CN107001431A - 基于细菌的蛋白质递送 - Google Patents

基于细菌的蛋白质递送 Download PDF

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CN107001431A
CN107001431A CN201580040391.2A CN201580040391A CN107001431A CN 107001431 A CN107001431 A CN 107001431A CN 201580040391 A CN201580040391 A CN 201580040391A CN 107001431 A CN107001431 A CN 107001431A
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塞西尔·阿里厄梅卢
西蒙·伊泰格
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Abstract

本发明涉及重组革兰氏阴性细菌菌株及其将异源蛋白递送到真核细胞中的用途。

Description

基于细菌的蛋白质递送
技术领域
本发明涉及重组革兰氏阴性细菌菌株及其将异源蛋白递送到真核细胞中的用途。
背景技术
瞬时转染技术已经在细胞生物学研究中应用多年以解决蛋白质功能。这些方法通常导致被研究的蛋白质大量过度表达，这可能产生过简化的信号模型[1]。对于控制短寿命信号过程的蛋白质，目标蛋白质存在的时间远长于它控制的信号事件[2]。甚至，基于DNA转染的瞬时过表达导致异质和不同步的细胞群，这使功能研究和阻碍组学方法复杂化。除此之外，将这些测定扩大到更大规模是非常昂贵的。以上提到的这些点由现有技术覆盖，如显微注射或纯化蛋白的蛋白转染、诱导型转运策略快速靶向质粒出生的小GTP酶到细胞膜[2]或添加融合ω细胞可渗透细菌毒素的纯化蛋白[3]。但是这些技术都很耗时且麻烦，并且根据我们的知识，没有一个满足所有提到的标准。
细菌已经进化形成不同的机制来直接注射蛋白质到靶细胞[4]。由耶尔森氏菌属、志贺氏菌属和沙门氏菌等细菌使用的III型分泌系统(T3SS)的功能类似于将所谓的细菌效应蛋白注射到宿主细胞中的纳米注射器。通过T3SS分泌的细菌蛋白，称为效应物，含有短的N-末端分泌信号[6]。在细菌内，有些效应物被伴侣结合。伴侣可能掩盖毒性结构域[7]，它们有助于分泌信号的暴露[8，9]，并保持底物在分泌能力的构象[10]，因此促进分泌。在诱导分泌时，邻近T3SS的ATP酶去除伴侣蛋白[11]，并且效应物通过针头展开或仅部分折叠[10]，并在宿主细胞质中重折叠一次。
T3S已经被用来将杂交肽和蛋白质递送到靶细胞中。异源细菌T3SS效应器已经被传递，以防所研究的细菌难以通过遗传学获得(如沙眼衣原体[12])。通常将报告蛋白融合到可能的T3SS分泌信号以研究对T3SS依赖性蛋白递送的需求，例如百日咳博德特氏菌腺苷酸环化酶[13]、鼠DHFR[10]或可磷酸化标签[14]。肽递送主要以疫苗接种为目的进行。这包括病毒表位[15，16]、细菌表位(李斯特菌溶血素，[17])以及代表人类癌细胞表位的肽[18]。在少数情况下，如纳米抗体[19]、核蛋白(Cre重组酶，MyoD)[20，21]或I110和ILlra[22]所做的那样，功能性真核蛋白已被递送以调节宿主细胞。上述系统中没有一个允许单蛋白递送，因为在每种情况下一个或多个内源效应子蛋白仍然被编码。此外，使用的载体没有设计出能简单克隆编码所选蛋白质的其它DNA片段，阻碍了这种系统的广泛应用。
因此，发明一种能在生理浓度下目标蛋白质的可扩展的、快速的、同步同质和可调节的递送的廉价和简单的方法对于许多细胞生物学家是非常有益的。
发明内容
本发明一般涉及重组革兰氏阴性细菌菌株及其用于将异源蛋白质递送到真核细胞中的用途。本发明提供革兰氏阴性细菌菌株及其用途，其允许各种病毒蛋白的III型效应器，也允许IV型效应器，和最重要的功能性真核蛋白转运。
本发明提供了用于荧光跟踪递送，用于重新定位至细胞核并且特别是用于在递送至宿主细胞之后去除细菌附属物的装置。这允许第一次仅使用T3SS将几乎天然蛋白质递送到真核细胞中。所呈现的基于T3SS的系统导致目标蛋白质可扩展、快速、同步同质和可调节的递送。本发明的递送系统适合于在活动物中注射真核蛋白质并且可以用于治疗目的。
在第一方面，本发明涉及选自耶尔森氏菌属、埃希氏菌属、沙门氏菌属和假单胞菌属的重组革兰氏阴性细菌菌株，其中所述革兰氏阴性细菌菌株用载体转化，所述载体在5'至3'方向包含：
启动子；
编码来自细菌T3SS效应蛋白的递送信号的第一DNA序列，可操作地连接到所述启动子上；
框内融合到所述第一DNA序列的3'末端的编码异源蛋白质的第二DNA序列，其中所述异源蛋白质选自参与凋亡或凋亡调节的蛋白质。
在另一方面，本发明涉及用载体转化的重组革兰氏阴性细菌菌株，其在5'至3'方向包含：
启动子；
编码来自细菌T3SS效应蛋白的递送信号的第一DNA序列，可操作地连接到所述启动子；
与框内融合到所述第一DNA序列的3'末端的编码异源蛋白质的第二DNA序列；和
编码蛋白酶切割位点的第三DNA序列，其中所述第三DNA序列位于所述第一DNA序列的3'端和所述第二DNA序列的5'端之间。
在另一方面，本发明涉及重组革兰氏阴性细菌菌株，其中所述重组革兰氏阴性细菌菌株是耶尔森氏菌菌株，并且其中所述耶尔森氏菌菌株是野生型或对于至少一种T3SS效应蛋白的产生是缺陷的，并用载体转化，所述载体在5'至3'方向：
启动子；
可操作地连接到所述启动子的编码来自细菌T3SS效应蛋白的递送信号的第一DNA序列，其中来自细菌T3SS效应蛋白的递送信号包含小肠结肠炎耶尔森氏菌YopE效应蛋白的N末端138个氨基酸；以及
框内融合到所述第一DNA序列的3'末端的编码异源蛋白质的第二DNA序列。
在另一方面，本发明涉及重组革兰氏阴性细菌菌株，其中所述重组革兰氏阴性细菌菌株是沙门氏菌菌株，并且其中所述沙门氏菌菌株是野生型或对于至少一种T3SS效应蛋白的产生是缺陷的，并用载体转化，所述载体在5'至3'方向包含：
启动子；
可操作地连接到所述启动子的编码来自细菌T3SS效应蛋白的递送信号的第一DNA序列，其中来自细菌T3SS效应蛋白的递送信号包含肠炎沙门氏菌SteA效应蛋白或肠炎沙门氏菌SopE效应蛋白的N-末端81或105氨基酸；以及
框内融合到所述第一DNA序列的3'末端的编码异源蛋白质的第二DNA序列。
在另一方面，本发明涉及一种载体，其在5'至3'方向包含：
启动子；
编码来自细菌T3SS效应蛋白的递送信号的第一DNA序列，可操作地连接到所述启动子；
框内融合到所述第一DNA序列的3'末端的编码异源蛋白质的第二DNA序列；
编码蛋白酶切割位点的第三DNA序列，其中所述第三DNA序列位于所述第一DNA序列的3'端和所述第二DNA序列的5'端之间。
本发明还涉及将异源蛋白质递送到真核细胞中的方法，包括以下步骤：
i)培养革兰氏阴性细菌菌株；以及
ii)将真核细胞与i)的革兰氏阴性细菌菌株接触，其中包含来自细菌T3SS效应蛋白的递送信号和异源蛋白质的融合蛋白由革兰氏阴性细菌菌株表达，并且被转运进入到真核细胞。
本发明还涉及将异源蛋白质递送到真核细胞中的方法，包括以下步骤：
i)培养革兰氏阴性细菌菌株；
ii)使真核细胞与i)的革兰氏阴性细菌菌株接触，其中包含来自细菌T3SS效应蛋白的递送信号和异源蛋白的融合蛋白由革兰氏阴性细菌菌株表达，并被转运进入真核细胞；和
iii)切割融合蛋白，使得异源蛋白与细菌T3SS效应蛋白的递送信号切割开来。
本发明还涉及纯化异源蛋白质的方法，包括培养革兰氏阴性细菌菌株，使得包含来自细菌T3SS效应蛋白的递送信号和异源蛋白质的融合蛋白质被表达并分泌到培养基的上清液中。
在另一方面，本发明涉及革兰氏阴性细菌菌株的文库，其中由革兰氏阴性细菌菌株的表达载体的第二DNA序列编码的异源蛋白质是人或鼠蛋白，并且其中由革兰氏阴性菌株表达的每个人或鼠的氨基酸序列不同。
附图说明
图1：T3SS蛋白递送的表征。(A)T3SS依赖性蛋白分泌到周围介质(体外分泌)(左侧)或真核细胞(右侧)。I：表示3型分泌系统。II表示分泌到周围介质中的蛋白质，III蛋白通过膜递送到真核细胞的细胞质中(VII)。VI表示T3SS被插入的两个细菌膜的拉伸和里面的细菌细胞溶质。IV是连接到YopE1-138N-末端片段(V)的融合蛋白(B)I：肠道沙门氏菌E40野生型的体外分泌，II：小肠结肠炎耶尔森氏菌ΔHOPEMT asd或III：使用抗YopE抗体通过免疫印迹法在总细菌裂解物(IV)和沉淀的培养上清液(V)上显示出小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBadSi_2。
图2：T3SS蛋白递送进入上皮细胞的表征。(A)对用MOI为100感染1小时的HeLa细胞用I：小肠结肠炎耶尔森氏菌ΔHOPEMT asd或II：小肠结肠炎耶尔森氏菌△HOPEMT asd+pBad_Si2的抗Myc免疫荧光染色。(B)定量来自(A)的HeLa细胞内的抗Myc免疫荧光染色强度。从n＝20个位点合并数据，指示的误差条是平均值的标准误差。I：未感染，II：小肠结肠炎耶尔森氏菌ΔHOPEMT asd或III：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBad_Si2。Y轴表示抗Myc染色强度[任意单位]，x轴表示感染时间(min)(C)细胞内抗Myc免疫荧光染色强度的定量。将HeLa细胞用在x轴上指示的MOI处的肠炎沙门氏菌ΔHOPEMTasd+pBad_Si2感染1小时。从n＝20个位点合并数据，指示的误差条是平均值的标准误差。Y轴表示抗Myc染色强度[a.u.]。
图3：基于T3SS的蛋白质递送的修饰允许YopE1-138融合蛋白(EGFP)的核定位。在被以下感染的HeLa细胞中的EGFP信号：I：小肠结肠炎耶尔森氏菌ΔHOPEMT asd或II：MOI为100的小肠结肠炎耶尔森氏菌ΔHOPEMT asdΔyopB携带质粒III：+YopE1-138-EGFP或IV：+YopE1-138-EGFP-NLS。EGFP信号显示在“a”中，核在“b”中染色用于定位比较。
图4：基于T3SS的蛋白质递送的修饰允许去除YopE1-138附属物。HeLa细胞同时用两种不同的小肠结肠炎耶尔森氏菌菌株感染，这是通过两种细菌悬浮液的简单混合而达到的。一个菌株递送与YopE1-138融合的TEV蛋白酶，而另一个菌株递送利用含有双TEV蛋白酶切割位点的接头与YopE1-138融合的目标蛋白质。在蛋白质递送到真核细胞中后，TEV蛋白酶将从目标蛋白质上切割YopE1-138附属物。(A)对未感染的毛地黄皂苷裂解的Hela细胞(II)或用I：小肠结肠炎耶尔森氏菌ΔHOPEMT asd和III：+pBadSi_2，IV：+YopE1-138-2x TEV切割位点-Flag INK4C，V：+YopE1-138-2x TEV切割位点-Flag INK4C，并用纯化的TEV蛋白酶进一步过夜处理，以及VI：+YopE1-138TEV切割位点-Flag-INK4C和第二菌株+YopE1-138-TEV进行感染(MOI为100)2小时后的毛地黄皂苷裂解的Hela细胞采用免疫印迹抗INK4C(以“a”表示)分析YopE1-138-2xTEV切割位点-Flag-INK4C或其裂解形式Flag-INK4C的存在。免疫印迹抗肌动蛋白作为加载对照(以“b”表示)。在情况(V)下，裂解的细胞与纯化的TEV蛋白酶温育过夜。(B)来自(A)的全长YopE1-138-2x TEV切割位点-Flag-INK4C的抗-INK4C染色强度的肌动蛋白标准化定量(在y轴上显示为[au])，其中将样品IV设定为100％。I：小肠结肠炎耶尔森氏菌ΔHOPEMT asd和IV：+YopE1-138-2x TEV切割位点Flag-INK4C，V：+YopE1-138-2x TEV切割位点Flag-INK4C，并用纯化的TEV蛋白酶进一步过夜处理，和VI：+YopE1-138-2x TEV切割位点Flag-INK4C和第二菌株+YopE 1-138-TEV。将n＝2个独立实验的数据合并，误差条表示平均标准误差。(C)对未感染(II)的毛地黄皂苷裂解的HeLa细胞或用I：小肠结肠炎耶尔森氏菌ΔHOPEMT asd和III：+pBadSi_2，IV：+YopE1-138-2x TEV切割位点-ET1-Myc，V：+YopE1-138-2xTEV切割位点-ET1-Myc，并用纯化的TEV蛋白酶过夜处理，和VI：+YopE1-138-2x TEV切割位点-ET1-Myc和第二菌株+YopE1-138-TEV进行感染(MOI为100)2小时后的毛地黄皂苷裂解的HeLa细胞通过免疫印迹抗Myc分析YopE1-138_2xTEV切割位点-ET1-Myc或其切割形式ET1-Myc的存在(以“a”表示)。作为加载对照，进行抗肌动蛋白的免疫印迹(以“b”表示)。在情况(V)下，裂解的细胞与纯化的TEV蛋白酶温育过夜。
图5：将细菌效应蛋白递送到真核细胞中。(A)将HeLa细胞用携带II：pBad_Si2或III：YopE1-138-SopE的I：小肠结肠炎耶尔森氏菌ΔHOPEMT asd感染，MOI为100，时间如图上所示(2、10或60min)。固定后，对细胞进行肌动蛋白细胞骨架染色(B)。不对HeLa细胞进行感染(II)或将HeLa细胞用携带III：YopE1-138-SopE-Myc的I：小肠结肠炎耶尔森氏菌ΔHOPEMTasd进行感染1小时(在一些情况下用IV：YopE1-138-SptP以在菌株下方所示的MOI(MOI50；MOI50：MOI50或MOI50：Mol100)共感染)。固定后，对细胞进行肌动蛋白细胞骨架染色(以“a”表示)，并且通过染色抗Myc跟踪YopE1-138-SopE-Myc融合蛋白的存在(以“b”表示)。
图6：将细菌效应蛋白递送到真核细胞中。(A)对未处理的HeLa细胞或以MOI为100用携带III：pBad_Si2或IV：YopE1-138-OspF的小肠结肠炎耶尔森氏菌ΔHOPEMT asd感染75分钟的HeLa细胞进行Phospho-p38(“a”)、总p38(“b”)和肌动蛋白(“c”)免疫印迹分析。在所示(+表示加入TNFα，-表示未用TNFα处理)感染的最后30分钟用TNFα刺激细胞。(B)对未处理(II)的HeLa细胞或以MOI为100用携带III：pBad_Si2、IV：YopE1-138-SopE或V:YopE1-138-SopB的I:小肠结肠炎耶尔森氏菌ΔHOPEMT asd感染22.5或45分钟的HeLa细胞进行Phospho-AktT308(“a”)、S473(“b”)和肌动蛋白(“c”)免疫印迹分析。(C)在未处理的HeLa细胞(I)或以MOI为100用V：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YopE1-138-BepA、VI：小肠结肠炎耶尔森氏菌ΔHOPEMTasd+YopE1-138-BepAE305-e、VII：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YopE1-138-BepGBid或VIII小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBad_Si2感染2.5小时后的HeLa细胞中的cAMP水平(在y轴上以fmol/孔表示)。将霍乱毒素(CT)作为阳性对照加入样品II(15μg/ml)、III(25μg/ml)、IV(50μg/ml)。从n＝3个独立实验合并数据，指示的误差条是平均值的标准误差。使用不成对的双尾t检验进行统计分析(ns表示非显著变化，**表示p<0.01，***表示p<0.001)。
图7：将人tBid递送到真核细胞中诱导大量凋亡。(A)对未处理的HeLa细胞或以MOI为100用携带III：pBad_Si2、IV：YopEl-138-Bid或V：YopE1-138-t-bid的I：小肠结肠炎耶尔森氏菌ΔHOPEMT asd感染60分钟后的HeLa细胞进行裂解的Caspase3p17(“a”)和肌动蛋白(“b”)蛋白质印迹分析。在有些情况下，细胞用VI：0.5μM星形孢菌素或VII：1μM星状孢菌素处理。(B)对未处理的毛地黄皂苷裂解HeLa细胞或以MOI为100用携带III：pBad_Si2、IV：YopEl-138-Bid或V：YopE1-138-t-bid的I：小肠结肠炎耶尔森氏菌ΔHOPEMT asd感染1小时后的毛地黄皂苷裂解HeLa细胞进行免疫印迹anti-Bid(a)分析，允许将内源性Bid水平(Z标记)与转运的YopE 1-138-Bid(X标记)或YopE1-138-t-Bid(Y标记)水平进行比较。免疫印迹抗肌动蛋白作为加载对照(以“b”表示)。在有些情况下，细胞用VI：0.5μM星形孢菌素或VII：1μM星形孢菌素处理。(C)HeLa细胞不处理(I)或以MOI为100下用II：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBad_Si2、III：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YopE1-138-Bid、IV：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YopE1-138-tBid感染。在有些情况下，用V：0.5μM星形孢菌素或VI：1μM星形孢菌素处理细胞。固定后，将细胞进行肌动蛋白细胞骨架染色(灰色)。
图8：斑马鱼的T3SS依赖性递送BIM：诱导斑马鱼胚胎中的凋亡。(A)通过注射约400个细菌进入后脑区用表达小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBad_Si1对照菌株(1)或zBIM转位菌株(II：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YopE1-138-zBIM)感染2个dpf斑马鱼胚胎。5.5小时后，将胚胎固定，对活化的Caspase 3(裂解的Caspase 3，p17；以“c”表示)染色并分析细菌的存在(EGFP信号，以“b”表示)。最大强度z投影以荧光图像显示。亮场z投影以“a”表示。(B)记录的(A)z-堆叠图像在最大强度z投影上的自动图像分析。简要地，通过GFP通道检测细菌，围绕细菌斑点的每个区域产生半径为10像素的圆，重叠区域在连接构件之间平均分开。在紧密围绕细菌的那些区域中，测量Caspase 3p17染色强度并在y轴上绘图(如[a.u.])使用Mann-Whitney检验进行统计分析(***表示p<0.001)。对于小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBad_Si1对照菌株(I)，采取n＝14数据合并或对于II：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YopE1-138-zB1M感染的动物，采取n＝19数据合并，指示的误差条是标准误差均值。
图9：tBiD依赖性磷酸蛋白体：HeLa细胞用小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YopE1-138-t-Bid以MOI为100感染3分钟，用小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBad_Si2作为对照。(A)tBID磷酸化蛋白的图示。含有以tBid依赖性方式(灰色)(q<0.01)显著调节的磷酸肽蛋白以及已知凋亡相关蛋白(深灰色)在已知和预测的蛋白质-蛋白质相互作用的STRING网络中列出(高可信度，得分0.7)。在STRING中至少有一个连接的蛋白质被列出。(B)用小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBad_Si2(I)或小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YOpE1-138-t-Bid(II)感染的HeLa细胞的共焦图像显示在tBid递送时诱导凋亡表型。细胞用Hoechst(“a”)对核染色，用鬼笔环肽对F-肌动蛋白染色(“b”)、用20抗微管蛋白抗体对微管蛋白染色(“c”)以及用mitotracker对线粒体染色。比例尺表示40μm。
图10：基于III型分泌物递送工具箱的描述。(A)用于产生具有YopE1-138融合构建体的克隆质粒pBad_Si1和pBad_Si2的载体图谱。分子伴侣SycE和YopE1-138-融合体在天然Y肠道凝胶启动子下。这两种质粒仅在存在于pBad_Si1上的阿拉伯糖诱导型EGFP存在时不同。(B)多克隆位点直接在pBad_Si1和pBad_Si2质粒上的YOpE1-138片段之后。
图11：T3SS蛋白递送到各种细胞系中的表征。在Swiss 3T3成纤维细胞('γ)、Jurkat细胞(“b”)和未处理(II)或用小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBad_Si2(I)以上面图像中所示的MOI(HUVECs的MOI25、50、100和200)感染1小时的HUVEC细胞(“c”)的抗Myc免疫荧光染色。
图12：细菌效应蛋白递送到真核细胞中的T3SS依赖性。用免疫印迹抗Myc分析以MOI为100用小肠结肠炎耶尔森氏菌ΔHOPEMT asdΔyobB+YopE1-138-SopE-Myc(I)或小肠结肠炎耶尔森氏菌ΔHOPEMT asdΔyobB+YopE1-138-SopE-Myc(II)感染上面所示印迹点(0、5、15、10、60和120min)时间后的毛地黄皂苷裂解的HeLa细胞。对应于YopE1-138-SopE-Myc的大小用“a”标记，而内源性c-Myc蛋白的大小用“b”标记。
图13和14：T3SS依赖性分泌各种其他蛋白质到培养物上清液中。体外分泌实验1：小肠结肠炎耶尔森氏菌ΔHOPEMT asdΔyobB+YopE1-138融合到所示蛋白质。使用抗YopE11Au抗体通过免疫印迹分析总细菌裂解物(“A”)和沉淀的培养上清液(“B”)的蛋白质含量。数字表示在相应高度的分子量(kDa)。
图15A至M：用于本研究的小肠结肠炎耶尔森氏菌和肠道沙门氏菌菌株。本研究中使用的小肠结肠炎耶尔森氏菌和肠道沙门氏菌菌株列表提供了编码在相应质粒上针对T3SS依赖性递送的背景菌株、质粒和蛋白质的信息。此外，提供了关于用于构建相应质粒、主链质粒和抗生素抗性的寡核苷酸的信息。
图16：将鼠tBid、鼠Bid BH3和鼠Bax BH3递送到B16F10细胞中诱导大量凋亡。B16F10细胞未感染(I)或用小肠结肠炎耶尔森氏菌ΔHOPEMT asd和II：+pBadSi_2、III：+YopE1-138-小肠结肠炎耶尔森氏菌密码子优化的鼠tBid、IV：+YopE1-138-小肠结肠炎耶尔森氏菌密码子优化的鼠Bid BH3或V：+YopEl-138-小肠结肠炎耶尔森氏菌密码子优化的鼠BaxBH3感染2.5小时后(MOI为50)。固定后，对细胞进行肌动蛋白细胞骨架和细胞核染色(两者均为灰色)。
图17：将鼠tBid、鼠Bid BH3和鼠Bax BH3递送到D2A1细胞中诱导大量凋亡。D2A1细胞未感染(I)或用小肠结肠炎耶尔森氏菌ΔHOPEMT asd和II：+pBadSi_2、III：+YopE1-138-小肠结肠炎耶尔森氏菌密码子优化的鼠tBid、IV：+YopE1-138-小肠结肠炎耶尔森氏菌密码子优化的鼠Bid BH3或V：+YopEl-138-小肠结肠炎耶尔森氏菌密码子优化的鼠Bax BH3感染2.5小时后(MOI为50)。固定后，对细胞进行肌动蛋白细胞骨架和细胞核染色(两者均为灰色)。
图18：将鼠tBid、鼠BH3和鼠Bax BH3递送到HeLa细胞中诱导大量凋亡。HeLa细胞未感染(I)或用小肠结肠炎耶尔森氏菌ΔHOPEMT asd和II：+pBadSi_2、III：+YopE1-138-小肠结肠炎耶尔森氏菌密码子优化的鼠tBid、IV：+YopE1-138-小肠结肠炎耶尔森氏菌密码子优化的鼠Bid BH3或V：+YopEl-138-小肠结肠炎耶尔森氏菌密码子优化的鼠Bax BH3感染2.5小时后(MOI为50)。固定后，对细胞进行肌动蛋白细胞骨架和细胞核染色(两者均为灰色)。
图19：将鼠tBid、鼠Bid BH3和鼠Bax BH3递送到4T1细胞中诱导大量凋亡。4T1细胞未感染(1)或用小肠结肠炎耶尔森氏菌ΔHOPEMT asd和II：+pBadSi_2、III：+YopE1-138-小肠结肠炎耶尔森氏菌密码子优化的鼠tBid、IV：+YopE1-138-小肠结肠炎耶尔森氏菌密码子优化的鼠Bid BH3或V：+YopEl-138-小肠结肠炎耶尔森氏菌密码子优化的鼠Bax BH3感染2.5小时后(MOI为50)。固定后，对细胞进行肌动蛋白细胞骨架和细胞核染色(两者均为灰色)。
图20：在SPI-1T3SS诱导条件下生长的肠杆菌向真核细胞递送鼠tBid诱导凋亡。对未处理(I)和或用III：携带IV：SteA1-20-t-Bid、V：SteAFL-Bid、VI：SopE1-81-t-Bid或VII：SopE1-105-t-Bid，MOI为100时用肠道沙门氏菌感染4小时的HeLa细胞进行裂解Caspase 3p17免疫印迹分析.对于该实验，所有肠道沙门氏菌aroA菌株在SPI-1T3SS诱导条件下生长。在有些情况下，用II：1μM星形孢菌素处理细胞。数字表示在相应高度的分子量(kDa)。
图21：在SPI-2T3SS诱导条件下生长的肠杆菌向真核细胞递送鼠tBid诱导凋亡。对未处理(I)和或用III：携带IV：SteA1-20-t-Bid、V：SteAFL-Bid、VI：SopE1-81-t-Bid或VII：SopE1-105-t-Bid，MOI为100时用肠道沙门氏菌感染4小时的HeLa细胞进行裂解Caspase 3p17免疫印迹分析.对于该实验，所有肠道沙门氏菌aroA菌株在SPI-2T3SS诱导条件下生长。在有些情况下，用II：1μM星形孢菌素处理细胞。数字表示在相应高度的分子量(kDa)。
图22：肠炎沙门氏菌T3SS依赖性分泌各种细胞周期蛋白到培养上清液中。肠炎沙门氏菌aroA+与如下所列的蛋白质融合的SteAFL(I、III、V、VII)或SopE1-105(II、IV、VI、VIII)的体外分泌实验I和II：Ink4a MycHis；III和IV：Ink4c-MycHis；V和VI：Mad2-MycHis；VII和VIII：Cdk1-MycHis。使用抗myc抗体通过免疫印迹分析沉淀的培养上清液(“A”)和总细菌素(“B”)的蛋白质含量。数字表示在相应高度的分子量(kDa)。
图23：各种已知细胞周期干扰肽T3SS依赖性分泌到培养上清液中。小肠结肠炎耶尔森氏菌ΔHOPEMT asd+pBad_Si2的体外分泌实验。II-VII：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YopE1-138，融合到下面所列的肽上：II：Ink4A84-103；III：p107/RBL1657-662；IV：p21141-160D149A；V：p21145-160D149A；VI：p2117-33；VII：细胞周期蛋白D2139-147。使用抗YopE抗体通过免疫印迹分析沉淀的培养基上清液(“A”)和总细菌裂解物(“B”)的蛋白质含量。数字表示在相应高度的分子量(kDa)。
图24：T3SS递送的蛋白质与泛素的融合允许去除YOpE1-138附属物。将HeLa细胞用递送目标蛋白菌株感染，其中目标蛋白用一个直接融合的泛素(YopE1-138-Ubi)融合到YopE1-138。在蛋白质递送到真核细胞中后，内源性泛素特异性蛋白酶将从目标蛋白质切割YopE1-138-Ubi附属物。在未感染(II)或用II：小肠结肠炎耶尔森氏菌ΔHOPEMT asd+YopE1-138-标志INK4C-MycHis或III：+YopE1-138-Flag-Ubiquitin-1，感染1小时(MOI为100)后通过免疫印迹抗INK4C分析IV：YopE1-138-Flag-Ubiquitin-INK4C-MycHis或V：YopE1-138-Flag-INK4C-MycHis，裂解形式VI：INK4C-MycHis和VII：内源性INK4C的存在。
具体实施方式
本发明提供了重组革兰氏阴性细菌菌株及其用于将异源蛋白质递送到真核细胞中的用途。
出于解释本说明书的目的，将应用以下定义，并且在适当时，以单数使用的术语也将包括复数，反之亦然。应当理解，本文所使用的术语仅用于描述特定实施例目的，而不是限制性的。
