CN112759653A - 一种pkek融合蛋白及其制备方法与应用 - Google Patents

一种pkek融合蛋白及其制备方法与应用 Download PDF

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CN112759653A
CN112759653A CN201911075149.5A CN201911075149A CN112759653A CN 112759653 A CN112759653 A CN 112759653A CN 201911075149 A CN201911075149 A CN 201911075149A CN 112759653 A CN112759653 A CN 112759653A
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刘合栋
曹春来
杨晓纯
杨祺
邹鑫
刘学明
梁雄基
王康钊
肖炳文
黄有泉
周翠
何秀仪
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Abstract

本发明公开了一种PKEK融合蛋白及其制备方法与应用。该融合蛋白通过将若干柔性连接肽与PKEK依次串联构成；其在微生物细胞内以包涵体蛋白的形式表达，同时通过将若干柔性连接肽与美白多肽PKEK依次串联构成融合蛋白，能够有效减少酶切过程中产生的错切杂质，进而高效获得高纯度美白多肽PKEK，且纯度可达95.4％。此外，利用本申请所述基因工程技术生产美白多肽PEKE的制备方法，在12h内即可大量获得PKEK融合蛋白包涵体，且表达量最高可达41.5g/L，工艺简单高效且易于放大，有利于工业化生产。

Description

一种PKEK融合蛋白及其制备方法与应用
技术领域
本发明属于基因工程领域，特别涉及一种PKEK融合蛋白及其制备方法与应用。
背景技术
皮肤最重要的功能是保护身体免受过多水分流失，也能够有效防止有害化学品或细菌以及太阳辐射的进入。如果人皮肤长时间暴露于太阳辐射环境中，皮肤通过形成黑色素来应对UV辐射，并最终形成光诱导的皮肤老化和色素疾病，太阳光谱中存在户外紫外线UVB(280～320nm)，UVB照射下皮肤局部色素沉淀过度容易产生雀斑、黄褐斑、褐黄斑等问题。皮肤的色素沉淀基本上由皮肤自身的黑色素决定，黑色素由表皮中特化的细胞，即黑色素细胞形成。在黑色素合成期间，作为起始酶的酪氨酸酶起到决定性作用；在角质形成细胞中，UV-A和UV-B辐射通过形成NO来激发黑色素细胞中黑色素的形成。黑色素细胞通过增加细胞生长来响应NO，形成更高程度的分支并增加黑色素合成，同时黑色素的生成受色素沉淀调控子(α-MSH)的控制。目前已开发出各种方法来降低皮肤的色素沉着，最常用的皮肤和毛发增亮剂是氢醌或氢醌糖苷熊果苷。然而，这些化合物对黑色素细胞具有细胞毒性作用且对皮肤具有刺激性作用。专利W0/2008/08M94和W0/2009/068351中描述了一种新型的美白多肽PKEK，体外细胞实验证实人角质细胞在紫外线UVB照射下，PKEK可有效减少白介素-6、白介素-8和TNF-a的表达，并显著降低色素沉淀调控子(α-MSH)的表达。在另外一组随机双盲人体皮肤功效测试试验中，10名健康受试者每天一次连续使用PKEK四周后，与对照组相比，紫外线UVB照射下PKEK受试组皮肤白介素-1α、白介素-6、白介素-8、肿瘤坏死因子-α以及酪氨酸酶表达显著下调；39名高加索女性白人将PKEK与维他命C磷酸钠合用6周后，面部斑点明显褪去，而单独使用PKEK或维他命C磷酸钠斑点褪去均不显著；由此可见，上述体内和体外实验均可证实PKEK能够有效减少因户外紫外线导致的面部色素沉淀。
目前PKEK主要采用液相合成或固相合成方法(SPPS)生产，由于化学合成所用的原料赖氨酸和谷氨酸侧链均要进行保护，转肽反应结束后需进行脱帽工艺，再进行结晶，操作比较繁琐，而且化学合成过程中易于形成消旋手性杂质，导致高纯度PKEK的制备成本居高不下。因此，通过基因工程技术制备多肽成为大势所趋。
专利申请CN107881187A公开了利用EEEEK、EDK、DDDDK作为连接肽，通过D或E的侧链羧基与K的侧链氨基形成盐键，以减少K位点的酰化修饰，然而在K前面引入D或E形成的连接肽，并没有影响蛋白酶的酶切效率，仍不能解决后续酶切工艺中PKEK的错切问题。
专利CN104725485A公开了一种重组活性肽的制备方法，具体地，利用类弹性蛋白纯化标签ELPs和SUMO多肽融合一个或多个降血压肽单体和抗氧化肽单体表达，对包涵体表达的目标蛋白先进行变性与复性再进行标签技术纯化；通过ELPs标签纯化技术纯化融合蛋白以及采用SUMO蛋白酶裂解及加盐离心去除二联体融合标签ELPs-SUMO，获得重组活性肽，然后再采用胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶酶解重组活性肽超滤得小肽混合体。该方法虽然可制备得到小肽，但是步骤工艺繁琐，需要昂贵且酶切效率低的SUMO蛋白酶，而且酶切前需要对包涵体进行变性复性。另外，由于SUMO蛋白酶不耐受变性液，故需要在酶切前利用层析技术去除变性复性液，导致通过生物合成法制备的小肽成本与化学合成法相比并无显著优势。
专利CN106560475A公布了一种活性短肽的基因工程生物合成工艺，通过将活性短肽串联后与纯化标签在微生物细胞内以可溶性蛋白或包涵体蛋白表达，并采用高特异性的蛋白酶降解重组蛋白质最终形成单个活性短肽。虽然该方法在制备单个活性短肽方面具有一定的优势，但由于PKEK含有多个碱性氨基酸，将多个PKEK串联后等电点较高，如10个串联PKEK多肽理论等电点为9.92，这类含有多个碱性氨基酸的多肽在微生物细胞中通常以可溶性蛋白形式表达极难形成包涵体蛋白，这样不仅易于被降解，而且游离PKEK串联多肽中的赖氨酸侧链氨基易于被乙酰化修饰，而且该方法仍无法解决PKEK串联多肽在酶切工艺中的错切问题，导致最终得到美白多肽PKEK的概率大大降低。
因此，目前亟需一种高效方法生产美白多肽PKEK。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种PKEK融合蛋白，所述融合蛋白在微生物细胞内以包涵体蛋白的形式表达，同时通过将若干柔性连接肽与美白多肽PKEK依次串联构成融合蛋白，能够有效减少酶切过程中产生的错切杂质，进而高效获得高纯度美白多肽PKEK，有利于工业化生产。
本申请的第二个目的是提供一种上述PKEK融合蛋白的编码基因。
本申请的第三个目的是提供上述PKEK融合蛋白的编码基因的应用。
本发明的目的通过下述技术方案实现：
一种PKEK融合蛋白，包括将若干柔性连接肽与美白多肽PKEK依次串联构成的蛋白，其结构式如下：(B-PKEK)n；其中：B为柔性连接肽、n为1～30的整数。
所述的PKEK融合蛋白，还包括伴侣蛋白，伴侣蛋白与一个连接肽连接，结构式如下：A-(B-PKEK)n；其中：A为伴侣蛋白；B为柔性连接肽；n为1～30的整数；所述的n优选为10～30；更优选为10～20；进一步优选为12～15。
所述的伴侣蛋白为KSI、PagP、PaP3.30、TAF12、mKSI、mPagP、mPaP3.30或mTAF12，m表示突变体。伴侣蛋白的作用是辅助本申请所述融合蛋白在大肠杆菌中形成包涵体，促进包涵体的形成，提高包涵体产量和质量，降低被降解和侧链修饰的风险；且便于下游的分离纯化。现有野生型醛固酮异构酶KSI片段、野生型PagP蛋白片段、野生型TAF12蛋白片段、野生型PaP3.30蛋白片段均具有高度疏水性，在大肠杆菌中易形成包涵体，并且能引导连接在其C端的小肽进行高效表达，因此选用KSI、PagP、PaP3.30或TAF12蛋白作为伴侣蛋白；另外，由于上述野生型分子伴侣蛋白有多个赖氨酸残基及精氨酸残基，在后续的酶切工艺中会形成过多的错误酶切片段，若将上述野生型分子伴侣蛋白中的赖氨酸、精氨酸突变为其他氨基酸，能够有效减少杂质的形成，显著提高酶切纯度，故所述伴侣蛋白还可以是KSI、PagP、PaP3.30或TAF12蛋白的突变体，即mKSI、mPagP、mPaP3.30或mTAF12；优选为KSI、PagP或PaP3.30。
所述的柔性连接肽为如Xm---X2-X1所示的肽序列；其中，m为0至10的整数，优选为1至7的整数；X1为蛋白酶酶切位点；X2至Xm为柔性片段。
所述的蛋白酶酶切位点为Arg或Lys；优选为Lys。
所述的柔性片段中的氨基酸不能为Pro、Thr或Trp；优选为Gly、Ala或Ser。
所述的柔性连接肽优选为AK、GK、AAK、AAGK、AAGGK、GGAAK、GGSASK或AAGGSGAGK。
所述的伴侣蛋白为KSI时，所述的柔性连接肽优选为AK、GK、AAK、AAGK、AAGGK、GGAAK或GGSASK；更优选为GK、AAK、AAGK、GGAAK或GGSASK；最优选为AAGK或GGAAK；所述的n优选10～15；更优选为12～15。
所述的伴侣蛋白为PagP时，所述的柔性连接肽优选为AK、GK、AAK、AAGK、AAGGK、GGAAK、GGSASK或AAGGSGAGK；更优选为AK、GK、AAKAAGK、AAGGK、GGAAK或GGSASK；所述的n优选为15～20。
所述的伴侣蛋白为PaP3.30时，所述的柔性连接肽优选为GGAAK、AAK；所述的n优选为10～30。
一种编码上述PKEK融合蛋白的基因，可通过密码子规则得到；更优选为依据宿主菌密码子使用偏好性设计得到的序列，如SEQ ID NO.37、SEQ ID NO.39、SEQ ID NO.40、SEQID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQID NO.56、SEQ ID NO.58、SEQ ID NO.60、SEQ ID NO.64所示的序列。
一种重组载体，含有上述PKEK融合蛋白的编码基因；是将上述PKEK融合蛋白的编码基因克隆入表达载体得到。
所述的表达载体上的启动子包括T7启动子、T7 lac启动子、Tac启动子、Lac启动子、Trp启动子；优选为T7启动子和T7lac启动子。
所述的表达载体优选为pET系列载体；更优选为pET-3a、pET-9a、pET-28a(+)、pET-22b(+)、pET-26b(+)或pET-31b(+)；最优选为pET-31b(+)和pET28a。
一种重组表达菌株，含有上述重组载体；是将上述重组载体转入基因工程菌株获得。
所述的基因工程菌株选自细菌、酵母和真菌；优选的是细菌；更优选的是大肠杆菌(Escherichia coli)。
上述PKEK融合蛋白可通过化学合成方法得到，也可通过基因工程方法表达得到；优选为通过基因工程方法表达得到，制备方法优选包括如下步骤：发酵培养上述重组表达菌株，收集融合蛋白的包涵体，得到PKEK融合蛋白。
上述PKEK融合蛋白在制备美白多肽PEKE中的应用，具体包括如下步骤：溶解、酶切融合蛋白的包涵体，分离纯化即得活性美白多肽PKEK。
一种利用基因工程技术生产美白多肽PEKE的制备方法，包括如下步骤：
S1、获得包含上述PKEK融合蛋白编码基因的重组载体，其编码的融合蛋白形如A-(B-PKEK)n，其中：A为伴侣蛋白；B为柔性连接肽；n为1至30的整数；
S2、将步骤S1获得的重组载体转入基因工程菌株中，得到重组表达菌株；发酵培养重组表达菌株，收集融合蛋白的包涵体；
S3、溶解、酶切融合蛋白的包涵体，分离纯化即得活性美白多肽PKEK。
步骤S2中所述的基因工程菌株为细菌、酵母或真菌，以将含有美白多肽的重组表达载体导入基因工程菌株内，避免引入新杂质，方便后续工业化生产。
所述的细菌优选为大肠杆菌。所述PKEK融合蛋白可以在大肠杆菌中以包涵体形式进行表达，融合蛋白无需复性，只需在合适条件下溶解后、直接稀释酶切即可得到小分子蛋白或多肽，工艺简单且易于放大，适于工业化生产。
步骤S2中所述的发酵中使用的发酵培养基由有机氮源、碳源和无机盐组成；具体地，所述发酵培养基(总体积为8L)由以下成分组成：酵母粉24g、蛋白胨40g、NaCl 8.0g、KH2PO4 24.0g、Na2HPO4 26.0g、MgSO4 8.0g、(NH4)2SO4 48.0g、CaCl2·2H2O 0.11g、甘油33.0g、微量元素7mL；灭菌后用氨水调pH至7.0。
其中，微量元素混合液(总体积100mL)由以下成分组成：FeCl2·4H2O 2.287g、ZnCl2 0.131g、CoCl2·6H2O0.2g、Na2Mo4·2H2O 0.2g、CaCl2·2H2O 0.1g、CuCl2·2H2O0.125g、H3BO4 0.05g、MnSO4·H2O 0.217g、37wt％浓盐酸10mL。
所述的发酵的方式为高密度发酵。
步骤S2中所述的包涵体是通过高压匀浆法破碎菌体细胞得到。
步骤S3中所述的酶切中使用的蛋白酶为牛胰蛋白酶、猪胰蛋白酶或赖氨酰特异性内切酶中的任一种或多种。
步骤S3中所述的溶解、酶切融合蛋白的包涵体的具体做法为：先将收集的融合蛋白的包涵体用高浓度尿素溶液溶解，稀释后用蛋白酶进行酶切。
所述的尿素溶液的浓度为5M以上，更优选为8M。
所述的尿素溶液的用量优选为1g包涵体：5.5～6.5mL尿素溶液；更优选为1g包涵体：6mL尿素溶液。
上述利用基因工程技术生产美白多肽PKEK的制备方法在工业化制备美白多肽PEKE中的应用。该利用基因工程技术生产美白多肽PKEK的制备方法简单、高效，制备得到的美白多肽纯度高，适于工业化生产。
本发明相对于现有技术具有如下优点及有益效果：
发明人通过创造性劳动不经意发现：当酶切位点羧基端氨基酸为小侧链柔性氨基酸即PKEK后面连接有连接肽时，酶切效率有显著提升。发明人通过将柔性连接肽与PKEK依次串联，使PKEK中的第二个赖氨酸的酶切效率显著高于第一个赖氨酸的酶切效率，最终得到大量的PKEK完整多肽分子，错切杂质PK、EK显著降低；同时由于引入连接肽，使部分错切杂质以连接肽+PK、EK+连接肽形式存在，导致错切杂质与完整PKEK美白多肽的理化性质有显著差别，这样通过层析工艺即可有效去除杂质，将错切杂质控制在较低水平。
本发明提供了一种PKEK融合蛋白，所述融合蛋白在微生物细胞内以包涵体蛋白的形式表达，同时通过将若干柔性连接肽与美白多肽PKEK依次串联构成融合蛋白，能够有效减少酶切过程中产生的错切杂质，进而高效获得高纯度美白多肽PKEK，且纯度可达95.4％；此外，利用本申请所述基因工程技术生产美白多肽PEKE的制备方法，在12h内即可大量获得PKEK融合蛋白包涵体，且表达量最高可达41.5g/L，工艺简单高效且易于放大，有利于工业化生产。
附图说明
图1是实施例1所述重组质粒pET31b-KSI-(AAK-PKEK)10的质粒图谱图。
图2是实施例1提供的基因工程菌株BL21(DE)/pET31b-KSI-(AAK-PKEK)10摇瓶发酵诱导3h得到的样品SDS-PAGE图谱图；其中，泳道M为蛋白marker、泳道1为阴性对照、泳道2～7代表不同的单克隆。
图3是实施例1提供的基因工程菌株BL21(DE)/pET31b-KSI-(AAK-PKEK)10摇瓶发酵诱导15h得到的样品SDS-PAGE图谱图；其中，泳道M为蛋白marker、泳道8为阴性对照、泳道9～14代表不同单克隆。
图4是实施例1提供的基因工程菌株BL21(DE)/pET31b-KSI-(AAK-PKEK)10发酵试验SDS-PAGE图谱图；其中，泳道1和泳道3为诱导0h得到的融合蛋白产量，泳道2、4为诱导12h得到的融合蛋白产量。
图5是实施例1提供的PEKE纯化后的收集液的HPLC图谱图。
图6是实施例1制备得到的美白多肽PKEK的高分辨质谱分析结果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
材料
限制性内切酶AlwNI和XhoI购于TaKaRa公司；(AAK-PKEK)10融合基因序列(如SEQID NO:1所示)委托广州Invitrogen公司合成；琼脂糖凝胶回收试剂盒购于TIANGEN公司；DNA Ligation Kit购于TaKaRa公司；大肠杆菌DH5α购于Life Technologies公司；质粒提取试剂盒购于TIANGEN公司；鉴定含有(AAK-PKEK)10基因片段的克隆委托Life Technologies公司测序；大肠杆菌BL21(DE3)购于Life Technologies公司；改良型Lowry法蛋白浓度测定试剂盒购于生工(生物工程)上海股份有限公司；搅拌式发酵罐购于东方生工生物有限公司；重组牛胰蛋白酶购于上海雅心生物技术有限公司；重组猪胰蛋白酶购于Roche罗氏试剂；重组赖氨酰内切酶购于日本和光纯药株式会社；Uni PMM30-500填料购于苏州纳微科技有限公司。
实施例1
一种利用基因工程技术生产美白多肽PKEK的制备方法，包括如下步骤：
1、构建重组质粒pET31b-KSI-(AAK-PKEK)10
1.1人工合成含连接肽和PKEK基因序列(AAK-PKEK)10
