CN110627889A - 重组蜘蛛丝蛋白及其制备方法和产业化应用 - Google Patents

重组蜘蛛丝蛋白及其制备方法和产业化应用 Download PDF

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CN110627889A
CN110627889A CN201810648563.XA CN201810648563A CN110627889A CN 110627889 A CN110627889 A CN 110627889A CN 201810648563 A CN201810648563 A CN 201810648563A CN 110627889 A CN110627889 A CN 110627889A
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马永
赵百学
王安良
孙文秀
江辰阳
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Jiangsu Jingsen Biomedical New Materials Technology Co Ltd
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Abstract

本发明公开了一类重组蜘蛛丝蛋白系列，及其编码基因、表达、纯化方法，以及所述重组蛛丝蛋白系列在缝合线、生物修复膜、止血材料等生物材料方面的应用。本发明的重组蜘蛛丝蛋白系列，其由N末端非重复区域(N‑NRT)、核心重复区域(REP)、C末端非重复区域(C‑NRT)组成，其中核心重复区域由1‑30个RepA和/或1‑15个RepB组成，单个RepA氨基酸序列如SEQ ID NO：1所示，单个RepB氨基酸序列如SEQ ID NO：2所示。本发明设计的一系列新型的原核系统可溶表达的蛛丝蛋白，具有较高的发酵产量和蛋白纯度，以该蛛丝蛋白系列经过简单的静纺或湿纺工艺制备的蛛丝膜、蛛丝纤维分别具有2.5‑6MPa、40‑280MPa的力学强度，且显示出良好的细胞相容性和止血性能，具备医疗领域产业化应用的前景。

Description

重组蜘蛛丝蛋白及其制备方法和产业化应用
技术领域
本发明涉及一类重组蜘蛛丝蛋白系列，及其编码基因、表达、纯化方法，以及所述重组蛛丝蛋白系列在成丝、成纳米膜等医用制品方面的应用。
背景技术
蜘蛛丝既有钢般的坚硬，又有橡胶般的弹性。其突出的性能主要表现在：高强度、高弹性、高断裂功，可以说是迄今为止最强韧的材料，被誉为“生物钢”。随着对蜘蛛丝的研究深入，发现蜘蛛丝还具有生物可降解性、超收缩性、耐高温、耐低温及与生物组织的相容性等特性。由于蜘蛛丝这些独特的物理和生物学特性，它在医学、材料、军事和纺织等方面都有着广泛的应用前景。瑞典科学家Jan Johansson利用无刺激性化学物质制造出的人工蜘蛛丝具有良好的生物相容性，可应用于脊髓修复或帮助干细胞生长来修复损伤的心脏组织等再生医学研究中，亦可用于自身防护用具等纺织工业应用中(Nature ChemicalBiology,DOI:10.1038/nchembio.2269)。
鉴于蜘蛛丝蛋白巨大的潜在应用性，国内外学者加强对蜘蛛丝的研究，希望蜘蛛丝能够像蚕丝那样大规模地应用于实际。由于蜘蛛无法驯养和天然蜘蛛丝产量少等原因，惟有通过基因工程手段才能大量获取蜘蛛丝，满足蜘蛛丝潜在的应用需求。目前研究人员可以借助大肠杆菌，酵母，昆虫细胞，哺乳动物细胞等表达系统进行蜘蛛丝蛋白的生物工程制备。其中，大肠杆菌表达系具有生长快、产量高、生产规模大、成本低、培养条件简单、遗传背景清楚等多重优点，目前被广泛应用于蜘蛛丝蛋白的重组表达研究中。但是，天然蜘蛛丝分子量非常大(>300KDa)，而大肠杆菌表达的外源蛋白大于60KDa时就会出现表达量降低的现象，天然的蜘蛛丝蛋白分子量过大，几乎不可能表达成功。例如：Fahnestock等人就报道了在大肠杆菌中表达了蜘蛛牵引丝蛋白，但发现大于3kb的基因表达效率降低，并有基因删节表达现象。
研究表明，蜘蛛丝蛋白有四种通用的氨基酸模块：(l)GPGXX；(2)GGx；(3)An/(GA)n；(4)间隔区(spacer)。GPGXX(主要是GPGGx和GPGQQ两种形式)是一个五聚肽，主要存在于牵引丝蛋白和鞭毛丝蛋白，形成β成转角结构，一系列的β系转角串联折叠在一起，类似弹簧结构。而这个“弹簧”结构与蜘蛛丝蛋白的弹性相关，蛛丝蛋白弹性的大小与该模块的含量正相关。GGX形成310-螺旋结构，即每三个氨基酸组成一个螺旋。An/(GA)n形成β成片层结构，结构片层之间的分子作用决定了蜘蛛丝蛋白的强度，也是导致蜘蛛丝不溶于水的原因之一。间隔区是一个保守区域，用来间隔富含甘氨酸的区域。总的来说：(l)蜘蛛丝的化学本质是蛋白质；(2)蜘蛛丝蛋白氨基酸序列高度重复性；(3)蜘蛛丝蛋白中含量较多的氨基酸主要为丙氨酸、甘氨酸和丝氨酸；(4)富含聚丙氨酸，形成β成片层刚性结构；(5)脯氨酸形成的β氨转角结构为蜘蛛丝提供良好的弹性。
研究人员可以依据上述蜘蛛丝蛋白特性设计重组的蜘蛛丝蛋白，重组蜘蛛丝蛋白一级结构含有上述决定蜘蛛丝蛋白力学性能的模块，并且可以根据不同材料的研发用途进行个性化的序列设计，这样通过人工的序列设计获得的人工蛛丝蛋白不仅具有力学性能可控性而且可以控制蛋白分子量以便实现蛋白在大肠杆菌表达系统中的高效表达。
申请人经过研究发现，在既有的蛛丝蛋白设计方案和制备工艺的基础上，虽能满足一般工业领域应用所需，但在医疗制品等领域不仅需要常规的拉力强度，更需要高纯度、低免疫原性等应用需求时却无法满足。
发明内容
为克服上述技术问题，本申请发明人重新设计了一系列衍生自天然蜘蛛丝的蛋白序列，同时将对应的一系列编码重组蜘蛛丝蛋白序列的基因导入到宿主细胞中进行蛋白表达，并建立高效的重组蜘蛛丝蛋白表达和制备工艺。在此基础上研究了该类重组蜘蛛丝蛋白系列在静电纺丝和湿法纺丝中的应用。
本发明第一目的在于提供重组蜘蛛丝蛋白，其由N末端非重复区域(N-NRT)、核心重复区域(REP)、C末端非重复区域(C-NRT)组成，其中核心重复区域由1-30个RepA和/或1-15个RepB组成，单个RepA氨基酸序列如SEQ ID NO：1所示，单个RepB氨基酸序列如SEQ IDNO：2所示。
RepA和RepB可以以自身串联体或者两者的杂交串联体作为核心重复区。以自身串联体作为核心重复区时，优选含有5-30个RepA作为核心重复区或者含有5-15个RepB作为核心重复区。
以两者杂交串联体作为核心重复区时，优选1-5个RepA与1-5个RepB的串联体作为核心重复区。更优选地所述1-5个RepA的串联体位于整个融合蛋白的靠近N端位置，而所述1-5个RepB的串联体位于整个融合蛋白的C端位置。
上述N末端非重复区域(N-NRT)，氨基酸序列如SEQ ID NO：3所示，C末端非重复区域(C-NRT)，氨基酸序列如SEQ ID NO：4所示。
本申请中发明人将RepA以5个串联体的形式(RepA5)进行基因合成，同时也将RepB以5个串联体的形式(RepB5)进行基因合成。另外为了便于后续的基因操作，编码RepA5及RepB5的核苷酸的5’端和3’端分别引入了同尾酶酶切位点BamHI和BglII。
本发明所涉及的部分序列如表1所示：
表1
所述核苷酸序列均经过针对大肠杆菌表达系统进行密码子优化，从而提高在大肠杆菌中的表达量。上述串联体核苷酸序列可由PCR的方法制备，也可由基因合成的方法制备。
本发明还提供了包含上述核苷酸序列的表达载体，该载体只要与大肠杆菌表达系统相匹配即可，优选具有拷贝数高、表达效率高的载体，如pET21b、pET28a、pBV220等。
本发明还提供了包含上述表达载体的大肠杆菌菌株，优选BL21(DE3)、BL21(DE3)plys、Rosetta(DE3)、Transetta(DE3)等。
本发明还提供了重组蜘蛛丝蛋白高密度发酵生产方法，具体包括：发酵种子活化、发酵一级种子液制备、发酵二级种子制备、高密度发酵，所述高密度发酵具体包括如下步骤：
将制备好的二级种子液按照5-15％的接种量接入到含分批发酵培养基的灭菌后的发酵罐中；
设定发酵温度为37℃、pH为6.8-7.2之间、DO设定在30-40％之间；
发酵开始后定期取样进行OD600和菌体湿重的测定，待DO曲线出现急剧上升的时候表明分批培养基中葡萄糖耗尽，开始进行补料培养(补料培养基的流加速度维持在8-12g/L/h)；
待菌体生长至OD600至40-60之间，使得发酵温度至20-37℃，待温度稳定后向发酵罐中加入终浓度为0.2-1.0mM的IPTG进行诱导表达；诱导表达8-12h结束培养。
所述分批发酵培养基成分包括：一水合柠檬酸0.5-3g/L，磷酸二氢钾8-15g/L，磷酸氢二铵3-7g/L，葡萄糖10-20g/L或者含有甘油15-30g/L，七水合硫酸镁1-3g/L。在发酵接种之前还要向分批发酵培养基中加入1/1000(V/V)的微量元素母液用于维持菌体正常生长和代谢。
所述补料培养基主要成分包括：甘油1024g/L，七水合硫酸镁10-20g/L，酵母粉30-60g/L，在补料开始之前还要在补料培养基中加入1/1000(V/V)的微量元素母液用于维持菌体正常生长和代谢。
所述微量元素母液成分包括：FeSO4.7H2O 10g/L、ZnSO4.7H2O 2.25g/L、CuSO4.5H2O15g/L、MnSO4.5H2O 5g/L、CaCl2.7H2O 1g/L、CoCl.6H2O 1g/L、Na2MoO4.2H2O 1.125g/L、H3BO30.0625g/L、HCl 41.75ml、Biotin 0.5g/L。
所述发酵种子活化方法具体为：将构建好的重组蜘蛛丝蛋白大肠杆菌菌株冻存管使用三区划线的方法接种到LB固体培养基中，37℃过夜培养进行活化。
所述发酵一级种子液制备方法具体为：从固体培养基上挑取形状饱满、大小适中的单菌落接种到LB液体培养基中，37℃，220rpm摇床培养8-10h，此为一级种子液。
所述发酵二级种子制备方法具体为：将一级种子液按照1％的接种量转接到新鲜的LB液体培养基中，37℃，220rpm摇床培养至OD600≈3-5之间，此为二级种子液制备。
上述任何用于菌体培养的器皿和培养基在使用前均应进行过滤或者湿热灭菌，所述任何培养基在灭菌结束冷却后使用前均应加入终浓度为50μg/ml的卡那霉素以保证纯种培养。
本发明还提供了重组蜘蛛丝蛋白纯化方法，包括如下步骤：
1、取一定量的高密度发酵菌体使用bufferA(50mMTris、100mM氯化钠、1mM EDTA，pH8.0)重悬至100g/L，将上述菌体悬浮液使用高压均质机进行菌体破碎。
2、将上述菌体裂解液使用低温离心机10000g离心30min，丢弃沉淀，收集上清。
3、离心收集第2步中收集的菌体裂解物上清液，放入到预热到75℃的水浴条件下保温10-50min，保温处理过程中要不断的温和搅拌，该过程称之为菌体裂解物的热处理。
4、将步骤3中热处理后的菌体裂解物离心收集，去除沉淀，收集上清液，上清液使用0.8μm水系膜进行过滤除去颗粒性杂质。
5、IMAC亲和层析纯化即得目的蛋白。
本申请发明人根据己报道的蜘蛛丝蛋白的氨基酸序列，结合蜘蛛丝蛋白的模块结构特性和功能的关系，优化设计了一系列新型蜘蛛丝蛋白序列，它集高强度、高弹性等蜘蛛丝主要特性于一体。并且，通过基因工程技术将这些序列分别导入到大肠杆菌并建立相应的高密度发酵工艺成功实现了蛛丝蛋白的原核高效可溶表达。而以往为了实现蛛丝蛋白的工业化应用，所设计的蛛丝蛋白往往在保证性能的前提下更多的是为了制备量的考虑，但对于医疗制品等需要精纯产品的特定领域，以往设计的蛛丝蛋白却有着先天的不足，即使能获得精纯产品，也需要付出巨大的投入。鉴于此，发明人根据蜘蛛丝蛋白的性质设计出了一系列可以用原核系统可溶表达的蛛丝蛋白，经过精纯后重组蜘蛛丝蛋白纯度(电泳纯)可以达到95％左右，以该蛛丝蛋白系列经过静纺或湿纺工艺制备的蛛丝膜、蛛丝纤维就可以有2.5-6MPa、40-280MPa的力学强度，并且具有良好的细胞相容性和止血效果，这完全可以满足了医用领域的特殊要求，具有进一步开发的价值。
附图说明
图1重组蜘蛛丝蛋白大肠杆菌菌株构建原理
图2不同设计方案的重组蜘蛛丝蛋白纯化后电泳图
图2-1 RepA5纯化样品电泳检测，其中M：蛋白分子量标准；lane1：RepA5纯化样品电泳；
图2-2RepA10纯化样品电泳检测，其中M：蛋白分子量标准；lane1-2：RepA10纯化样品电泳；
图2-3RepA20纯化样品电泳检测，其中M：蛋白分子量标准；lane1：RepA20纯化样品电泳；
图2-4RepB5纯化样品电泳检测，其中M：蛋白分子量标准；lane1：RepB5纯化样品电泳；
图2-5RepB10纯化样品电泳检测，其中M：蛋白分子量标准；lane1：RepB10纯化样品电泳；
图2-6RepB15纯化样品电泳检测，其中M：蛋白分子量标准；lane1：RepB15纯化样品电泳；
图2-7RepA5RepB5纯化样品电泳检测，其中M：蛋白分子量标准；lane1：RepA5RepB5纯化样品电泳；
图2-8 RepB5金属离子鳌合层析纯化样品电泳检测，其中M：蛋白分子量标准；lane1：层析纯化前；lane2：层析纯化穿透；lane3-4：层析纯化洗脱样品；
图3重组蜘蛛丝蛋白静电纺丝样品扫描电镜图，其中上一：RepA5，上二：RepA10；上三：RepA20；中一：RepB5；中二：RepB10；中三：RepB15；下一：RepA5RepB5；下二：RepA5RepB5-RepA5RepB5
图4重组蜘蛛丝蛋白静电纺丝纤维膜力学测试结果，其中图4-1至图4-8依次对应的重组蜘蛛丝蛋白样品为：RepB5、RepB10、RepB15、RepA5、RepA10、RepA20、RepA5RepB5、RepA5RepB5-RepA5RepB5
图5重组蜘蛛丝蛋白湿法纺丝纤维丝力学性能测定，图5-1至图5-6分别为RepB5、RepB10、RepB15、RepA5、RepA10、RepA20湿法纺丝纤维丝的力学性能测定结果
图6重组蜘蛛丝蛋白湿法纺丝纤维丝电镜观测图，其中图6-1为RepA5湿法纺丝的电镜观察，图6-2为RepA5纤维丝拉断面观察
图7小鼠成纤维细胞(L929)在重组蜘蛛丝蛋白静电纺丝纤维膜以及牛跟腱胶原蛋白(I型)膜上的贴附效果图，其中7-1为胶原蛋白膜，7-2为RepB5蛋白膜；
图8-1小鼠成纤维细胞(L929)在两种静电纺丝纤维膜上的增殖检测