本文所用的术语“革兰氏阴性细菌菌株”包括以下细菌：杀鲑气单胞菌、嗜水气单胞菌、维氏气单胞菌、厌氧粘细菌、支气管炎博德特氏菌、支气管炎博德特氏菌、副百日咳杆菌、百日咳杆菌、大豆慢生根瘤菌、伯克霍尔德新洋葱伯克霍尔德杆菌、洋葱伯克霍尔德菌、鼻疽伯克霍尔德氏菌、鼻疽杆菌、沙眼衣原体肺炎、沙眼衣原体、流产衣原体、衣原体肺炎、紫色杆菌、枸橼酸杆菌啮齿类、普通脱硫弧菌、爱德华氏菌、Endozoicomonas elysicola、梨火疫病菌、阿尔伯蒂埃希氏杆菌、大肠杆菌、细胞内劳森菌、根瘤菌、黄色粘球菌、成团泛菌、美人鱼发光杆菌、发光杆菌、Photorabdus temperate、海绵假交替单胞菌、绿脓杆菌、变形假单胞菌、丁香假单胞菌、青枯雷尔氏菌、根瘤菌、沙门氏杆菌和其他沙门菌，志贺氏菌和其他志贺氏菌、Sodalis glossinidius、溶藻弧菌、Vibrio azureus、坎氏弧菌，Vibriocaribbenthicus、哈维氏弧菌、副溶血性弧菌、Vibrio tasmaniensis、Vibrio tubiashii、地毯草黄单胞菌、野油菜黄单胞菌、白叶枯病菌、小肠结肠炎耶尔森氏菌、耶尔森氏菌、假结核菌。本发明优选的革兰氏阴性细菌菌是肠杆菌科和假单胞菌科包含的革兰氏阴性细菌菌株。本发明的革兰氏阴性细菌菌株通常被用于由细菌T3SS递送异源蛋白质到体外和体内的真核细胞。
本文使用的术语“重组革兰氏阴性细菌菌株”是指用载体遗传转化的革兰氏阴性细菌菌株。本发明的有用载体是例如表达载体，用于染色体或毒力质粒插入的载体或用于染色体或毒力质粒插入的DNA片段。
术语“对产生生长必需氨基酸缺陷的革兰氏阴性细菌菌株”和“营养缺陷型突变体”在本文中可互换使用，指在不存在至少一种外源提供的必需氨基酸或其前体的情况下不能生长的革兰氏阴性细菌菌株。所述菌株缺陷产生的氨基酸是例如天冬氨酸，内消旋-2,6-二氨基庚二酸，芳香族氨基酸或亮氨酸-精氨酸[23]。这种应变可以通过例如缺失天冬氨酸-β-半醛脱氢酶基因(Δasd)。这样的营养缺陷突变体不能在不存在外源性内消旋-2,6-二氨基庚二酸的情况下生长[24]。突变，例如缺失天冬氨酸-β-半醛脱氢酶基因，优选用于本发明的对产生生长必需的氨基酸缺陷的革兰氏阴性细菌菌株。
术语“对产生结合真核细胞表面或细胞外基质的粘附蛋白缺陷的革兰氏阴性细菌菌株”是指与由相应野生型表达的粘附蛋白相比不表达至少一种粘附蛋白的突变革兰氏阴性菌株型。粘附蛋白可以包括例如扩展的聚合物粘附分子如菌毛或非菌毛粘附素。菌毛粘附素包括1型菌毛(例如具有FimH粘附素的大肠杆菌菌毛)、P型菌毛(例如具有来自大肠杆菌的PapG粘附素的Pap-菌毛)、4型菌毛(例如来自铜绿假单胞菌的菌毛)或curli(Csg蛋白与来自肠炎沙门氏菌的CsgA粘附素)。非菌毛粘附包括三聚体自转运粘附素，例如来自小肠结肠炎耶尔森氏菌YadA、BpaA(假单胞菌)、Hia(流感嗜血杆菌)、BadA(B.nselae)、NadA(脑膜炎奈瑟氏球菌)或UspA1以及其它自转运粘附素如AIDA-1(大肠杆菌)以及其他粘附素/侵袭素如来自小肠结肠炎耶尔森氏菌或Intimin(大肠杆菌)的InvA或Dr-家族或Afa-家族的成员(大肠杆菌)。本文所用的术语YadA和InvA是指来自小肠结肠炎耶尔森氏菌的蛋白质。自体转运蛋白YadA[25，26]结合不同的胶原蛋白以及纤连蛋白，而侵袭素InvA[27-29]结合到真核细胞膜中的β-整合素。如果革兰氏阴性细菌菌株是小肠结肠炎耶尔森氏菌菌株，那么该菌株优选是InvA和/或YadA缺陷的。
如本文所用，术语“肠杆菌科家族”包括在土壤、水、植物和动物中发现的革兰氏阴性、杆状、兼性厌氧细菌家族，其经常如脊椎动物中的病原体发生。该家族的细菌共享相似的生理学并且证明在相应基因组的功能元件和基因内的保守性。除氧化酶阴性外，该家族的所有成员都是葡萄糖发酵，大多数是硝酸盐还原剂。
本发明的肠杆菌科细菌可以是来自该家族的任何细菌，具体包括但不限于以下属的细菌：埃希氏菌属、志贺氏菌属、爱德华氏菌属、沙门氏菌属、柠檬酸杆菌属、克雷伯菌属、肠杆菌属、沙雷氏菌属、变形杆菌属、摩根(氏)菌属、普罗威登斯菌属或耶尔森氏鼠疫杆菌属。在更具体的实施方案中，细菌是大肠杆菌、大肠杆菌、弗氏杆菌、埃氏肠杆菌、埃希氏菌、肠炎沙门氏菌、痢疾杆菌、志贺氏菌、志贺氏菌、志贺氏菌、志贺氏菌、产气肠杆菌、肠杆菌、肠杆菌镰刀菌、变形杆菌、普通变形杆菌、变形杆菌、普通变形杆菌、金黄色葡萄球菌或摩氏摩根氏菌。优选地，革兰氏阴性细菌菌株选自耶尔森氏菌属、埃希氏菌属、沙门氏菌属、志贺氏菌属、假单胞菌属、衣原体属、欧文氏菌属、泛菌属、弧菌属、伯克霍尔德氏菌属、兰氏菌属、黄单胞菌属、色杆菌属、Sodalis、柠檬酸杆菌属、爱德华氏菌属、根瘤菌属、气单胞菌属、光杆菌属、博德特氏菌属和脱硫弧菌属、更优选来自耶尔森氏菌属、埃希氏菌属、沙门氏菌属和假单胞菌属、最优选来自耶尔森氏菌属和沙门氏菌属。
本文所用的术语“耶尔森氏菌”包括耶尔森氏菌的所有物种，包括小肠结肠炎耶尔森氏菌、假结核耶尔森氏菌和鼠疫耶尔森氏菌。优选的是小肠结肠炎耶尔森氏菌。
本文使用的术语“沙门氏菌”包括沙门氏菌的所有物种，包括肠道沙门氏菌和沙门氏菌。优选为肠道沙门氏菌。
本文所用的“启动子”是指调节转录单位表达的核酸序列。“启动子区”是能够结合细胞中的RNA聚合酶并启动下游(3'方向)编码序列的转录的调节区。在启动子区域内将发现转录起始位点(通过用核酸酶S1作图方便定义)，以及负责RNA聚合酶结合的蛋白质结合结构域(共有序列)，例如推定的-35区域和Pribnow框。当描述两个DNA区之间的关系时，术语“可操作地连接”仅仅意味着它们彼此功能相关，并且它们位于相同的核酸片段上。如果启动子控制基因的转录并且其位于与基因相同的核酸片段上，则启动子可操作地连接到结构基因。通常启动子在所述革兰氏阴性细菌菌株中是有功能的，即启动子能够表达本发明的融合蛋白，即启动子能够表达本发明的融合蛋白，而不进一步基因工程或进一步表达蛋白质。此外，功能性启动子不能对细菌T3SS天然地反调节。
本文使用的术语“递送”是指将蛋白质从重组革兰氏阴性菌株运输到真核细胞，包括在重组革兰氏阴性菌株中表达异源蛋白质的步骤，从这种革兰氏阴性细菌菌株分泌表达的蛋白质，并通过这种革兰氏阴性细菌菌株将分泌的蛋白质转移到真核细胞的胞质溶胶中。因此，本文可互换使用的术语“递送信号”或“分泌信号”是指可以被革兰氏阴性细菌菌株分泌和转运系统识别并指导从革兰氏阴性细菌菌株递送蛋白质到的真核细胞的多肽序列。
如本文所用，蛋白质的“分泌”是指异源蛋白质跨越重组革兰氏阴性细菌菌株的细胞膜的运输。蛋白质的“转位”是指异源蛋白质从重组革兰氏阴性细菌菌株穿过真核细胞的质膜递送到这种真核细胞的胞质溶胶中。
本文所用的术语“真核细胞”包括以下真核细胞：Hi-5、HeLa、Hek、HUVEC、3T3、CHO、Jurkat、Sf-9、HepG2、Vera、MDCK、Mefs、THP-1、J774、RAW、Caco2、NCI60、DU145、Lncap、MCF-7、MDA-MB-438、PC3、T47D、A549、U87、SHSY5Y、Ea.Hy926、Saos-2、4T1、D2A1、B16F10和原代人肝细胞。本文使用的“真核细胞”也称为“靶细胞”或“靶真核细胞”。
本文所用的术语“T3SS效应蛋白”是指通过T3S系统天然注射到真核细胞的细胞溶质中的蛋白质和由T3S系统天然分泌的蛋白质，其可能例如形成孔进入真核细胞膜(包括成孔递送器(如耶尔森氏菌YopB和YopD)和尖端蛋白(如Yersinia LcrV)。优选使用通过T3S系统天然注射到真核细胞的细胞质中的蛋白质。这些剧毒因子将使真核细胞麻痹或重编程以获得病原体的益处。T3S效应物具有大量的生物化学活性和调节关键宿主调节分子的功能[5，30]，包括AvrA、AvrB、AvrBs2、AvrBS3、AvrBsT、AvrD、AvrD1、AvrPphB、AvrPphC、AvrPphEPto、AvrPpiBPto、AvrPto、AvrPtoB、AvrRpmL、AvrRpt2、AvrXv3、CigR、EspF、EspG、EspH、EspZ、ExoS、ExoT、GogB、GtgA、GtgE、GALA家族蛋白、HopAB2、HopAO1、Hopl1、HopM1、HopN1、HopPtoD2、HopPtoE、HopPtoF、IpB、IpgB、IpgB2、IpgD、LcrV、Map、OspC1、OspE2、OspF、OspG、Osp1、PipB、PipB2、PopB、PopP2、PthXol、PthXo6、PthXo7、SifA、SifB、SipA/SspA、SipB、SipC/SspC、SipD/SspD、SlrP、SopA、SopB/SigD、SopD、SopE、SopE2、SpiC/SsaB、SptP、SpvB、SpvC、SrfJ、Sse、SseB、SseC、SseD、SseF、SseG、Ssel/SrfH、SseJ、SseK1、SseK2、SseK3、SseL、SspH1、SspH2、SteA、SteB、SteC、SteD、SteE、TccP2、Tir、VirA、VirPphA、VopF、XopD、YopB、YopD、YopE、YopH、YopJ、YopM、YopO、YopP、YopT、YpkA。
耶尔森氏菌的T3SS效应基因已经从YopE、YopH、YopM、YopO、YopP/YopJ和YopT[31]。各自的效应基因可以从弗氏志贺氏菌(例如OspF、IpgD、IpgB1)、肠沙门氏菌(例如SopE、SopB、SptP)、铜绿假单胞菌(例如ExoS、ExoT、ExoU、ExoY)或大肠杆菌(Map、EspF、EspG、EspH、EspZ)克隆。这些基因的核酸序列对于本领域技术人员是可获得的，例如在Genebank数据库(来自NC 002120GL 10955536的yopH、yopO、yopE、yopP、yopM、yopT、；来自AF386526.1GI 18462515的福氏志贺菌杆菌效应蛋白；来自NCO16810.1GI378697983或FQ312003.1GI301156631的肠道沙门菌；来自AE004091.2GI：110227054或CP000438.1GI：115583796的绿脓杆菌效应子和来自NC O 11601.1GL215485161的大肠杆菌效应子蛋白)。
为了本发明的目的，基因用小写字母表示，斜体表示以区别于蛋白质。在基因(由小写字母和斜体字母表示)遵循细菌物种名称(如大肠杆菌)的情况下，它们是指相应细菌物种中相应基因的突变。例如，YopE是指由yopE基因编码的效应子蛋白。小肠结肠炎耶尔森氏菌yopE代表在yopE基因中具有突变的肠道小肠结肠炎耶尔森氏菌。
如本文所用，术语“多肽”、“肽”、“蛋白质”、“多肽的”和“肽的”可互换使用，表示通过α-氨基和羧基之间的肽键彼此连接的一系列氨基酸残基相邻残基的组。优选具有包含至少10个氨基酸，更优选至少20个氨基酸的氨基酸序列的蛋白质。根据本发明，“异源蛋白质”包括天然存在的蛋白质或其部分，并且还包括人工改造的蛋白质或其部分。如本文所用，术语“异源蛋白质”是指除了其可融合的T3SS效应蛋白或其N末端片段之外的蛋白质或其部分。特别地，本文使用的异源蛋白质是指不属于蛋白质组的蛋白质或其部分，即本发明提供和使用的特定重组革兰氏阴性细菌菌株的完整天然蛋白质互补物，例如，其不属于蛋白质组，即耶尔森氏菌属、埃希氏菌属、沙门氏菌属或假单胞菌属的特定细菌菌株的完整天然蛋白质补体。通常异源蛋白质是动物来源的，包括人来源的。优选地，异源蛋白质是人蛋白质。更优选异源蛋白质选自参与凋亡或凋亡调节的蛋白质、细胞周期调节剂、锚蛋白重复蛋白、细胞信号转导蛋白、报告蛋白、转录因子、蛋白酶、小GTP酶、GPCR相关蛋白、纳米体融合构建体和纳米抗体、细菌T3SS效应子、细菌T4SS效应子和病毒蛋白。特别优选异源蛋白质选自参与凋亡或凋亡调节的蛋白质、细胞周期调节剂、锚蛋白重复蛋白、报告蛋白、小GTP酶、GPCR相关蛋白、纳米抗体融合构建体、细菌T3SS效应子、细菌T4SS效应子和病毒蛋白。甚至更特别优选的是选自参与凋亡或凋亡调节的蛋白质、细胞周期调节剂和锚蛋白重复蛋白的异源蛋白质。最优选的是参与凋亡或凋亡调节的蛋白质，如动物，优选参与凋亡或凋亡调节的人异源蛋白质。
在一些实施方案中，本发明的革兰氏阴性细菌菌株的载体包含相互独立地框内融合到所述第一DNA序列的3'端的编码相同或两种不同异源蛋白质的两个第二DNA序列。在一些实施方案中，本发明的革兰氏阴性细菌菌株的载体包含相互独立地框内融合到所述第一DNA序列的3'端的编码相同或三种不同异源蛋白质的三个第二DNA序列。
由重组革兰氏阴性细菌菌株表达的异源蛋白质通常具有1-150kD，优选1-120kD，更优选在100kDa，最优选15-100kDa之间的分子量。由重组革兰氏阴性细菌菌株表达的异源蛋白质通常具有1-150kD，优选1-120kD，更优选在100kDa，最优选15-100kDa之间的分子量。
根据本发明，“参与凋亡或凋亡调节的蛋白质”包括但不限于Bad、Bcl2、Bak、Bmt、Bax、Puma、Noxa、Bim、Bcl-xL、Apaf1、Caspase 9、Caspase 3、Caspase 6、Caspase 7、Caspase 10、DFFA、DFFB、ROCK1、APP、CAD、ICAD、CAD、EndoG、AIF、HtrA2、Smac/Diablo、Arts、ATM、ATR、Bok/Mtd、Bmf、Mcl-)、IAP家族、LC8、PP2B、14-3-3蛋白、PKA、PKC、PI3K、Erk1/2、p90RSK、TRAF2、TRADD、FADD、Daxx、Caspase8、Caspase2、RIP、RAIDD、MKK7、JNK、(p16(Ink4a)、p15(Ink4b)、p18(Ink4c)、p19(Ink4d))和Cip1/Waf1/Kipl-2-家族(p21(Cip1c))的FKHR、GSK3、CDKs和它们的抑制剂/Waf1)、p27(Kipl)、p57(Kip2)。优选Bad、Bmt、Bcl2、Bak、Bax、Puma、Noxa、Bim、Bcl-xL、Caspase9、Caspase3、Caspase6、Caspase7、Smac/Diablo、Bok/Mtd、Bmf、Mcl-TRADD、Daxx、Caspase8、Caspase2、RIP、RAIDD、FKHR、CDK及其抑制剂如INK4-家族(p16(Ink4a)、p15(Ink4b)、p18(Ink4c)、p19(Ink4d))，最优选BIM、截短的Bid、FADD、胱天蛋白酶3(及其亚基)、Bax、Bad、Akt、CDK及其抑制剂如INK4-家族(p16(Ink4a)、p15(Ink4b)、p18(Ink4c)、p19[32-34]。此外，参与细胞凋亡或凋亡调节的蛋白质包括DIVA、Bcl-Xs、Nbk/Bik、Hrk/Dp5、Bid和tBid、Eg1-1、Bcl-Gs、细胞色素C、Beclin、CED-13、BNIP1、BNIP3、Bcl-B、Bcl-W、Ced-9、A1、NR13、Bfl-1、半胱天冬酶1、半胱天冬酶2、半胱天冬酶4、半胱天冬酶5、半胱天冬酶8.参与凋亡或凋亡调节的蛋白质选自促凋亡蛋白质、抗凋亡蛋白、凋亡预防途径的抑制剂和促存活信号或途径的抑制剂。促凋亡蛋白包含选自Bax、Bak、Diva、Bcl-Xs、Nbk/Bik、Hrk/Dp5、Bmf、Noxa、Puma、Bim、Bad、Bid和tBid、Bok、Apaf1、Smac/Diablo、BNIP1、BNIP3、Bcl-Gs、Beclin1、Egl-1和CED-13、细胞色素C、FADD、Caspase家族和CDKs及其抑制剂如INK4家族(p16(Ink4a)、p15(Ink4b)、pkl、Bmf、Noxa、Puma、Bim、Bad、Bid和tBid组成的组中选择的一个或多个氨基酸序列(例如、p18(Ink4c)、p19(Ink4d)Bok、Egl-1、Apaf1、Smac/Diablo、BNIP1、BNIP3、Bcl-Gs、Beclin1、Egl-1和CED-13、细胞色素C、FADD和Caspase家族。优选的是Bax、Bak、Diva、Bcl-Xs、Nbk/Bik、Hrk/Dp5、Bmf、Noxa、Puma、Bim、Bad、Bid和tBid、Bok、Egl-1、Apaf1、BNIP1、BNIP3、Bcl-Beclin 1、Egl-1和CED-13、Smac/Diablo、FADD、Caspase家族、CDK及其抑制剂如INK4-家族(p16(Ink4a)、p15(Ink4b)、p18(Ink4c)、p19(Ink4d))。同样优选的是Bax、Bak、Diva、Bcl-Xs、Nbk/Bik、Hrk/Dp5、Bmf、Noxa、Puma、Bim、Bad、Bid和tBid、Bok、Apaf1、BNIP1、BNIP3、Bcl-Gs、Beclin1、Egl-1和CED-13、Smac/Diablo、FADD、Caspase家族。
抗凋亡蛋白包含选自Bcl-2、Bcl-Xl、Bcl-B、Bcl-W、Mcl-1、Ced-9、A1、NR13、IAP家族和Bfi-1的蛋白质。优选的是Bcl-2、Bcl-XI、Bcl-B、Bcl-W、Mcl-1、Ced-9、A1、NR13和Bfl-1。
细胞凋亡预防途径的抑制剂包含选自Bad、Noxa和Cdc25A的蛋白质。优选Bad和Noxa。
促生存信号传导或通路的抑制剂包含选自PTEN、ROCK、PP2A、PHLPP、JNK，p38的蛋白质。优选PTEN、ROCK、PP2A和PHLPP。
在有些实施方案中，参与凋亡或凋亡调节的异源蛋白选自唯BH3蛋白、胱天蛋白酶和凋亡的死亡受体控制的细胞内信号传导蛋白。
唯BH3蛋白包含选自Bad、BIM、Bid和tBid、Puma、Bik/Nbk、Bod、Hrk/Dp5、BNIP1、BNIP3、Bmf、Noxa、Mcl-1、Bcl-Gs、Beclin1、Egl-1和CED-13的蛋白。优选Bad、BIM、Bid和tBid。
半胱天冬酶包含选自半胱天冬酶1、半胱天冬酶2、半胱天冬酶3、半胱天冬酶4、半胱天冬酶5、半胱天冬酶6、半胱天冬酶7、半胱天冬酶8、半胱天冬酶9、半胱天冬酶10的蛋白质。优选半胱天冬酶3、半胱天冬酶8和半胱天冬酶9的蛋白。
细胞死亡受体控制的细胞内信号蛋白包括选自FADD、TRADD、ASC、BAP31、GULP1/CED-6、CIDEA、MFG-E8、CIDEC、RIPK1/RIP1、CRADD、RIPK3/RIP3、Crk、SHB、CrkL、DAXX、14-3-3家族、FLIP、DFF40和45、PEA-15、SODD。优选的是FADD和TRADD。
在有些实施方案中，参与凋亡或凋亡调节的两种异源蛋白包含在本发明的革兰氏阴性细菌菌株的载体中，其中一种蛋白是促凋亡蛋白，而另一种蛋白是凋亡预防途径的抑制剂或其中一种蛋白是促凋亡蛋白，而另一种蛋白是促生存信号传导或通路的抑制剂。
本发明包括的促凋亡蛋白质通常具有α螺旋结构，优选由两亲性螺旋环绕的疏水螺旋，通常包含BH1、BH2、BH3或BH4结构域中的至少一个，优选包含至少一个BH3结构域。通常由本发明包括的促凋亡蛋白质不具有酶活性。
本文所用的术语“蛋白酶切割位点”是例如指蛋白质或融合蛋白氨基酸内的特定氨基酸基序。在蛋白质或融合蛋白的氨基酸序列内，其被识别氨基酸基序的特异性蛋白酶切割。综述见[35]。蛋白酶切割位点的实例是被选自肠激酶(轻链)、肠肽酶、解离蛋白酶、人鼻病毒蛋白酶(HRV 3C)、TEV蛋白酶、TVMV蛋白酶、FactorXa蛋白酶和凝血酶一组的蛋白酶切割的氨基酸基序。
以下氨基酸基序被相应的蛋白酶识别：
-Asp-Asp-Asp-Asp-Lys：肠激酶(轻链)/肠肽酶
-Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro：PreScission蛋白酶/人鼻病毒蛋白酶(HRV3C)
-Glu-Asn-Leu-Tyr-Phe-Gln-Ser和基于Glu-XX的修饰基序Tyr-X-Gln-Gly/Ser(其中X是任意氨基酸)被TEV蛋白酶(烟草蚀纹病毒)
-Glu-Thr-Val-Arg-Phe-Gln-Ser：TVMV蛋白酶
-Ile-(Glu或Asp)-Gly-Arg：FactorXa蛋白酶
-Leu-Val-Pro-Arg/Gly-Ser：凝血酶。
本文所用的蛋白酶切割位点包括泛素。因此，在一些优选的实施方案中，泛素用作蛋白酶切割位点，即第三DNA序列编码泛蛋白如蛋白酶切割位点，其可以在N-末端位点被特异性泛素加工蛋白酶切割。其可以被特异性遍在蛋白加工蛋白酶切割，所述蛋白酶在融合蛋白已被递送至的细胞中在N末端位点内源性地称为去泛素化酶。泛素在其C末端由一组内源性泛素特异性C末端蛋白酶(去泛素化酶，DUB)加工。通过DUB的遍在蛋白的切割被认为发生在泛素的非常C末端(在G76之后)。“个体”，“受试者”或“患者”是脊椎动物。在某些实施方案中，脊椎动物是哺乳动物。哺乳动物包括但不限于灵长类(包括人和非人灵长类动物)和啮齿动物(例如小鼠和大鼠)。在某些实施方案中，哺乳动物是人。
术语“突变”在本文中用作一般术语，并且包括单个碱基对和多个碱基对的改变。这样的突变可以包括取代、移码突变、缺失、插入和截短。
本文所用的术语“标记分子或标记分子的受体位点”是指结合特定氨基酸序列的小化合物，这样导致结合的化合物产生荧光，结合的化合物优选香豆素连接酶/香豆素受体位点(及其衍生物)、试卤灵连接酶/试卤灵受体位点(及其衍生物)和四半胱氨酸基模序(如Cys-Cys-Pro-Gly-Cys-Cys及其衍生物)与FlAsH/ReAsH染料蛋白(life technologies)或如增强型绿色荧光蛋白(EGFP)的荧光蛋白使用。
本文所用的术语“核定位信号”是指标记导入真核细胞核中的蛋白质的氨基酸序列，并且优选包括病毒核定位信号，例如SV40大T抗原衍生的NLS(PPKK RKV)。
本文所用的术语“多克隆位点”是指含有几个限制性位点的短DNA序列，用于通过限制性内切核酸酶如Ac11、HindIII、Sspl、MLuCI、Tsp509I、Pcil、Agel、BspMI、BfuAI、SexAI、MLuI、BceAI、HpyCH4IV、HpyCH4III、Bael、BsaXI、Aflff1、SpeI、Bsr1、Bmr1、BglII、AfeI、Alul、StuI、Seal、Clal、BspDI、PI-SceI、NsiI、Asel、Swal、CspCI、MfeI、BssSI、BmgBI、PmLl、Drall1、Alel、EcoP15I、PvuII、AlwNI、BtsIMutI、TspRI、NdeI、NlallI、CviAII、Fatl、MslI、FspEI、XcmL、BstXI、PflMI、Bed、Ncol、BseYI、Faul、Smal、Xmal、TspMI、Nt.CviPII、LpnPI、Acil、SacII、BsrBI、Msp1、Hpall、ScrFI、BssKI、StyD4I、BsaJI、BslI、BtgI、Neil、AvrII、MnII、BbvCI、Nb.BbvCI、Nt.BbvCI、SbfI、BpuI1、Bsu36I、EcoNI、HpyAV、BstNI、PspGI、Styl、Bcgl、PvuI、BstUI、EagI、RsrII、BsiEI、BsiWI、BsmBI、Hpy99I、MspAll、MspJI、SgrAI、Bfal、BspCN1、XhoI、Earl、Acul、PstI、BpmL、Ddel、Sfc1、Afl1、BpuEI、SmII、Aval、BsoBI、MboII、BbsI、XmnI、BsmL、Nb.BsmI、EcoRI、Hgal、AatII、Zral、Tth11I、PfF1、PshAI、Ahd1、Drd1、Eco53kI、Sad、BseRI、Plel、Nt BbI、BtsI、BmbI、BtsI、NbBtsI、BstAPI、SphII、SphI、NmeAIII、NaeI、NgoMIV、BglII、BglII、BglII、BglII、BglII、AsiSI、BtgZI、HinPlI、Hhal、BssHII、NotI、Fnu4H1、Cac8I、Mwol、NheI、Bmt1、SapI、BspQI、Nt.BspQI、Blp1、Tsel、ApeK1、Bsp12861、Alw1、Nt.AlwI、BamHI、FokI、BtsCI、HaelII、Phol、FseI、SfI、NarI、KasI、Sfol、PluTI、AscI、Ecil、BsmFI、ApaI、PspOMI、Sau96I、NlalV、KpnI、Acc65I、BsaI、HphI、BstEII、Avail、Ban1、BaeGI、BsaHI、、Rsal、CviQI、BstZ17I、BciVI、SalI、Nt.BsmAI、BsmAI、BcoDI、ApaLI、Bsgl、Accl、Hpyl66II、Tsp45I、Hpal、Pmel、Hindi、BsiHKAI、ApoI、Nspl、BsrFI、BstYI、Haell、CviKI-、EcoRI109I、PpuMI、I-CeuI、SnaBI、I-SceI、BspHI、BspEI、MmeI、Taqal、Nrul、Hpyl 881、Hpyl88III、XbaI、Bell、HpyCH4V、FspI、PI-PspI、MscI、BsrGI、Psil、BstBI、Dral、PspXI、BsaWI、BsaAI、Eael、优选Xhol、XbaI、HindIII、NcoI、NotI、EcoRI、EcoRV、BamHI、NheI、SacI、SalI、BstBI。本文所用的术语“多克隆位点”还指用于重组事件的短DNA序列，例如在Gateway克隆策略中或用于诸如Gibbson装配或拓扑克隆的方法。
本文所用的术语“耶尔森氏菌野生型菌株”是指天然存在的变体(如小肠结肠炎耶尔森氏菌E40)或含有允许使用载体的遗传修饰的天然存在的变体，例如限制性内切核酸酶或抗生素抗性基因中的缺失突变(如小肠结肠炎耶尔森氏菌MRS40、小肠结肠炎耶尔森氏菌E40的氨苄青霉素敏感型衍生物)。这些菌株含有染色体DNA以及未修饰的毒力质粒(称为pYV)。
术语“包含”通常在包括的意义上使用，也就是说允许一个或多个特征或组件的存在。
在一个实施方案中，本发明提供了重组革兰氏阴性细菌菌株，其中所述革兰氏阴性细菌菌株选自耶尔森氏菌属、埃希氏菌属、沙门氏菌属和假单胞菌属。在一个实施方案中，本发明提供了一种重组革兰氏阴性细菌菌株，其中所述革兰氏阴性细菌菌株选自耶尔森氏菌属和沙门氏菌属。优选地，革兰氏阴性细菌菌株是耶尔森氏菌菌株，更优选地耶氏酵母菌株。最优选的是小肠结肠炎耶尔森氏菌E40[13]或氨苄青霉素敏感性衍生物，如[36]中所述的小肠结肠炎耶尔森氏菌MRS40。还优选地，革兰氏阴性细菌菌株是沙门氏菌菌株，更优选沙门氏菌肠道菌株。最优选的是如Public health England culture collection(NCTC 13347)所述的鼠伤寒沙门氏菌SL1344。
在本发明的一个实施方案中，来自细菌T3SS效应蛋白的递送信号包含细菌T3SS效应蛋白或其N末端片段，其中T3SS效应蛋白或其N末端片段可包含伴侣结合位点。包含分子伴侣结合位点的T3SS效应子蛋白或其N末端片段在本发明中特别用作递送信号。优选的T3SS效应蛋白或其N末端片段选自SopE、SopE2、SptP、YopE、ExoS、SipA、SipB、SipD、SopA、SopB、SopD、IpgB1、IpgD、SipC、SifA、SseJ、Sse、SrfH、YopJ、AvrA、AvrBsT、YopT、YopH、YpkA、Tir、EspF、TccP2、IpgB2、OspF、Map、OspG、Ospl、IpaH、SspH1、VopF、ExoS、ExoT、HopAB2、XopD、AvrRpt2、HopAO1、HopPtoD2、HopU1、GALA蛋白家族、AvrBs2、AvrD1、AvrBS3、YopO、YopP、YopE、YopM、YopT、EspG、EspH、EspZ、IpaA、IpaB、IpaC、VirA、IcsB、OspC1、OspE2、IpaH9.8、IpaH7.8、AvrB、AvrD、AvrPphB、AvrPphC、AvrPphEPto、AvrPpiBPto、AvrPto、AvrPtoB、VirPphA、AvrRpmL、HopPtoE、HopPtoF、HopPtoN、PopB、PopP2、AvrBs3、XopD和AvrXv3。更优选的T3SS效应蛋白或其N末端片段选自SopE、SptP、YopE、ExoS、SopB、IpgB1、IpgD、YopJ、YopH、EspF、OspF、ExoS、YopO、YopP、YopE、YopM、YopT、其中最优选的T3SS效应蛋白或其N末端片段选自IpgB1、SopE、SopB、SptP、OspF、IpgD、YopH、YopO、YopP、YopE、YopM、YopT、特别是YopE或其N末端片段。同样优选的T3SS效应蛋白或其N末端片段选自SopE、SopE2、SptP、SteA、SipA、SipB、SipD、SopA、SopB、SopD、IpgB1、IpgD、SipC、SifA、SifB、SseJ、Sse、SrfH、YopJ、AvrA、AvrBsT、YopH、YpkA、Tir、EspF、TccP2、IpgB2、OspF、Map、OspG、Ospl、IpaH、VopF、ExoS、ExoT、HopAB2、AvrRpt2、HopAO1、HopU1、GALA蛋白家族、AvrBs2、AvrD1、YopO、YopP、YopE、YopT、EspG、EspH、EspZ、IpaA、IpaB、IpaC、VirA、IcsB、OspC1、OspE2、IpaH9.8、IpaH7.8、AvrB、AvrD、AvrPphB、AvrPphC、AvrPphEP、AvrPpiBPto、AvrPto、AvrPtoB、VirPphA、AvrRpmL、HopPtoD2、HopPtoE、HopPtoF、HopPtoN、PopB、PopP2、AvrBs3、XopD和AvrXv3。同样更优选的T3SS效应蛋白或其N末端片段选自SopE、SptP、SteA、SifB、SopB、IpgB1、IpgD、YopJ、YopH、EspF、OspF、ExoS、YopO、YopP、YopE、YopT、其中同样最优选的T3SS效应蛋白或其N末端片段选自IpgB1、SopE、SopB、SptP、SteA、SifB、OspF、IpgD、YopH、YopO、YopP、YopE和YopT、特别是SopE、SteA或YopE或其N末端片段、更特别是SteA或YopE或其N末端片段、最特别是YopE或其N末端片段。