设计连接肽和PKEK基因序列(AAK-PKEK)10的融合基因片段，依据密码子表将这些氨基酸序列转换为核苷酸序列，在转换过程中根据大肠杆菌的密码子使用偏好性，选用使用频率较高的密码子，调整其GC含量，去掉影响基因转录的顺式作用元件和重复序列而优化得到，同时在基因序列的3’端引入双终止密码子TAATGA，为了便于基因操作，在(AAK-PKEK)10融合基因序列的5’端引入AlwNI酶切位点序列CAGATGCTG，因此在N端延伸肽前引入ML两个氨基酸，在(AAK-PKEK)10融合基因3’端引入XhoI酶切位点CTCGAG，优化后的的(AAK-PKEK)10融合基因序列如SEQ ID NO:1所示。
1.2pET31b-KSI-(AAK-PKEK)10重组表达载体的构建
用限制性内切酶AlwNI和XhoI对质粒pET-31b(+)进行双酶切，取5μL酶切产物用质量体积比0.8％琼脂糖凝胶电泳进行分析，确认酶切完全后，将全部酶切产物进行质量体积比0.8％琼脂糖电泳，割下含约5.7kb的DNA片段的凝胶，用琼脂糖凝胶回收试剂盒将凝胶中的DNA片段纯化到30μL去离子水中，就得到带粘性末端的质粒片段。同理，用AlwNI和XhoI对(AAK-PKEK)10融合基因片段进行双酶切，将酶切后的DNA片段纯化到20μL去离子水中，就得到带粘性末端的基因片段。用DNA连接试剂盒(DNA Ligation Kit)将基因片段和质粒片段进行连接，连接条件为16℃过夜，取10μL过夜连接的产物，加入到80μL用CaCl2法(美国冷泉港实验室出版的《分子克隆实验指南》第三版)制备的大肠杆菌DH5α感受态细胞中，42℃处理90s，迅速加入300μL经过37℃预热的LB液体培养基(其中，蛋白胨10g/L、酵母粉5g/L、氯化钠5g/L，pH7.0～7.5)，在37℃摇床中低速(100～150rpm)振荡培养45min，然后取100μL培养物涂布添加了氨苄青霉素(终浓度为100μg/ml)的LB固体培养基(其中，蛋白胨10g/L、酵母粉5g/L、氯化钠5g/L、琼脂粉15g/L，pH7.0～7.5)。在37℃培养箱中倒置培养约18h，直到长出单菌落，随机挑取部分单菌落进行菌液PCR鉴定，PCR反应的条件为：94℃5min；94℃30s、55℃30s、72℃30s，30个循环；72℃5min；引物为：T7启动子引物和T7终止子引物。
T7启动子引物：5’-TAATACGACTCACTATAGGG-3’(SEQ ID NO:35)；
T7终止子引物：5’-TGCTAGTTATTGCTCAGCGG-3’(SEQ ID NO:36)。
将初步筛选得到的阳性克隆用质粒提取试剂盒，并按说明书进行抽提质粒，通过NdeI和XhoI进行酶切鉴定，得到约590bp的基因片段和约5.3kb的载体片段的克隆，即为含有(AAK-PKEK)10基因片段的克隆。将鉴定含有(AAK-PKEK)10基因片段的克隆进行测序。将测序证明没有碱基突变和读码框移位的单克隆，接种至50mL添加了氨苄青霉素(终浓度为100μg/mL)的LB液体培养基，37℃、250rpm培养18h，获得的培养物用质粒提取剂盒抽提质粒，即为重组表达载体，命名为pET31b-KSI-(AAK-PKEK)10，如图1所示。
2、BL21(DE3)/pET31b-KSI-(AAK-PKEK)10表达菌株的筛选及产物鉴定
按照美国冷泉港实验室出版的《分子克隆实验指南》第三版提供的氯化钙法，制备大肠杆菌BL21(DE3)感受态细胞。取1μL实施例1中的重组表达载体pET31b-KSI-(AAK-PKEK)10转化上述大肠杆菌感受态细胞，转化方法同样按照1.2中的氯化钙法进行。将转化液分别涂布至添加了氨苄青霉素(终浓度为100μg/mL)的LB固体培养基，37℃倒置培养直到出现单菌落，即获得了PKEK融合蛋白表达菌种库，并命名为BL21(DE3)/pET31b-KSI-(AAK-PKEK)10。同时，以pET-31b(+)转化大肠杆菌BL21(DE3)感受态细胞，得到BL21(DE3)/pET31b菌，作为阴性对照。
用无菌牙签分别挑取上述工程菌在LB固体培养基形成的单菌落，挑取3个单克隆接种至50mL LB液体培养基，37℃、250rpm振荡培养，当菌液OD600＝0.5～1.0时，取3～6mL菌液接种至100mL LB液体培养基，并取3～6mL BL21(DE3)/pET31b菌液接种至100mL LB液体培养基作为阴性对照，37℃、250rpm振荡培养至OD600＝0.6～0.8时添加IPTG(异丙基-β-D-硫代吡喃半乳糖苷，终浓度为1mmol/L)开始诱导，并分别取诱导3h、15h菌液(4mL)，12000rpm离心1分钟，去掉上清液，将菌体保存在-20℃备用。
取出-20℃冻存的菌体，加入5mL浓度为8mol/L的尿素溶液重悬菌体，在冰水混合物中超声波破碎10分钟(超声3秒、停5秒，如此循环)。取15μL破碎液，加入15μL上样缓冲液，充分混匀后取10μL进行SDS-PAGE(其中浓缩胶含体积百分比5％的丙烯酰胺-甲叉双丙烯酰胺(按照体积比29:1配制)、分离胶含体积百分比15％的丙烯酰胺-甲叉双丙烯酰胺。电泳条件为：浓缩胶设定电流为11mA，分离胶设定电流为22mA。电泳结束后，取出凝胶，用考马斯亮蓝染色液(每升含0.6g考马斯亮蓝R-250、450mL乙醇、100mL冰醋酸，余量为纯化水)染色过夜，用脱色液(每升含250mL乙醇、80mL冰醋酸，余量为纯化水)脱色直到背景透明，随后将凝胶在透明背景下拍照提取图像，如图2和图3所示，用生物图像处理软件Gel-Pro Analyzer4.0进行灰度扫描分析目的条带占总条带的比例，即为PKEK融合蛋白在总蛋白质中的纯度。使用改良型Lowry法蛋白浓度测定试剂盒按说明书进行操作，测定菌体破碎液的总蛋白质浓度，再通过稀释倍数换算得到菌液的总蛋白浓度。根据PKEK融合蛋白在总蛋白质中的纯度和菌液的总蛋白浓度，换算得到PKEK融合蛋白的表达量。
根据电泳结果发现，基因工程菌BL21(DE3)/pET31b-KSI-(AAK-PKEK)10在诱导前不会表达目的蛋白质，这可以有效降低目的蛋白质对宿主细胞的毒性，有利于延长蛋白的表达时间。不含有PKEK基因的对照菌BL21(DE3)/pET-31b(+)经IPTG诱导后可表达分子量大小约为14kDa的醛固酮异构酶，而基因工程菌BL21(DE3)/pET31b-KSI-(AAK-PKEK)10经IPTG诱导后可表达大小约为22kDa的蛋白质，这与PKEK融合蛋白的理论分子量21.2KDa(使用在线软件http://web.expasy.org/compute_pi/预测得到)相符，说明基因工程菌中已成功导入PKEK基因。
将上述基因工程菌BL21(DE3)/pET31b-KSI-(AAK-PKEK)10，在加有终浓度为100μg/mL的氨苄青霉素新鲜LB固体培养基上划线进行纯培养，挑取单菌落用LB液体培养基培养至OD600＝0.8～1.0，制成含体积百分比15％甘油的菌液，存放至-70℃冰箱备用。
3、PKEK融合蛋白基因工程菌BL21(DE3)/pET31b-KSI-(AAK-PKEK)10发酵试验
3.1制备种子培养物
取20μL在-70℃冷冻保存的菌种BL21(DE3)/pET31b-KSI-(AAK-PKEK)10，接种至50mL添加了氨苄青霉素(终浓度为100μg/mL)的LB液体培养基，在28℃、250rpm的摇床中培养16小时，从而活化菌种，再将50mL活化过的菌种接种到400mL的LB液体培养基中，在28℃、250rpm的条件下继续培养3h，获得种子培养物，控制菌体浓度OD600在2.0～3.0之间。
3.2 20L发酵罐中的发酵培养
使用20L搅拌式发酵罐，按照表1所示的发酵培养基的配方经灭菌后用氨水调pH至7.0后进行投料，投料体积为8L，严格控制发酵条件：温度控制为37℃，pH控制为7.0，溶解氧(DO)控制在5～50％的最大氧饱和度之间(氧饱和度”0”的校正条件是：饱和亚硫酸钠溶液；最大氧饱和度的校正条件是：转速为150rpm，空气流速4L/min)，发酵转速控制在150～700rpm之间，具体发酵转速根据DO的变化进行调控，空气流速控制在4～20L/min之间，当培养至碳源耗尽时开始补料，其中每千克补料液含有：500g甘油、33g酵母浸粉、66g酵母蛋白胨、3.5mL微量元素、余量为水，全程以0.4mL·min-1·L-1的补料速率进行匀速补料。当培养至菌体浓度OD600约等于80时，开始添加IPTG至终浓度0.3mM，开始诱导。
表1：发酵培养基配方
组分 用量(g)
Yeast Extract(酵母粉) 24.0
Tryptone(蛋白胨) 40.0
NaCl 8.0
KH<sub>2</sub>PO<sub>4</sub> 24.0
Na<sub>2</sub>HPO<sub>4</sub> 26.0
MgSO<sub>4</sub> 8.0
(NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> 48.0
CaCl<sub>2</sub>·2H<sub>2</sub>O 0.11
甘油 33.0
微量元素混合液(见表2) 7mL
表2：微量元素混合液(100mL)配方表
组分 用量(g)
FeCl<sub>2</sub>·4H<sub>2</sub>O 2.287
ZnCl<sub>2</sub> 0.131
CoCl<sub>2</sub>·6H<sub>2</sub>O 0.2
Na<sub>2</sub>Mo<sub>4</sub>·2H<sub>2</sub>O 0.2
CaCl<sub>2</sub>·2H<sub>2</sub>O 0.1
CuCl<sub>2</sub>·2H<sub>2</sub>O 0.125
H<sub>3</sub>BO<sub>4</sub> 0.05
MnSO<sub>4</sub>·H<sub>2</sub>O 0.217
37wt％浓盐酸 10mL
诱导12h停止发酵，分别在诱导0h和12h取1mL发酵液，用纯化水稀释10倍后，测定(AAK-PKEK)10融合蛋白的表达量(两个平行批次)。
基因工程菌株BL21(DE)/pET31b-KSI-(AAK-PKEK)10发酵试验的SDS-PAGE图谱如图4所示，PKEK纯化后的收集液的液相检测结果如图5所示，换算后含有融合蛋白包涵体的平均产量为41.5g/L，其中融合蛋白包涵体中的融合蛋白含量为35％，即14.5g/L。
4、PKEK融合蛋白包涵体的提取和酶切
4.1包涵体的分离提取
取上述步骤3中的发酵液以4500rpm离心30分钟收集菌体，以质量体积比为1:7的比例将菌体重悬于破碎液(每升含有6.05g Tris和1.46g EDTA，余量为纯化水，调pH为8.0)中，水浴下高压(900bar)匀浆破碎菌体，离心收集沉淀，再以质量体积比为1:15的比例将收集沉淀溶于洗涤缓冲液(每升含有6.05g Tris、1.46g EDTA、2.9g NaCl、10mL TritionX-100，余量为纯化水)进行洗涤，离心收集包涵体沉淀。
4.2包涵体变性液酶切
将上述收集的包涵体2.5g用15mL变性液(每升含有8mol尿素，余量为纯化水)溶解后，再用5mM Tris溶液稀释1倍，调pH为9.0，室温下搅拌2～5h。
采用重组牛胰蛋白酶按照说明书进行酶切，经酶切即可得到PKEK单体粗品。
5、酶切后的PKEK粗品纯化
将100mL按照上述步骤4中制备的经酶切后的PKEK粗品(融合蛋白序列为SEQ IDNO:14上样到装有10mL Uni PMM30-500填料的层析柱上(其预先用含0.1％(v/v)TFA的20％异丙醇的缓冲液平衡)，上样结束后用平衡缓冲液(含0.1％(v/v)TFA的20％异丙醇的缓冲液)冲洗至基线平稳。再以100mL浓度为20％(v/v)异丙醇A液(含0.1％(v/v)TFA缓冲液)和100mL浓度为40％(v/v)异丙醇B液(含0.1％(v/v)TFA缓冲液)线性梯度洗脱，洗脱流速≤2倍柱体积每小时。主峰分段收集，约20％峰高开始收集，检测波长为280nm，即得到PKEK。纯化工艺制备的PKEK如图6所示，纯度达到95.4％。
PKEK液相检测条件：BEH-C8(1.7μm粒径，2.1×100mm column)分析柱，柱温为35℃，0.2mL/min流速。流动相A：含0.1％(v/v)TFA的水溶液；流动相B：含0.09％(v/v)TFA的乙腈；梯度：0～25min，B相从30％升至80％；25～38min：B相从80％升至100％。
质谱鉴定：赛默飞高分辨液质联用仪取PEKE主峰进行高分辨质谱分析，使用Thermo Biopharma Finder2.0解卷积，采集范围：MS100～1000。结果显示本发明制备得到的PKEK分子量大小为500.3Da，如图6所示，与理论分子量完全一致，说明通过上述方法制备的多肽即为高纯度美白多肽PKEK。
实施例2
本实施例与实施例1所述方法基本相同，不同之处在于，本实施例用于酶切的蛋白酶为重组猪胰蛋白酶，16℃反应2h后调酸终止反应，经酶切即可得到PKEK单体粗品。
通过上述方法高效制备得到了高纯度美白多肽PKEK。
实施例3
本实施例与实施例1所述方法基本相同，不同之处在于，本实施例用于酶切的蛋白酶为重组赖氨酰内切酶，25℃反应2h后调酸终止反应，经酶切即可得到PKEK单体粗品。
实施例4
本实施例与实施例1所述方法基本相同，不同之处在于，本实施例选用的重组表达载体中含有的分子伴侣、连接肽和PKEK串联数量不同。不同分子伴侣和连接肽融合蛋白的相对产量如表3所示，修饰酶切后的PKEK相对纯度如表4所示。
表3：不同分子伴侣和连接肽融合蛋白发酵产量试验
氨基酸序列 核酸序列 分子伴侣 连接肽 连接肽-PKEK串联数 相对产量
SEQ ID NO:6 SEQ ID NO:1 无 AAK 10 无包涵体
SEQ ID NO:7 SEQ ID NO:47 无 无 10 无包涵体
SEQ ID NO:8 SEQ ID NO:48 无 无 20 无包涵体
SEQ ID NO:9 SEQ ID NO:49 KSI 无 1 6％
SEQ ID NO:10 SEQ ID NO:50 KSI 无 10 42％
SEQ ID NO:11 SEQ ID NO:51 KSI AK 1 9％
SEQ ID NO:12 SEQ ID NO:52 KSI AK 10 91％
SEQ ID NO:13 SEQ ID NO:53 KSI GK 10 103％
SEQ ID NO:14 SEQ ID NO:40 KSI AAK 10 100％
SEQ ID NO:15 SEQ ID NO:54 KSI PPK 10 31％
SEQ ID NO:16 SEQ ID NO:41 KSI AAGK 12 120％
SEQ ID NO:17 SEQ ID NO:55 KSI AAGGK 10 98％
SEQ ID NO:18 SEQ ID NO:42 KSI GGAAK 15 132％
SEQ ID NO:19 SEQ ID NO:56 KSI GGSASK 10 101％
SEQ ID NO:20 SEQ ID NO:57 KSI AAGGSGAGK 10 89％
SEQ ID NO:21 SEQ ID NO:37 PagP AK 20 121％
SEQ ID NO:22 SEQ ID NO:58 PagP GK 20 145％
SEQ ID NO:23 SEQ ID NO:43 PagP AAK 20 140％
SEQ ID NO:24 SEQ ID NO:59 PagP WTK 20 55％
SEQ ID NO:25 SEQ ID NO:44 PagP AAGK 15 174％
SEQ ID NO:26 SEQ ID NO:45 PagP AAGGK 20 155％
SEQ ID NO:27 SEQ ID NO:46 PagP GGAAK 15 162％
SEQ ID NO:28 SEQ ID NO:60 PagP GGSASK 20 130％
SEQ ID NO:29 SEQ ID NO:61 PagP AAGGSGAGK 20 99％
SEQ ID NO:30 SEQ ID NO:38 TAF12 GGAAK 10 84％
SEQ ID NO:31 SEQ ID NO:62 TAF12 AAK 20 71％
SEQ ID NO:32 SEQ ID NO:39 PaP3.30 GGAAK 10 102％
SEQ ID NO:33 SEQ ID NO:63 PaP3.30 AAK 20 96％
SEQ ID NO:34 SEQ ID NO:64 PaP3.30 AAK 30 112％
说明：上表中的相对产量仅指融合蛋白分子中PKEK部分的相对产量，计算时已扣除分子伴侣和连接肽。
上表中，SEQ ID NO:9～SEQ ID NO:20蛋白分子的重组载体酶切位点为AlwNI、XhoI，其余蛋白分子酶切位点均为NdeI、XhoI。SEQ ID NO:7～SEQ ID NO:10由于无连接肽，为保证后续酶切可获得完整PKEK，在分子伴侣与第一个PKEK间***Lys。KSI分子伴侣的氨基酸序列如SEQ ID NO:2所示；PagP分子伴侣的氨基酸序列如SEQ ID NO:3所示；TAF12分子伴侣的氨基酸序列如SEQ ID NO:4所示；PaP3.30分子伴侣的氨基酸序列如SEQ ID NO:5所示；PagP分子伴侣和连接肽融合蛋白分子(氨基酸序列如SEQ ID NO:21)的核苷酸序列如SEQ ID NO:37所示；TAF12分子伴侣和连接肽融合蛋白分子(氨基酸序列如SEQ ID NO:30)的核苷酸序列如SEQ ID NO:38所示；PaP3.30分子伴侣和连接肽融合蛋白分子(氨基酸序列如SEQ ID NO:32)的核苷酸序列如SEQ ID NO:39所示；不同分子伴侣和连接肽融合蛋白分子的氨基酸序列如SEQ ID NO:6～SEQ ID NO:34所示，不同分子伴侣和连接肽融合蛋白分子的核酸序列如SEQ ID NO:37～SEQ ID NO:64所示，其中，为方便对比分析，SEQ ID NO:14所用的重组表达载体通过实施例1制备得到。本发明中所有的核酸序列5’端均为酶切位点AlwNI或NdeI，3’端为终止密码子(taatga)及酶切位点XhoI，所有氨基酸序列均未体现酶切位点。