图8-2人脐带血间充质干细胞(MSC)在两种静电纺丝纤维膜上的增殖检测
图9-1 RepB5和牛跟腱胶原蛋白静电纺丝纤维膜的皮肤修复试验，第1天和第22天动物创面照片
图9-2 RepB5和牛跟腱胶原蛋白静电纺丝纤维膜的皮肤修复试验检测结果
图10RepB5和牛跟腱胶原蛋白体外凝血试验检测结果
具体实施方式
以下实施例是针对本专利进行的进一步说明，需要说明的是实施例所述内容只是用于解释本专利并不构成对本专利发明内容的限制。
实施例1重组蜘蛛丝蛋白大肠杆菌表达菌株构建
本发明涉及到多个重组蜘蛛丝蛋白菌株的构建。重复模块的拼接(重复模块是指RepB5、RepA5)通过同尾酶的分子操作实现，方案流程如图1所示。下面以RepA5与RepB5串联体形成的杂交串联体(RepA5RepB5)为例进行说明，具体如下：
1、参考GenBank中已经公布的ArgiopetrifasciataMaSp2拖丝蛋白mRNA序列(GenBank基因登录号为：AH015065.2)，从中截取核心重复区域获得RepA和RepB核苷酸序列，通过人工拼装和密码子优化后形成RepA5和RepB5核苷酸序列，分别如SEQ ID NO：5和SEQ ID NO：6所示。为了方便后续的分子操作，在不改变RepA5和RepB5的氨基酸序列条件下，在合成的RepA5和RepB5人工核苷酸序列的5’端和3’端非别引入了酶切位点BamHI和BglII。RepA5和RepB5人工核苷酸序列被分别克隆至pUC57质粒，形成重组质粒pUC57-RepA5和pUC57-RepB5。
2、将N-NRT与C-NRT基因序列(如SEQ ID NO：16和如SEQ ID NO：17所示)人工合成，其中N-NRT的5’端含有XbaI酶切位点，3’端含有BamHI酶切位点(该酶切位点作为N-NRT重组核苷酸的一部分，不改变N-NRT的氨基酸序列)；C-NRT的5’端含有BglII酶切位点(该酶切位点作为N-NRT重组核苷酸的一部分，不改变N-NRT的氨基酸序列)，3’端含有XhoI酶切位点；N-NRT与C-NRT对应的核苷酸以融合的形式合成，即N-NRT-C-NRT(5’→3’)。N-NRT-C-NRT通过酶切、酶连的方式插入pET28aplus载体(将pET28a原始载体去除原有BglII改造而成)，记为pET28aplus-N-NRT-C-NRT。
4、pUC57-RepA5和pUC57-RepB5使用BamHI和BglII酶切获取带有相应粘性末端的RepA5和RepB5片段。将pET28aplus-N-NRT-C-NRT使用BamHI和BglII酶切获取带有相应粘性末端的载体骨架片段。将酶切获取的粘性片段酶连后转化到大肠杆菌Transetta(DE3)中形成重组表达菌株。
5、从步骤4中的转化平板中挑取单克隆进行培养，提取质粒后使用BamHI和BglII酶切进行鉴定，根据同尾酶的切割原理分析鉴定获得的阳性克隆，阳性克隆即为Transetta(DE3)-RepA5和Transetta(DE3)-RepB5,对应的阳性质粒为pET28aplus-N-NRT-RepA5-C-NRT和pET28aplus-N-NRT-RepB5-C-NRT。
6、pET28aplus-N-NRT-RepA5-C-NRT使用BglII酶切获取带有相应粘性末端的载体骨架片段，pUC57-RepA5使用BamHI和BglII酶切获取带有相应粘性末端的RepA5片段。将2种粘性片段连后转化到大肠杆菌Transetta(DE3)中形成重组表达菌株，鉴定后得到Transetta(DE3)-RepA10(含有10个RepA的串联体)，对应的阳性质粒为pET28aplus-N-NRT-RepA10-C-NRT。同理，对pET28aplus-N-NRT-RepA10-C-NRT进行重复操作即可获得含有n个RepA5的串联体(如含有5、10、20、30…个RepA的串联体)重组大肠杆菌菌株，即为Transetta(DE3)-n×RepA5。
7、同理，将pUC57-RepB5和pET28aplus-N-NRT-RepB5-C-NRT分别按照步骤4-6中的操作，即可获得Transetta(DE3)-n×RepB5(含有n个RepB5的串联体)。
8、将pET28aplus-N-NRT-RepA5-C-NRT质粒使用BglII酶切获取带有相应粘性末端的载体骨架片段，将pET28aplus-N-NRT-RepB5-C-NRT使用BamHI和BglII酶切获取带有相应粘性末端的RepB5片段。将带有粘性末端的RepB5与带有粘性末端的pET28aplus-N-NRT-RepA5-C-NRT酶连后转化到大肠杆菌Transetta(DE3)中形成重组表达菌株。从转化平板中挑取单克隆进行培养，提取质粒后使用BamHI和BglII酶切进行鉴定，根据同尾酶的切割原理分析鉴定获得的阳性克隆，阳性克隆即为Transetta(DE3)-RepA5RepB5，对应的阳性质粒为pET28aplus-N-NRT-RepA5-RepB5-C-NRT。
9、将上述酶切鉴定为阳性的菌株测序验证序列是否正确。
实施例2重组蜘蛛丝蛋白大肠杆菌菌株高密度发酵
蜘蛛丝蛋白含有大量的重复氨基酸序列，这一特性导致其在大肠杆菌中重组表达时会遇到tRNA池枯竭、基因复制障碍、蛋白翻译截断等问题，最终导致表达量不高。有研究表明，随着人工蛛丝蛋白的分子量增大(大于60KDa)，蛋白的表达难度会极大上升，并且可能出现表达失败的问题。而天然蛛丝蛋白的分子量可以达到200KDa以上，虽然没有直接研究表明蛛丝蛋白的分子量和其力学性能呈正比例关系，但仍需要使人工设计的蛛丝蛋白在分子量上与天然蛛丝蛋白尽可能相近。所以，重组蜘蛛丝蛋白就要在分子量和大肠杆菌表达量上做好平衡，分子量过大则表达量降低或者表达失败，分子量过小则对天然序列的仿生程度低。
本部分阐述了发明人关于重组蜘蛛丝蛋白的高密度发酵工艺，以Transetta(DE3)-RepA20的发酵工艺进行说明(RepA20分子量在实施例1中的蛛丝蛋白构建体中分子量较大，达到88KDa，对于工艺产量的评价具有一定代表性)。
1、将构建好的重组重组蜘蛛丝蛋白大肠杆菌菌株冻存管使用三区划线的方法接种到LB固体培养基中，37℃，220rpm摇床过夜培养进行活化；
2、从固体培养基上挑取形状饱满、大小适中的单菌落接种到LB液体培养基中，37℃，220rpm摇床培养8-10h，此为一级种子液。
3、将一级种子液按照1％的接种量转接到新鲜的LB液体培养基中，37℃，220rpm摇床培养至OD600≈3-5之间，此为二级种子液制备。
4、将制备好的二级种子液接种到发酵培养基中开始补料-分批高密度发酵培养，该高密度发酵工艺可以实现重组蜘蛛丝蛋白的大量表达。所述分批发酵培养基成分包括：一水合柠檬酸1.7g/L，磷酸二氢钾12g/L，磷酸氢二铵4g/L，甘油30g/L，七水合硫酸镁1.2g/L，在发酵接种之前还要向分批发酵培养基中加入1/1000(V/V)的微量元素母液用于维持菌体正常生长和代谢，所述微量元素母液成分包括：FeSO4.7H2O 10g/L、ZnSO4.7H2O 2.25g/L、CuSO4.5H2O 15g/L、MnSO4.5H2O 5g/L、CaCl2.7H2O 1g/L、CoCl.6H2O 1g/L、Na2MoO4.2H2O1.125g/L、H3BO30.0625g/L、HCl 41.75ml、Biotin 0.5g/L。所述补料培养基主要成分包括：甘油1024g/L，七水合硫酸镁20g/L，酵母粉50g/L。在补料开始之前还要补料培养基中加入1/1000(V/V)的上述微量元素母液用于维持菌体正常生长和代谢。所述的高密度发酵方法步骤包括：将制备好的二级种子液按照5-15％的接种量接入到灭菌后的发酵罐中→设定发酵温度为37℃、pH为6.8-7.0之间、DO设定在30-40％之间(通过转速/空气/高纯氧的关联进行控制)→发酵开始后定期取样进行OD600和菌体湿重的测定，待DO曲线出现急剧上升的时候表明分批培养基中葡萄糖耗尽，开始进行补料培养(补料培养基的流加速度维持在12g/L/h(补料培养基质量/发酵液初始体积/时间))→待菌体生长至0D600≈40-55之间，降低发酵温度至30℃，待温度稳定后向发酵罐中加入终浓度为0.5的IPTG进行诱导表达→诱导表达6h结束培养。
上述任何用于菌体培养的器皿和培养基在使用前均应进行过滤或者湿热灭菌，所述任何培养基在灭菌结束冷却后使用前均应加入终浓度为50μg/ml的卡那霉素以保证纯种培养。重组蜘蛛丝蛋白含有大量的氨基酸重复序列，实施例1获得的菌株采用该高密度发酵工艺可实现有效表达，部分菌株可以达到0.5g/L以上，为重组蜘蛛丝的大规模应用奠定了基础。
实施例3重组蜘蛛丝蛋白纯化制备
此部分内容，阐述了蜘蛛丝蛋白的非层析的蛋白纯化手段，经过简单纯化后重组蜘蛛丝蛋白纯度(电泳纯)即可以达到75％以上，需要明确的是，其残余的杂质对于后续的纺丝等操作无明显影响，该纯度蛛丝蛋白已能满足绝大部分的工业化应用。而由于本发明中的人工蛛丝蛋白在N端含有6×His标签，并且所设计分子为可溶表达，因此使得本专利中的人工蛛丝蛋白通过IMAC亲和层析的方法纯化更可以获得95％以上的电泳纯度，达到了精制纯化的要求，完全可以满足医疗等特殊用途需要。本实施例使用实施例2制备的发酵菌体分别采用简易纯化与精制纯化两种工艺处理，具体纯化操作如下：
1、取一定量的高密度发酵菌体使用bufferA(50mMTris、100mM氯化钠、1mM EDTA，pH8.0)重悬至100g/L，将上述菌体悬浮液使用高压均质机进行菌体破碎。
2、将上述菌体裂解液使用低温离心机10000g离心30min，丢弃沉淀，收集上清。
3、离心收集第2步中收集的菌体裂解物上清液，放入到预热到75℃的水浴条件下保温20min，保温处理过程中要不断的温和搅拌，该过程称之为菌体裂解物的热处理。
4、将步骤3中热处理后的菌体裂解物离心收集，去除沉淀，收集上清液，上清液使用0.8μm水系膜进行过滤除去颗粒性杂质。
5、将第4步中过滤处理后的上清液加入3M硫酸铵水溶液进行盐析，硫酸铵加入时要缓慢，并且盐析体系处于快速搅拌混合的状态。随着硫酸铵的加入，待盐析体系出现浑浊时，停止加入硫酸铵，将盐析体系放于4℃搅拌放置5h。
6、将第5步中的盐析体系离心处理，收集盐析沉淀，沉淀中即含有人工蛛丝蛋白。
7、将步骤6中的人工蛛丝蛋白盐析沉淀使用20-50％的乙醇水溶液反复的洗涤，直至硫酸铵被基本去除(洗涤液的电导率不高于0.05mS/cm)，此时收集蛛丝蛋白沉淀，将该蛋白冻干后即获得蛛丝蛋白原料。
取部分沉淀蛋白使用实施例1中的方法进行SDS-PAGE分析，分析结果如图2所示，电泳检测结果使用灰度分析各种人工蛛丝蛋白均可以获得75％以上电泳纯度。最终离心收集得到的蛋白可用冷冻干燥、热风干燥、喷雾干燥等方法进行干燥处理即可得到纯化后的蜘蛛丝蛋白，干燥后的蛛丝蛋白称重后，根据纯度和发酵体积核算重组蜘蛛丝蛋白RepA20的发酵产量为0.45g/L。
同时，发明人还选择RepB5进行了IMAC亲和层析纯化的研究，亲和层析使用本实施例步骤4中加热后的样品作为起始待纯化样品(样品中EDTA应被去除)，纯化方法参考Gehealthcare的镍离子鳌合层析填料的纯化方案开展，纯化后样品电泳分析如图2-8所示。分析可知，目标蛋白可以有效地与层析填料结合，并且经过纯化可以获得95％以上的电泳纯度，达到了精制纯化的要求。
申请人采用实施例2、实施例3的制备发酵、纯化工艺分别对部分构建的重组蜘蛛丝蛋白大肠杆菌菌株进行发酵和纯化制备，通过结果核算，不同重组蜘蛛丝蛋白菌株蛋白表达量如表2所示。
表2
实施例4重组蜘蛛丝蛋白静电纺丝原液制备和静电纺丝
1、取干燥后的重组蜘蛛丝蛋白样品使用HFIP(六氟异丙醇)进行溶解，投料量为10％(wt％)，室温搅拌溶解48h以上，至溶液均匀透亮。
2、取上述纺丝原液2ml加入到注射器中，选择注射针为27G针头，设定注射速率为1ml/h，设定电压为16KV，设定接收距离为15cm，纺丝头左右摆幅宽度为8-12cm，接受基板为锡箔纸，进行静电纺丝制备纤维膜。
3、将上述静电纺丝制备的纤维膜，使用扫描电镜进行检测分析，分析结果如图3所示。
4、将步骤2中制备的纤维膜使用裁刀裁剪(去除纺丝辐射的边缘部位)成宽度为1cm的条状。
5、将步骤4中制备的纤维膜从锡箔纸上剥离，将纤维膜使用数字式螺旋测微仪测定厚度，然后使用岛津制万能拉力试验机进行拉力测试(使用50N传感器，测试速度为10mm/min，相对湿度为40％，测试距离为2cm，预加载为0.1N)，测试结果如图4所示。由图3、4可见，采用本专利的蜘蛛丝蛋白可满足静电纺丝对蛋白的可纺性要求，宏观上纤维膜均匀完整，扫描电镜显示微观上具有典型的纤维形貌，同时力学测定结果显示静电纺丝制备的重组蜘蛛丝蛋白纤维膜力学强度在2.5-6MPa之间。
6、目前可以用于医用的人工皮肤要求材料的强度应在2-17MPa之间，因此所述静电纺丝获得的纤维膜在力学强度上具有皮肤类医用材料的应用潜力。
实施例5重组蜘蛛丝蛋白湿法纺丝原液制备和湿法纺丝
1、取干燥后的蛋白样品使用HFIP(六氟异丙醇)溶剂进行溶解，蛋白浓度为20％(w/v％)，室温搅拌溶解10h以上至溶液均匀透亮。
2、将步骤1中的纺丝液去除泡沫，然后注入1ml注射器，使用27G注射针头从注射器中推出，纺丝液从针头中挤出后浸入到乙醇中被固化形成纤维丝。
3、将步骤2中的固化纤维丝取出，在80℃蒸汽中拉伸3-5倍，然后在150℃环境中暴露30s，获得成熟纤维丝。
4、将步骤3中的成熟纤维丝使用岛津制万能拉力试验机进行拉力测试(使用50N传感器，测试速度为10mm/min，相对湿度为40％，测试距离为2cm，预加载为0.1N)，测试结果如图5所示，力学强度在220～280Mpa之间。
5、将步骤3中的成熟纤维丝和步骤4中力学测定中拉断的纤维丝在扫描电镜下观察纤维表面和断面，结果如图6所示。
6、所述方法制备的纤维丝为较为规则的圆柱形，表面光滑为明显的沟槽，并且拉断面比较密实。相比较于目前报道的人工重组蜘蛛丝纤维丝的力学强度(40-280MPa)，本发明人设计的几种重组蜘蛛丝蛋白对应的纤维丝力学强度属于较高水平。
实施例6重组蜘蛛丝蛋白静电纺丝膜细胞相容性评价
本发明人静电纺丝法制备的重组蜘蛛丝蛋白纤维膜在物理形貌上具备静电纺丝纤维膜的特点，因此为了评价该纤维膜的生物学相容性，我们使用了人脐带血间充质干细胞(MSC)以及小鼠成纤维细胞(L929)的贴附和增值来进行评价。同时业内公认，天然的胶原蛋白具备优秀的生物相容性、利于皮肤修复和促凝血作用，体现在促进细胞的贴附、增值等特点上，因此我们在实验中采用了牛跟腱胶原蛋白制备的静电纺丝膜来进行比较。牛跟腱胶原蛋白静电纺丝膜制备参考实施例5中的方法，并在该方法的基础上调整了蛋白溶解的溶剂选择，所制备的两种蛋白静电纺丝膜在厚度上保证一致。具体的细胞相容性评价方法如下：