在有些实施方案中，由第一DNA序列编码的细菌T3SS效应蛋白的递送信号包含细菌T3SS效应蛋白或其N末端片段，其中其N末端片段包括至少前10个，优选至少前20个，更优选至少前100个氨基酸。
一些实施方案中，由第一DNA序列编码的细菌T3SS效应蛋白的递送信号包含细菌T3SS效应蛋白或其N末端片段，其中细菌T3SS效应蛋白或其N末端片段包含伴侣结合位点。
优选的包含伴侣结合位点的T3SS效应蛋白或其N末端片段包含伴侣伴侣结合位点和T3SS效应蛋白或其N末端片段的以下组合：SycE-YopE、InvB-SopE、SicP-SptP、SycT-YopT、SycO-YopO、SycN/YscB-YopN、SycH-YopH、SpcS-ExoS、CesF-EspF、SycD-YopB、SycD-YopD。更优选的是SycE-YopE、InvB-SopE、SycT-YopT、SycO-YopO、SycN/YscB-YopN、SycH-YopH、SpcS-ExoS、CesF-EspF。最优选的是YopE或其包含SycE伴侣结合位点的N-末端片段，例如YopE效应蛋白的N-末端片段，其含有YopE效应蛋白的N-末端138个氨基酸，本文称为YopE1-138，如图所示在SEQ ID NO.2或SopE或其N末端片段，其包含InvB伴侣结合位点，如SopE效应蛋白的N-末端片段，其含有SopE效应蛋白的N-末端81或105个氨基酸，本文称为SopE1-81或SopE1-81，如SEQ ID NO.142或143所示。
在本发明的一个实施方案中，重组革兰氏阴性细菌菌株是耶尔森氏菌菌株，并且由第一DNA序列编码的细菌T3SS效应蛋白的递送信号，包含YopE效应子蛋白或N末端部分，优选为小肠结肠炎耶尔森氏菌菌株YopE效应蛋白或其N末端部分。优选地，SycE结合位点包含在YopE效应蛋白的N末端部分内。在这方面，YopE效应子蛋白的N末端片段可以包含N末端的12、16、18、52、53、80或138个氨基酸[10，37，38]。最优选的是如在Forsberg和Wolf-Watz[39]中所描述的，在本文中称为YopE1-138以及如SEQ ID NO.2所示的YopE效应蛋白的N-末端片段，其含有YopE效应蛋白的N末端138个氨基酸。
在本发明的一个实施方案中，重组革兰氏阴性细菌菌株是沙门氏菌菌株，并且由第一DNA序列编码的细菌T3SS效应蛋白的递送信号包含SopE或SteA效应蛋白或其N末端部分，优选肠沙门氏菌SopE或SteA效应蛋白或其N末端部分。优选地，伴侣结合位点包含在SopE效应蛋白的N末端部分内。在这方面，SopE效应蛋白的N末端片段可以包含N末端81或105个氨基酸。最优选的是全长SteA和含有如SEQ ID NO.142或143所描述的效应蛋白的N-末端105个氨基酸的SopE效应蛋白的N末端片段。
本领域技术人员熟悉鉴定能够递送蛋白质的效应子蛋白的多肽序列的方法。例如，由Sory等人[13]描述的一种这样的方法。简言之，Yop蛋白的各种部分可以框内融合到报告子酶，例如百日咳博德特氏菌脂环化酶的钙调蛋白激活的腺苷酸环化酶结构域(或Cya)。Yop-Cya杂合蛋白到真核细胞的胞质溶胶中的递送通过在感染的真核细胞中出现导致cAMP积累的环化酶活性来指示。通过采用这种方法，如果需要，本领域技术人员可以确定能够递送蛋白质的最小序列需求，即最短长度的连续氨基酸序列，参见例如[13]。因此，本发明的优选递送信号至少由能够递送蛋白质的T3SS效应蛋白的最小氨基酸序列组成。在一个实施方案中，本发明提供突变体重组革兰氏阴性细菌菌株，特别是重组革兰氏阴性细菌菌株，其对于至少一种T3SS功能性效应蛋白的产生是缺陷的。
根据本发明，这种突变体革兰氏阴性细菌菌株如这样的突变型耶尔森氏菌菌株可以通过将至少一个突变引入至少一种效应物编码基因中来产生。优选地，就耶尔森氏菌菌株而言，这种效应物编码基因包括YopE、YopH、YopO/YpkA、YopM、YopP/YopJ和YopT。
优选地，就沙门氏菌菌株而言，这些效应物编码基因包括AvrA、CigR、GogB、GtgA、GtgE、PipB、SifB、SipA/SspA、SipB、SipC/SspC、SipD/SspD、SlrP、SopB/SigD、SopA、SpiC/SsaB、SseB、SseC、SseD、SseF、SseG、Ssel/SrfH、SopD、SopE、SopE2、SspH1、SspH2、PipB2、SifA、SopD2、SseJ、SseK1、SseK2、SseK3、SseL、SteC、SteA、SteB、SteD、SteE、SpvB、SpvC、SpvD、SrfJ、SptP。最优选地，所有效应物编码基因都缺失。本领域技术人员可以使用任何数量的标准技术在这些T3SS效应基因中产生突变。Sambrook等描述了这样的技术。参见Sambrook等[40]。
根据本发明，突变可以在效应物编码基因的启动子区产生，使得这种效应基因的表达被消除。
突变也可以在效应物编码基因的编码区中产生，使得编码的效应子蛋白的催化活性被消除。效应蛋白的“催化活性”通常指效应蛋白的抗靶细胞功能，即毒性。这种活性由效应子蛋白的催化结构域中的催化基序控制。用于鉴定效应蛋白的催化结构域和/或催化基序的方法是本领域技术人员公知的。例如参见[41，42]。
因此，本发明的一个优选的突变是整个催化结构域的缺失。另一个优选的突变是效应物编码基因中的移码突变，使得催化结构域不存在于由这种“移码”基因表达的蛋白质产物中。最优选的突变是具有效应子蛋白的整个编码区缺失的突变。本发明还涵盖其它突变，例如小缺失或碱基对取代，其在效应子蛋白的催化基序中产生，导致破坏给定效应子蛋白的催化活性。
在T3SS功能效应蛋白的基因中产生的突变可以通过许多方法引入特定菌株。一种这样的方法包括将突变基因克隆到“自杀”载体中，该载体能够通过等位基因交换将突变序列引入菌株。这种“自杀”载体的一个例子描述在[43]中。
以这种方式，可将多个基因中产生的突变连续地引入产生多突变体的革兰氏阴性细菌菌株中，例如六重突变重组菌株。这些突变序列的引入顺序并不重要。在有些情况下，可能需要仅突变一些但不是所有的效应基因。因此，本发明进一步考虑除六突变型耶尔森氏菌以外的多突变体耶尔森氏菌，例如双突变体、三突变体、四突变体和五重突变株。为了递送蛋白质的目的，本发明突变株的分泌和转运系统需要是完整的。
本发明优选的重组革兰氏阴性细菌菌株是六联突变型耶尔森氏菌菌株，其中所有效应物编码基因都被突变，使得所得的耶尔森氏菌不再产生任何功能效应蛋白。对于小肠结肠炎耶尔森氏菌，这种六联突变型耶尔森氏菌菌株被命名为ΔyopH、O、P、E、M、T。如实例，这样的六联突变体可以从肠道结肠炎耶尔森氏菌MRS40菌株产生，产生小肠结肠炎耶尔森氏菌MRS40ΔyopH、O、P、E、M、T，这是优选的。
本发明的另一方面涉及用于与重组革兰氏阴性细菌菌株组合用于将期望的蛋白质递送到真核细胞中的载体，其中所述载体沿5'至3'方向包含：
启动子；
编码细菌T3SS效应蛋白的递送信号的第一DNA序列，可操作地连接到所述启动子；
框内融合到所述第一DNA序列的3'末端的编码异源蛋白质的第二DNA序列；和作为选择的
编码蛋白酶切割位点的第三DNA序列，其中所述第三DNA序列位于所述第一DNA序列的3'末端和所述第二DNA序列的5'末端之间。
如上所述的启动子，异源蛋白和蛋白酶切割位点可用于革兰氏阴性细菌菌株的载体。
根据本发明可以使用的载体取决于本领域技术人员已知的革兰氏阴性细菌菌株。根据本发明可以使用的载体包括表达载体、用于染色体或毒力质粒插入的载体和用于染色体或毒力质粒插入的DNA片段。在例如耶尔森氏菌属、埃希氏菌属、沙门氏菌属或假单胞菌属菌株上有用的表达载体是pUC、pBad、pACYC、pUCP20和pET质粒。在例如耶尔森氏菌属、埃希氏菌属、沙门氏菌属或假单胞菌属菌株上有用的用于染色体或毒力质粒插入的载体是例如pKNG101。用于染色体或毒力质粒插入的DNA片段是指在例如耶尔森氏菌属、埃希氏菌属、沙门氏菌属或假单胞菌属菌株如λ-红色基因工程。
用于染色体或毒力质粒插入的载体或用于染色体或毒力质粒插入的DNA片段可以插入本发明的第一、第二和/或第三DNA序列，使得第一、第二和/或第三DNA序列可操作地连接到内源重组革兰氏阴性细菌菌株的启动子。因此，如果使用用于染色体或毒力质粒插入的载体或用于染色体或毒力质粒插入的DNA片段，可以在内源细菌DNA(染色体或质粒DNA)上编码内源启动子，并且仅第一和第二DNA序列是由用于染色体或毒力质粒插入的工程化载体或用于染色体或毒力质粒插入的DNA片段提供。因此，用于转化重组革兰氏阴性细菌菌株的载体不必包含启动子，即本发明的重组革兰氏阴性细菌菌株可以用不包含启动子的载体转化。优选地，使用表达载体。本发明的载体通常用于通过细菌T3SS将异源蛋白质在体外和体内递送到真核细胞中。
用于耶尔森氏菌的优选表达载体选自pBad_Si1和pBad_Si2。通过将含有用于YopE和SycE的内源启动子的SycE-YopE1-138片段从纯化的pYV40克隆到pBad-MycHisA(Invitrogen)的KpnI/HindIII位点中构建pBad_Si2。另外的修饰包括通过消化、Klenow片段处理和重连接去除pBad-MycHisA的NcoI/BgUI片段。此外在YopE1-138的3'端，添加以下切割位点：XbaI-XhoI-BstBI-(HindIII)。pBad Sil等于pBad_Si2，但编码从pEGFP-Cl(Clontech)在阿拉伯糖诱导型启动子下的NcoI/BgUI位点扩增的EGFP。
沙门氏菌的优选表达载体选自pSi_266、pSi_267、pSi_268和pSi_269。从肠炎沙门氏菌(S.enterica)SL1344中扩增含有相应的内源性启动子和SteA1-20片段(pSi_266)、全长SteA序列(pSi_267)、SopE1-81片段(pSi_268)或SopE1-105片段(pSi_269)的质粒pSi_266、pSi_267、pSi_268和pSi_269基因组DNA并克隆到pBad-MycHisA(Invitrogen)的NcoI/KpnI位点。
本发明的载体可包括其它序列元件，例如3'终止序列(包括终止密码子和poly A序列)或赋予药物抗性的基因，其允许选择已接受本载体的转化体。本发明的载体可以通过许多已知的方法转化到重组革兰氏阴性细菌菌株中。为了本发明的目的，用于导入载体的转化方法包括但不限于电穿孔、磷酸钙介导的转化、缀合或其组合。例如，可以通过标准电穿孔程序将载体转化入第一细菌菌株。随后，这样的载体可以通过缀合，也称为“移动”的过程从第一细菌菌株转移到期望的菌株中。可以用例如抗生素选择转化体(即：吸收载体的革兰氏阴性细菌菌株)。这些技术是本领域公知的。参见例如[13]。
根据本发明，本发明的重组革兰氏阴性细菌菌株的表达载体的启动子可以是相应菌株的T3SS效应蛋白的天然启动子或相容的细菌菌株或用于表达载体的启动子其可用于例如耶尔森氏菌属、埃希氏菌属、沙门氏菌属或假单胞菌属菌株，例如pUC和pBad。这样的启动子是T7启动子，Plac启动子或Ara-bad启动子。如果重组革兰氏阴性细菌菌株是耶尔森氏菌菌株，则启动子可以来自耶尔森氏病毒virulon基因。“耶尔森氏病毒virulon基因”是指在耶尔森氏菌pYV质粒上的基因，其表达受温度和通过与靶细胞接触两者控制。这些基因包括编码分泌机制元件(Ysc基因)的基因、编码转运蛋白(YopB，YopD和LcrV)的基因、编码控制元件的基因(YopN，TyeA和LcrG)、编码T3SS效应子伴侣(SycD、SycE、SycH、SycN、SycO和SycT)以及编码效应子(YopE、YopH、YopO/YpkA、YopM、YopT和YopP/YopJ)的基因以及其他pYV编码的蛋白如VirF和YadA。在本发明的优选实施方案中，启动子是T3SS功能效应物编码基因的天然启动子。如果重组革兰氏阴性细菌菌株是耶尔森氏菌菌株，则启动子选自YopE、YopH、YopO/Y pkA、YopM和YopP/YopJ中的任一个。更优选地、启动子来自YopE或SycE。
如果重组革兰氏阴性细菌菌株是沙门氏菌菌株，则启动子可以来自Spil或Spill致病性岛或来自其他地方编码的效应蛋白。这些基因包括编码分泌机制元件的基因、编码转运蛋白的基因、编码控制元件的基因、编码T3SS效应子伴侣的基因、编码效应子的基因以及由SPI-1或SPI-2编码的其它蛋白。在本发明的一个优选实施方案中，启动子是T3SS功能效应物编码基因的天然启动子。如果重组革兰氏阴性细菌菌株是沙门氏菌菌株，则启动子选自任何一种效应蛋白。更优选地，启动子来自SopE、InvB或SteA。
在优选的实施方案中，表达载体包含编码蛋白酶切割位点的DNA序列。功能性和通常适用的切割位点的产生允许在转运后切割递送信号。由于递送信号可干扰靶细胞内转运蛋白的正确定位和/或功能，在递送信号和目标蛋白之间引入蛋白酶切割位点提供了几乎天然蛋白首次递送到真核细胞中。优选地，蛋白酶切割位点是被选自肠激酶(轻链)、肠肽酶、解离蛋白酶、人鼻病毒蛋白酶3C、TEV蛋白酶、TVMV蛋白酶、因子Xa的蛋白酶或其催化结构域切割的氨基酸基序蛋白酶和凝血酶，更优选由TEV蛋白酶切割的氨基酸基序。同样优选的蛋白酶切割位点是被选自肠激酶(轻链)、肠肽酶、解离蛋白酶、人类鼻病毒蛋白酶3C、TEV蛋白酶、TVMV蛋白酶、因子Xa蛋白酶、泛素加工蛋白酶(称为去泛素化酶)和凝血酶一组的蛋白酶或催化结构域切割的氨基酸基序。最优选的是被TEV蛋白酶或泛素加工蛋白酶切割的氨基酸基序。因此，在本发明的另一个实施方案中，异源蛋白质通过蛋白酶从细菌T3SS效应蛋白的递送信号裂解。优选的裂解方法是这样的方法，其中：
a)蛋白酶通过本文所述的重组革兰氏阴性细菌菌株转移到真核细胞中，其表达包含来自细菌T3SS效应蛋白的递送信号和作为异源蛋白的蛋白酶的融合蛋白；或
b)蛋白酶在真核细胞中为组成型或瞬时表达。
通常，用于将期望的蛋白质递送到真核细胞中的重组革兰氏阴性细菌菌株和将蛋白酶转运到真核细胞中的重组革兰氏阴性菌株是不同的。
在本发明的一个实施方案中，载体包含编码标记分子或标记分子的受体位点的另外的DNA序列。编码标记分子或标记分子的受体位点的另外的DNA序列通常与第二DNA序列的5'端或3'端融合。用于标记分子的优选的标记分子或受体位点选自增强的绿色荧光蛋白(EGFP)、香豆素、香豆素连接酶受体位点、试卤灵、resurofm连接酶受体位点、与FlAsH一起使用的四半胱氨酸基序/ReAsH染料(life technologies)。最优选的是试卤灵和resurofm连接酶受体位点或EGFP。标记分子或受体位点对标记分子的使用将导致标记分子与目标异源蛋白质的连接，然后将其原样传递到真核细胞中，使得能够通过例如活细胞显微镜追踪蛋白质。在本发明的一个实施方案中，载体包含编码肽标签的另外的DNA序列。编码肽标签的另外的DNA序列通常融合到第二DNA序列的5'端或3'端。优选的肽标签选自Myc标签、His标签、Flag标签、HA标签、Strep标签或V5标签或这些组中的两个或多个标签的组合。最优选的是Myc标签、Flag标签、His标签和组合的Myc-和His-标签。使用肽标签将导致例如通过使用抗标签抗体的免疫荧光或Western印迹使得标记蛋白质具有可追踪性。此外，使用肽标签允许在分泌到培养物上清液中之后或在转移到真核细胞中之后亲和纯化期望的蛋白质，在这两种情况下，使用适合相应标签的纯化方法(例如使用的金属螯合亲和纯化与Flag标签一起使用的基于His标签或抗Flag抗体的纯化)。
在本发明的一个实施方案中，载体包含编码核定位信号(NLS)的另外的DNA序列。编码核定位信号(NLS)的另外的DNA序列通常融合到第二DNA序列的5'端或3'端，其中所述另外的DNA序列编码核定位信号(NLS)。优选的NLS选自SV40大T抗原NLS及其衍生物[44]以及其它病毒NLS。最优选的是SV40大T抗原NLS及其衍生物。
在本发明的一个实施方案中，载体包含多克隆位点。多克隆位点通常位于第一DNA序列的3'端和/或第二DNA序列的5'端或3'端。载体可包含一个或多于一个多克隆位点。优选的多克隆位点选自由XhoI、XbaI、HindIII、NcoI、NotI、EcoRI、EcoRV、BamHI、NheI、SacI、SalI、BstBI组成的限制酶组。最优选的是XbaI、XhoI、BstBI和HindIII。
从载体的融合的第一和第二和任选的第三DNA序列表达的蛋白质也称为“融合蛋白”或“杂合蛋白”，即递送信号和异源蛋白的融合蛋白或杂合物。融合蛋白也可以包括例如递送信号和两种或更多种不同的异源蛋白。
本发明涉及用于将如上所述的异源蛋白质递送到细胞培养物以及体内的真核细胞中的方法。因此,在一个实施方案中，递送异源蛋白质的方法包括
i)培养如本文所述的革兰氏阴性细菌菌株；
ii)用i)中的革兰氏阴性细菌菌株接触真核细胞，其中包含来自细菌T3SS效应蛋白的递送信号和异源蛋白的融合蛋白由革兰氏阴性细菌菌株表达并转移入真核细胞；和任选地
iii)切割所述融合蛋白使得异源蛋白与菌T3SS效应蛋白的递送信号切割开来。
在有些实施方案中，包含来自细菌T3SS效应蛋白的递送信号和异源蛋白的至少两种融合蛋白由革兰氏阴性细菌菌株表达并通过本发明的方法转移到真核细胞中。
可以根据本领域已知的方法(例如：FDA、Bacteriological Analytical Manual(BAM)、第8章：小肠结肠炎耶尔森氏菌)培养重组革兰氏阴性细菌菌株从而表达包含来自细菌T3SS效应蛋白的递送信号和异源蛋白的融合蛋白。优选地、重组革兰氏阴性细菌菌株可以在脑心浸液肉汤中在如在28℃时培养。为了诱导T3SS以及如YopE/SycE启动子依赖基因的表达，细菌可以在37℃生长。
在优选的实施方案中，使真核细胞与i)的两种革兰氏阴性细菌菌株接触，其中第一革兰氏阴性细菌菌株表达第一融合蛋白，其包含来自细菌T3SS效应蛋白的递送信号和第一异源蛋白，并且第二革兰氏阴性细菌菌株表达第二融合蛋白，其包含来自细菌T3SS效应蛋白的递送信号和第二异源蛋白，使得第一和第二融合蛋白转运到真核细胞中。该实施方案提供了例如真核细胞与两种细菌菌株共感染如递送例如两个不同的杂交蛋白质进入细胞以解决它们的功能性互作的有效方法。本发明涉及广范围的真核细胞，其可以被本发明的重组革兰氏阴性细菌菌株靶向。Hi-5(BTI-TN-5B1-4；life technologiesB855-02)、HeLa细胞、HeLa Ccl2(如ATCC No.CCL-2)、成纤维细胞、例如3T3成纤维细胞(ATCC号CCL-92)或Mef(ATCC号SCRC-1040)、Hek(ATCC号CRL-1573)、HUVEC(ATCC号PCS-100-013)、CHO如ATCCNo.CCL-61)、Jurkat(如ATCC No.TIB-152)、Sf-9(如ATCC No.CRL-1711)、HepG2(如ATCCNo.HB-8065)、Vera CCL-81)、MDCK(ATCC编号CCL-34)、THP-1(ATCC编号TIB-202)、J774(编号TIB-67)、RAW(编号TIB-、Caco2(如ATCC No.HTB-37)、NCI细胞系(如ATCC No.HTB-182)、DU145(如ATCC No.HTB-81)、Lncap(如ATCC No.CRL-1740)、MCF-7(ATCC No.HTB-22)、MDA-MB细胞系(如ATCC No.HTB-128)、PC3(如ATCC No.CRL-1435)、T47D(如ATCC No.CRL-2865)、A549ATCC No.CCL-185)、U87(如ATCC No.HTB-14)、SHSY5Y(如ATCC No.CRL-2266s)、Ea.Hy926(如ATCC No.CRL-2922)、Saos-2HTBH-85)、4T1(如ATCC No.CRL-2539)、B16F10(如ATCC No.CRL-6475)或原代人肝细胞(如life technologiesHMCPIS)、优选HeLa、Hek、HUVEC、3T3、CHO、Jurkat、Sf-9、HepG2Vera、THP-1、Caco2、Mef、A549,4T1、B16F10和原代人肝细胞，最优选HeLa、Hek、HUVEC、3T3、CHO、Jurkat、THP-1、A549和Mef。“靶”是指重组革兰氏阴性细菌菌株对真核细胞的细胞外粘附。
根据本发明，蛋白质的递送可以通过在适当的条件下使真核细胞与重组革兰氏阴性细菌菌株接触来实现。关于诱导virulon基因的表达和转运的条件，包括期望的温度、Ca++浓度、添加刚果红的诱导剂。本领域技术人员通常可获得各种参考文献和技术，其中重组将革兰氏阴性细菌菌株和靶细胞混合等。参见例如[45]。条件可以根据待靶向的真核细胞的类型和待使用的重组细菌菌株而变化。这些变化可以由本领域技术人员使用常规技术来解决。
本领域技术人员还可以使用多种测定来确定融合蛋白的递送是否成功。例如：可以使用识别融合标签(如Myc标签)的抗体通过免疫荧光检测融合蛋白。测定还可以基于被递送的蛋白质的酶活性如[13]所述的测定。
在一个实施方案中，本发明提供了纯化异源蛋白的方法，包括培养如本文所述的革兰氏阴性细菌菌株，使得包含来自细菌T3SS效应蛋白的递送信号和异源蛋白的融合蛋白被表达和分泌培养物的上清液。所表达的融合蛋白还可以在来自细菌T3SS效应蛋白的递送信号和异源蛋白之间包含蛋白酶切割位点和/或可以进一步包含肽标签。
因此，在一个具体实施方案中，纯化异源蛋白质的方法包括：
i)培养本文所述的革兰氏阴性细菌菌株，使得包含来自细菌T3SS效应蛋白的递送信号、异源蛋白和在细菌T3SS效应蛋白的递送信号和异源蛋白之间蛋白酶裂解位点的融合蛋白表达并分泌到培养物的上清液中；
ii)向培养物的上清液中加入蛋白酶，其中所述蛋白酶切割融合蛋白，使得异源蛋白质与来自细菌T3SS效应蛋白的递送信号分割；
iii)任选地从培养物的上清液中分离异源蛋白质。
因此，在另一个具体实施方案中，纯化异源蛋白质的方法包括：
i)如本文所述培养革兰氏阴性细菌菌株，使得包含来自细菌T3SS效应蛋白的递送信号、异源蛋白和肽标签的融合蛋白被表达并分泌到培养物的上清液中；
ii)靶向肽标签，例如通过上清液的亲和柱纯化。
因此，在另一个具体实施方案中，纯化异源蛋白质的方法包括：
i)培养如本文所述的革兰氏阴性细菌菌株，使得包含来自细菌T3SS效应蛋白的递送信号、异源蛋白质和在细菌T3SS效应蛋白的递送信号与异源蛋白质之间的肽标签的融合蛋白表达并分泌到培养物的上清液中；
ii)向培养物的上清液中加入蛋白酶，其中所述蛋白酶切割融合蛋白，使得异源蛋白质与来自细菌T3SS效应蛋白的递送信号切割；
iii)靶向肽标签，例如通过上清液的亲和柱纯化。
在上述特定实施方案中，蛋白酶可以以例如纯化的蛋白酶蛋白的形式加入到培养物的上清液中，或通过向培养物的上清液中加入表达和分泌蛋白酶的细菌菌株。其它步骤可包括除去蛋白酶如通过亲和柱纯化。
在一个实施方案中，本发明提供本文所述的重组革兰氏阴性细菌菌株用于药物。
在一个实施方案中，本发明提供了本文所述的重组革兰氏阴性细菌菌株，其用于将异源蛋白质如药物或如疫苗递送至受试者。异源蛋白质可以通过使革兰氏阴性细菌菌株与真核细胞例如真菌细胞接触而如疫苗递送给受试者，例如与活的动物在体内这样异源蛋白质转移到活动物中，然后产生针对异源蛋白质的抗体。产生的抗体可以直接使用或分离和纯化并用于诊断、研究以及治疗用途。包含抗体或其中含有的DNA序列的B细胞可以用于进一步产生特异性抗体用于诊断、研究以及治疗用途。
在一个实施方案中，本发明提供了递送异源蛋白质的方法，其中异源蛋白质在体外递送到真核细胞中。在另一个实施方案中，本发明提供了递送异源蛋白质的方法，其中真核细胞是活的动物，其中活体动物与革兰氏阴性细菌菌株在体内接触使得融合蛋白转运到活的动物中。优选的动物是哺乳动物，更优选人。
在另一个实施方案中，本发明提供了如上所述的重组革兰氏阴性细菌菌株用于由递送的异源蛋白质引发的细胞途径或事件的抑制剂的高通量筛选的用途。
在另一个实施方案中，本发明提供革兰氏阴性细菌菌株的文库，其中由革兰氏阴性细菌菌株的表达载体的第二DNA序列编码的异源蛋白质是人或鼠蛋白质、优选人蛋白质。其中由革兰氏阴性细菌菌株表达的每种人或胞壁质蛋白在氨基酸序列上不同。可能的库可以包含Addgene人激酶Orf收集物的560蛋白(Addgene No.1000000014)。如用于表达的克隆载体可以使用上述表达载体。在另一个实施方案中，本发明提供了试剂盒，其包含本文所述的载体及表达和分泌能够切割载体包含的蛋白酶裂解位点的蛋白酶的细菌菌株。特别有用的载体是与细菌菌株组合用于将所需蛋白质递送到如上所述的真核细胞中的载体，其中所述载体沿5'至3'方向包含：
启动子；
编码来自细菌T3SS效应蛋白的递送信号的第一DNA序列，可操作地连接到所述启动子；
框内融合到所述第一DNA序列的3'末端的编码异源蛋白质的第二DNA序列；
编码蛋白酶切割位点的第三DNA序列，其中所述第三DNA序列位于所述第一DNA序列的3'末端和所述第二DNA序列的5'末端之间。
实施例
实施例1：
A)材料和方法
细菌菌株及其生长条件。本研究中使用的菌株列于图15A至M。用于质粒纯化和克隆的大肠杆菌Top 10以及用于接合的大肠杆菌Sm10λ以及用于繁殖pKG101的大肠杆菌BW19610[46]，常规地在37℃下在LB琼脂平板上和在LB肉汤中生长。安非他酮以200μg/mL(Yersinia)或100μg/mL(大肠杆菌)的浓度使用以选择表达载体。链霉素以100μg/mL的浓度使用以选择自杀载体。肠炎克雷伯菌MRS40[36]非安非他明抗性E40衍生物[13]及其衍生菌株在脑心浸液(BHI；Difco)中常规生长。向所有小肠结肠炎耶尔森氏菌菌株中加入萘啶酸(35μg/mL)，并且所有的肠炎杆菌菌株另外补充100μg/mL内消旋-2,6-二氨基庚二酸(mDAP，Sigma Aldrich)。肠杆菌SL1344在37℃下常规地在LB琼脂平板上和在LB肉汤中生长。使用浓度为100μg/mL的安非他酮以选择肠炎沙门氏菌中的表达载体。
小肠结肠炎耶尔森氏菌的遗传调控。小肠结肠炎耶尔森氏菌的遗传调控已经被描述[47，48]。简言之，通过2-片段重叠PCR使用纯化的pYV40质粒或基因组DNA作为模板构建用于修饰或缺失pYV质粒或染色体上的基因修饰或缺失的突变体，生成相应基因缺失或修饰部分两侧200-250bp的侧翼。将所得片段克隆在大肠杆菌BW19610[46]中的pKNG101[43]中。将序列验证的质粒转化到大肠杆菌Sm10λpir中，从中将质粒转移到相应的小肠结肠炎耶尔森氏菌株中。携带整合载体的突变体在无选择压力的情况下繁殖一代。然后使用蔗糖选择失去载体的克隆。最后通过菌落PCR鉴定突变体。
质粒的构建。质粒pBad_Si2或pBad Sil(图10)用于克隆具有YopE(SEQ ID No.2)的N末端138个氨基酸的融合蛋白。通过将包含用于YopE和SycE的内源启动子的SycE-YopE1-138片段从纯化的pYV40克隆到pBad-MycHisA(Invitrogen)的KpnI/HindIII位点中构建pBad_Si2。另外的修饰包括通过消化，Klenow片段处理和重新连接除去pBad-MycHisA的NcoI/BgUI片段。将双向转录终止子(BBa_B1006；iGEM foundation)克隆到KpnI切割和Klenow处理(pBad_Si2)或BgIII切割位点(pBad Sil)中。此外，在YopE1-138的3'端，添加以下切割位点：XbaI-XhoI-BstBI-(HindIII)(图10B)。pBad Sil等于pBad_Si2，但编码从pEGFP-Cl(Clontech)在阿拉伯糖诱导型启动子下的NcoI/BgUI位点扩增的EGFP。从肠炎沙门氏菌(S.enterica)SL1344中扩增含有相应的内源性启动子和SteA1-20片段(pSi_266)、全长SteA序列(pSi_267)、SopE1-81片段(pSi_268)或将SopE1-105片段(pSi_269)的质粒pSi_266、pSi_267、pSi_268和pSi_269基因组DNA并克隆到pBad-MycHisA(Invitrogen)的NcoI/KpnI位点。
全长基因或其片段用下表I中列出的特异性引物扩增，并作为与YopE1-138的融合物克隆到质粒pBad_Si2中或在z-BIM(SEQ ID No.21)的情况下克隆到pBad Sil中(参见下表II)。对于与SteA或SopE的融合，合成的DNA构建体被KpnI/HindIII切割并分别克隆到pSi_266、pSi_267、pSi_268或pSi_269中。在细菌种类的基因的情况下，使用纯化的基因组DNA作为模板(S.flexneri M90T、肠道沙门氏菌亚种鼠伤寒沙门氏菌SL1344、汉赛巴尔通体ATCC49882)。对于人基因，如果没有另外说明，使用通用cDNA文库(Clontech)(图15A-M)，从cDNA文库(M.Affolter的赠品)扩增斑马鱼基因。将连接的质粒克隆到大肠杆菌Top 10中。使用如标准大肠杆菌电穿孔的设置将测序的质粒电穿孔到所需的肠道肠杆菌或肠杆菌菌株中。