表4：不同分子伴侣和连接肽融合蛋白修饰酶切后的PKEK相对纯度
Figure BDA0002262207610000071
Figure BDA0002262207610000081
说明：上表中所有组酶切时包涵体中PKEK含量相同，已扣除分子伴侣和连接肽。
表4的结果说明无连接肽时，酶切获得的单体PKEK远低于有连接肽的分子。同时，当连接肽中的Xm---X2为AA、GG、GGAA、GGSAS等小侧链疏水柔性多肽时，其酶切后获得的PKEK单体远高于WT、PP等大侧链疏水性多肽。WT、PP等大侧链疏水性多肽为连接肽，酶切后的PKEK单体产量仅仅与无连接肽组相当。可见，本发明提供的连接肽可大幅提高PKEK单体酶切收率。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 珠海联邦制药股份有限公司
<120> 一种PKEK融合蛋白及其制备方法与应用
<160> 64
<170> SIPOSequenceListing 1.0
<210> 1
<211> 228
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> (AAK-PKEK)10
<400> 1
catatggctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 60
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 120
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaagctgct 180
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaataat gactcgag 228
<210> 2
<211> 127
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI分子伴侣
<400> 2
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu
115 120 125
<210> 3
<211> 165
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP分子伴侣
<400> 3
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu
165
<210> 4
<211> 96
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> TAF12分子伴侣
<400> 4
Met Ser Pro Glu Asn Asn Gln Val Leu Thr Lys Lys Lys Leu Gln Asp
1 5 10 15
Leu Val Arg Glu Val Asp Pro Asn Glu Gln Leu Asp Glu Asp Val Glu
20 25 30
Glu Met Leu Leu Gln Ile Ala Asp Asp Phe Ile Glu Ser Val Val Thr
35 40 45
Ala Ala Cys Gln Leu Ala Arg His Arg Lys Ser Ser Thr Leu Glu Val
50 55 60
Lys Asp Val Gln Leu His Leu Glu Arg Gln Trp Asn Met Trp Ile Pro
65 70 75 80
Gly Phe Gly Ser Glu Glu Ile Arg Pro Tyr Lys Lys Ala Gln Met Leu
85 90 95
<210> 5
<211> 166
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PaP3.30分子伴侣
<400> 5
Met Leu Ser Lys Ser Leu Glu Asp His Glu Ala Gly Val Cys Pro Leu
1 5 10 15
Gly Cys Pro Tyr Cys Leu Ala Asp Tyr Glu Arg Ile Trp Gly Val Arg
20 25 30
Val Val Ser Ser Val Ala Ala Ser Asn Asp Lys Val Glu Val Asp Pro
35 40 45
Asn Gly Ile Lys Ala Gly Asp Pro Gly Ala Lys Leu Asp Lys Gly Lys
50 55 60
Val Asp Val Gly Ile Ile Phe Glu Ala Phe Pro Arg Ala Leu Tyr Ala
65 70 75 80
Val Ala Gln Val Ala Asn Phe Gly Ala Ser Lys Tyr Ser Arg Gly Gly
85 90 95
Trp Arg Phe Val Glu Asn Gly Ile Gln Arg Tyr Asp Ala Ala Phe Gly
100 105 110
Arg His Leu Leu Glu Arg His Lys Gly Glu Val Leu Asp Pro Gln Ser
115 120 125
Ser Leu Pro His Arg Tyr His Glu Val Trp Asn Ala Leu Ala Ala Leu
130 135 140
Glu Leu Val Ile Gln Gln Glu Glu Gly Ser Asn Gly Thr Ser Thr Gly
145 150 155 160
Ser Glu Gly Gln Met Leu
165
<210> 6
<211> 70
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> (AAK-PKEK)10
<400> 6
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
1 5 10 15
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
20 25 30
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
35 40 45
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
50 55 60
Ala Lys Pro Lys Glu Lys
65 70
<210> 7
<211> 41
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> K-(PKEK)10
<400> 7
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
1 5 10 15
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
20 25 30
Lys Pro Lys Glu Lys Pro Lys Glu Lys
35 40
<210> 8
<211> 81
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> K-(PKEK)20
<400> 8
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
1 5 10 15
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
20 25 30
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
35 40 45
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
50 55 60
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
65 70 75 80
Lys
<210> 9
<211> 132
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-K-PKEK
<400> 9
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Lys
115 120 125
Pro Lys Glu Lys
130
<210> 10
<211> 168
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-K-(PKEK)10
<400> 10
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Lys
115 120 125
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
130 135 140
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
145 150 155 160
Pro Lys Glu Lys Pro Lys Glu Lys
165
<210> 11
<211> 133
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-(AK-PKEK)
<400> 11
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Lys Pro Lys Glu Lys
130
<210> 12
<211> 187
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-(AK-PKEK)10
<400> 12
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu
130 135 140
Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro
145 150 155 160
Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala
165 170 175
Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys
180 185
<210> 13
<211> 187
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-(GK-PKEK)10
<400> 13
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Gly
115 120 125
Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu
130 135 140
Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro
145 150 155 160
Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly
165 170 175
Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys
180 185
<210> 14
<211> 197
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-(AAK-PKEK)10
<400> 14
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
130 135 140
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
145 150 155 160
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
165 170 175
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
180 185 190
Lys Pro Lys Glu Lys
195
<210> 15
<211> 197
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-(PPK-PKEK)10
<400> 15
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Pro
115 120 125
Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys
130 135 140
Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys
145 150 155 160
Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys
165 170 175
Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro
180 185 190
Lys Pro Lys Glu Lys
195
<210> 16
<211> 223
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-(AAGK-PKEK)12
<400> 16
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
130 135 140
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
145 150 155 160
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
165 170 175
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
180 185 190
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
195 200 205
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys
210 215 220
<210> 17
<211> 217
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-(AAGGK-PKEK)10
<400> 17
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu
130 135 140
Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro
145 150 155 160
Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
165 170 175
Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala
180 185 190
Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys
195 200 205
Ala Ala Gly Gly Lys Pro Lys Glu Lys
210 215
<210> 18
<211> 262
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-(GGAAK-PKEK)15
<400> 18
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Gly
115 120 125
Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu
130 135 140
