nm,优选地10-70nm。
11.上述权利要求的一项或多项的方法,其中步骤a)中提供的蜘蛛丝蛋白质是改造的以便引入容许组织特异性蛋白酶解的蛋白酶特异性识别序列。
12.上述权利要求的一项或多项的方法,其中在所述纳米胶囊或微胶囊形成前后将试剂偶联到所述蜘蛛丝蛋白质,以便将所述胶囊定向到特定的细胞或组织。
13.权利要求12的方法,其中所述试剂选自交联到所述蜘蛛丝蛋白质的RGD肽、细胞或组织特异性抗体、偶联到所述蜘蛛丝蛋白质的细胞或组织特异性受体。
14.通过权利要求1-13的一项或多项的方法可以获得的纳米胶囊和微胶囊。
15.含有权利要求14的纳米胶囊和微胶囊和药用载体的药物组合物。
16.含有权利要求14的纳米胶囊和微胶囊的化妆品或食物产品。
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EP05016712A EP1757276B1 (en) | 2005-08-01 | 2005-08-01 | Method of producing nano- and microcapsules of spider silk protein |
EP05016712.1 | 2005-08-01 | ||
PCT/EP2006/007608 WO2007014755A1 (en) | 2005-08-01 | 2006-08-01 | Method of producing nano- and microcapsules of spider silk protein |
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EP (1) | EP1757276B1 (zh) |
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JP2972877B1 (ja) * | 1998-07-24 | 1999-11-08 | 農林水産省蚕糸・昆虫農業技術研究所長 | 高分子素材のドープ、高分子素材からなるマイクロビーズおよびそのビーズの製造方法 |
JP2001163899A (ja) | 1999-12-09 | 2001-06-19 | Natl Inst Of Sericultural & Entomological Science | 機能性絹フィブロインの製造方法とその利用 |
US6608242B1 (en) | 2000-05-25 | 2003-08-19 | E. I. Du Pont De Nemours And Company | Production of silk-like proteins in plants |
WO2003045336A1 (fr) * | 2001-11-29 | 2003-06-05 | National Institute Of Agrobiological Sciences | Emulsifiant et procede d'elaboration correspondant |
US20040132978A1 (en) * | 2002-11-12 | 2004-07-08 | Fahnestock Stephen R. | Method for purifying and recovering silk proteins in soluble form and uses thereof |
CN1288193C (zh) * | 2004-03-10 | 2006-12-06 | 复旦大学 | 蚕丝蛋白纳米微球及其制备方法 |
CA2608862C (en) | 2004-06-11 | 2020-05-05 | Trustees Of Tufts College | Silk-based drug delivery system |
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CN104684540A (zh) * | 2012-07-13 | 2015-06-03 | 塔夫茨大学 | 在丝纤蛋白生物材料中对不相混溶的相的包封 |
CN108186391A (zh) * | 2012-07-13 | 2018-06-22 | 塔夫茨大学 | 在丝纤蛋白生物材料中香料和/或调味剂的包封 |
CN104927777A (zh) * | 2015-06-02 | 2015-09-23 | 清华大学深圳研究生院 | 相变储能微胶囊制备方法及应用 |
CN104927777B (zh) * | 2015-06-02 | 2018-04-24 | 清华大学深圳研究生院 | 相变储能微胶囊制备方法及应用 |
CN109329910A (zh) * | 2018-09-28 | 2019-02-15 | 青岛大学 | 一种蜘蛛丝组合物口服液的制备方法 |
CN114144160A (zh) * | 2019-07-12 | 2022-03-04 | 保尔特纺织品公司 | 重组蜘蛛丝挤出物制剂 |
CN111214385A (zh) * | 2020-03-13 | 2020-06-02 | 珀莱雅化妆品股份有限公司 | 一种负载皮肤营养剂的纳米粒乳液及其制备方法 |
CN111214385B (zh) * | 2020-03-13 | 2022-05-17 | 珀莱雅化妆品股份有限公司 | 一种负载皮肤营养剂的纳米粒乳液及其制备方法 |
Also Published As
Publication number | Publication date |
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EP1757276B1 (en) | 2007-09-26 |
US20100056438A1 (en) | 2010-03-04 |
PL1757276T3 (pl) | 2008-02-29 |
JP5129746B2 (ja) | 2013-01-30 |
CA2617530C (en) | 2013-10-01 |
CA2617530A1 (en) | 2007-02-08 |
DE602005002654T2 (de) | 2008-06-19 |
US8729238B2 (en) | 2014-05-20 |
DE602005002654D1 (de) | 2007-11-08 |
US20130136779A1 (en) | 2013-05-30 |
ATE374018T1 (de) | 2007-10-15 |
ES2294606T3 (es) | 2008-04-01 |
CN101309676B (zh) | 2012-02-29 |
PT1757276E (pt) | 2008-01-02 |
WO2007014755A1 (en) | 2007-02-08 |
EP1757276A1 (en) | 2007-02-28 |
JP2009502492A (ja) | 2009-01-29 |
DK1757276T3 (da) | 2008-02-04 |
US8372436B2 (en) | 2013-02-12 |
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