表I(引物Nr.Si_：序列)
285:CATACCATGGGAGTGAGCAAGGGCGAG
286:GGAAGATCTttACTTGTACAGCTCGTCCAT
287:CGGGGTACCTCAACTAAATGACCGTGGTG
288:GTTAAAGCTTttcgaatctagactcgagCGTGGCGAACTGGTC
292:C AGTctcgagC AAATTCTAAAC AAAATACTTCC AC
293:cagtTTCGAATTAATTTGTATTGCTTTGACGG
296:C AGTctcgagACTAAC AT AAC ACTATCC ACCC AG
297:GTTAAAGCTTTCAGGAGGCATTCTGAAG
299:CAGTctcgagCAGGCCATCAAGTGTGTG
300:cagtTTCGAATCATTTTCTCTTCCTCTTCTTCA
301:CAGTctcgagGCTGCCATCCGGAA
302:cagtTTCGAATCACAAGACAAGGCACCC
306:GTTAAAGCTTGGAGGCATTCTGAAGatacttatt
307:CAGTctcgagCAAATACAGAGCTTCTATCACTCAG
308:GTTAAAGCTTTCAAGATGTGATTAATGAAGAAATG
317:cagtTTCGAACCCATAAAAAAGCCCTGTC
318:GTTAAAGCTTCTACTCTATCATCAAACGATAAAATGg
324:CAGTctcgagTTCACTCAAGAAACGCAAA
339:cagtTTCGAATTTTCTCTTCCTCTTCTTCAcg
341:cgtaTCTAGAAAAATGATGAAAATGGAGACTG
342:GTT AAAGCTTttaGCTGG AG AC GGTG AC
346:CAGTctcgagTTCCAGATCCCAGAGTTTG
347:GTTAAAGCTTTCACTGGGAGGGGG
351:CAGTctcgagctcgagTTATCTACTCATAGAAACTACTTTTGCAG
352:cgcGGATCCtcagtgtctctgcggcatta
353:CATTTATTCCTCCTAGTTAGTCAcagcaactgctgctcctttc
354:gaaaggagcagcagttgctgTGACTAACTAGGAGGAATAAATG
355:cgattcacggattgctttctCATTATTCCCTCCAGGTACTA
356:TAGTACCTGGAGGGAATAATGagaaagcaatccgtgaatcg
357:cgtaTCTAGAcggctttaagtgcgacattc
364:cgtaTCTAGACTAAAGTATGAGGAGAGAAAATTGAA
365:GTTAAAGCTTTCAGCTTGCCGTCGT
367:CGTAtctagaGACCCGTTCCTGGTGC
369:cgtaTCTAGAccccccaagaagaagc
373:GTTAAAGCTTGCTGGAGACGGTGACC
386:CGTAtctagaTCAGGACGCTTCGGAGGTAG
387:CGTAtctagaATGGACTGTGAGGTCAACAA
389:CGTAtctagaGGCAACCGCAGCA
391:GTTAAAGCTTTCAGTCCATCCCATTTCTg
403:CGTAtctagatctggaatatccctggaca
406:GTT AAAGCTTgtctgtctcaatgccacagt
410:CAGTctcgagATGTCCGGGGTGGTg
413:cagtTTCGAATCACTGCAGCATGATGTC
417:CAGTctcgagAGTGGTGTTGATGATGACATG
420:cagtTTCGAATTAGTGATAAAAATAGAGTTCTTTTGTGAG
423:C AGTctcgagATGCAC ATAACTAATTTGGGATT
424:cagtTTCGAATTATACAAATGACGAATACCCTTT
425:GTTAAAGCTTttacaccttgcgcttcttcttgggcggGCTGGAGACGGTGAC
428:CGTAtctagaATGGACTTCAACAGGAACTTT
429:CGTAtctagaGGACATAGTCCACCAGCG
430:GTTAAAGCTTTCAGTTGGATCCGAAAAAC
433:CGTAtctagaGAATTAAAAAAAACACTCATCCCA
434:CGTAtctagaCCAAAGGCAAAAGCAAAAA
435:GTTAAAGCTTTTAGCTAGCCATGGCAAGC
436:CGTAtctagaATGCCCCGCCCC
437:GTTAAAGCTTCTACCCACCGTACTCGTCAAT
438:CGTAtctagaATGTCTGAC ACGTCC AGAGAG
439:GTTAAAGCTTTCATCTTCTTCGCAGGAAAAAG
445:cgcGGATCCttatgggttctcacagcaaaa
446:C ATTT ATTCCTCCT AGTTAGTC Aaggcaacagccaatcaagag
447:ctcttgattggctgttgcctTGACT AACT AGGAGG AATAAATG
448:ttgattgcagtgacatggtgCATTATTCCCTCC AGGTACTA
449:TAGT ACCTGGAGGGAATAATGcaccatgtcactgcaatcaa
450:cgtaTCTAGAtagccgcagatgttggtatg
451:CGTAtctagaGATCAAGTCCAACTGGTGG
463:C AGTctcgaggaaagcttgtttaaggggc
464:cagtTTCGAAttagcgacggcgacg
476:GTT AAAGCTTttACTTGTAC AGCTCGTCC AT
477:CGTAtctagaGTGAGCAAGGGCGAG
478:CAGTctcgagATGGAAGATTATACCAAAATAGAGAAA
479:GTTAAAGCTTCTACATCTTCTTAATCTGATTGTCCa
482:CGTAtctagaATGGCGCTGCAGCt
483:GTTAAAGCTTTCAGTCATTGACAGGAATTTTg
486:CGTAtctagaATGGAGCCGGCGGCG
487:GTTAAAGCTTTCAATCGGGGATGTCTg
492:CGTAtctagaATGCGCGAGGAGAACAAGGG
493:GTTAAAGCTTTCAGTCCCCTGTGGCTGTGc
494:CGTAtctagaATGGCCGAGCCTTG
495:GTTAAAGCTTttaTTGAAGATTTGTGGCTCC
504:CGTAtctagaGAAAATCTGTATTTTC AAAGTGAAAATCTGT ATTTTC AAAGTATGCCCCGCCCC
505:GTTAAAGCTTCCCACCGTACTCGTCAATtc
508:CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAG TATGGCCGAGCCTTG
509:GTTAAAGCTTTTGAAGATTTGTGGCTCCc
511:CGTAtctagaGAAAATCTGTATTTTC AAAGTGAAAATCTGT ATTTTC AAAGTGTGAGCAAGGGCGAG
512:CGTAtctagaGAAAATCTGTATTTTC AAAGTGAAAATCTGT ATTTTC AAAGTCCGCCGAAAAAAAAACGTAAAGTTGTGAGCAAGGGCGAG
513:GTTAAAGCTTttAAACTTTACGTTTTTTTTTCGGCGGCTTGTACAGCTCGTCCAT
515:GTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTCAAAGTGATTATAAAGATGATGATGATAAAATGGCCGAGCCTTG
558:CGT ATCTAGAATGACC AGTTTTGAAGATGC
559:GTTAAAGCTTTC AT GACTC ATTTTC ATCC AT
561:CGTATCTAGAATGAGTCTCTTAAACTGTGAGAACAG
562:GTTAAAGCTTCT AC ACCCCCGC ATC A
580:catgccatggATTTATGGTC ATAGATATGACCTC
585:CAGTctcgagATGCAGATCTTCGTCAAGAC
586:GTTAAAGCTTgctagcttcgaaACCACCACGTAGACGTAAGAC
588:cagtTTCGAAGATTATAAAGATGATGATGATAAAATGGCCGAGCCTTG
612:CGGGGTACCatgaggtagcttatttcctgataaag
613:CGGGGTACCataattgtccaaatagttatggtagc
614:catgccatggCGGCAAGGCTCCTC
615:cggggtaccTTTATTTGTCAAC ACTGCCC
616:cggggtaccTGCGGGGTCTTTACTCG
677:TTACTATTCGAAGAAATTATTCATAATATTGCCCGCCATCTGGCCCAAATTGGTGATGAAATGGATCATTAAGCTTGGAGTA
678:TACTCCAAGCTTAATGATCCATTTCATCACCAATTTGGGCCAGATGGCGGGCAATATTATGAATAATTTCTTCGAATAGTAA
682:TTACTACTCGAGAAAAAACTGAGCGAATGTCTGCGCCGCATTGGTGATGAACTGGATAGCTAAGCTTGGAGTA
683:TACTCCAAGCTTAGCTATCCAGTTCATCACCAATGCGGCGCAGACATTCGCTCAGTTTTTTCTCGAGTAGTAA
表II：克隆的融合蛋白
Yop分泌。通过在BHI-Ox(允许分泌条件)中将培养物转移至37℃进行顶部调节子的诱导[49]。添加葡萄糖作为碳源(4mg/mL)。
通过在4℃下以20800g离心10min来分离总细胞和上清液部分。将细胞沉淀作为总细胞级分。上清液中的蛋白质用10％(w/v)三氯乙酸在4℃沉淀1小时。在离心(20800g，15min)并除去上清液后，将所得沉淀在冰冷的丙酮中洗涤过夜。再次离心样品，弃去上清液，将沉淀物空气干燥并重悬于1x SDS装载染料中。
通过SDS-PAGE分析分泌的蛋白。在每种情况下，每个泳道加载由3×108个细菌分泌的蛋白质。使用12.5％SDS-PAGE凝胶进行免疫印迹检测特异性分泌蛋白。为了检测总细胞中的蛋白质，如果没有另外说明，每个泳道加载2×108个细菌，并且在免疫印迹检测之前在12.5％SDS-PAGE凝胶上分离蛋白质。
使用针对YopE的大鼠单克隆抗体(MIPA193-13A9；1:1000，[50])进行免疫印迹。将针对小肠结肠炎耶尔森氏菌ΔΗΟΡΕΜΤasd的抗血清过夜预先吸附两次以减少背景染色。在用ECL化学发光底物(LumiGlo，KPM)显色之前，用针对大鼠抗体并与辣根过氧化物酶缀合的二抗(1：5000；Southern biotech)进行检测。
细胞培养和感染。HeLa Ccl2、瑞士3T3成纤维细胞、4T1、B16F10和D2A1在补充有10％FCS和2mM L-谷氨酰胺(cDMEM)的Dulbecco’s改进的Eagle培养基(DMEM)中培养。分离HUVEC并如所述[51]进行培养。Jurkat和4T1细胞在补充有10％FCS和2mM L-谷氨酰胺的RPMI1640中培养。使小肠结肠炎耶尔森氏菌在含有添加剂的BHI中在室温下生长过夜，在新鲜BHI中稀释至OD600为0.2，并在室温下生长2小时，然后温度调至37℃下再水浴振荡30min或在EGFP递送的情况下振荡1小时。最后，通过离心(6000rcf，30秒)收集细菌，并用补充有10mM HEPES和2mM L-谷氨酰胺的DMEM洗涤一次。肠杆菌在含有添加剂的LB中在37℃下生长过夜，并且在新鲜LB中1:40稀释并在37℃生长2.5小时(Spil T3SS诱导条件)，或者将过夜培养物在37℃下进一步温育(溢出T3SS诱导条件)。最后，通过离心(6000rcf，30秒)收集细菌，并用补充有10mM HEPES和2mM L-谷氨酰胺的DMEM洗涤一次。将接种在96孔(用于免疫荧光)或6孔(用于免疫印迹)板中的细胞以指定的MOI在补充有10mM HEPES和2mM L-谷氨酰胺的DMEM中感染。在添加细菌后，将板以1750rpm离心1min，并在37℃放置指定的时间段。如果需要，细胞外细菌用庆大霉素(100mg/mL)杀死。在免疫荧光分析的情况下，通过4％PFA固定来终止感染测定。对于免疫印迹分析，用冰冷的PBS洗涤细胞两次，加入磷酸安全裂解缓冲液(Novagen)以裂解细胞。在冰上温育后，将细胞离心(16000rcf，25min，4℃)。收集上清液并通过Bradford BCA测定法(Pierce)在SDS PAGE和免疫印迹之前使用anti-phospho-Akt(Ser473和T308，均为Cell Signaling)、抗肌动蛋白(Millipore)、anti-Bid(CellSignaling)、anti-Myc(Santa Cruz)、anti-p38(Cell Signaling)、anti-phospho-p38(Thr80/Tyr182；Cell Signaling)、anti-Caspase-3pl7(Cell Signaling)和anti-Ink4C(Cell Signaling)抗体。
肠炎沙门氏菌的分泌分析。为了诱导肠炎沙门氏菌的蛋白质分泌，将肠杆菌在定轨振荡器(设定为150rpm)上在含有0.3M NaCl的LB中培养过夜。然后将肠杆菌在含有0.3MNaCl的新鲜LB中以1:50稀释，并在37℃下振荡生长4小时。
通过在4℃下以80000g离心20min来分离总细胞和上清液部分。将细胞沉淀作为总细胞级分。上清液中的蛋白质用三氯乙酸10％(w/v)最终在4℃沉淀1小时。在离心(20800g，15min)并除去上清液后，将所得沉淀在冰冷的丙酮中洗涤过夜。将样品再次离心，弃去上清液，将沉淀物空气干燥并重悬于1x SDS装载染料中。
通过SDS-PAGE分析分泌的蛋白。在每种情况下，每个泳道加载由3×108个细菌分泌的蛋白质。使用12.5％SDS-PAGE凝胶进行免疫印迹检测特异性分泌蛋白。为了检测总细胞中的蛋白质，如果没有另外说明，每个泳道加载2×108个细菌，并且在免疫印迹检测之前在12.5％SDS-PAGE凝胶上分离蛋白质。免疫印迹使用抗Myc抗体(Santa Cruz)进行。
来自感染细胞的T3SS转运蛋白的免疫印迹。如上所述，以100的MOI感染6孔板中的HeLa细胞。在用TEV蛋白酶转位的小肠结肠炎耶尔森氏菌菌株共感染的情况下，设定菌株的OD600，并将两种细菌悬浮液以1：1的比例(如果没有另外说明)在管中混合，然后加入细胞。在感染结束时，将细胞用冰冷的PBS洗涤两次，并通过在小体积的冰冷PBS中刮取来收集。离心(16000rcf，5min，4℃)后，将沉淀溶解于补充有蛋白酶抑制剂混合物(Roche complete，Roche)的0.002％的洋地黄皂苷中。将溶解的沉淀物在冰上孵育5min，然后离心(16000rcf，25min，4℃)。收集上清液并通过Bradford BCA测定(Pierce)并在SDS PAGE和使用anti-Myc(Santa Cruz，9E11)或anti-Ink4C(Cell Signaling)抗体的Western印迹之前分析总蛋白含量。
免疫荧光。如上所述感染接种在96孔板(Corning)中的细胞，并在用4％PFA固定后，用PBS洗涤细胞三次。然后使用5％山羊血清在PBS 0.3％Triton X-100中在室温下封闭孔1小时。将一级抗体(anti-Myc，Santa Cruz，1:100)在含有1％BSA和0.3％Triton X-100的PBS中稀释，并将细胞在4℃孵育过夜。在用含有1％BSA和0.3％Triton X-100的PBS稀释二抗(AF 488抗小鼠，寿命技术，1:250)之前，用PBS洗涤细胞4次。如果需要，进行HoechstDNA染色(寿命技术，1:2500)和/或肌动蛋白染色(Dy647-Phalloidin，DyeOmics)。在有些情况下，在洗涤PFA之后仅直接施用DNA和/或肌动蛋白染色。将细胞在室温下孵育1小时，用PBS洗涤3次，并通过如下所述的自动图像分析进行分析。
自动显微镜和图像分析。使用ImageXpress Micro(Molecular devices，Sunnyvale，USA)自动获得图像。使用MetaXpress(Molecular devices，Sunnyvale，USA)进行抗-Myc染色强度的定量。手动选择除了核区域的细胞内的区域和含有细菌的区域(具有40个像素的面积的圆圈)并记录平均强度。
TNFa刺激和磷酸-p38的免疫印迹。如上所述，以100的MOI感染接种在6孔板中的HeLa细胞。30min后加入庆大霉素以及45分钟后加入TNFα(10ng/mL)。1小时15分钟后将细胞用冰冷的PBS洗涤两次，并加入磷酸安全裂解缓冲液(Novagen)以裂解细胞。在冰上温育后，将细胞离心(16000rcf，25min，4℃)。收集上清液并通过Bradford BCA测定法(Pierce)在SDS PAGE和免疫印迹之前使用anti-Phospho-p38、总p38抗体(Cell Signaling)和抗肌动蛋白抗体(Millipore)。
感染的HeLa细胞的cAMP水平测定。如上所述感染接种在96孔板中的HeLa细胞。在感染前30分钟，将cDMEM改变为补充有10mM HEPES和2mML-谷氨酰胺和100μM 3-异丁基-1-甲基黄嘌呤(IBMX，Sigma Aldrich)的DMEM。60min后，加入庆大霉素并将细胞在37℃下再温育90分钟。根据制造商的说明书(Amersham，cAMP Biotrak，RPN225)，使用竞争性ELISA进行cAMP的测定。作为阳性对照，向补充有10mM HEPES和2mM L-谷氨酰胺和100μM IBMX的DMEM中向细胞中加入指定剂量的霍乱毒素(C8052，Sigma Aldrich)1小时。
斑马鱼胚胎感染、成像和自动图像定量。所有动物实验根据批准的指南进行。斑马鱼保持在标准条件下[52]。胚胎在28.5℃下按小时受精后(hpf)分期[53]。在本研究中使用以下斑马鱼系：野生型鱼(AB/EK和EK/TL)。感染方案遵循[54]中给出的指南。将12hpf胚胎保持在含有0.2mM N-苯基硫脲(PTU)的E3培养基中以防止色素形成。受精2天(dpi)的胚胎用0.2mg/mL三卡因麻醉并使用发环工具在E3中的1％琼脂板上对齐[54]。使小肠结肠炎耶尔森氏菌在补充有0.4％阿拉伯糖和抗生素的BHI和mDap中生长，并在室温下过夜，用含有0.5％阿拉伯糖和其他添加剂的新鲜BHI稀释至OD 600为0.2，在室温下生长2小时，然后温度转变为37℃水浴振荡45分钟。最后，通过离心(6000rcf，30秒)收集细菌，并用PBS洗涤一次。在含有mDAP的PBS中OD 600设定为2。使用Femtojet Microinjector(Eppendorf)使用Femtotips II(Eppendorf)将1-2nL的该悬浮液注射到对齐的斑马鱼胚胎的后脑中，其中针尖用细镊子折断。注射时间设置为0.2s，补偿压力为15hPa(Eppendorf，Femtojet)，注射压力调节在600和800hPa之间。通过显微镜检查和通过对照板检查液滴大小以及相应的接种物。显微注射后，将鱼收集在含有Tricaine和PTU的E3中，并在37℃孵育30分钟，并在28℃下孵育另外5小时。荧光双目(Leica)用于在斑马鱼后脑中感染后1小时观察细菌EGFP荧光，并且丢弃未正确注射的胚胎。在感染结束时，将鱼用2％冰冷的PFA在冰上固定1小时，然后在4℃下用新鲜冰冷的PFA固定过夜。如前所述进行抗体染色[55,56]。毫无疑问，胚胎用0.1％吐温PBS洗涤4次，每次洗涤5分钟，并在室温下用PBS-T+0.5％Triton X-100透化30分钟。将胚在封闭溶液(PBS 0.1％Tween 0.1％TritonX-100 5％山羊血清和1％BSA)中在4℃封闭过夜。将抗体(裂解的胱天蛋白酶-3(Asp 175)，Cell Signaling)在封闭溶液中以1:100稀释，并在4℃下在黑暗中振荡孵育。将鱼用0.1％吐温PBS洗涤7次，持续30分钟，然后加入在封闭溶液中稀释的二抗(山羊抗兔AF647，Invitrogen，1：500)，并在4℃温育过夜。将幼虫用0.1％Tween的PBS在4℃下洗涤4次，每次30分钟，并洗涤过夜，并进一步洗涤3-4次。使用40<x>水浸物镜用Leica TCS SP5共聚焦显微镜拍摄图像。使用Imaris(Bitplane)和ImageJ软件(http://imagej.nih.gov/ij/)分析图像。
通过CellProfiler[57]对记录的z-堆叠图像的最大强度z投影执行图像分析(对于pBad_Si2n＝14或对于z-BIM n＝19)。简而言之，通过GFP通道检测细菌。围绕细菌斑点的每个区域创建具有10个像素的半径的圆。重叠区域在连接构件之间相等地分开。在紧密围绕细菌的那些区域中，测量Caspase 3p17染色强度。
磷酸化蛋白的样品制备。对于每种条件，将HeLa CCL-2细胞的两个6孔板生长至汇合。如上所述感染细胞30分钟。在指定的时间点，将板置于冰上，用冰冷的PBS洗涤两次。然后将样品收集在脲溶液[8M尿素(AppliChem)、0.1M碳酸氢铵(Sigma)、0.1％RapiGest(Waters)、1xPhosSTOP(Roche)中。将样品短暂涡旋，在4℃(Hielscher)超声处理，在热混合器(Eppendorf)上振荡5分钟，并在4℃和16000g下离心20分钟。收集上清液并储存在-80℃用于进一步处理。使用BCA蛋白测定(Pierce)测量蛋白浓度。
磷酸肽富集。用终浓度为10mM的三(2-羧乙基)膦在37℃下将二硫键还原1小时。游离巯基用20mM碘乙酰胺(Sigma)在室温下在黑暗中烷基化30分钟。在室温下用N-乙酰半胱氨酸以终浓度25mM淬灭过量的碘乙酰胺10分钟。加入Lys-C内肽酶(Wako)至最终酶/蛋白质比例为1:200(w/w)，并在37℃温育4小时。随后将溶液用0.1M碳酸氢铵(Sigma)稀释至终浓度低于2M脲，并在37℃下用测序级修饰的胰蛋白酶(Promega)以50:1的蛋白质-酶比消化过夜。在C18Sep-Pak柱(Waters)上脱盐并在真空下干燥。如前所述，从2mg总肽质量中用TiO 2分离磷酸肽[58]。简言之，将干燥的肽溶解在用邻苯二甲酸饱和的80％乙腈(ACN)-2.5％三氟乙酸(TFA)溶液中。将肽加入到在封端的Mobicol离心柱(MoBiTec)中的相同量的平衡的TiO2(5-μm珠尺寸，GL Sciences)中，其在旋转振荡器上温育30分钟。将柱用饱和邻苯二甲酸溶液洗涤两次，用80％ACN和0.1％TFA洗涤两次，最后用0.1％TFA洗涤两次。用0.3MNH4OH溶液洗脱肽。用5％TFA溶液和2M HCl将洗脱液的pH调节至低于2.5。磷酸肽再次用微型C18柱(Harvard Apparatus)脱盐。
LC-MS/MS分析。使用配备有内部填充了1.9μmC18树脂(Reprosil-AQPur，Dr.Maisch)的加热的RP-HPLC柱(75μm×45cm)的EASY nano-LC系统(Thermo FisherScientific)进行肽的色谱分离。使用从98％溶剂A(0.15％甲酸)和2％溶剂B(98％乙腈，2％水，0.15％甲酸)的线性梯度，每个LC-MS/MS运行分析1μg总磷酸肽样品的等分试样至30％溶剂B，历时120分钟，流速为200nl/min。在装备有纳米电喷雾离子源(均为ThermoFisher Scientific)的双压力LTQ-Orbitrap质谱仪上进行质谱分析。每个MSI扫描(在Orbitrap中获得)之后是20个最丰富的前体离子的碰撞诱导解离(CID，在LTQ中获得)，动态排除30秒。对于磷酸肽分析，10种最丰富的前体离子经受能够进行多级激活的CID。总循环时间约为2秒。对于MSI，在最大300ms的时间内在轨道阱电池中累积10 6个离子，并在60,000FWHM(400m/z)的分辨率下扫描。使用正常扫描模式，10 4离子的目标设置和25ms的累积时间获取MS2扫描。带有未分配电荷状态的单电荷离子和离子被排除触发MS2事件。归一化的碰撞能量设定为32％，并且对于每个光谱获得一个微量。无标记定量和数据库搜索。将获得的原始文件导入到Progenesis软件工具(Nonlinear Dynamics，版本4.0)中，以使用默认参数进行无标记定量。MS2光谱直接从Progenesis以mgf格式导出，并使用MASCOT算法(Matrix Science，Version 2.4)针对包含智人的预测SwissProt条目的正向和反向序列的诱骗数据库[59]进行搜索(www.ebi.ac。uk，发布日期16/05/2012)和使用来自MaxQuant软件(版本1.0.13.13)的SequenceReverser工具产生的常见的污染物(总共41,250个序列)。为了鉴定源自小肠结肠炎耶尔森氏菌的蛋白质，针对上述相同数据库搜索非磷酸肽富集的样品，包括预期的肠道沙门氏菌的SwissProt条目(www.ebi.ac.uk，发布日期15/08/2013)前体离子耐受性设定为10ppm，并且将碎片离子耐受性设定为0.6Da。搜索标准设置如下：需要完全胰蛋白酶特异性(在赖氨酸或精氨酸残基之后切割，除非随后是脯氨酸)，允许2次错过切割，将脲基甲基化(C)设定为固定修饰，并将磷酸化(S，T，Y)或氧化(M)分别作为富集TiO 2或未富集样品的可变修饰。最后，数据库搜索结果导出为xmL文件，并导入回到Progenesis软件以进行MSI功能分配。对于磷酸肽定量，输出包含所有检测到的特征的MSI峰丰度的csv文件，并且对于未富集的样品，创建包含基于每种蛋白质的所有鉴定的肽的总和特征强度的所有蛋白质测量的csv文件。重要的是，Progenesis软件设定由相似组的肽识别的蛋白质被分组在一起，并且仅数据库中具有用于单个蛋白质的特定序列的非冲突肽被用于蛋白质定量。使用内部开发的SafeQuant v1.O R脚本(未发布的数据，可从https://github.com/eahrne/SafeQuant/获得)进一步处理这两个文件。简而言之，软件将识别水平假发现率设置为1％(基于诱饵蛋白序列数据库命中的数量)，并且归一化所有样品中鉴定的MS 1峰丰度(提取的离子色谱图，XIC)，即总和XIC的所有确信识别的肽特征被缩放为对于所有LC-MS运行相等。接下来，基于每个时间点的中值XIC，为每个时间点分配所有定量的磷酸肽/蛋白质的丰度比。每个比率的统计显着性由其q值(假发现率调整的p值)给出，通过计算修正的t统计p值[60]和调整多重测试[61]获得。MASCOT自动分配磷酸化残基的位置(得分>10)。所有注释的光谱以及所用的MS原始文件和搜索参数将通过PRIDE合作伙伴库[62]保存到ProteomeXchange Consortium(http：//proteomecentral.proteomexchange。org)。
序列比对使用基于EMBL-EBI web的ClustalW2多序列比对工具在http://www.ebi.ac.uk/Tools/msa/clustalw2/进行。
B)结果
基于YopE融合蛋白的3型分泌的蛋白质递送系统
虽然肠道粘杆菌T3SS效应子YopE(SEQ ID No.1)的N-末端含有足以使异源蛋白质转运的分泌信号[10]，但其伴侣蛋白(SycE)的伴侣结合位点(CBS)不包括在内[63]。我们选择YopE(SEQ ID No.2)的N-末端138个氨基酸与待递送的蛋白质融合，因为已经显示其对于其他异源T3S底物的递送提供最佳结果[38]。由于YopE的这些N-末端138个氨基酸含有CBS，我们进一步决定共表达SycE。从纯化的小肠结肠炎耶尔森氏菌pYV40毒力质粒克隆的SycE-YopE1-138片段含有YopE和其伴侣SycE的内源性启动子(图10)。因此，SycE和任何YopE1-138融合蛋白通过从在RT至37℃的生长的快速温度转换诱导。在37℃的培养时间将影响细菌中存在的融合蛋白量。在YopE1-138(图10B)的3'端添加多克隆位点(MCS)，随后是Myc和6xHis标签和终止密码子。
仔细选择背景应变。首先，为了限制内源效应子的转运，我们使用了对所有已知效应物Yop H，O，P，E，M和T(命名为ΔΗΟΡΕΜΤ)删除的肠道沙门氏菌菌株。此外，我们使用的营养缺陷突变体不能在外源性内消旋-2,6-二氨基庚二酸不存在下生长[65]。该菌株缺失天冬氨酸-β-半醛脱氢酶基因(Aasd)，并由瑞士安全机构归类为生物安全水平1(对AO10088/2的修改)。此外，我们删除粘附蛋白YadA和/或InvA，以提供更大的背景菌株的选择。虽然使用yadA或yadA/invA菌株减少背景信号诱导[66]，所传递的蛋白质量也受到影响[67]。