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro
145 150 155 160
Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala
165 170 175
Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly
180 185 190
Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
195 200 205
Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys
210 215 220
Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys
225 230 235 240
Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala
245 250 255
Ala Lys Pro Lys Glu Lys
260
<210> 19
<211> 227
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-( GGSASK-PKEK)10
<400> 19
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Gly
115 120 125
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
130 135 140
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser
145 150 155 160
Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu
165 170 175
Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser
180 185 190
Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
195 200 205
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
210 215 220
Lys Glu Lys
225
<210> 20
<211> 257
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> KSI-(AAGGSGAGK-PKEK)10
<400> 20
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
130 135 140
Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala
145 150 155 160
Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro
165 170 175
Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys
180 185 190
Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
195 200 205
Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly
210 215 220
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys
225 230 235 240
Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu
245 250 255
Lys
<210> 21
<211> 285
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP-(AK-PKEK)20
<400> 21
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu
165 170 175
Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro
180 185 190
Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala
195 200 205
Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu
210 215 220
Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro
225 230 235 240
Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala
245 250 255
Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu
260 265 270
Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys
275 280 285
<210> 22
<211> 285
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP-(GK-PKEK)20
<400> 22
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu
165 170 175
Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro
180 185 190
Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly
195 200 205
Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu
210 215 220
Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro
225 230 235 240
Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly
245 250 255
Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu
260 265 270
Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys
275 280 285
<210> 23
<211> 305
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP-(AAK-PKEK)20
<400> 23
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
165 170 175
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
180 185 190
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
195 200 205
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
210 215 220
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
225 230 235 240
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
245 250 255
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
260 265 270
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
275 280 285
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
290 295 300
Lys
305
<210> 24
<211> 305
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP-(WTK-PKEK)20
<400> 24
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro
165 170 175
Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu
180 185 190
Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp
195 200 205
Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys
210 215 220
Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys
225 230 235 240
Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys
245 250 255
Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr
260 265 270
Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro
275 280 285
Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu
290 295 300
Lys
305
<210> 25
<211> 285
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP-(AAGK-PKEK)15
<400> 25
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
165 170 175
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
180 185 190
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
195 200 205
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
210 215 220
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
225 230 235 240
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
245 250 255
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
260 265 270
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys
275 280 285
<210> 26
<211> 345
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP-(AAGGK-PKEK)20
<400> 26
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala
165 170 175
Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys
180 185 190
Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys
195 200 205
Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys
210 215 220
Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly
225 230 235 240
Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala
245 250 255
Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu
260 265 270
Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro
275 280 285
Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
290 295 300
Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala
305 310 315 320
Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys
325 330 335
Ala Ala Gly Gly Lys Pro Lys Glu Lys
340 345
<210> 27
<211> 300
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP-(GGAAK-PKEK)15
<400> 27
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly
165 170 175
Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
180 185 190
Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys
195 200 205
Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys
210 215 220
Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala
225 230 235 240
Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly
245 250 255
Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu
260 265 270
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro
275 280 285
Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
290 295 300
<210> 28
<211> 365
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP-(GGSASK-PKEK)20
<400> 28
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
165 170 175
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
180 185 190
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser
195 200 205
Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu
210 215 220
Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser
225 230 235 240
Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
245 250 255
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
260 265 270
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser
275 280 285
Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu
290 295 300
Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser
305 310 315 320
Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
325 330 335
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
340 345 350
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys
355 360 365
<210> 29
<211> 425
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PagP-(AAGGSGAGK-PKEK)20
<400> 29
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys
165 170 175
Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala
180 185 190
Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
195 200 205
Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala
210 215 220
Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro
225 230 235 240
Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys
245 250 255
Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
260 265 270
Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly
275 280 285
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys
290 295 300
Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu
305 310 315 320
Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala
325 330 335
Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser
340 345 350
Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly
355 360 365
Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys
370 375 380
Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala
385 390 395 400
Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
405 410 415
Ser Gly Ala Gly Lys Pro Lys Glu Lys
420 425
<210> 30
<211> 186
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> TAF12-(GGAAK-PKEK)10
<400> 30
Met Ser Pro Glu Asn Asn Gln Val Leu Thr Lys Lys Lys Leu Gln Asp
1 5 10 15
Leu Val Arg Glu Val Asp Pro Asn Glu Gln Leu Asp Glu Asp Val Glu
20 25 30
Glu Met Leu Leu Gln Ile Ala Asp Asp Phe Ile Glu Ser Val Val Thr
35 40 45
Ala Ala Cys Gln Leu Ala Arg His Arg Lys Ser Ser Thr Leu Glu Val
50 55 60
Lys Asp Val Gln Leu His Leu Glu Arg Gln Trp Asn Met Trp Ile Pro
65 70 75 80
Gly Phe Gly Ser Glu Glu Ile Arg Pro Tyr Lys Lys Ala Gln Met Leu
85 90 95
Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys
100 105 110
Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys
115 120 125
Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala
130 135 140
Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly
145 150 155 160
Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu
165 170 175
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
180 185
<210> 31
<211> 236
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> TAF12-(AAK-PKEK)20
<400> 31
Met Ser Pro Glu Asn Asn Gln Val Leu Thr Lys Lys Lys Leu Gln Asp
1 5 10 15
Leu Val Arg Glu Val Asp Pro Asn Glu Gln Leu Asp Glu Asp Val Glu
20 25 30
Glu Met Leu Leu Gln Ile Ala Asp Asp Phe Ile Glu Ser Val Val Thr
35 40 45
Ala Ala Cys Gln Leu Ala Arg His Arg Lys Ser Ser Thr Leu Glu Val
50 55 60
Lys Asp Val Gln Leu His Leu Glu Arg Gln Trp Asn Met Trp Ile Pro
65 70 75 80
Gly Phe Gly Ser Glu Glu Ile Arg Pro Tyr Lys Lys Ala Gln Met Leu
85 90 95
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
100 105 110
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
115 120 125
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
130 135 140
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
145 150 155 160
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
165 170 175
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
180 185 190
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
195 200 205
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
210 215 220
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
225 230 235
<210> 32
<211> 256
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PaP3.30-(GGAAK-PKEK)10
<400> 32
Met Leu Ser Lys Ser Leu Glu Asp His Glu Ala Gly Val Cys Pro Leu
1 5 10 15
Gly Cys Pro Tyr Cys Leu Ala Asp Tyr Glu Arg Ile Trp Gly Val Arg
20 25 30
Val Val Ser Ser Val Ala Ala Ser Asn Asp Lys Val Glu Val Asp Pro
35 40 45
Asn Gly Ile Lys Ala Gly Asp Pro Gly Ala Lys Leu Asp Lys Gly Lys
50 55 60
Val Asp Val Gly Ile Ile Phe Glu Ala Phe Pro Arg Ala Leu Tyr Ala
65 70 75 80
Val Ala Gln Val Ala Asn Phe Gly Ala Ser Lys Tyr Ser Arg Gly Gly
85 90 95
Trp Arg Phe Val Glu Asn Gly Ile Gln Arg Tyr Asp Ala Ala Phe Gly
100 105 110
Arg His Leu Leu Glu Arg His Lys Gly Glu Val Leu Asp Pro Gln Ser
115 120 125
Ser Leu Pro His Arg Tyr His Glu Val Trp Asn Ala Leu Ala Ala Leu
130 135 140
Glu Leu Val Ile Gln Gln Glu Glu Gly Ser Asn Gly Thr Ser Thr Gly
145 150 155 160
Ser Glu Gly Gln Met Leu Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly
165 170 175
Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu
180 185 190
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro
195 200 205
Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala
210 215 220
Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly
225 230 235 240
Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
245 250 255
<210> 33
<211> 306
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PaP3.30-(AAK-PKEK)20
<400> 33
Met Leu Ser Lys Ser Leu Glu Asp His Glu Ala Gly Val Cys Pro Leu
1 5 10 15
Gly Cys Pro Tyr Cys Leu Ala Asp Tyr Glu Arg Ile Trp Gly Val Arg
20 25 30
Val Val Ser Ser Val Ala Ala Ser Asn Asp Lys Val Glu Val Asp Pro
35 40 45
Asn Gly Ile Lys Ala Gly Asp Pro Gly Ala Lys Leu Asp Lys Gly Lys
50 55 60
Val Asp Val Gly Ile Ile Phe Glu Ala Phe Pro Arg Ala Leu Tyr Ala
65 70 75 80
Val Ala Gln Val Ala Asn Phe Gly Ala Ser Lys Tyr Ser Arg Gly Gly
85 90 95
Trp Arg Phe Val Glu Asn Gly Ile Gln Arg Tyr Asp Ala Ala Phe Gly
100 105 110
Arg His Leu Leu Glu Arg His Lys Gly Glu Val Leu Asp Pro Gln Ser
115 120 125
Ser Leu Pro His Arg Tyr His Glu Val Trp Asn Ala Leu Ala Ala Leu
130 135 140
Glu Leu Val Ile Gln Gln Glu Glu Gly Ser Asn Gly Thr Ser Thr Gly
145 150 155 160
Ser Glu Gly Gln Met Leu Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
165 170 175
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
180 185 190
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
195 200 205
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
210 215 220
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
225 230 235 240
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
245 250 255
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
260 265 270
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
275 280 285
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
290 295 300
Glu Lys
305
<210> 34
<211> 376
<212> PRT
<213> 人工序列(Artificial Sequence)
<223> PaP3.30-(AAK-PKEK)30
<400> 34
Met Leu Ser Lys Ser Leu Glu Asp His Glu Ala Gly Val Cys Pro Leu
1 5 10 15
Gly Cys Pro Tyr Cys Leu Ala Asp Tyr Glu Arg Ile Trp Gly Val Arg
20 25 30
Val Val Ser Ser Val Ala Ala Ser Asn Asp Lys Val Glu Val Asp Pro
35 40 45
Asn Gly Ile Lys Ala Gly Asp Pro Gly Ala Lys Leu Asp Lys Gly Lys
50 55 60
Val Asp Val Gly Ile Ile Phe Glu Ala Phe Pro Arg Ala Leu Tyr Ala
65 70 75 80
Val Ala Gln Val Ala Asn Phe Gly Ala Ser Lys Tyr Ser Arg Gly Gly
85 90 95
Trp Arg Phe Val Glu Asn Gly Ile Gln Arg Tyr Asp Ala Ala Phe Gly
100 105 110
Arg His Leu Leu Glu Arg His Lys Gly Glu Val Leu Asp Pro Gln Ser
115 120 125
Ser Leu Pro His Arg Tyr His Glu Val Trp Asn Ala Leu Ala Ala Leu
130 135 140
Glu Leu Val Ile Gln Gln Glu Glu Gly Ser Asn Gly Thr Ser Thr Gly
145 150 155 160
Ser Glu Gly Gln Met Leu Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
165 170 175
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
180 185 190