1、取刚纺制完成的牛跟腱胶原蛋白和重组蜘蛛丝蛋白RepB5静电纺丝初生膜抽真空12h去除纤维膜内未挥发干净的溶剂，前者使用化学交联法进行交联固定，后者使用物理法固定，固定的目的是降低两种蛋白的水溶性。
2、将步骤1中固定完毕的纤维膜按照细胞培养皿孔的直径大小，裁切成合适的大小，保证纤维膜可以均匀、完全的覆盖培养皿底部。
3、将步骤2裁切完成的两种蛋白的圆形纤维膜分别使用75％乙醇浸泡2h后，取出分别铺于24孔板底部，晾干。胶原蛋白膜分别铺2块板，每块板6个孔，重组蛛丝蛋白RepB5膜分别铺2块板，每块板6个孔。晾干后，紫外照射1小时。
4、L929细胞铺板
将T75细胞培养瓶中的培养液吸出，PBS清洗一遍后，加入1mL胰酶，轻轻晃动培养瓶，使胰酶接触到整个细胞培养瓶底部所有细胞后，将胰酶吸出，将T75细胞培养瓶放至37℃，消化3min后取出培养瓶，观察细胞是否已经与细胞瓶底部分离。培养瓶中加入10mL含有10％FBS的1640培养基，轻轻吹打混匀后，将10mL细胞悬液吸出后加入到50mL离心管中，取少量细胞悬液于1.5ml离心管中，使用血球计数板进行计数。将其配置成4×104个/ml的细胞悬液。24孔板中，铺好膜的孔和未铺膜的孔，每孔加入200ul细胞悬液然后补加300μl培养基进行培养。
5、MSC细胞铺板
将T75细胞培养瓶中的培养液吸出，PBS清洗一遍后，加入1mL胰酶，轻轻晃动培养瓶，使胰酶接触到整个细胞培养瓶底部所有细胞后，将胰酶吸出，将T75细胞培养瓶放至37℃，消化3min后取出培养瓶，观察细胞是否已经与细胞瓶底部分离。培养瓶中加入10mL含有10％FBS的DMEM/F-12培养基，轻轻吹打混匀后，将10mL细胞悬液吸出后加入到50mL离心管中，取少量细胞悬液于1.5ml离心管中，使用血球计数板进行计数。将其配置成4×104个/ml的细胞悬液。24孔板中，铺好膜的孔和未铺膜的孔，每孔加入200ul细胞悬液然后补加300μl培养基进行培养。
6、细胞在牛跟腱胶原蛋白膜和蛛丝蛋白膜上细胞增殖情况检测
第3,4,5天分别取出L929细胞1块24孔细胞培养板，使用显微镜进行观察并拍照，结果如图7所示，MSC细胞由于贴付在纤维膜上后会导致透光率下降而无法显微镜下成像，故而未拍照观测。从图7中可以看出，牛跟腱胶原蛋白膜和重组蜘蛛丝蛋白纤维膜均可以有效的实现L929的贴附，并且细胞形状良好。从增殖效果来看，对于重组蜘蛛丝蛋白纤维膜而言，其在L929和MSC细胞培养方面都表现出了明显优于胶原蛋白纤维膜的促增值作用。
取出的24孔细胞培养板中，每孔吸出所有培养基，然后在每个孔中加入200ul培养基，再加入20ulCCK-8溶液，37℃孵育1h后，使用酶标仪，在450mm处进行读数，对结果进行统计，分析细胞增殖状况，结果如图8-1和8-2所示。
因此可以判定，两种纤维膜相比，重组蜘蛛丝蛋白纤维膜可以更快的实现细胞的贴附和生长，因此具备良好的生物相容性。
实施例7重组蜘蛛丝蛋白静电纺丝膜皮肤再生试验
皮肤创面修复与再生是静电纺丝纤维膜的一个重要的潜在应用领域，本发明人将静电纺丝法制备的重组蜘蛛丝蛋白纤维膜与天然胶原蛋白膜一起评价了其在皮肤创面修复中的作用，具体评价方法与结果如下：
动物模型建立：采用自身对照的方法，大鼠在脊柱两侧约0.5cm处等距离选3个直径为12mm的圆形区域，每点间隔1cm。大鼠按照3.0mL/kg的剂量腹腔注射10％水合氯醛溶液，待翻正反射消失后在剥离圆形区域的全层皮肤组织，覆盖供试品RepB5和牛跟腱胶原蛋白纤维膜。实验后连续3d每只注射2.0×103U/d的氨苄西林钠，以防感染。术后观察创面愈合情况，术后3周，大鼠行安乐死，采集手术部位数字图像，利用软件计算术部创面/无毛区域的面积，以考察各组样品促进创面愈合的作用，结果如图9所示。
从皮肤创面修复与再生试验中可以看出重组蛛丝蛋白静电纺丝纤维膜的皮肤修复效果优于牛跟腱胶原蛋白纤维膜，手术后创面恢复更好。
实施例8重组蜘蛛丝蛋白体外凝血试验
目前市售比较高端的医用凝血产品包括粉状天然胶原蛋白，具备凝血快速和生物安全的特点，因此本发明人还考察了与天然胶原蛋白相比重组蛛丝蛋白的体外凝血效果，方法如下：
1、原材料获得：将重组蜘蛛丝蛋白冻干粉与牛跟腱胶原蛋白样品分别使用同样条件粉碎制粉，过50目筛，获得粒径基本一致的均一粉状供试品。
2、将等质量的牛跟腱胶原蛋白粉、RepB5粉分别置于采血管中，抽真空；空白对照管直接抽真空，每样3管。
3、取11～12周年龄，普通级新西兰白兔1只，雌雄不限，体重1.8～2.2kg，家兔按照3.5mL/kg的剂量腹腔注射10％水合氯醛溶液，待翻正反射消失后通过颈总主动脉采集3ml血液至采各血管。待血液加入真空管后开始计时，室温下每隔约5s倾斜试管一次，至血液不再流动时停止计时，即得全血凝固时间。计算结果如图10所示。
从试管凝血试验结果可以看出，同样的试验条件下蛛丝蛋白RepB5所制备的蛋白粉凝血时间短于牛跟腱胶原蛋白粉末，因此判断具备良好的促凝血潜力。
序列表
<110> 江苏众红生物工程创药研究院有限公司
<120> 重组蜘蛛丝蛋白及其制备方法和产业化应用
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ggtccgggtt acggtccggg tgcgggtggt cagcagggtc cgggtagcca gggtcagcaa 60
ggtggccaag gcagcggtca gcaaggcccg ggtagcggtg gtcagcaggg tcctggtagc 120
cagggcccgt acggtccgag cgctgcggcg gcggcggcgg cggcggggcc gggttatggc 180
ccgggcgcgg gtggtcagca gggtccaggt agccaaggtc agcaaggtgg ccagggcagc 240
ggtcagcaag gcccaggtag cggtggtcag cagggtcccg gtagccaagg cccgtacggt 300
ccttctgctg cggcggcggc ggcggcggcg ggtccgggtt atggccctgg cgcgggtggt 360
cagcagggtc caggtagcca gggtcagcaa ggtggccaag gcagcggtca gcaaggccct 420
ggtagcggtg gtcagcaggg tccaggtagc cagggcccgt acggtccatc cgctgcggcg 480
gcggcggcgg cggcgggccc gggttatggc cctggagcgg gtggtcagca gggtccaggt 540
agccagggtc agcaaggtgg ccaaggcagc ggtcagcaag gccccggtag cggtggtcag 600
cagggtccag gtagccaggg tccgtacggt ccttccgctg cggcggcggc ggcggcggcg 660
ggaccgggtt atggccctgg ggcgggtggt cagcagggtc caggtagcca gggtcagcaa 720
ggtggccaag gcagcggtca gcaaggccca ggtagcggtg gtcagcaggg tccaggtagc 780
cagggtccgt atggtccttc agctgcggcg gcggcggcgg cggcg 825
<210> 7
<211> 368
<212> PRT
<213> Argiope trifasciata
<400> 7
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Pro Gly Tyr Gly Pro Gly Ala
20 25 30
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
35 40 45
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
50 55 60
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
65 70 75 80
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
85 90 95
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
100 105 110
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
115 120 125
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
130 135 140
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
145 150 155 160
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
165 170 175
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
180 185 190
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
195 200 205
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
210 215 220
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
225 230 235 240
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly
245 250 255
Pro Gly Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ser Arg Leu Ser
260 265 270
Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val
275 280 285
Ser Ser Gly Pro Thr Asn Pro Ala Ser Leu Ser Asn Ala Ile Ser Ser
290 295 300
Val Val Ser Gln Val Ser Ser Ser Asn Pro Gly Leu Ser Gly Cys Asp
305 310 315 320
Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His Ile
325 330 335
Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ala Ala Ser Ser Gln
340 345 350
Tyr Ala Gln Leu Val Gly Gln Ser Leu Thr Gln Ala Leu Gly Leu Glu
355 360 365
<210> 8
<211> 588
<212> PRT
<213> Argiope trifasciata
<400> 8
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Pro Gly Tyr Gly Pro Gly Ala
20 25 30
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
35 40 45
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
50 55 60
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
65 70 75 80
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
85 90 95
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
100 105 110
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
115 120 125
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
130 135 140
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
145 150 155 160
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
165 170 175
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
180 185 190
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
195 200 205
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
210 215 220
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
225 230 235 240
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
245 250 255
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
260 265 270
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
275 280 285
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
290 295 300