YopE融合蛋白递送到真核细胞中的表征
在体外分泌测定(参见图1A)中，人工诱导蛋白分泌到周围液体中。在基于TCA的蛋白质沉淀后，使用抗YopE抗体的免疫印迹分析来确定分泌的蛋白质量(图1B)。虽然wt菌株分泌全长YopE，但AHOPEMT asd菌株没有。在存在YopE1-138-Myc-His(进一步称为YopE1-138-Myc；SEQ_ID_No._3)时，较小的YopE条带变得可见(图1B)。因此，YopE1-138片段在本文所述的装置中分泌良好。为了分析蛋白质转移到真核细胞的同源性，我们用YopE1-138-Myc编码菌株感染HeLa细胞，并通过IF染色Myc标签(图2A和B)。虽然在开始只有细菌被染色，在感染后30分钟(p.i.)，细胞轮廓开始可见，其在感染时间增加时增强(图2B)。这种趋势由HeLa细胞内的Myc标签染色强度很好地反映(图2A和B)。YopE1-138-Myc可以在细胞中的任何地方检测到(图2A)，除了核[68]。值得注意的是，大多数(如果不是所有)细胞通过这种方法以可比较的方式达到。由于小肠结肠炎已知感染许多不同的细胞类型[69]，我们遵循YopE1-138-Myc交付到各种细胞系。在感染的鼠成纤维细胞、Jurkat细胞和HUVEC中观察到相同的均一的抗Myc IF染色(图11)。甚至，将MOI向上或向下调节允许调节递送的蛋白质量(图2C)，而仍然大多数细胞保持靶向。低细菌数量不会导致少量具有大量递送蛋白质的细胞，而是大多数细胞含有少量递送蛋白质(图2C)。
T3SS递送蛋白质到细胞核的重定向
由于YopE本身定位于细胞质(图2A)，测试YopE1-138片段是否阻碍核融合蛋白的定位是特别有趣。因此，我们将SV40NLS添加到YopE1-138-EGFP(分别为SEQ ID No.39和SEQ IDNo.38)的C末端(和N末端，相似的结果)。虽然YopE1-138-EGFP(SEQ ID No.37)导致弱的细胞质染色，但是YopE1-138-EGFP-NLS在感染的HeLa细胞中产生更强的核EGFP信号(图3)。这表明YopE1-138片段与NLS的使用相容。虽然mCherry已经用于植物病原体[70]，这代表GFP类蛋白通过编码T3SS的人类或动物致病细菌的成功传递。这验证了SycE和YopE1-138依赖性策略对于选择的许多蛋白质的递送非常有希望。
去除融合蛋白转运到真核细胞后的YopE1-138附属物
而对于细菌递送，YopE1-138片段是非常有益的，它可能阻碍融合蛋白功能和/或定位。因此，其在蛋白质递送后的去除将是最佳的。为此，我们在YopE1-138和融合配偶体(转录调节因子ET1-Myc(SEQ ID No.36和41)[74]和人INK4C(SEQ ID No.3)之间插入两个TEV切割位点(ENLYFQS)[71-73]40和SEQ IDNo.43))。为了保持所提出的方法的优点，我们进一步将TEV蛋白酶(S219V变体；[75])与YopE1-138(SEQ ID No.42)在另一肠炎杆菌菌株中融合。同时用两种菌株感染HeLa细胞。为了允许分析蛋白质的转运部分，在2小时后用已知不裂解细菌([76]；参见图12作为对照)的洋地黄皂苷裂解感染的HeLa细胞(图4)。免疫印迹分析揭示了仅当细胞已经用相应的菌株感染时YopE1-138-2xTEV-切割位点-ET1-Myc或YopE1-138-2xTEV-切割位点Flag-INK4C-Myc的存在(图4A和C)。在用纯化的TEV蛋白酶过夜消化该细胞裂解物时，可以观察到移动带(图4A和C)。该条带对应于具有TEV切割位点的N-末端残基的ET1-Myc(图4C)或Flag-INK4C(图4A)，最可能只有一个丝氨酸。当细胞与递送TEV蛋白酶的菌株共同感染时，相同的裂解的ET1-Myc或Flag-INK4C片段变得可见，表明经由T3SS递送的TEV蛋白酶是功能性的，并且单个细胞已被两种细菌菌株感染(图4A和C)。虽然切割不完全，但大部分转运蛋白质在感染2小时后已经被切割，甚至用纯化的TEV蛋白酶过夜消化也不能产生更好的切割速率(图4B)。据报道，TEV蛋白酶依赖性切割可能需要依赖于融合蛋白的优化[77，78]。转移后TEV蛋白酶依赖性去除YopE1-138附属物因此提供了第一次几乎天然异源蛋白质的T3SS蛋白质递送，仅通过一个N-末端氨基酸改变氨基酸组成。
对YopE片段的TEV蛋白酶依赖性切割的替代方法包括将泛素掺入感兴趣的融合蛋白中。实际上，泛素在其C-末端由一组内源性泛素特异性C末端蛋白酶(去泛素化酶，DUB)加工。由于切割假定发生在泛素的C-末端(在G76之后)，目标蛋白应该不含额外的氨基酸序列。该方法在YopE1-138-泛素-Flag-INK4C-MycHis融合蛋白上测试。在受表达YopE1-138-Flag-INK4C-MycHis的细菌感染的对照细胞中，发现对应于YopE1-138-Flag-INK4C-MycHis的条带，指示融合蛋白的有效转运(图24)。当细胞用YopE1-138-泛素-Flag-INK4C-MycHis-表达细菌感染1小时时，可以看到对应于Flag-INK4C-MycHis大小的另外的条带，表明融合蛋白的一部分被切割。该结果显示，将泛素导入融合蛋白使得能够切割YopE1-138片段而不需要外源蛋白酶。
III型和IV型细菌效应子的转运
来自沙门氏菌的SopE是一种良好表征的鸟嘌呤核苷酸交换因子(GEF)，它与Cdc42相互作用，促进肌动蛋白细胞骨架重塑[79]。鉴于YopE1-138-Myc向HeLa细胞的转运没有影响，转运的YopE1-138-SopE(SEQ ID No.5和135)诱导肌动蛋白网络中的显著变化(图5A)。使用来自志贺氏菌(Shigella flexneri)的另一种GEF效应蛋白IpgB1(SEQ ID No.4)获得了类似的结果。引人注目的是，肌动蛋白细胞骨架的第一次变化在2分钟时被观察到(图5A)。因此，可以得出结论，T3SS依赖性蛋白质递送在通过离心启动感染后立即发生。为了证明严格的T3SS依赖性递送，一个形成到真核细胞膜的转运孔的T3SS蛋白之一被删除(YopB，参见[80])(图12)。
在沙门氏菌感染期间，SopE转运之后是SptP的转运，其作为Cdc42的GTP酶活化蛋白(GAP)[81]。尽管单独的YopE1-138-SopE-Myc(SEQ ID No.135)的转运触发了大量的F-肌动蛋白重排，但与表达YopE1-138-SptP(SEQ ID No.8)的细菌的共感染取消了这种剂量依赖性方式效果(图5B)。抗Myc染色表明这种抑制不是由于YopE1-138-SopE-Myc转运水平的降低(图5B)。这些结果一起表明，细胞与两种细菌菌株的共感染是将两种不同效应物递送到单细胞中以解决其功能相互作用的有效方法。
S.flexneri III型效应物OspF作为磷酸苏氨酸裂解酶，使MAP激酶p38和ER去磷酸化[82]。为了测试转运的YopE1-138-OspF(SEQ ID No.7)的功能，我们监测了用TNFα刺激后p38的磷酸化。在未感染的细胞或用表达YopE1-138-Myc的细菌感染的细胞中，TNFα诱导p38磷酸化。相比之下，在YopE1-138-OspF转运后，TNFα诱导的磷酸化被消除，这表明所递送的OspF对p38具有活性(图6A)。
在沙门氏菌感染期间，III型效应物SopB通过Akt的持续活化来保护上皮细胞免于凋亡[83]。尽管YopE1-138-Myc或YopE1-138-SopE的转运对Akt没有影响，但YopE1-138-SopB(SEQID No.6)的转运在T308和S473处诱导Akt的强烈磷酸化，反映了活性形式(图6B)。使用来自柔红霉素的SopB同源物(IpgD，SEQ ID No.9)获得了类似的结果。总而言之，我们的结果表明基于YopE1-138的递送系统对迄今测试的所有T3S效应器起作用，并且其允许研究涉及中央细胞功能包括细胞骨架、炎症和细胞存活的控制的蛋白质。
许多细菌，包括根癌土壤杆菌、嗜肺军团杆菌和汉赛巴尔通体，使用IV型分泌以将效应物注入细胞中。我们测试了使用我们的工具是否可以将来自henselae的IV型效应物BepA转运到HeLa细胞中。克隆含有C-末端Bid结构域的全长BepA(SEQ ID No.10)和BepAE305-end(SEQ ID No.11)，并用相应的菌株感染细胞。由于BepA显示诱导环AMP(cAMP)的产生[84]，在感染后测量HeLa细胞中cAMP的水平。尽管汉密氏酵母效应物BepG(SEQ IDNo.136)的Bid结构域的转运不能诱导cAMP、全长BepA和BepAE305末端触发的预期量的cAMP产生[84](图6C)。该结果表明，IV型效应物也可以通过基于YopE1-138的递送系统有效地递送到宿主细胞靶中，并且它们是功能性的。
真核蛋白转移到上皮细胞中
为了表明人蛋白质可以通过III型分泌递送，我们融合人细胞凋亡诱导剂用于由肠道沙门氏菌递送至YopE1-138或由肠杆菌递送至SteA1-20、SteA、SopE1-81或SopE1-105。然后我们监测了人BH3相互作用结构域死亡激动剂(BID，SEQ ID No.24)的转运，其是Bcl-2蛋白家族的促凋亡成员。它是由caspase-8(CASP8)诱导的线粒体损伤的介质。CASP8切割BID，并且截短的BID(tBID，SEQ IDNo.25)转运至线粒体，其中它触发细胞色素c释放。后者导致胱天蛋白酶3(CASP3)激活的固有模式，在此期间它被切割成17和12kDa亚基[85]。而用YopE1-138-Myc或YopE1-138-BID表达小肠结肠炎杆菌感染1小时不能诱导细胞凋亡，人tBID的转运比充分表征的细胞凋亡诱导剂星形孢菌素在更大程度上引发细胞死亡(图7A和C)。如所预期的，tBID的转运导致CASP3p17亚基产生，甚至在与星形孢菌素一样大的量(图7A)。为了能够将转运蛋白质量与内源Bid进行比较，将HeLa细胞用洋地黄皂苷裂解，并使用抗Bid抗体通过免疫印迹分析(图7B)。T3SS递送的YopE1-138-tBID在HeLa细胞中达到约内源性Bid水平，而递送的YopE1-138-BID以甚至更高的量(2.5倍)存在(图7B)。HeLa细胞的深度蛋白质组和转录组图谱估计每个细胞4.4×105拷贝BID[86]。因此，可以得出结论，T3SS依赖性人蛋白质递送达到每个细胞105至106个蛋白质。这些数字拟合通过大肠杆菌T3SS转位的纳米抗体的每个细胞的拷贝数[19]。假设对于MOI和对于感染持续时间的因子为10的水平，对于抗生素添加的时间点和对于在感染之前在37℃的培养时间，因子为3.2，递送的蛋白质拷贝/细胞可以从约1000个拷贝/细胞调节到约106个拷贝/细胞。总之，这些结果表明转运的tBID是功能性的并在相关水平递送。这验证了研究蛋白质在细胞凋亡的调节作用，细胞生物学的中心方面的作用的转运工具。
我们进一步将鼠tBID(针对小肠结肠炎耶尔森氏菌的密码子优化；SEQ IDNo.194)或小鼠tBID或小鼠BAX的BH3结构域(在这两种情况下针对小肠结肠炎耶尔森氏菌进行密码子优化；SEQ ID No.138和139)融合至YopE1-138用于由肠道沙门氏菌递送。而不传递蛋白质或YopE1-138-Myc的小肠结肠炎杆菌ΔHoPETMTasd不能诱导细胞凋亡，而小鼠tBID(密码子优化至肠炎沙门氏菌，SEQ ID No.194)的转位在B16F10中引发细胞死亡(图2)。16)，D2A1(图17)，HeLa(图18)和4T1(图19)细胞。还发现针对小肠结肠炎耶尔森氏菌(SEQID 138)优化的鼠BID密码子的BH3结构域或针对小肠结肠炎耶尔森氏菌(SEQ ID 139)优化的鼠BAX密码子的转运诱导在B16F10(图16)、D2A1(图17)、HeLa(图18)和4T1(图19)细胞上的大量细胞死亡。
然而用肠炎沙门氏菌(S.enterica)aroA细菌感染4小时不能诱导细胞凋亡，小鼠tBID的递送引发细胞凋亡，因为小鼠tBID的递送导致CASP3p17亚基产生(图20和21)。当使用Spil T3SS诱导条件(图20)时，SopE融合蛋白的细胞凋亡诱导程度更大，这反映了SopE排他地由Spil T3SS递送。SteA 1-20融合的鼠tBID不能诱导凋亡，非常可能是因为SteA的20个N-末端氨基酸内的分泌信号不足以允许递送融合蛋白(图20和21)。与全长SteA融合的小鼠tBID在Spil和Spill T3SS诱导条件下导致HeLa细胞中凋亡诱导(图20和21)，反映了SteA由T3SS两者递送的能力。必须注意的是，即使在溢出T3SS诱导条件下，Spil T3SS的部分活性也被期望，如在Spill T3SS诱导条件下SopE融合蛋白的活性所见(图21)。
除了这里功能上详细阐述的转运真核蛋白，使用这里描述的工具分泌了几种其他真核蛋白。这包括用于递送小肠结肠炎耶尔森氏菌蛋白的来自细胞周期调节(Mad2(SEQ IDNo.15)、CDK1(SEQ ID No.14)、INK4A(SEQ ID No.16)、INK4B(SEQ ID No.17)和INK4C(SEQID No.18))及其部件(INK4A 84-103(SEQ ID No.158)、p107 657-662(SEQ ID No.159)、p21 141-160(SEQ ID No.160)、p21145-160(SEQ ID No.161)、p2117-33(SEQ ID No.162)和细胞周期蛋白D2 139-147(SEQ ID No 163))、凋亡相关蛋白(Bad(SEQ ID No.29)、FADD(SEQ ID No.28)和Caspase 3p17(SEQ ID No.22)和p12(SEQ ID No.23)、zebrafish Bid(SEQ ID No.19)(SEQ ID No.13)、Bax BH3(SEQ ID No.139))，信号传导蛋白(鼠TRAF6(SEQID No.12))和其部件(tBid BH3、TIFA(SEQ ID No.13))、GPCR Ga亚基(GNA12，最短同种型，(SEQ ID No.30))、纳米抗体(vhhGFP4，(SEQ ID No.31))和纳米抗体融合构建体(图13和14)以及小的GTP酶(Rac1Q61E(SEQ ID No.26和137)和RhoA Q63L(SEQ ID No.12))(SEQ IDNo.32,33,34)[87]27)和来自人Akt的Pleckstrin同源结构域(SEQ ID No.35)。除了功能上详尽的凋亡相关蛋白(鼠tBid，SEQ ID No.144-147)之外，这还包括由肠杆菌递送(图22)来自细胞周期调节的蛋白质(Mad2(SEQ ID No.168-169)、CDK1(SEQ ID No.170-171)、INK4A(SEQ ID No.164-165)和INK4C(SEQ ID No.166-167))。虽然这些蛋白质没有被功能验证，但是T3SS依赖分泌的多种真核蛋白与可能删除YopE附录结合的可能性开放了T3SS在细胞生物学的广泛适用性的新观点。
在斑马鱼胚胎中截断的Bid的体内转运诱导凋亡
这种细菌工具的一个有趣的特点是活动物的潜在用途。斑马鱼在其胚胎状态可以保持透明允许荧光染色和显微镜[54,88,89]。已经详细描述了几种斑马鱼凋亡诱导剂，其中z-BIM是最有效的[90]。因此，我们决定将z-BIM克隆到我们的系统中。即使与人BIM弱同源，我们测定人上皮细胞中YopE1-138-z-BIM(SEQ ID No.21)的细胞凋亡诱导的效力。用菌株转运的YopE1-138-z-BIM感染1小时的HeLa细胞显示出细胞死亡的明显迹象。然后我们进行体内实验与受精后(dpf)斑马鱼胚胎，使用局部感染模型通过显微注射细菌进入后脑的[54]。在感染5.5小时后，将鱼固定，透化并对存在的CASP3p17染色。在用表达YopE1-138-Myc的菌株感染时，在后脑区域可见细菌(染色“b”，图8AA)检测到细菌周围的凋亡诱导(染色“c”，图8AA)。相比之下，在递送YopE1-138-z-BIM的菌株感染时，在细菌周围的区域中观察到存在裂解的CASP3的强烈增加(图8AA II)。在最大强度z投影上的自动图像分析证实，YopE1-138-z-BIM转运细菌远远超过对照细菌，在附近细胞中诱导细胞凋亡(图8B)。这表明z-BIM在细菌转运时在斑马鱼中有功能。这些结果进一步验证了T3SS在活的动物中的真核蛋白递送的用途。
磷蛋白组学揭示转运蛋白质对蛋白质磷酸化的全局影响
磷酸化是广泛的翻译后修饰，其可以激活或灭活生物过程，因此是研究信号传导事件的合适靶标[91，92]。尽管如此，目前还没有系统级分析细胞凋亡中的磷酸化。为了分析递送到HeLa细胞中的人tBid的影响，我们通过LC-MS/MS使用无标记的磷蛋白体方法。在三个独立实验中，将细胞保留未处理，用AHOPEMT asd+YopE1-138-Myc或用AHOPEMT asd+YopE1-138-tBid感染30分钟。裂解细胞，随后进行酶消化，磷酸肽的富集和单个磷肽的定量和鉴定。我们将用ΔΗΟΡΕΜΤasd+YopE1-138-Myc感染的细胞与用ΔΗΟΡΕΜΤasd+YopE1-138-tBid感染的细胞进行比较，从而允许我们鉴定363个tBid依赖性磷酸化事件。286个磷酸肽显示磷酸化增加，而77个在tBid递送时磷酸化较少，对应于243种不同的蛋白质，我们定义为tBid磷酸化蛋白质。STRING数据库用于创建tBid磷酸化蛋白的蛋白质-蛋白质相互作用网络[93](图9A)。另外，已知与线粒体凋亡相关的27种蛋白质被添加到网络，构建中心簇。有趣的是，只有来自tBid磷酸化蛋白质的少数蛋白质连接到该中心簇，表明许多蛋白质经历磷酸化的变化，其迄今未直接与凋亡蛋白相关。为了表征tBid磷酸化蛋白质所涵盖的生物功能，我们使用用于注释，可视化和综合发现的数据库的功能注释工具(DAVID，http://david.abcc.ncifcrf.gov/)进行了基因本体论分析[94，95]。鉴定的生物学功能显示不同的细胞过程受tBid影响。参与染色质重排和转录调节的许多蛋白质经历磷酸化的改变(即CBX3，CBX5，TRIM28，HDAC1)。HDAC1例如是在转录调节中起作用的组蛋白脱乙酰酶。已经显示HDAC1可以调节NF-kB的转录活性，NF-kB是也参与凋亡的蛋白质。我们另外确定了一个参与RNA加工的蛋白质簇，以前已经表明它在调节细胞凋亡中发挥重要作用[96]。FINRPK例如介导p53/TP53对DNA损伤的反应，并且是诱导凋亡所必需的[97]。此外，参与蛋白质翻译的蛋白质的磷酸化也受影响。几种真核起始因子(即EIF4E2、EIF4B、EIF3A、EIF4G2)经历磷酸化的变化，这与凋亡细胞中整体蛋白质合成减少的观察一致。有趣的是，参与细胞骨架重塑的许多蛋白质(例如PXN，MAP1B9)的磷酸化在tBid递送时被改变，这与细胞形态在tBid递送时显着改变的观察一致(图9B)。细胞收缩和损失接触反映在事实上，我们观察到粘附相关蛋白质如Z02和Paxillin的磷酸化。类似地，核的收缩伴随层状蛋白如LaminA/C和Lamin B1的磷酸化。总而言之，tBID递送诱导快速凋亡反应由线粒体完整性的破裂表示(图9B)，我们发现tBid诱导的细胞凋亡影响参与不同细胞过程的数百个磷酸化事件。虽然许多已鉴定的蛋白质与凋亡有关，但是已知仅有少数已知在凋亡时被磷酸化磷酸化蛋白质方法因此为进一步研究凋亡提供了有用的资源。
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Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Val Thr Asn Ile
130 135 140
Thr Leu Ser Thr Gln His Tyr Arg Ile His Arg Ser Asp Val Glu Pro
145 150 155 160
Val Lys Glu Lys Thr Thr Glu Lys Asp Ile Phe Ala Lys Ser Ile Thr
165 170 175
Ala Val Arg Asn Ser Phe Ile Ser Leu Ser Thr Ser Leu Ser Asp Arg
180 185 190
Phe Ser Leu His Gln Gln Thr Asp Ile Pro Thr Thr His Phe His Arg
195 200 205
Gly Asn Ala Ser Glu Gly Arg Ala Val Leu Thr Ser Lys Thr Val Lys
210 215 220
Asp Phe Met Leu Gln Lys Leu Asn Ser Leu Asp Ile Lys Gly Asn Ala
225 230 235 240
Ser Lys Asp Pro Ala Tyr Ala Arg Gln Thr Cys Glu Ala Ile Leu Ser
245 250 255
Ala Val Tyr Ser Asn Asn Lys Asp Gln Cys Cys Lys Leu Leu Ile Ser
260 265 270
Lys Gly Val Ser Ile Thr Pro Phe Leu Lys Glu Ile Gly Glu Ala Ala
275 280 285
Gln Asn Ala Gly Leu Pro Gly Glu Ile Lys Asn Gly Val Phe Thr Pro
290 295 300
Gly Gly Ala Gly Ala Asn Pro Phe Val Val Pro Leu Ile Ala Ser Ala
305 310 315 320
Ser Ile Lys Tyr Pro His Met Phe Ile Asn His Asn Gln Gln Val Ser
325 330 335
Phe Lys Ala Tyr Ala Glu Lys Ile Val Met Lys Glu Val Thr Pro Leu
340 345 350
Phe Asn Lys Gly Thr Met Pro Thr Pro Gln Gln Phe Gln Leu Thr Ile
355 360 365
Glu Asn Ile Ala Asn Lys Tyr Leu Gln Asn Ala Ser
370 375 380
<210> 6
<211> 701
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - SopB
<400> 6
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Met Gln Ile Gln
130 135 140
Ser Phe Tyr His Ser Ala Ser Leu Lys Thr Gln Glu Ala Phe Lys Ser
145 150 155 160
Leu Gln Lys Thr Leu Tyr Asn Gly Met Gln Ile Leu Ser Gly Gln Gly
165 170 175
Lys Ala Pro Ala Lys Ala Pro Asp Ala Arg Pro Glu Ile Ile Val Leu
180 185 190
Arg Glu Pro Gly Ala Thr Trp Gly Asn Tyr Leu Gln His Gln Lys Ala
195 200 205
Ser Asn His Ser Leu His Asn Leu Tyr Asn Leu Gln Arg Asp Leu Leu
210 215 220
Thr Val Ala Ala Thr Val Leu Gly Lys Gln Asp Pro Val Leu Thr Ser
225 230 235 240
Met Ala Asn Gln Met Glu Leu Ala Lys Val Lys Ala Asp Arg Pro Ala
245 250 255
Thr Lys Gln Glu Glu Ala Ala Ala Lys Ala Leu Lys Lys Asn Leu Ile
260 265 270
Glu Leu Ile Ala Ala Arg Thr Gln Gln Gln Asp Gly Leu Pro Ala Lys
275 280 285
Glu Ala His Arg Phe Ala Ala Val Ala Phe Arg Asp Ala Gln Val Lys
290 295 300
Gln Leu Asn Asn Gln Pro Trp Gln Thr Ile Lys Asn Thr Leu Thr His
305 310 315 320
Asn Gly His His Tyr Thr Asn Thr Gln Leu Pro Ala Ala Glu Met Lys
325 330 335
Ile Gly Ala Lys Asp Ile Phe Pro Ser Ala Tyr Glu Gly Lys Gly Val
340 345 350
Cys Ser Trp Asp Thr Lys Asn Ile His His Ala Asn Asn Leu Trp Met
355 360 365
Ser Thr Val Ser Val His Glu Asp Gly Lys Asp Lys Thr Leu Phe Cys
370 375 380
Gly Ile Arg His Gly Val Leu Ser Pro Tyr His Glu Lys Asp Pro Leu
385 390 395 400
Leu Arg His Val Gly Ala Glu Asn Lys Ala Lys Glu Val Leu Thr Ala
405 410 415
Ala Leu Phe Ser Lys Pro Glu Leu Leu Asn Lys Ala Leu Ala Gly Glu
420 425 430
Ala Val Ser Leu Lys Leu Val Ser Val Gly Leu Leu Thr Ala Ser Asn
435 440 445
Ile Phe Gly Lys Glu Gly Thr Met Val Glu Asp Gln Met Arg Ala Trp
450 455 460
Gln Ser Leu Thr Gln Pro Gly Lys Met Ile His Leu Lys Ile Arg Asn
465 470 475 480
Lys Asp Gly Asp Leu Gln Thr Val Lys Ile Lys Pro Asp Val Ala Ala
485 490 495
Phe Asn Val Gly Val Asn Glu Leu Ala Leu Lys Leu Gly Phe Gly Leu
500 505 510
Lys Ala Ser Asp Ser Tyr Asn Ala Glu Ala Leu His Gln Leu Leu Gly
515 520 525
Asn Asp Leu Arg Pro Glu Ala Arg Pro Gly Gly Trp Val Gly Glu Trp
530 535 540
Leu Ala Gln Tyr Pro Asp Asn Tyr Glu Val Val Asn Thr Leu Ala Arg
545 550 555 560
Gln Ile Lys Asp Ile Trp Lys Asn Asn Gln His His Lys Asp Gly Gly
565 570 575
Glu Pro Tyr Lys Leu Ala Gln Arg Leu Ala Met Leu Ala His Glu Ile
580 585 590
Asp Ala Val Pro Ala Trp Asn Cys Lys Ser Gly Lys Asp Arg Thr Gly
595 600 605
Met Met Asp Ser Glu Ile Lys Arg Glu Ile Ile Ser Leu His Gln Thr
610 615 620
His Met Leu Ser Ala Pro Gly Ser Leu Pro Asp Ser Gly Gly Gln Lys
625 630 635 640
Ile Phe Gln Lys Val Leu Leu Asn Ser Gly Asn Leu Glu Ile Gln Lys
645 650 655
Gln Asn Thr Gly Gly Ala Gly Asn Lys Val Met Lys Asn Leu Ser Pro
660 665 670
Glu Val Leu Asn Leu Ser Tyr Gln Lys Arg Val Gly Asp Glu Asn Ile
675 680 685
Trp Gln Ser Val Lys Gly Ile Ser Ser Leu Ile Thr Ser
690 695 700
<210> 7
<211> 383
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - OspF
<400> 7