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
195 200 205
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
210 215 220
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
225 230 235 240
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
245 250 255
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
260 265 270
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
275 280 285
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
290 295 300
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
305 310 315 320
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
325 330 335
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
340 345 350
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
355 360 365
Lys Ala Ala Lys Pro Lys Glu Lys
370 375
<210> 35
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> T7启动子引物
<400> 35
taatacgact cactataggg 20
<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> T7终止子引物
<400> 36
tgctagttat tgctcagcgg 20
<210> 37
<211> 870
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP-(AK-PKEK)20
<400> 37
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggc taaaccgaaa gaaaaagcta aaccgaaaga aaaagctaaa 540
ccgaaagaaa aagctaaacc gaaagaaaaa gctaaaccga aagaaaaagc taaaccgaaa 600
gaaaaagcta aaccgaaaga aaaagctaaa ccgaaagaaa aagctaaacc gaaagaaaaa 660
gctaaaccga aagaaaaagc taaaccgaaa gaaaaagcta aaccgaaaga aaaagctaaa 720
ccgaaagaaa aagctaaacc gaaagaaaaa gctaaaccga aagaaaaagc taaaccgaaa 780
gaaaaagcta aaccgaaaga aaaagctaaa ccgaaagaaa aagctaaacc gaaagaaaaa 840
gctaaaccga aagaaaaata atgactcgag 870
<210> 38
<211> 573
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> TAF12-(GGAAK-PKEK)10
<400> 38
catatgtctc cggaaaacaa ccaggttctg accaaaaaaa aactgcagga cctggttcgt 60
gaagttgacc cgaacgaaca gctggacgaa gacgttgaag aaatgctgct gcagatcgct 120
gacgacttca tcgaatctgt tgttaccgct gcttgccagc tggctcgtca ccgtaaatct 180
tctaccctgg aagttaaaga cgttcagctg cacctggaac gtcagtggaa catgtggatc 240
ccgggtttcg gttctgaaga aatccgtccg tacaaaaaag ctcagatgct gggtggtgct 300
gctaaaccga aagaaaaagg tggtgctgct aaaccgaaag aaaaaggtgg tgctgctaaa 360
ccgaaagaaa aaggtggtgc tgctaaaccg aaagaaaaag gtggtgctgc taaaccgaaa 420
gaaaaaggtg gtgctgctaa accgaaagaa aaaggtggtg ctgctaaacc gaaagaaaaa 480
ggtggtgctg ctaaaccgaa agaaaaaggt ggtgctgcta aaccgaaaga aaaaggtggt 540
gctgctaaac cgaaagaaaa ataatgactc gag 573
<210> 39
<211> 783
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PaP3.30-(GGAAK-PKEK)10
<400> 39
catatgctgt ctaaatctct ggaagaccac gaagctggtg tttgcccgct gggttgcccg 60
tactgcctgg ctgactacga acgtatctgg ggtgttcgtg ttgtttcttc tgttgctgct 120
tctaacgaca aagttgaagt tgacccgaac ggtatcaaag ctggtgaccc gggtgctaaa 180
ctggacaaag gtaaagttga cgttggtatc atcttcgaag ctttcccgcg tgctctgtac 240
gctgttgctc aggttgctaa cttcggtgct tctaaatact ctcgtggtgg ttggcgtttc 300
gttgaaaacg gtatccagcg ttacgacgct gctttcggtc gtcacctgct ggaacgtcac 360
aaaggtgaag ttctggaccc gcagtcttct ctgccgcacc gttaccacga agtttggaac 420
gctctggctg ctctggaact ggttatccag caggaagaag gttctaacgg tacctctacc 480
ggttctgaag gtcagatgct gggtggtgct gctaaaccga aagaaaaagg tggtgctgct 540
aaaccgaaag aaaaaggtgg tgctgctaaa ccgaaagaaa aaggtggtgc tgctaaaccg 600
aaagaaaaag gtggtgctgc taaaccgaaa gaaaaaggtg gtgctgctaa accgaaagaa 660
aaaggtggtg ctgctaaacc gaaagaaaaa ggtggtgctg ctaaaccgaa agaaaaaggt 720
ggtgctgcta aaccgaaaga aaaaggtggt gctgctaaac cgaaagaaaa ataatgactc 780
gag 783
<210> 40
<211> 606
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-(AAK-PKEK)10
<400> 40
catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat 60
gccggcgatc tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc 120
gtgggttccg agcccaggtc cggtacggct gcgattcgtg agttttacgc caactcgctc 180
aaactgcctt tggcggtgga gctgacgcag gaggtacgcg cggtcgccaa cgaagcggcc 240
ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga ccgtagttgc gcccatcgat 300
cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt tggcgagaag 360
aatattcacg catgccagat gctggctgct aaaccgaaag aaaaagctgc taaaccgaaa 420
gaaaaagctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 480
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 540
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaataatga 600
ctcgag 606
<210> 41
<211> 684
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-(AAGK-PKEK)12
<400> 41
catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat 60
gccggcgatc tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc 120
gtgggttccg agcccaggtc cggtacggct gcgattcgtg agttttacgc caactcgctc 180
aaactgcctt tggcggtgga gctgacgcag gaggtacgcg cggtcgccaa cgaagcggcc 240
ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga ccgtagttgc gcccatcgat 300
cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt tggcgagaag 360
aatattcacg catgccagat gctggctgct ggtaaaccga aagaaaaagc tgctggtaaa 420
ccgaaagaaa aagctgctgg taaaccgaaa gaaaaagctg ctggtaaacc gaaagaaaaa 480
gctgctggta aaccgaaaga aaaagctgct ggtaaaccga aagaaaaagc tgctggtaaa 540
ccgaaagaaa aagctgctgg taaaccgaaa gaaaaagctg ctggtaaacc gaaagaaaaa 600
gctgctggta aaccgaaaga aaaagctgct ggtaaaccga aagaaaaagc tgctggtaaa 660
ccgaaagaaa aataatgact cgag 684
<210> 42
<211> 801
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-(GGAAK-PKEK)15
<400> 42
catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat 60
gccggcgatc tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc 120
gtgggttccg agcccaggtc cggtacggct gcgattcgtg agttttacgc caactcgctc 180
aaactgcctt tggcggtgga gctgacgcag gaggtacgcg cggtcgccaa cgaagcggcc 240
ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga ccgtagttgc gcccatcgat 300
cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt tggcgagaag 360
aatattcacg catgccagat gctgggtggt gctgctaaac cgaaagaaaa aggtggtgct 420
gctaaaccga aagaaaaagg tggtgctgct aaaccgaaag aaaaaggtgg tgctgctaaa 480
ccgaaagaaa aaggtggtgc tgctaaaccg aaagaaaaag gtggtgctgc taaaccgaaa 540
gaaaaaggtg gtgctgctaa accgaaagaa aaaggtggtg ctgctaaacc gaaagaaaaa 600
ggtggtgctg ctaaaccgaa agaaaaaggt ggtgctgcta aaccgaaaga aaaaggtggt 660
gctgctaaac cgaaagaaaa aggtggtgct gctaaaccga aagaaaaagg tggtgctgct 720
aaaccgaaag aaaaaggtgg tgctgctaaa ccgaaagaaa aaggtggtgc tgctaaaccg 780
aaagaaaaat aatgactcga g 801
<210> 43
<211> 930
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP-(AAK-PKEK)20
<400> 43
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 540
gctgctaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 600
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 660
gaaaaagctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 720
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 780
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaagctgct 840
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct 900
gctaaaccga aagaaaaata atgactcgag 930
<210> 44
<211> 870
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP-(AAGK-PKEK)15
<400> 44
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggc tgctggtaaa ccgaaagaaa aagctgctgg taaaccgaaa 540
gaaaaagctg ctggtaaacc gaaagaaaaa gctgctggta aaccgaaaga aaaagctgct 600
ggtaaaccga aagaaaaagc tgctggtaaa ccgaaagaaa aagctgctgg taaaccgaaa 660
gaaaaagctg ctggtaaacc gaaagaaaaa gctgctggta aaccgaaaga aaaagctgct 720
ggtaaaccga aagaaaaagc tgctggtaaa ccgaaagaaa aagctgctgg taaaccgaaa 780
gaaaaagctg ctggtaaacc gaaagaaaaa gctgctggta aaccgaaaga aaaagctgct 840
ggtaaaccga aagaaaaata atgactcgag 870
<210> 45
<211> 1050
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP-(AAGGK-PKEK)20
<400> 45
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggc tgctggtggt aaaccgaaag aaaaagctgc tggtggtaaa 540