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
305 310 315 320
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
325 330 335
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
340 345 350
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
355 360 365
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
370 375 380
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
385 390 395 400
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
405 410 415
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
420 425 430
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
435 440 445
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
450 455 460
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln
465 470 475 480
Ala Pro Val Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro Gln Ala
485 490 495
Ser Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro
500 505 510
Thr Asn Pro Ala Ser Leu Ser Asn Ala Ile Ser Ser Val Val Ser Gln
515 520 525
Val Ser Ser Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln
530 535 540
Ala Leu Leu Glu Ile Val Ser Ala Leu Val His Ile Leu Gly Ser Ser
545 550 555 560
Ser Ile Gly Gln Ile Asn Tyr Ala Ala Ser Ser Gln Tyr Ala Gln Leu
565 570 575
Val Gly Gln Ser Leu Thr Gln Ala Leu Gly Leu Glu
580 585
<210> 9
<211> 1028
<212> PRT
<213> Argiope trifasciata
<400> 9
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Pro Gly Tyr Gly Pro Gly Ala
20 25 30
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
35 40 45
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
50 55 60
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
65 70 75 80
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
85 90 95
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
100 105 110
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
115 120 125
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
130 135 140
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
145 150 155 160
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
165 170 175
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
180 185 190
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
195 200 205
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
210 215 220
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
225 230 235 240
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
245 250 255
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
260 265 270
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
275 280 285
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
290 295 300
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
305 310 315 320
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
325 330 335
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
340 345 350
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
355 360 365
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
370 375 380
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
385 390 395 400
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
405 410 415
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
420 425 430
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
435 440 445
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
450 455 460
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
465 470 475 480
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
485 490 495
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
500 505 510
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
515 520 525
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
530 535 540
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
545 550 555 560
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
565 570 575
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
580 585 590
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
595 600 605
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
610 615 620
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
625 630 635 640
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
645 650 655
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
660 665 670
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
675 680 685
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
690 695 700
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
705 710 715 720
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
725 730 735
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
740 745 750
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
755 760 765
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
770 775 780
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
785 790 795 800
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
805 810 815
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
820 825 830
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
835 840 845
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
850 855 860
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
865 870 875 880
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
885 890 895
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
900 905 910
Gly Gln Gln Gly Pro Gly Ser Gln Ala Pro Val Ala Ser Ala Ala Ala
915 920 925
Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val
930 935 940
Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala Ser Leu Ser Asn
945 950 955 960
Ala Ile Ser Ser Val Val Ser Gln Val Ser Ser Ser Asn Pro Gly Leu
965 970 975
Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala
980 985 990
Leu Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ala
995 1000 1005
Ala Ser Ser Gln Tyr Ala Gln Leu Val Gly Gln Ser Leu Thr Gln Ala
1010 1015 1020
Leu Gly Leu Glu
1025
<210> 10
<211> 1468
<212> PRT
<213> Argiope trifasciata
<400> 10
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Pro Gly Tyr Gly Pro Gly Ala
20 25 30
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
35 40 45
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
50 55 60
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
65 70 75 80
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
85 90 95
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
100 105 110
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
115 120 125
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