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Phe Glu
130 135 140
Met Pro Ile Lys Lys Pro Cys Leu Lys Leu Asn Leu Asp Ser Leu Asn
145 150 155 160
Val Val Arg Ser Glu Ile Pro Gln Met Leu Ser Ala Asn Glu Arg Leu
165 170 175
Lys Asn Asn Phe Asn Ile Leu Tyr Asn Gln Ile Arg Gln Tyr Pro Ala
180 185 190
Tyr Tyr Phe Lys Val Ala Ser Asn Val Pro Thr Tyr Ser Asp Ile Cys
195 200 205
Gln Ser Phe Ser Val Met Tyr Gln Gly Phe Gln Ile Val Asn His Ser
210 215 220
Gly Asp Val Phe Ile His Ala Cys Arg Glu Asn Pro Gln Ser Lys Gly
225 230 235 240
Asp Phe Val Gly Asp Lys Phe His Ile Ser Ile Ala Arg Glu Gln Val
245 250 255
Pro Leu Ala Phe Gln Ile Leu Ser Gly Leu Leu Phe Ser Glu Asp Ser
260 265 270
Pro Ile Asp Lys Trp Lys Ile Thr Asp Met Asn Arg Val Ser Gln Gln
275 280 285
Ser Arg Val Gly Ile Gly Ala Gln Phe Thr Leu Tyr Val Lys Ser Asp
290 295 300
Gln Glu Cys Ser Gln Tyr Ser Ala Leu Leu Leu His Lys Ile Arg Gln
305 310 315 320
Phe Ile Met Cys Leu Glu Ser Asn Leu Leu Arg Ser Lys Ile Ala Pro
325 330 335
Gly Glu Tyr Pro Ala Ser Asp Val Arg Pro Glu Asp Trp Lys Tyr Val
340 345 350
Ser Tyr Arg Asn Glu Leu Arg Ser Asp Arg Asp Gly Ser Glu Arg Gln
355 360 365
Glu Gln Met Leu Arg Glu Glu Pro Phe Tyr Arg Leu Met Ile Glu
370 375 380
<210> 8
<211> 685
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - SptP
<400> 8
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Leu
130 135 140
Lys Tyr Glu Glu Arg Lys Leu Asn Asn Leu Thr Leu Ser Ser Phe Ser
145 150 155 160
Lys Val Gly Val Ser Asn Asp Ala Arg Leu Tyr Ile Ala Lys Glu Asn
165 170 175
Thr Asp Lys Ala Tyr Val Ala Pro Glu Lys Phe Ser Ser Lys Val Leu
180 185 190
Thr Trp Leu Gly Lys Met Pro Leu Phe Lys Asn Thr Glu Val Val Gln
195 200 205
Lys His Thr Glu Asn Ile Arg Val Gln Asp Gln Lys Ile Leu Gln Thr
210 215 220
Phe Leu His Ala Leu Thr Glu Lys Tyr Gly Glu Thr Ala Val Asn Asp
225 230 235 240
Ala Leu Leu Met Ser Arg Ile Asn Met Asn Lys Pro Leu Thr Gln Arg
245 250 255
Leu Ala Val Gln Ile Thr Glu Cys Val Lys Ala Ala Asp Glu Gly Phe
260 265 270
Ile Asn Leu Ile Lys Ser Lys Asp Asn Val Gly Val Arg Asn Ala Ala
275 280 285
Leu Val Ile Lys Gly Gly Asp Thr Lys Val Ala Glu Lys Asn Asn Asp
290 295 300
Val Gly Ala Glu Ser Lys Gln Pro Leu Leu Asp Ile Ala Leu Lys Gly
305 310 315 320
Leu Lys Arg Thr Leu Pro Gln Leu Glu Gln Met Asp Gly Asn Ser Leu
325 330 335
Arg Glu Asn Phe Gln Glu Met Ala Ser Gly Asn Gly Pro Leu Arg Ser
340 345 350
Leu Met Thr Asn Leu Gln Asn Leu Asn Lys Ile Pro Glu Ala Lys Gln
355 360 365
Leu Asn Asp Tyr Val Thr Thr Leu Thr Asn Ile Gln Val Gly Val Ala
370 375 380
Arg Phe Ser Gln Trp Gly Thr Cys Gly Gly Glu Val Glu Arg Trp Val
385 390 395 400
Asp Lys Ala Ser Thr His Glu Leu Thr Gln Ala Val Lys Lys Ile His
405 410 415
Val Ile Ala Lys Glu Leu Lys Asn Val Thr Ala Glu Leu Glu Lys Ile
420 425 430
Glu Ala Gly Ala Pro Met Pro Gln Thr Met Ser Gly Pro Thr Leu Gly
435 440 445
Leu Ala Arg Phe Ala Val Ser Ser Ile Pro Ile Asn Gln Gln Thr Gln
450 455 460
Val Lys Leu Ser Asp Gly Met Pro Val Pro Val Asn Thr Leu Thr Phe
465 470 475 480
Asp Gly Lys Pro Val Ala Leu Ala Gly Ser Tyr Pro Lys Asn Thr Pro
485 490 495
Asp Ala Leu Glu Ala His Met Lys Met Leu Leu Glu Lys Glu Cys Ser
500 505 510
Cys Leu Val Val Leu Thr Ser Glu Asp Gln Met Gln Ala Lys Gln Leu
515 520 525
Pro Pro Tyr Phe Arg Gly Ser Tyr Thr Phe Gly Glu Val His Thr Asn
530 535 540
Ser Gln Lys Val Ser Ser Ala Ser Gln Gly Glu Ala Ile Asp Gln Tyr
545 550 555 560
Asn Met Gln Leu Ser Cys Gly Glu Lys Arg Tyr Thr Ile Pro Val Leu
565 570 575
His Val Lys Asn Trp Pro Asp His Gln Pro Leu Pro Ser Thr Asp Gln
580 585 590
Leu Glu Tyr Leu Ala Asp Arg Val Lys Asn Ser Asn Gln Asn Gly Ala
595 600 605
Pro Gly Arg Ser Ser Ser Asp Lys His Leu Pro Met Ile His Cys Leu
610 615 620
Gly Gly Val Gly Arg Thr Gly Thr Met Ala Ala Ala Leu Val Leu Lys
625 630 635 640
Asp Asn Pro His Ser Asn Leu Glu Gln Val Arg Ala Asp Phe Arg Asp
645 650 655
Ser Arg Asn Asn Arg Met Leu Glu Asp Ala Ser Gln Phe Val Gln Leu
660 665 670
Lys Ala Met Gln Ala Gln Leu Leu Met Thr Thr Ala Ser
675 680 685
<210> 9
<211> 678
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - IpgD
<400> 9
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Met His Ile Thr
130 135 140
Asn Leu Gly Leu His Gln Val Ser Phe Gln Ser Gly Asp Ser Tyr Lys
145 150 155 160
Gly Ala Glu Glu Thr Gly Lys His Lys Gly Val Ser Val Ile Ser Tyr
165 170 175
Gln Arg Val Lys Asn Gly Glu Arg Asn Lys Gly Ile Glu Ala Leu Asn
180 185 190
Arg Leu Tyr Leu Gln Asn Gln Thr Ser Leu Thr Gly Lys Ser Leu Leu
195 200 205
Phe Ala Arg Asp Lys Ala Glu Val Phe Cys Glu Ala Ile Lys Leu Ala
210 215 220
Gly Gly Asp Thr Ser Lys Ile Lys Ala Met Met Glu Arg Leu Asp Thr
225 230 235 240
Tyr Lys Leu Gly Glu Val Asn Lys Arg His Ile Asn Glu Leu Asn Lys
245 250 255
Val Ile Ser Glu Glu Ile Arg Ala Gln Leu Gly Ile Lys Asn Lys Lys
260 265 270
Glu Leu Gln Thr Lys Ile Lys Gln Ile Phe Thr Asp Tyr Leu Asn Asn
275 280 285
Lys Asn Trp Gly Pro Val Asn Lys Asn Ile Ser His His Gly Lys Asn
290 295 300
Tyr Ser Phe Gln Leu Thr Pro Ala Ser His Met Lys Ile Gly Asn Lys
305 310 315 320
Asn Ile Phe Val Lys Glu Tyr Asn Gly Lys Gly Ile Cys Cys Ala Ser
325 330 335
Thr Arg Glu Arg Asp His Ile Ala Asn Met Trp Leu Ser Lys Val Val
340 345 350
Asp Asp Glu Gly Lys Glu Ile Phe Ser Gly Ile Arg His Gly Val Ile
355 360 365
Ser Ala Tyr Gly Leu Lys Lys Asn Ser Ser Glu Arg Ala Val Ala Ala
370 375 380
Arg Asn Lys Ala Glu Glu Leu Val Ser Ala Ala Leu Tyr Ser Arg Pro
385 390 395 400
Glu Leu Leu Ser Gln Ala Leu Ser Gly Lys Thr Val Asp Leu Lys Ile
405 410 415
Val Ser Thr Ser Leu Leu Thr Pro Thr Ser Leu Thr Gly Gly Glu Glu
420 425 430
Ser Met Leu Lys Asp Gln Val Ser Ala Leu Lys Gly Leu Asn Ser Lys
435 440 445
Arg Gly Gly Pro Thr Lys Leu Leu Ile Arg Asn Ser Asp Gly Leu Leu
450 455 460
Lys Glu Val Ser Val Asn Leu Lys Val Val Thr Phe Asn Phe Gly Val
465 470 475 480
Asn Glu Leu Ala Leu Lys Met Gly Leu Gly Trp Arg Asn Val Asp Lys
485 490 495
Leu Asn Asp Glu Ser Ile Cys Ser Leu Leu Gly Asp Asn Phe Leu Lys
500 505 510
Asn Gly Val Ile Gly Gly Trp Ala Ala Glu Ala Ile Glu Lys Asn Pro
515 520 525
Pro Cys Lys Asn Asp Val Ile Tyr Leu Ala Asn Gln Ile Lys Glu Ile
530 535 540
Val Asn Asn Lys Leu Gln Lys Asn Asp Asn Gly Glu Pro Tyr Lys Leu
545 550 555 560
Ser Gln Arg Val Thr Leu Leu Ala Tyr Thr Ile Gly Ala Val Pro Cys
565 570 575
Trp Asn Cys Lys Ser Gly Lys Asp Arg Thr Gly Met Gln Asp Ala Glu
580 585 590
Ile Lys Arg Glu Ile Ile Arg Lys His Glu Thr Gly Gln Phe Ser Gln
595 600 605
Leu Asn Ser Lys Leu Ser Ser Glu Glu Lys Arg Leu Phe Ser Thr Ile
610 615 620
Leu Met Asn Ser Gly Asn Met Glu Ile Gln Glu Met Asn Thr Gly Val
625 630 635 640
Pro Gly Asn Lys Val Met Lys Lys Leu Pro Leu Ser Ser Leu Glu Leu
645 650 655
Ser Tyr Ser Glu Arg Ile Gly Asp Pro Lys Ile Trp Asn Met Val Lys
660 665 670
Gly Tyr Ser Ser Phe Val
675
<210> 10
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - BepA
<400> 10
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Pro
130 135 140
Lys Ala Lys Ala Lys Thr Lys Asn Thr Glu Ile Ile Ser Pro His His
145 150 155 160
Tyr Val Tyr Pro Asn Thr Thr Thr Leu Lys Asn Lys Tyr Gly Ile Lys
165 170 175
Asn Leu Asn Ala Phe Leu Glu Lys Cys Ser His Asp Thr Ala Lys Ala
180 185 190
Met Ile Asn Leu Arg Glu Glu Ser Leu Pro Glu Tyr Phe Asp Thr Ala
195 200 205
Tyr Leu Cys His Ile His Gln Gln Leu Phe Lys Asn Thr Phe Glu Trp
210 215 220
Ala Gly Tyr Leu Arg His Ile Pro Phe Thr Phe Ala Asp Gly Thr Thr
225 230 235 240
Ala Ala Met Pro Glu Met Lys Arg Thr Gly Trp Lys Asn Ala Phe Ala
245 250 255
Ile Gly Asp Glu Ile Gln Glu Gly Leu Gln Arg Leu Asp Gln Thr Leu
260 265 270
Ala Glu Lys Asn Asn Leu Gln Gly Leu Thr Arg Glu Glu Phe Asn Ser
275 280 285
Glu Ala Ile Glu Leu Phe Asn Ser Leu Asn Gln Leu His Pro Phe Arg
290 295 300
Glu Gly Asn Gly Arg Thr Gln Arg Leu Phe Phe Glu Asn Leu Ala Lys
305 310 315 320
Ala Ala Gly His Gln Leu Asn Phe Ser Leu Ile Thr Lys Glu Arg Met
325 330 335
Met Val Ala Ser Val Ala Val Ala Glu Asn Gly Asp Leu Glu Pro Met
340 345 350
Gln His Leu Phe Glu Asp Ile Ser Asn Pro Glu Lys Ile Arg Leu Leu
355 360 365
Lys Glu Phe Met His Thr Met Lys Asn Thr Gly Arg Asn Val Asn Asp
370 375 380
Arg Pro Val Met Val Ala Lys Glu Gly Glu Thr Tyr Thr Gly Thr Tyr
385 390 395 400
Arg Gly Ala Gly Leu Glu Gly Phe Ala Leu Asn Val Lys Gly Ala Tyr
405 410 415
Ile Ile Gly Asn Ile Asp His Leu Pro Pro Glu Gln Leu Lys Ile Leu
420 425 430
Lys Pro Gly Asp Lys Ile Thr Phe Thr Ala Pro Lys Ala Glu
435 440 445
<210> 11
<211> 284
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - BepA E305-end
<400> 11
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Glu Gly
130 135 140
Asn Gly Arg Thr Gln Arg Leu Phe Phe Glu Asn Leu Ala Lys Ala Ala
145 150 155 160
Gly His Gln Leu Asn Phe Ser Leu Ile Thr Lys Glu Arg Met Met Val
165 170 175
Ala Ser Val Ala Val Ala Glu Asn Gly Asp Leu Glu Pro Met Gln His
180 185 190
Leu Phe Glu Asp Ile Ser Asn Pro Glu Lys Ile Arg Leu Leu Lys Glu
195 200 205
Phe Met His Thr Met Lys Asn Thr Gly Arg Asn Val Asn Asp Arg Pro
210 215 220
Val Met Val Ala Lys Glu Gly Glu Thr Tyr Thr Gly Thr Tyr Arg Gly
225 230 235 240
Ala Gly Leu Glu Gly Phe Ala Leu Asn Val Lys Gly Ala Tyr Ile Ile
245 250 255
Gly Asn Ile Asp His Leu Pro Pro Glu Gln Leu Lys Ile Leu Lys Pro
260 265 270
Gly Asp Lys Ile Thr Phe Thr Ala Pro Lys Ala Glu
275 280
<210> 12
<211> 672
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - murine Traf6
<400> 12
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Ser
130 135 140
Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Ser Ser Asp
145 150 155 160
Cys Cys Ala Ala Met Ala Ala Ser Cys Ser Ala Ala Val Lys Asp Asp
165 170 175
Ser Val Ser Gly Ser Ala Ser Thr Gly Asn Leu Ser Ser Ser Phe Met
180 185 190
Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu Glu Ser
195 200 205
Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala Val Gln
210 215 220
Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys Ser Ile
225 230 235 240
Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu Leu Glu
245 250 255
Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu Ser Leu
260 265 270
Thr Val Lys Cys Pro Asn Lys Gly Cys Leu Gln Lys Met Glu Leu Arg
275 280 285
His Leu Glu Asp His Gln Val His Cys Glu Phe Ala Leu Val Asn Cys
290 295 300
Pro Gln Cys Gln Arg Pro Phe Gln Lys Cys Gln Val Asn Thr His Ile
305 310 315 320
Ile Glu Asp Cys Pro Arg Arg Gln Val Ser Cys Val Asn Cys Ala Val
325 330 335
Ser Met Ala Tyr Glu Glu Lys Glu Ile His Asp Gln Ser Cys Pro Leu
340 345 350
Ala Asn Ile Ile Cys Glu Tyr Cys Gly Thr Ile Leu Ile Arg Glu Gln
355 360 365
Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile Pro Cys
370 375 380
Thr Phe Ser Val Phe Gly Cys His Gln Lys Met Gln Arg Asn His Leu
385 390 395 400
Ala Arg His Leu Gln Glu Asn Thr Gln Leu His Met Arg Leu Leu Ala
405 410 415
Gln Ala Val His Asn Val Asn Leu Ala Leu Arg Pro Cys Asp Ala Ala
420 425 430
Ser Pro Ser Arg Gly Cys Arg Pro Glu Asp Pro Asn Tyr Glu Glu Thr
435 440 445
Ile Lys Gln Leu Glu Ser Arg Leu Val Arg Gln Asp His Gln Ile Arg
450 455 460
Glu Leu Thr Ala Lys Met Glu Thr Gln Ser Met Tyr Val Gly Glu Leu
465 470 475 480
Lys Arg Thr Ile Arg Thr Leu Glu Asp Lys Val Ala Glu Met Glu Ala
485 490 495
Gln Gln Cys Asn Gly Ile Tyr Ile Trp Lys Ile Gly Lys Phe Gly Met
500 505 510
His Leu Lys Ser Gln Glu Glu Glu Arg Pro Val Val Ile His Ser Pro
515 520 525
Gly Phe Tyr Thr Gly Arg Pro Gly Tyr Lys Leu Cys Met Arg Leu His
530 535 540
Leu Gln Leu Pro Thr Ala Gln Arg Cys Ala Asn Tyr Ile Ser Leu Phe
545 550 555 560
Val His Thr Met Gln Gly Glu Tyr Asp Ser His Leu Pro Trp Pro Phe
565 570 575
Gln Gly Thr Ile Arg Leu Thr Ile Leu Asp Gln Ser Glu Ala Leu Ile
580 585 590
Arg Gln Asn His Glu Glu Val Met Asp Ala Lys Pro Glu Leu Leu Ala
595 600 605
Phe Gln Arg Pro Thr Ile Pro Arg Asn Pro Lys Gly Phe Gly Tyr Val
610 615 620
Thr Phe Met His Leu Glu Ala Leu Arg Gln Gly Thr Phe Ile Lys Asp
625 630 635 640
Asp Thr Leu Leu Val Arg Cys Glu Val Ser Thr Arg Phe Asp Met Gly
645 650 655
Gly Leu Arg Lys Glu Gly Phe Gln Pro Arg Ser Thr Asp Ala Gly Val
660 665 670
<210> 13
<211> 326
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - TIFA
<400> 13
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Thr
130 135 140
Ser Phe Glu Asp Ala Asp Thr Glu Glu Thr Val Thr Cys Leu Gln Met
145 150 155 160
Thr Val Tyr His Pro Gly Gln Leu Gln Cys Gly Ile Phe Gln Ser Ile
165 170 175
Ser Phe Asn Arg Glu Lys Leu Pro Ser Ser Glu Val Val Lys Phe Gly
180 185 190
Arg Asn Ser Asn Ile Cys His Tyr Thr Phe Gln Asp Lys Gln Val Ser
195 200 205
Arg Val Gln Phe Ser Leu Gln Leu Phe Lys Lys Phe Asn Ser Ser Val
210 215 220
Leu Ser Phe Glu Ile Lys Asn Met Ser Lys Lys Thr Asn Leu Ile Val
225 230 235 240
Asp Ser Arg Glu Leu Gly Tyr Leu Asn Lys Met Asp Leu Pro Tyr Arg
245 250 255
Cys Met Val Arg Phe Gly Glu Tyr Gln Phe Leu Met Glu Lys Glu Asp
260 265 270