ccgaaagaaa aagctgctgg tggtaaaccg aaagaaaaag ctgctggtgg taaaccgaaa 600
gaaaaagctg ctggtggtaa accgaaagaa aaagctgctg gtggtaaacc gaaagaaaaa 660
gctgctggtg gtaaaccgaa agaaaaagct gctggtggta aaccgaaaga aaaagctgct 720
ggtggtaaac cgaaagaaaa agctgctggt ggtaaaccga aagaaaaagc tgctggtggt 780
aaaccgaaag aaaaagctgc tggtggtaaa ccgaaagaaa aagctgctgg tggtaaaccg 840
aaagaaaaag ctgctggtgg taaaccgaaa gaaaaagctg ctggtggtaa accgaaagaa 900
aaagctgctg gtggtaaacc gaaagaaaaa gctgctggtg gtaaaccgaa agaaaaagct 960
gctggtggta aaccgaaaga aaaagctgct ggtggtaaac cgaaagaaaa agctgctggt 1020
ggtaaaccga aagaaaaata atgactcgag 1050
<210> 46
<211> 915
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP-(GGAAK-PKEK)15
<400> 46
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggg tggtgctgct aaaccgaaag aaaaaggtgg tgctgctaaa 540
ccgaaagaaa aaggtggtgc tgctaaaccg aaagaaaaag gtggtgctgc taaaccgaaa 600
gaaaaaggtg gtgctgctaa accgaaagaa aaaggtggtg ctgctaaacc gaaagaaaaa 660
ggtggtgctg ctaaaccgaa agaaaaaggt ggtgctgcta aaccgaaaga aaaaggtggt 720
gctgctaaac cgaaagaaaa aggtggtgct gctaaaccga aagaaaaagg tggtgctgct 780
aaaccgaaag aaaaaggtgg tgctgctaaa ccgaaagaaa aaggtggtgc tgctaaaccg 840
aaagaaaaag gtggtgctgc taaaccgaaa gaaaaaggtg gtgctgctaa accgaaagaa 900
aaataatgac tcgag 915
<210> 47
<211> 141
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> K-(PKEK)10
<400> 47
1 catatgaaac cgaaagaaaa accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac
71 cgaaagaaaa accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaat aatgactcga
141 g
<210> 48
<211> 261
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> K-(PKEK)20
<400> 48
1 catatgaaac cgaaagaaaa accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac
71 cgaaagaaaa accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac cgaaagaaaa
141 accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac cgaaagaaaa accgaaagaa
211 aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaat aatgactcga g
<210> 49
<211> 411
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-K-PKEK
<400> 49
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgaaaccg aaagaaaaat aatgactcga g
<210> 50
<211> 519
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-K-（PKEK）10
<400> 49
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgaaaccg aaagaaaaac cgaaagaaaa accgaaagaa
421 aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac cgaaagaaaa accgaaagaa aaaccgaaag
491 aaaaaccgaa agaaaaataa tgactcgag
<210> 51
<211> 414
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-AK-PKEK
<400> 51
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctggctaaa ccgaaagaaa aataatgact cgag
<210> 52
<211> 576
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-（AK-PKEK）10
<400> 52
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctggctaaa ccgaaagaaa aagctaaacc gaaagaaaaa
421 gctaaaccga aagaaaaagc taaaccgaaa gaaaaagcta aaccgaaaga aaaagctaaa ccgaaagaaa
491 aagctaaacc gaaagaaaaa gctaaaccga aagaaaaagc taaaccgaaa gaaaaagcta aaccgaaaga
561 aaaataatga ctcgag
<210> 53
<211> 576
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-（GK-PKEK）10
<400> 53
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgggtaaa ccgaaagaaa aaggtaaacc gaaagaaaaa
421 ggtaaaccga aagaaaaagg taaaccgaaa gaaaaaggta aaccgaaaga aaaaggtaaa ccgaaagaaa
491 aaggtaaacc gaaagaaaaa ggtaaaccga aagaaaaagg taaaccgaaa gaaaaaggta aaccgaaaga
561 aaaataatga ctcgag
<210> 54
<211> 606
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-（PPK-PKEK）10
<400> 54
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgccgccg aaaccgaaag aaaaaccgcc gaaaccgaaa
421 gaaaaaccgc cgaaaccgaa agaaaaaccg ccgaaaccga aagaaaaacc gccgaaaccg aaagaaaaac
491 cgccgaaacc gaaagaaaaa ccgccgaaac cgaaagaaaa accgccgaaa ccgaaagaaa aaccgccgaa
561 accgaaagaa aaaccgccga aaccgaaaga aaaataatga ctcgag
<210> 55
<211> 666
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-（AAGGK-PKEK）10
<400> 55
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctggctgct ggtggtaaac cgaaagaaaa agctgctggt
421 ggtaaaccga aagaaaaagc tgctggtggt aaaccgaaag aaaaagctgc tggtggtaaa ccgaaagaaa
491 aagctgctgg tggtaaaccg aaagaaaaag ctgctggtgg taaaccgaaa gaaaaagctg ctggtggtaa
561 accgaaagaa aaagctgctg gtggtaaacc gaaagaaaaa gctgctggtg gtaaaccgaa agaaaaagct
631 gctggtggta aaccgaaaga aaaataatga ctcgag
<210> 56
<211> 696
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-（GGSASK-PKEK）10
<400> 56
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgggtggt tctgcttcta aaccgaaaga aaaaggtggt
421 tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta
491 aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga
561 aaaaggtggt tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga aaaaggtggt
631 tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga aaaataatga ctcgag
<210> 57
<211> 915
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> KSI-（AAGGSGAGK-PKEK）10
<400> 57
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctggctgct ggtggttctg gtgctggtaa accgaaagaa
421 aaagctgctg gtggttctgg tgctggtaaa ccgaaagaaa aagctgctgg tggttctggt gctggtaaac
491 cgaaagaaaa agctgctggt ggttctggtg ctggtaaacc gaaagaaaaa gctgctggtg gttctggtgc
561 tggtaaaccg aaagaaaaag ctgctggtgg ttctggtgct ggtaaaccga aagaaaaagc tgctggtggt
631 tctggtgctg gtaaaccgaa agaaaaagct gctggtggtt ctggtgctgg taaaccgaaa gaaaaagctg
701 ctggtggttc tggtgctggt aaaccgaaag aaaaagctgc tggtggttct ggtgctggta aaccgaaaga
771 aaaataatga ctcgag
<210> 58
<211> 870
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP-（GK-PKEK）20
<400> 58
1 catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag cagccggaac
71 actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac gacaaagaaa aaaccgaccg
141 ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct cgttgggacg aaaaaggtaa ctggcacggt
211 ctgtacgcta tggctttcaa agactcttgg aacaaatggg aaccgatcgc tggttacggt tgggaatcta
281 cctggcgtcc gctggctgac gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga
351 caactggaac tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc
421 cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt ttccagttcc
491 agatgctggg taaaccgaaa gaaaaaggta aaccgaaaga aaaaggtaaa ccgaaagaaa aaggtaaacc
561 gaaagaaaaa ggtaaaccga aagaaaaagg taaaccgaaa gaaaaaggta aaccgaaaga aaaaggtaaa
631 ccgaaagaaa aaggtaaacc gaaagaaaaa ggtaaaccga aagaaaaagg taaaccgaaa gaaaaaggta
701 aaccgaaaga aaaaggtaaa ccgaaagaaa aaggtaaacc gaaagaaaaa ggtaaaccga aagaaaaagg
771 taaaccgaaa gaaaaaggta aaccgaaaga aaaaggtaaa ccgaaagaaa aaggtaaacc gaaagaaaaa
841 ggtaaaccga aagaaaaata atgactcgag
<210> 59
<211> 930
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PagP-（WTK-PKEK）20
<400> 59
1 catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag cagccggaac
71 actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac gacaaagaaa aaaccgaccg
141 ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct cgttgggacg aaaaaggtaa ctggcacggt
211 ctgtacgcta tggctttcaa agactcttgg aacaaatggg aaccgatcgc tggttacggt tgggaatcta
281 cctggcgtcc gctggctgac gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga
351 caactggaac tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc
421 cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt ttccagttcc
491 agatgctgtg gaccaaaccg aaagaaaaat ggaccaaacc gaaagaaaaa tggaccaaac cgaaagaaaa
561 atggaccaaa ccgaaagaaa aatggaccaa accgaaagaa aaatggacca aaccgaaaga aaaatggacc
631 aaaccgaaag aaaaatggac caaaccgaaa gaaaaatgga ccaaaccgaa agaaaaatgg accaaaccga