130 135 140
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
145 150 155 160
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
165 170 175
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
180 185 190
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
195 200 205
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
210 215 220
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
225 230 235 240
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
245 250 255
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
260 265 270
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
275 280 285
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
290 295 300
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
305 310 315 320
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
325 330 335
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
340 345 350
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
355 360 365
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
370 375 380
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
385 390 395 400
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
405 410 415
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
420 425 430
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
435 440 445
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
450 455 460
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
465 470 475 480
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
485 490 495
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
500 505 510
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
515 520 525
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
530 535 540
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
545 550 555 560
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
565 570 575
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
580 585 590
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
595 600 605
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
610 615 620
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
625 630 635 640
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
645 650 655
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
660 665 670
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
675 680 685
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
690 695 700
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
705 710 715 720
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
725 730 735
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
740 745 750
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
755 760 765
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
770 775 780
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
785 790 795 800
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
805 810 815
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
820 825 830
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
835 840 845
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
850 855 860
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
865 870 875 880
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
885 890 895
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
900 905 910
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
915 920 925
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
930 935 940
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
945 950 955 960
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
965 970 975
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
980 985 990
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
995 1000 1005
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
1010 1015 1020
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
1025 1030 1035 1040
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
1045 1050 1055
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
1060 1065 1070
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
1075 1080 1085
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
1090 1095 1100
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
1105 1110 1115 1120
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
1125 1130 1135
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
1140 1145 1150
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
1155 1160 1165
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
1170 1175 1180
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
1185 1190 1195 1200
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
1205 1210 1215
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
1220 1225 1230
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
1235 1240 1245
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
1250 1255 1260
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
1265 1270 1275 1280
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
1285 1290 1295
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
1300 1305 1310
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
1315 1320 1325
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
1330 1335 1340
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln
1345 1350 1355 1360
Ala Pro Val Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro Gln Ala
1365 1370 1375
Ser Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro
1380 1385 1390
Thr Asn Pro Ala Ser Leu Ser Asn Ala Ile Ser Ser Val Val Ser Gln
1395 1400 1405
Val Ser Ser Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln
1410 1415 1420
Ala Leu Leu Glu Ile Val Ser Ala Leu Val His Ile Leu Gly Ser Ser
1425 1430 1435 1440
Ser Ile Gly Gln Ile Asn Tyr Ala Ala Ser Ser Gln Tyr Ala Gln Leu
1445 1450 1455
Val Gly Gln Ser Leu Thr Gln Ala Leu Gly Leu Glu
1460 1465
<210> 11
<211> 427
<212> PRT
<213> Argiope trifasciata
<400> 11
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Ser Gly Pro Gly Tyr Gly Pro
20 25 30
Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly
35 40 45
Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro
50 55 60
Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala
65 70 75 80
Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly
85 90 95
Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly
100 105 110