Gly Glu Ser Leu Glu Phe Phe Glu Thr Gln Phe Ile Leu Ser Pro Arg
275 280 285
Ser Leu Leu Gln Glu Asn Asn Trp Pro Pro His Arg Pro Ile Pro Glu
290 295 300
Tyr Gly Thr Tyr Ser Leu Cys Ser Ser Gln Ser Ser Ser Pro Thr Glu
305 310 315 320
Met Asp Glu Asn Glu Ser
325
<210> 14
<211> 437
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Cdk1
<400> 14
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Met Glu Asp Tyr
130 135 140
Thr Lys Ile Glu Lys Ile Gly Glu Gly Thr Tyr Gly Val Val Tyr Lys
145 150 155 160
Gly Arg His Lys Thr Thr Gly Gln Val Val Ala Met Lys Lys Ile Arg
165 170 175
Leu Glu Ser Glu Glu Glu Gly Val Pro Ser Thr Ala Ile Arg Glu Ile
180 185 190
Ser Leu Leu Lys Glu Leu Arg His Pro Asn Ile Val Ser Leu Gln Asp
195 200 205
Val Leu Met Gln Asp Ser Arg Leu Tyr Leu Ile Phe Glu Phe Leu Ser
210 215 220
Met Asp Leu Lys Lys Tyr Leu Asp Ser Ile Pro Pro Gly Gln Tyr Met
225 230 235 240
Asp Ser Ser Leu Val Lys Ser Tyr Leu Tyr Gln Ile Leu Gln Gly Ile
245 250 255
Val Phe Cys His Ser Arg Arg Val Leu His Arg Asp Leu Lys Pro Gln
260 265 270
Asn Leu Leu Ile Asp Asp Lys Gly Thr Ile Lys Leu Ala Asp Phe Gly
275 280 285
Leu Ala Arg Ala Phe Gly Ile Pro Ile Arg Val Tyr Thr His Glu Val
290 295 300
Val Thr Leu Trp Tyr Arg Ser Pro Glu Val Leu Leu Gly Ser Ala Arg
305 310 315 320
Tyr Ser Thr Pro Val Asp Ile Trp Ser Ile Gly Thr Ile Phe Ala Glu
325 330 335
Leu Ala Thr Lys Lys Pro Leu Phe His Gly Asp Ser Glu Ile Asp Gln
340 345 350
Leu Phe Arg Ile Phe Arg Ala Leu Gly Thr Pro Asn Asn Glu Val Trp
355 360 365
Pro Glu Val Glu Ser Leu Gln Asp Tyr Lys Asn Thr Phe Pro Lys Trp
370 375 380
Lys Pro Gly Ser Leu Ala Ser His Val Lys Asn Leu Asp Glu Asn Gly
385 390 395 400
Leu Asp Leu Leu Ser Lys Met Leu Ile Tyr Asp Pro Ala Lys Arg Ile
405 410 415
Ser Gly Lys Met Ala Leu Asn His Pro Tyr Phe Asn Asp Leu Asp Asn
420 425 430
Gln Ile Lys Lys Met
435
<210> 15
<211> 347
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Mad2
<400> 15
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Ala
130 135 140
Leu Gln Leu Ser Arg Glu Gln Gly Ile Thr Leu Arg Gly Ser Ala Glu
145 150 155 160
Ile Val Ala Glu Phe Phe Ser Phe Gly Ile Asn Ser Ile Leu Tyr Gln
165 170 175
Arg Gly Ile Tyr Pro Ser Glu Thr Phe Thr Arg Val Gln Lys Tyr Gly
180 185 190
Leu Thr Leu Leu Val Thr Thr Asp Leu Glu Leu Ile Lys Tyr Leu Asn
195 200 205
Asn Val Val Glu Gln Leu Lys Asp Trp Leu Tyr Lys Cys Ser Val Gln
210 215 220
Lys Leu Val Val Val Ile Ser Asn Ile Glu Ser Gly Glu Val Leu Glu
225 230 235 240
Arg Trp Gln Phe Asp Ile Glu Cys Asp Lys Thr Ala Lys Asp Asp Ser
245 250 255
Ala Pro Arg Glu Lys Ser Gln Lys Ala Ile Gln Asp Glu Ile Arg Ser
260 265 270
Val Ile Arg Gln Ile Thr Ala Thr Val Thr Phe Leu Pro Leu Leu Glu
275 280 285
Val Ser Cys Ser Phe Asp Leu Leu Ile Tyr Thr Asp Lys Asp Leu Val
290 295 300
Val Pro Glu Lys Trp Glu Glu Ser Gly Pro Gln Phe Ile Thr Asn Ser
305 310 315 320
Glu Glu Val Arg Leu Arg Ser Phe Thr Thr Thr Ile His Lys Val Asn
325 330 335
Ser Met Val Ala Tyr Lys Ile Pro Val Asn Asp
340 345
<210> 16
<211> 298
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Ink4A
<400> 16
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Glu
130 135 140
Pro Ala Ala Gly Ser Ser Met Glu Pro Ser Ala Asp Trp Leu Ala Thr
145 150 155 160
Ala Ala Ala Arg Gly Arg Val Glu Glu Val Arg Ala Leu Leu Glu Ala
165 170 175
Gly Ala Leu Pro Asn Ala Pro Asn Ser Tyr Gly Arg Arg Pro Ile Gln
180 185 190
Val Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu Leu His
195 200 205
Gly Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg Pro Val
210 215 220
His Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val Leu His
225 230 235 240
Arg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg Leu Pro
245 250 255
Val Asp Leu Ala Glu Glu Leu Gly His Arg Asp Val Ala Arg Tyr Leu
260 265 270
Arg Ala Ala Ala Gly Gly Thr Arg Gly Ser Asn His Ala Arg Ile Asp
275 280 285
Ala Ala Glu Gly Pro Ser Asp Ile Pro Asp
290 295
<210> 17
<211> 280
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Ink4B
<400> 17
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Arg
130 135 140
Glu Glu Asn Lys Gly Met Pro Ser Gly Gly Gly Ser Asp Glu Gly Leu
145 150 155 160
Ala Ser Ala Ala Ala Arg Gly Leu Val Glu Lys Val Arg Gln Leu Leu
165 170 175
Glu Ala Gly Ala Asp Pro Asn Gly Val Asn Arg Phe Gly Arg Arg Ala
180 185 190
Ile Gln Val Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu
195 200 205
Leu His Gly Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg
210 215 220
Pro Val His Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val
225 230 235 240
Leu His Arg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg
245 250 255
Leu Pro Val Asp Leu Ala Glu Glu Arg Gly His Arg Asp Val Ala Gly
260 265 270
Tyr Leu Arg Thr Ala Thr Gly Asp
275 280
<210> 18
<211> 310
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Ink4C
<400> 18
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Ala
130 135 140
Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly Asp Leu
145 150 155 160
Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn Ala Gln
165 170 175
Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly Asn Pro
180 185 190
Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp Leu Lys
195 200 205
Asp Arg Thr Gly Phe Ala Val Ile His Asp Ala Ala Arg Ala Gly Phe
210 215 220
Leu Asp Thr Leu Gln Ala Leu Pro Glu Phe Gln Ala Asp Val Asn Ile
225 230 235 240
Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys Glu Gly
245 250 255
His Leu Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser Asn Val
260 265 270
Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala Arg Leu
275 280 285
Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly Ala Gly
290 295 300
Gly Ala Thr Asn Leu Gln
305 310
<210> 19
<211> 329
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - z-Bid
<400> 19
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Asp
130 135 140
Phe Asn Arg Asn Phe Asp His Ile Pro His Thr Ser Leu Val Leu Leu
145 150 155 160
Ser Phe Leu Asn Gln Lys Asp Cys Gln Asn Gly Glu Ser Gly Arg Val
165 170 175
Phe Asp Tyr Arg Glu Asp Asn Leu Ser Thr Asn His Ile Asp Ser Asp
180 185 190
Gly Asp Ile Glu Thr Asp Gly His Ser Pro Pro Ala Thr Tyr Arg Asp
195 200 205
Leu Leu His Glu Leu Gln His Glu Val Gln Pro Gly Leu Ser Val Asn
210 215 220
Ala Glu Glu Ala Arg Ala Ala Arg Glu Met Ala Ala Glu Leu Ile Arg
225 230 235 240
Ile Ala Asp Leu Leu Glu Gln Ser Val Leu Ser Gln Ala Ala Glu Ser
245 250 255
Leu Thr Lys Lys Leu Arg Ser Phe Gln Glu Gln Val Trp Ala Ser His
260 265 270
Leu Ser Lys Gly Val Gln Thr Leu Leu Gln His Val Ala Ala Ala Lys
275 280 285
Glu Phe Lys Lys Glu Leu Val Glu Met Ala Phe Thr Phe Met Leu Met
290 295 300
Lys Thr Val Cys Glu Arg Thr Pro Asp Phe Leu Phe Gly Leu Tyr Gly
305 310 315 320
Thr Val Val Gln Phe Phe Gly Ser Asn
325
<210> 20
<211> 273
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - z-t-Bid
<400> 20
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Gly His
130 135 140
Ser Pro Pro Ala Thr Tyr Arg Asp Leu Leu His Glu Leu Gln His Glu
145 150 155 160
Val Gln Pro Gly Leu Ser Val Asn Ala Glu Glu Ala Arg Ala Ala Arg
165 170 175
Glu Met Ala Ala Glu Leu Ile Arg Ile Ala Asp Leu Leu Glu Gln Ser
180 185 190
Val Leu Ser Gln Ala Ala Glu Ser Leu Thr Lys Lys Leu Arg Ser Phe
195 200 205
Gln Glu Gln Val Trp Ala Ser His Leu Ser Lys Gly Val Gln Thr Leu
210 215 220
Leu Gln His Val Ala Ala Ala Lys Glu Phe Lys Lys Glu Leu Val Glu
225 230 235 240
Met Ala Phe Thr Phe Met Leu Met Lys Thr Val Cys Glu Arg Thr Pro
245 250 255
Asp Phe Leu Phe Gly Leu Tyr Gly Thr Val Val Gln Phe Phe Gly Ser
260 265 270
Asn
<210> 21
<211> 318
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - z-BIM
<400> 21
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Ser
130 135 140
Asp Thr Ser Arg Glu Gln Thr Leu Ala Asn Gly Pro Ala Ser Gln Gly
145 150 155 160
Ser Gly Glu Ser Thr Gly Gly Gly Val Val Leu Pro Ala Gly His Phe
165 170 175
Asp Phe Pro Gln Pro Gly Glu Gly Asp Pro Leu Arg Gly Gly Ile Ser
180 185 190
Met Ser Asn Asn Gln Ser Arg Ser Pro Met Asn Arg Thr Phe Ser Arg
195 200 205
Ser Ser Ser Gly Tyr Phe Ser Val Asp Ser Asp Ser Val Pro Gly Ser
210 215 220
Pro Leu Met Pro Asn Ile Ser Glu Ala Gln Asp Gly Gln Asn Asp Glu
225 230 235 240
Val Trp Leu Ser Glu His Ser His Gln His Leu Gln Met Ala Ala Pro
245 250 255
Val Ala Ala Leu Pro Pro Glu Met Val Val Ala Arg Glu Leu Arg Arg
260 265 270
Ile Gly Asp Glu Phe Asn Arg Leu Tyr Cys Glu Ala Gly Ala Gly Val
275 280 285
Asn Gln Leu Arg Ala Pro Asn Glu His Ala Ile Val Leu Trp Met Asn
290 295 300
Val Ile Ile Gly Arg Leu Val His Phe Phe Leu Arg Arg Arg
305 310 315
<210> 22
<211> 289
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Caspase3 p17
<400> 22
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Ser Gly
130 135 140
Ile Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu
145 150 155 160
Cys Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr
165 170 175
Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe
180 185 190
Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu
195 200 205
Glu Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys
210 215 220
Arg Ser Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile
225 230 235 240
Ile Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe
245 250 255
Phe Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe
260 265 270
Ile Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr
275 280 285
Asp
<210> 23
<211> 242
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Caspase3 p10/12
<400> 23
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Gly Val Asp
130 135 140
Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp Phe Leu Tyr
145 150 155 160
Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn Ser Lys Asp
165 170 175
Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys Gln Tyr Ala
180 185 190
Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn Arg Lys Val
195 200 205
Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe His Ala Lys
210 215 220
Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu Leu Tyr Phe
225 230 235 240
Tyr His
<210> 24
<211> 337
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 -人Bid
<400> 24
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Asp
130 135 140
Cys Glu Val Asn Asn Gly Ser Ser Leu Arg Asp Glu Cys Ile Thr Asn
145 150 155 160
Leu Leu Val Phe Gly Phe Leu Gln Ser Cys Ser Asp Asn Ser Phe Arg
165 170 175
Arg Glu Leu Asp Ala Leu Gly His Glu Leu Pro Val Leu Ala Pro Gln
180 185 190
Trp Glu Gly Tyr Asp Glu Leu Gln Thr Asp Gly Asn Arg Ser Ser His
195 200 205
Ser Arg Leu Gly Arg Ile Glu Ala Asp Ser Glu Ser Gln Glu Asp Ile
210 215 220
Ile Arg Asn Ile Ala Arg His Leu Ala Gln Val Gly Asp Ser Met Asp
225 230 235 240
Arg Ser Ile Pro Pro Gly Leu Val Asn Gly Leu Ala Leu Gln Leu Arg
245 250 255
Asn Thr Ser Arg Ser Glu Glu Asp Arg Asn Arg Asp Leu Ala Thr Ala
260 265 270
Leu Glu Gln Leu Leu Gln Ala Tyr Pro Arg Asp Met Glu Lys Glu Lys
275 280 285
Thr Met Leu Val Leu Ala Leu Leu Leu Ala Lys Lys Val Ala Ser His
290 295 300
Thr Pro Ser Leu Leu Arg Asp Val Phe His Thr Thr Val Asn Phe Ile
305 310 315 320
Asn Gln Asn Leu Arg Thr Tyr Val Arg Ser Leu Ala Arg Asn Gly Met
325 330 335
Asp
<210> 25
<211> 277
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 -人t-Bid
<400> 25
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Gly Asn
130 135 140
Arg Ser Ser His Ser Arg Leu Gly Arg Ile Glu Ala Asp Ser Glu Ser
145 150 155 160
Gln Glu Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala Gln Val Gly
165 170 175
Asp Ser Met Asp Arg Ser Ile Pro Pro Gly Leu Val Asn Gly Leu Ala
180 185 190
Leu Gln Leu Arg Asn Thr Ser Arg Ser Glu Glu Asp Arg Asn Arg Asp
195 200 205
Leu Ala Thr Ala Leu Glu Gln Leu Leu Gln Ala Tyr Pro Arg Asp Met
210 215 220
Glu Lys Glu Lys Thr Met Leu Val Leu Ala Leu Leu Leu Ala Lys Lys
225 230 235 240
Val Ala Ser His Thr Pro Ser Leu Leu Arg Asp Val Phe His Thr Thr
245 250 255
Val Asn Phe Ile Asn Gln Asn Leu Arg Thr Tyr Val Arg Ser Leu Ala
260 265 270
Arg Asn Gly Met Asp
275
<210> 26
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Rac1 Q61E
<400> 26
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Gln Ala Ile Lys
130 135 140
Cys Val Val Val Gly Asp Gly Ala Val Gly Lys Thr Cys Leu Leu Ile
145 150 155 160
Ser Tyr Thr Thr Asn Ala Phe Pro Gly Glu Tyr Ile Pro Thr Val Phe
165 170 175
Asp Asn Tyr Ser Ala Asn Val Met Val Asp Gly Lys Pro Val Asn Leu
180 185 190
Gly Leu Trp Asp Thr Ala Gly Glu Glu Asp Tyr Asp Arg Leu Arg Pro
195 200 205
Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Ile Cys Phe Ser Leu Val
210 215 220
Ser Pro Ala Ser Phe Glu Asn Val Arg Ala Lys Trp Tyr Pro Glu Val
225 230 235 240
Arg His His Cys Pro Asn Thr Pro Ile Ile Leu Val Gly Thr Lys Leu
245 250 255
Asp Leu Arg Asp Asp Lys Asp Thr Ile Glu Lys Leu Lys Glu Lys Lys