701 aagaaaaatg gaccaaaccg aaagaaaaat ggaccaaacc gaaagaaaaa tggaccaaac cgaaagaaaa
771 atggaccaaa ccgaaagaaa aatggaccaa accgaaagaa aaatggacca aaccgaaaga aaaatggacc
841 aaaccgaaag aaaaatggac caaaccgaaa gaaaaatgga ccaaaccgaa agaaaaatgg accaaaccga
911 aagaaaaata atgactcgag
<210> 60
<211> 1110
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP-（GGSASK-PKEK）20
<400> 60
1 catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag cagccggaac
71 actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac gacaaagaaa aaaccgaccg
141 ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct cgttgggacg aaaaaggtaa ctggcacggt
211 ctgtacgcta tggctttcaa agactcttgg aacaaatggg aaccgatcgc tggttacggt tgggaatcta
281 cctggcgtcc gctggctgac gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga
351 caactggaac tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc
421 cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt ttccagttcc
491 agatgctggg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg
561 tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct
631 tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga
701 aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg
771 tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct
841 tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga
911 aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg
981 tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct
1051 tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaata atgactcgag
<210> 61
<211> 1290
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP-（AAGGSGAGK-PKEK）20
<400> 61
1 catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag cagccggaac
71 actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac gacaaagaaa aaaccgaccg
141 ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct cgttgggacg aaaaaggtaa ctggcacggt
211 ctgtacgcta tggctttcaa agactcttgg aacaaatggg aaccgatcgc tggttacggt tgggaatcta
281 cctggcgtcc gctggctgac gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga
351 caactggaac tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc
421 cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt ttccagttcc
491 agatgctggc tgctggtggt tctggtgctg gtaaaccgaa agaaaaagct gctggtggtt ctggtgctgg
561 taaaccgaaa gaaaaagctg ctggtggttc tggtgctggt aaaccgaaag aaaaagctgc tggtggttct
631 ggtgctggta aaccgaaaga aaaagctgct ggtggttctg gtgctggtaa accgaaagaa aaagctgctg
701 gtggttctgg tgctggtaaa ccgaaagaaa aagctgctgg tggttctggt gctggtaaac cgaaagaaaa
771 agctgctggt ggttctggtg ctggtaaacc gaaagaaaaa gctgctggtg gttctggtgc tggtaaaccg
841 aaagaaaaag ctgctggtgg ttctggtgct ggtaaaccga aagaaaaagc tgctggtggt tctggtgctg
911 gtaaaccgaa agaaaaagct gctggtggtt ctggtgctgg taaaccgaaa gaaaaagctg ctggtggttc
981 tggtgctggt aaaccgaaag aaaaagctgc tggtggttct ggtgctggta aaccgaaaga aaaagctgct
1051 ggtggttctg gtgctggtaa accgaaagaa aaagctgctg gtggttctgg tgctggtaaa ccgaaagaaa
1121 aagctgctgg tggttctggt gctggtaaac cgaaagaaaa agctgctggt ggttctggtg ctggtaaacc
1191 gaaagaaaaa gctgctggtg gttctggtgc tggtaaaccg aaagaaaaag ctgctggtgg ttctggtgct
1261 ggtaaaccga aagaaaaata atgactcgag
<210> 62
<211> 723
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> TAF12-（AAK-PKEK）20
<400> 62
1 catatgtctc cggaaaacaa ccaggttctg accaaaaaaa aactgcagga cctggttcgt gaagttgacc
71 cgaacgaaca gctggacgaa gacgttgaag aaatgctgct gcagatcgct gacgacttca tcgaatctgt
141 tgttaccgct gcttgccagc tggctcgtca ccgtaaatct tctaccctgg aagttaaaga cgttcagctg
211 cacctggaac gtcagtggaa catgtggatc ccgggtttcg gttctgaaga aatccgtccg tacaaaaaag
281 ctcagatgct ggctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
351 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
421 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
491 cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
561 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
631 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
701 cgaaagaaaa ataatgactc gag
<210> 63
<211> 933
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP3.30-（AAK-PKEK）20
<400> 63
1 catatgctgt ctaaatctct ggaagaccac gaagctggtg tttgcccgct gggttgcccg tactgcctgg
71 ctgactacga acgtatctgg ggtgttcgtg ttgtttcttc tgttgctgct tctaacgaca aagttgaagt
141 tgacccgaac ggtatcaaag ctggtgaccc gggtgctaaa ctggacaaag gtaaagttga cgttggtatc
211 atcttcgaag ctttcccgcg tgctctgtac gctgttgctc aggttgctaa cttcggtgct tctaaatact
281 ctcgtggtgg ttggcgtttc gttgaaaacg gtatccagcg ttacgacgct gctttcggtc gtcacctgct
351 ggaacgtcac aaaggtgaag ttctggaccc gcagtcttct ctgccgcacc gttaccacga agtttggaac
421 gctctggctg ctctggaact ggttatccag caggaagaag gttctaacgg tacctctacc ggttctgaag
491 gtcagatgct ggctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
561 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
631 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
701 cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
771 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
841 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
911 cgaaagaaaa ataatgactc gag
<210> 64
<211> 1143
<212> DNA
<213> 人工序列(Artificial Sequence)
<223> PagP3.30-（AAK-PKEK）30
<400> 64
1 catatgctgt ctaaatctct ggaagaccac gaagctggtg tttgcccgct gggttgcccg tactgcctgg
71 ctgactacga acgtatctgg ggtgttcgtg ttgtttcttc tgttgctgct tctaacgaca aagttgaagt
141 tgacccgaac ggtatcaaag ctggtgaccc gggtgctaaa ctggacaaag gtaaagttga cgttggtatc
211 atcttcgaag ctttcccgcg tgctctgtac gctgttgctc aggttgctaa cttcggtgct tctaaatact
281 ctcgtggtgg ttggcgtttc gttgaaaacg gtatccagcg ttacgacgct gctttcggtc gtcacctgct
351 ggaacgtcac aaaggtgaag ttctggaccc gcagtcttct ctgccgcacc gttaccacga agtttggaac
421 gctctggctg ctctggaact ggttatccag caggaagaag gttctaacgg tacctctacc ggttctgaag
491 gtcagatgct ggctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
561 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
631 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
701 cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
771 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
841 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
911 cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
981 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
1051 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
1121 cgaaagaaaa ataatgactc gag

Claims (10)

1.一种PKEK融合蛋白，其特征在于：包括将若干柔性连接肽与美白多肽PKEK依次串联构成的蛋白，其结构式如下：(B-PKEK)n；其中：B为柔性连接肽、n为1～30的整数；
所述的柔性连接肽为如Xm---X2-X1所示的肽序列，m为0至10的整数；X1为蛋白酶酶切位点，X2至Xm为柔性片段；
所述的柔性片段中的氨基酸不能为Pro、Thr或Trp。
2.根据权利要求1所述的PKEK融合蛋白，其特征在于：还包括伴侣蛋白，伴侣蛋白与一个连接肽连接，结构式如下：A-(B-PKEK)n；其中：A为伴侣蛋白、B为柔性连接肽、n为1～30的整数。
3.根据权利要求2所述的PKEK融合蛋白，其特征在于：所述的伴侣蛋白为KSI、PagP、PaP3.30、TAF12、mKSI、mPagP、mPaP3.30或mTAF12，m表示突变体。
4.根据权利要求1～3任一项所述的PKEK融合蛋白，其特征在于：
所述的蛋白酶酶切位点为Arg或Lys；
所述的柔性片段中的氨基酸为Gly、Ala或Ser。
5.根据权利要求4所述的PKEK融合蛋白，其特征在于：所述的柔性连接肽为AK、GK、AAK、AAGK、AAGGK、GGAAK、GGSASK或AAGGSGAGK。
6.编码权利要求1～5任一项所述的PKEK融合蛋白的基因。
7.一种重组载体，其特征在于：含有权利要求6所述的基因。
8.一种重组表达菌株，其特征在于，含有权利要求7所述的重组载体。
9.一种利用基因工程技术生产美白多肽PEKE的制备方法，其特征在于，包括如下步骤：
S1、获得包含权利要求6所述的基因的重组载体，其编码的融合蛋白形如A-(B-PKEK)n，其中：A为伴侣蛋白；B为柔性连接肽；n为1至30的整数；
S2、将步骤S1获得的重组载体转入基因工程菌株中，得到重组表达菌株；发酵培养重组表达菌株，收集融合蛋白的包涵体；
S3、溶解、酶切融合蛋白的包涵体，分离纯化即得活性美白多肽PKEK。
10.权利要求9所述利用基因工程技术生产美白多肽PEKE的制备方法在工业化制备美白多肽PEKE中的应用。
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Cited By (2)

* Cited by examiner, † Cited by third party
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CN113616807A (zh) * 2021-07-26 2021-11-09 中山大学 一种靶向线粒体的多肽及其制备方法与应用
CN113616807B (zh) * 2021-07-26 2023-07-21 中山大学 一种靶向线粒体的多肽及其制备方法与应用

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