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
115 120 125
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
130 135 140
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly
145 150 155 160
Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser
165 170 175
Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
180 185 190
Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln
195 200 205
Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly
210 215 220
Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala
225 230 235 240
Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly
245 250 255
Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly
260 265 270
Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly
275 280 285
Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Arg Ser Gly
290 295 300
Pro Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Ala
305 310 315 320
Pro Val Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro Gln Ala Ser
325 330 335
Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr
340 345 350
Asn Pro Ala Ser Leu Ser Asn Ala Ile Ser Ser Val Val Ser Gln Val
355 360 365
Ser Ser Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala
370 375 380
Leu Leu Glu Ile Val Ser Ala Leu Val His Ile Leu Gly Ser Ser Ser
385 390 395 400
Ile Gly Gln Ile Asn Tyr Ala Ala Ser Ser Gln Tyr Ala Gln Leu Val
405 410 415
Gly Gln Ser Leu Thr Gln Ala Leu Gly Leu Glu
420 425
<210> 12
<211> 706
<212> PRT
<213> Argiope trifasciata
<400> 12
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Ser Gly Pro Gly Tyr Gly Pro
20 25 30
Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly
35 40 45
Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro
50 55 60
Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala
65 70 75 80
Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly
85 90 95
Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly
100 105 110
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
115 120 125
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
130 135 140
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly
145 150 155 160
Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser
165 170 175
Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
180 185 190
Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln
195 200 205
Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly
210 215 220
Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala
225 230 235 240
Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly
245 250 255
Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly
260 265 270
Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly
275 280 285
Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Arg Ser Gly
290 295 300
Ser Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly
305 310 315 320
Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly
325 330 335
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
340 345 350
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
355 360 365
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly
370 375 380
Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser
385 390 395 400
Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
405 410 415
Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln
420 425 430
Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly
435 440 445
Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala
450 455 460
Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly
465 470 475 480
Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly
485 490 495
Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly
500 505 510
Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
515 520 525
Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln
530 535 540
Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln
545 550 555 560
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
565 570 575
Ala Ala Ala Ala Arg Ser Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gln
580 585 590
Gln Gly Pro Gly Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ser Arg
595 600 605
Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val Ser Thr
610 615 620
Leu Val Ser Ser Gly Pro Thr Asn Pro Ala Ser Leu Ser Asn Ala Ile
625 630 635 640
Ser Ser Val Val Ser Gln Val Ser Ser Ser Asn Pro Gly Leu Ser Gly
645 650 655
Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val
660 665 670
His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ala Ala Ser
675 680 685
Ser Gln Tyr Ala Gln Leu Val Gly Gln Ser Leu Thr Gln Ala Leu Gly
690 695 700
Leu Glu
705
<210> 13
<211> 985
<212> PRT
<213> Argiope trifasciata
<400> 13
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Ser Gly Pro Gly Tyr Gly Pro
20 25 30
Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly
35 40 45
Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro
50 55 60
Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala
65 70 75 80
Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly
85 90 95
Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly
100 105 110
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
115 120 125
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
130 135 140
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly
145 150 155 160
Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser
165 170 175
Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
180 185 190
Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln
195 200 205
Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly
210 215 220
Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala
225 230 235 240
Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly
245 250 255
Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly
260 265 270
Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly
275 280 285
Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Arg Ser Gly
290 295 300
Ser Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly
305 310 315 320
Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly
325 330 335
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
340 345 350
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
355 360 365
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly
370 375 380
Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser
385 390 395 400
Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
405 410 415
Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln
420 425 430
Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly
435 440 445
Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala
450 455 460
Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly
465 470 475 480
Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly
485 490 495
Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly
500 505 510
Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
515 520 525
Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln
530 535 540
Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln
545 550 555 560
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
565 570 575
Ala Ala Ala Ala Arg Ser Gly Ser Gly Pro Gly Tyr Gly Pro Gly Ala
580 585 590
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly
595 600 605
Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser
610 615 620
Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
625 630 635 640
Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln
645 650 655
Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly
660 665 670
Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala
675 680 685
Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly
690 695 700
Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly
705 710 715 720
Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly
725 730 735
Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
740 745 750
Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln
755 760 765
Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln
770 775 780
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
785 790 795 800
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
805 810 815
Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln
820 825 830
Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr
835 840 845
Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Arg Ser Gly Pro Gly
850 855 860
Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Ala Pro Val
865 870 875 880
Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg
885 890 895
Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro
900 905 910
Ala Ser Leu Ser Asn Ala Ile Ser Ser Val Val Ser Gln Val Ser Ser
915 920 925
Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu
930 935 940
Glu Ile Val Ser Ala Leu Val His Ile Leu Gly Ser Ser Ser Ile Gly
945 950 955 960
Gln Ile Asn Tyr Ala Ala Ser Ser Gln Tyr Ala Gln Leu Val Gly Gln
965 970 975
Ser Leu Thr Gln Ala Leu Gly Leu Glu
980 985
<210> 14
<211> 647
<212> PRT
<213> Argiope trifasciata
<400> 14
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Pro Gly Tyr Gly Pro Gly Ala
20 25 30
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
35 40 45
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
50 55 60
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
65 70 75 80
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
85 90 95
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
100 105 110
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
115 120 125
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
130 135 140
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
145 150 155 160
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
165 170 175
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
180 185 190
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
195 200 205
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
210 215 220
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
225 230 235 240
Ala Ala Ala Ala Gly Ser Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly
245 250 255
Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly
260 265 270
Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly
275 280 285
Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
290 295 300
Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln
305 310 315 320
Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln
325 330 335
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
340 345 350
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
355 360 365
Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln
370 375 380
Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr
385 390 395 400
Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly
405 410 415
Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly
420 425 430
Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly
435 440 445
Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala
450 455 460
Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro
465 470 475 480
Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro
485 490 495
Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro
500 505 510
Ser Ala Ala Ala Ala Ala Ala Ala Ala Arg Ser Gly Pro Gly Tyr Gly
515 520 525
Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Ala Pro Val Ala Ser
530 535 540
Ala Ala Ala Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser
545 550 555 560
Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala Ser
565 570 575
Leu Ser Asn Ala Ile Ser Ser Val Val Ser Gln Val Ser Ser Ser Asn
580 585 590
Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile
595 600 605
Val Ser Ala Leu Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile
610 615 620
Asn Tyr Ala Ala Ser Ser Gln Tyr Ala Gln Leu Val Gly Gln Ser Leu
625 630 635 640
Thr Gln Ala Leu Gly Leu Glu
645
<210> 15
<211> 1146
<212> PRT
<213> Argiope trifasciata
<400> 15
Met His His His His His His His His His His Ser Ser Gly Ser Ser
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Gly Pro Gly Tyr Gly Pro Gly Ala
20 25 30
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
35 40 45
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
50 55 60
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
65 70 75 80
Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
85 90 95
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
100 105 110
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
115 120 125
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
130 135 140
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr
145 150 155 160
Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly
165 170 175
Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser
180 185 190
Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala
195 200 205
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln
210 215 220
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
225 230 235 240
Ala Ala Ala Ala Gly Ser Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly
245 250 255
Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly
260 265 270
Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly
275 280 285
Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
290 295 300
Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln
305 310 315 320
Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln
325 330 335
Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala
340 345 350
Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln
355 360 365
Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln
370 375 380
Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr
385 390 395 400
Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly
405 410 415
Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly
420 425 430
Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly
435 440 445
Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala
450 455 460
Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro
465 470 475 480
Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro
485 490 495
Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro
500 505 510
Ser Ala Ala Ala Ala Ala Ala Ala Ala Arg Ser Gly Pro Gly Tyr Gly
515 520 525
Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser
530 535 540
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala
545 550 555 560
Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly
565 570 575
Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln
580 585 590
Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala
595 600 605
Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly
610 615 620
Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser
625 630 635 640
Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly
645 650 655
Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln
660 665 670
Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr
675 680 685
Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly
690 695 700
Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser
705 710 715 720
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala
725 730 735
Ala Ala Ala Ala Ala Ala Ala Gly Ser Gly Pro Gly Tyr Gly Pro Gly
740 745 750
Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln
755 760 765
Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly
770 775 780
Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala
785 790 795 800
Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser
805 810 815
Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser
820 825 830
Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala
835 840 845
Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly
850 855 860
Gly Gln Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser
865 870 875 880
Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln
885 890 895
Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro
900 905 910
Gly Tyr Gly Pro Gly Ala Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly
915 920 925
Gln Gln Gly Gly Gln Gly Ser Gly Gln Gln Gly Pro Gly Ser Gly Gly
930 935 940
Gln Gln Gly Pro Gly Ser Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala
945 950 955 960
Ala Ala Ala Ala Ala Gly Pro Gly Tyr Gly Pro Gly Ala Gly Gly Gln
965 970 975
Gln Gly Pro Gly Ser Gln Gly Gln Gln Gly Gly Gln Gly Ser Gly Gln
980 985 990
Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro
995 1000 1005
Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Arg Ser Gly Pro
1010 1015 1020
Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Ala Pro
1025 1030 1035 1040
Val Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser
1045 1050 1055
Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn
1060 1065 1070
Pro Ala Ser Leu Ser Asn Ala Ile Ser Ser Val Val Ser Gln Val Ser
1075 1080 1085
Ser Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu
1090 1095 1100
Leu Glu Ile Val Ser Ala Leu Val His Ile Leu Gly Ser Ser Ser Ile
1105 1110 1115 1120
Gly Gln Ile Asn Tyr Ala Ala Ser Ser Gln Tyr Ala Gln Leu Val Gly
1125 1130 1135
Gln Ser Leu Thr Gln Ala Leu Gly Leu Glu
1140 1145
<210> 16
<211> 72
<212> DNA
<213> Argiope trifasciata
<400> 16
atgcatcacc atcaccatca ccatcaccat cactcttccg gttcatcgtt agaagttttg 60
tttcaaggcc ct 72
<210> 17
<211> 366
<212> DNA
<213> Argiope trifasciata
<400> 17
ggtccgggtt acggtccggg cgcgggtcag caaggcccgg gtagccaggc gccggtggcg 60
agcgcggcgg cgagccgtct gagcagcccg caagcgagca gccgtgtgag cagcgcggtt 120
agcaccctgg tgagcagcgg tccgaccaac ccggcgagcc tgagcaacgc gatcagcagc 180
gtggttagcc aagtgagcag cagcaacccg ggcctgagcg gttgcgacgt gctggttcaa 240
gcgctgctgg agatcgttag cgcgctggtg cacattctgg gtagcagcag catcggtcaa 300
attaactacg cggcgagcag ccagtatgcg caactggtgg gccagagcct gacccaagcg 360
ctgggt 366

Claims (10)

1.重组蜘蛛丝蛋白，其氨基酸序列由N末端非重复区域、核心重复区域、C末端非重复区域组成，其中核心重复区域由1-30个RepA和/或1-15个RepB组成，单个RepA氨基酸序列如SEQ ID NO：1所示，单个RepB氨基酸序列如SEQ ID NO：2所示；所述N末端非重复区域，氨基酸序列如SEQ ID NO：3所示；所述C末端非重复区域，氨基酸序列如SEQ ID NO：4所示。
2.根据权利要求1所述的重组蜘蛛丝蛋白，其特征在于：所述核心重复区域由5-30个RepA或5-15个RepB组成。
3.根据权利要求1所述的重组蜘蛛丝蛋白，其特征在于：所述核心重复区域由1-5个RepA和1-5个RepB的串联体组成。
4.根据权利要求3所述的重组蜘蛛丝蛋白，其特征在于：所述1-5个RepA的串联体位于整个融合蛋白的靠近N端位置，所述1-5个RepB的串联体位于整个融合蛋白的C端位置。
5.根据权利要求1至4任一项所述的重组蜘蛛丝蛋白，其特征在于：所述核心重复区域由5个RepA串联体、10个RepA串联体、20个RepA串联体、30个RepA串联体、5个RepB串联体、10个RepB串联体、15个RepB串联体、5个RepA加5个RepB串联体，或者2个RepA5RepB5的串联体组成。
6.一种包含如权利要求5所述的核苷酸序列的载体。
7.一种包含如权利要求6所述的载体的大肠杆菌菌株。
8.一种表达重组蜘蛛丝蛋白的方法，其特征在于包括如下步骤：
(1)将如权利要求7所述的大肠杆菌菌落接入发酵罐中，设定发酵温度为37℃、pH为6.8-7.2之间、DO设定在30-40％之间；
(2)发酵开始后定期取样进行OD600和菌体湿重的测定，待DO曲线出现急剧上升时，开始进行补料培养，补料培养基的流加速度维持在8-12g/L/h；
(3)待菌体生长至OD600≈45-55之间，降低发酵温度至25-30℃，待温度稳定后向发酵罐中加入终浓度为0.2-1.0mM的IPTG进行诱导表达；诱导表达8-12h结束培养。
9.一种重组蜘蛛丝蛋白的纯化方法，其特征在于包括如下步骤：
(1)取表达重组蜘蛛丝蛋白的大肠杆菌，经菌体破碎、收集裂解物上清液；
(2)取上述裂解物上清液膜过滤去杂；
(3)IMAC亲和层析纯化即得目的蛋白。
10.如权利要求1至5任一项所述的重组蜘蛛丝蛋白在制作蛋白类缝合线、蛋白类生物修复膜、蛋白类止血材料中的应用。
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