260 265 270
Leu Thr Pro Ile Thr Tyr Pro Gln Gly Leu Ala Met Ala Lys Glu Ile
275 280 285
Gly Ala Val Lys Tyr Leu Glu Cys Ser Ala Leu Thr Gln Arg Gly Leu
290 295 300
Lys Thr Val Phe Asp Glu Ala Ile Arg Ala Val Leu Cys Pro Pro Pro
305 310 315 320
Val Lys Lys Arg Lys Arg Lys
325
<210> 27
<211> 333
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - RhoA Q63L
<400> 27
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Met Ala Ala Ile
130 135 140
Arg Lys Lys Leu Val Ile Val Gly Asp Gly Ala Cys Gly Lys Thr Cys
145 150 155 160
Leu Leu Ile Val Phe Ser Lys Asp Gln Phe Pro Glu Val Tyr Val Pro
165 170 175
Thr Val Phe Glu Asn Tyr Val Ala Asp Ile Glu Val Asp Gly Lys Gln
180 185 190
Val Glu Leu Ala Leu Trp Asp Thr Ala Gly Leu Glu Asp Tyr Asp Arg
195 200 205
Leu Arg Pro Leu Ser Tyr Pro Asp Thr Asp Val Ile Leu Met Cys Phe
210 215 220
Ser Ile Asp Ser Pro Asp Ser Leu Glu Asn Ile Pro Glu Lys Trp Thr
225 230 235 240
Pro Glu Val Lys His Phe Cys Pro Asn Val Pro Ile Ile Leu Val Gly
245 250 255
Asn Lys Lys Asp Leu Arg Asn Asp Glu His Thr Arg Arg Glu Leu Ala
260 265 270
Lys Met Lys Gln Glu Pro Val Lys Pro Glu Glu Gly Arg Asp Met Ala
275 280 285
Asn Arg Ile Gly Ala Phe Gly Tyr Met Glu Cys Ser Ala Lys Thr Lys
290 295 300
Asp Gly Val Arg Glu Val Phe Glu Met Ala Thr Arg Ala Ala Leu Gln
305 310 315 320
Ala Arg Arg Gly Lys Lys Lys Ser Gly Cys Leu Val Leu
325 330
<210> 28
<211> 350
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - FADD
<400> 28
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Asp
130 135 140
Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser Ser Ser
145 150 155 160
Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly Lys Arg
165 170 175
Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met Leu Leu
180 185 190
Glu Gln Asn Asp Leu Glu Pro Gly His Thr Glu Leu Leu Arg Glu Leu
195 200 205
Leu Ala Ser Leu Arg Arg His Asp Leu Leu Arg Arg Val Asp Asp Phe
210 215 220
Glu Ala Gly Ala Ala Ala Gly Ala Ala Pro Gly Glu Glu Asp Leu Cys
225 230 235 240
Ala Ala Phe Asn Val Ile Cys Asp Asn Val Gly Lys Asp Trp Arg Arg
245 250 255
Leu Ala Arg Gln Leu Lys Val Ser Asp Thr Lys Ile Asp Ser Ile Glu
260 265 270
Asp Arg Tyr Pro Arg Asn Leu Thr Glu Arg Val Arg Glu Ser Leu Arg
275 280 285
Ile Trp Lys Asn Thr Glu Lys Glu Asn Ala Thr Val Ala His Leu Val
290 295 300
Gly Ala Leu Arg Ser Cys Gln Met Asn Leu Val Ala Asp Leu Val Gln
305 310 315 320
Glu Val Gln Gln Ala Arg Asp Leu Gln Asn Arg Ser Gly Ala Met Ser
325 330 335
Pro Met Ser Trp Asn Ser Asp Ala Ser Thr Ser Glu Ala Ser
340 345 350
<210> 29
<211> 308
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Bad
<400> 29
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Met Phe Gln Ile
130 135 140
Pro Glu Phe Glu Pro Ser Glu Gln Glu Asp Ser Ser Ser Ala Glu Arg
145 150 155 160
Gly Leu Gly Pro Ser Pro Ala Gly Asp Gly Pro Ser Gly Ser Gly Lys
165 170 175
His His Arg Gln Ala Pro Gly Leu Leu Trp Asp Ala Ser His Gln Gln
180 185 190
Glu Gln Pro Thr Ser Ser Ser His His Gly Gly Ala Gly Ala Val Glu
195 200 205
Ile Arg Ser Arg His Ser Ser Tyr Pro Ala Gly Thr Glu Asp Asp Glu
210 215 220
Gly Met Gly Glu Glu Pro Ser Pro Phe Arg Gly Arg Ser Arg Ser Ala
225 230 235 240
Pro Pro Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu Arg Arg
245 250 255
Met Ser Asp Glu Phe Val Asp Ser Phe Lys Lys Gly Leu Pro Arg Pro
260 265 270
Lys Ser Ala Gly Thr Ala Thr Gln Met Arg Gln Ser Ser Ser Trp Thr
275 280 285
Arg Val Phe Gln Ser Trp Trp Asp Arg Asn Leu Gly Arg Gly Ser Thr
290 295 300
Ala Pro Ser Gln
305
<210> 30
<211> 309
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - GPCR GNA12
<400> 30
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Met Ser Gly Val
130 135 140
Val Gly Pro Met Gln Glu Pro Gly Ala Leu Asp Val Gly Gly Leu Arg
145 150 155 160
Ser Gln Arg Gln Lys Trp Phe Gln Cys Phe Asp Gly Ile Thr Ser Ile
165 170 175
Leu Phe Met Val Ser Ser Ser Glu Tyr Asp Gln Val Leu Met Glu Asp
180 185 190
Arg Arg Thr Asn Arg Leu Val Glu Ser Met Asn Ile Phe Glu Thr Ile
195 200 205
Val Asn Asn Lys Leu Phe Phe Asn Val Ser Ile Ile Leu Phe Leu Asn
210 215 220
Lys Met Asp Leu Leu Val Glu Lys Val Lys Thr Val Ser Ile Lys Lys
225 230 235 240
His Phe Pro Asp Phe Arg Gly Asp Pro His Arg Leu Glu Asp Val Gln
245 250 255
Arg Tyr Leu Val Gln Cys Phe Asp Arg Lys Arg Arg Asn Arg Ser Lys
260 265 270
Pro Leu Phe His His Phe Thr Thr Ala Ile Asp Thr Glu Asn Val Arg
275 280 285
Phe Val Phe His Ala Val Lys Asp Thr Ile Leu Gln Glu Asn Leu Lys
290 295 300
Asp Ile Met Leu Gln
305
<210> 31
<211> 259
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - VhH4 nanobody recognizing EGFP
<400> 31
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Asp
130 135 140
Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly
145 150 155 160
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn Arg Tyr
165 170 175
Ser Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
180 185 190
Ala Gly Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu Asp Ser Val
195 200 205
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr
210 215 220
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Asn Val Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr
245 250 255
Val Ser Ser
<210> 32
<211> 458
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Slmb1-VhH4
<400> 32
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Lys Met
130 135 140
Met Lys Met Glu Thr Asp Lys Ile Met Asp Glu Thr Asn Ser Asn Ala
145 150 155 160
Gln Ala Phe Thr Thr Thr Met Leu Tyr Asp Pro Val Arg Lys Lys Asp
165 170 175
Ser Ser Pro Thr Tyr Gln Thr Glu Arg Glu Leu Cys Phe Gln Tyr Phe
180 185 190
Thr Gln Trp Ser Glu Ser Gly Gln Val Asp Phe Val Glu His Leu Leu
195 200 205
Ser Arg Met Cys His Tyr Gln His Gly Gln Ile Asn Ala Tyr Leu Lys
210 215 220
Pro Met Leu Gln Arg Asp Phe Ile Thr Leu Leu Pro Ile Lys Gly Leu
225 230 235 240
Asp His Ile Ala Glu Asn Ile Leu Ser Tyr Leu Asp Ala Glu Ser Leu
245 250 255
Lys Ser Ser Glu Leu Val Cys Lys Glu Trp Leu Arg Val Ile Ser Glu
260 265 270
Gly Met Leu Trp Lys Lys Leu Ile Glu Arg Lys Val Arg Thr Asp Ser
275 280 285
Leu Trp Arg Gly Leu Ala Glu Arg Arg Asn Trp Met Gln Tyr Leu Phe
290 295 300
Lys Pro Arg Pro Gly Gln Thr Gln Arg Pro His Ser Phe His Arg Glu
305 310 315 320
Leu Phe Pro Lys Ile Met Asn Asp Ile Asp Ser Ile Glu Asn Asn Trp
325 330 335
Arg Thr Gly Arg His Met Asp Gln Val Gln Leu Val Glu Ser Gly Gly
340 345 350
Ala Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
355 360 365
Gly Phe Pro Val Asn Arg Tyr Ser Met Arg Trp Tyr Arg Gln Ala Pro
370 375 380
Gly Lys Glu Arg Glu Trp Val Ala Gly Met Ser Ser Ala Gly Asp Arg
385 390 395 400
Ser Ser Tyr Glu Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
405 410 415
Asp Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu
420 425 430
Asp Thr Ala Val Tyr Tyr Cys Asn Val Asn Val Gly Phe Glu Tyr Trp
435 440 445
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
450 455
<210> 33
<211> 470
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - NLS-Slmb1-VhH4
<400> 33
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Pro Pro
130 135 140
Lys Lys Lys Arg Lys Val Gln Phe Lys Met Met Lys Met Glu Thr Asp
145 150 155 160
Lys Ile Met Asp Glu Thr Asn Ser Asn Ala Gln Ala Phe Thr Thr Thr
165 170 175
Met Leu Tyr Asp Pro Val Arg Lys Lys Asp Ser Ser Pro Thr Tyr Gln
180 185 190
Thr Glu Arg Glu Leu Cys Phe Gln Tyr Phe Thr Gln Trp Ser Glu Ser
195 200 205
Gly Gln Val Asp Phe Val Glu His Leu Leu Ser Arg Met Cys His Tyr
210 215 220
Gln His Gly Gln Ile Asn Ala Tyr Leu Lys Pro Met Leu Gln Arg Asp
225 230 235 240
Phe Ile Thr Leu Leu Pro Ile Lys Gly Leu Asp His Ile Ala Glu Asn
245 250 255
Ile Leu Ser Tyr Leu Asp Ala Glu Ser Leu Lys Ser Ser Glu Leu Val
260 265 270
Cys Lys Glu Trp Leu Arg Val Ile Ser Glu Gly Met Leu Trp Lys Lys
275 280 285
Leu Ile Glu Arg Lys Val Arg Thr Asp Ser Leu Trp Arg Gly Leu Ala
290 295 300
Glu Arg Arg Asn Trp Met Gln Tyr Leu Phe Lys Pro Arg Pro Gly Gln
305 310 315 320
Thr Gln Arg Pro His Ser Phe His Arg Glu Leu Phe Pro Lys Ile Met
325 330 335
Asn Asp Ile Asp Ser Ile Glu Asn Asn Trp Arg Thr Gly Arg His Leu
340 345 350
Glu Met Asp Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln
355 360 365
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val
370 375 380
Asn Arg Tyr Ser Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg
385 390 395 400
Glu Trp Val Ala Gly Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu
405 410 415
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn
420 425 430
Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
435 440 445
Tyr Tyr Cys Asn Val Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr
450 455 460
Gln Val Thr Val Ser Ser
465 470
<210> 34
<211> 466
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Slmb1-VhH4-NLS
<400> 34
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Lys Met
130 135 140
Met Lys Met Glu Thr Asp Lys Ile Met Asp Glu Thr Asn Ser Asn Ala
145 150 155 160
Gln Ala Phe Thr Thr Thr Met Leu Tyr Asp Pro Val Arg Lys Lys Asp
165 170 175
Ser Ser Pro Thr Tyr Gln Thr Glu Arg Glu Leu Cys Phe Gln Tyr Phe
180 185 190
Thr Gln Trp Ser Glu Ser Gly Gln Val Asp Phe Val Glu His Leu Leu
195 200 205
Ser Arg Met Cys His Tyr Gln His Gly Gln Ile Asn Ala Tyr Leu Lys
210 215 220
Pro Met Leu Gln Arg Asp Phe Ile Thr Leu Leu Pro Ile Lys Gly Leu
225 230 235 240
Asp His Ile Ala Glu Asn Ile Leu Ser Tyr Leu Asp Ala Glu Ser Leu
245 250 255
Lys Ser Ser Glu Leu Val Cys Lys Glu Trp Leu Arg Val Ile Ser Glu
260 265 270
Gly Met Leu Trp Lys Lys Leu Ile Glu Arg Lys Val Arg Thr Asp Ser
275 280 285
Leu Trp Arg Gly Leu Ala Glu Arg Arg Asn Trp Met Gln Tyr Leu Phe
290 295 300
Lys Pro Arg Pro Gly Gln Thr Gln Arg Pro His Ser Phe His Arg Glu
305 310 315 320
Leu Phe Pro Lys Ile Met Asn Asp Ile Asp Ser Ile Glu Asn Asn Trp
325 330 335
Arg Thr Gly Arg His Met Asp Gln Val Gln Leu Val Glu Ser Gly Gly
340 345 350
Ala Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
355 360 365
Gly Phe Pro Val Asn Arg Tyr Ser Met Arg Trp Tyr Arg Gln Ala Pro
370 375 380
Gly Lys Glu Arg Glu Trp Val Ala Gly Met Ser Ser Ala Gly Asp Arg
385 390 395 400
Ser Ser Tyr Glu Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
405 410 415
Asp Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu
420 425 430
Asp Thr Ala Val Tyr Tyr Cys Asn Val Asn Val Gly Phe Glu Tyr Trp
435 440 445
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Pro Pro Lys Lys Lys Arg
450 455 460
Lys Val
465
<210> 35
<211> 246
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Akt PH-domain
<400> 35
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Ala Ile
130 135 140
Val Lys Glu Gly Trp Leu His Lys Arg Gly Glu Tyr Ile Lys Thr Trp
145 150 155 160
Arg Pro Arg Tyr Phe Leu Leu Lys Asn Asp Gly Thr Phe Ile Gly Tyr
165 170 175
Lys Glu Arg Pro Gln Asp Val Asp Gln Arg Glu Ala Pro Leu Asn Asn
180 185 190
Phe Ser Val Ala Gln Cys Gln Leu Met Lys Thr Glu Arg Pro Arg Pro
195 200 205
Asn Thr Phe Ile Ile Arg Cys Leu Gln Trp Thr Thr Val Ile Glu Arg
210 215 220
Thr Phe His Val Glu Thr Pro Glu Glu Arg Glu Glu Trp Thr Thr Ala
225 230 235 240
Ile Gln Thr Val Ala Asp
245
<210> 36
<211> 465
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - ET1
<400> 36
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Pro
130 135 140
Arg Pro Lys Leu Lys Ser Asp Asp Glu Val Leu Glu Ala Ala Thr Val
145 150 155 160
Val Leu Lys Arg Cys Gly Pro Ile Glu Phe Thr Leu Ser Gly Val Ala
165 170 175
Lys Glu Val Gly Leu Ser Arg Ala Ala Leu Ile Gln Arg Phe Thr Asn
180 185 190
Arg Asp Thr Leu Leu Val Arg Met Met Glu Arg Gly Val Glu Gln Val
195 200 205
Arg His Tyr Leu Asn Ala Ile Pro Ile Gly Ala Gly Pro Gln Gly Leu
210 215 220
Trp Glu Phe Leu Gln Val Leu Val Arg Ser Met Asn Thr Arg Asn Asp
225 230 235 240
Phe Ser Val Asn Tyr Leu Ile Ser Trp Tyr Glu Leu Gln Val Pro Glu
245 250 255
Leu Arg Thr Leu Ala Ile Gln Arg Asn Arg Ala Val Val Glu Gly Ile
260 265 270
Arg Lys Arg Leu Pro Pro Gly Ala Pro Ala Ala Ala Glu Leu Leu Leu
275 280 285
His Ser Val Ile Ala Gly Ala Thr Met Gln Trp Ala Val Asp Pro Asp
290 295 300
Gly Glu Leu Ala Asp His Val Leu Ala Gln Ile Ala Ala Ile Leu Cys
305 310 315 320
Leu Met Phe Pro Glu His Asp Asp Phe Gln Leu Leu Gln Ala His Ala
325 330 335
Ser Ala Tyr Ser Arg Ala Arg Thr Lys Asn Asn Tyr Gly Ser Thr Ile
340 345 350
Glu Gly Leu Leu Asp Leu Pro Asp Asp Asp Ala Pro Glu Glu Ala Gly
355 360 365
Leu Ala Ala Pro Arg Leu Ser Phe Leu Pro Ala Gly His Thr Arg Arg
370 375 380
Leu Ser Thr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His
385 390 395 400
Leu Asp Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp
405 410 415
Phe Asp Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe
420 425 430
Thr Pro His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe
435 440 445
Glu Phe Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly
450 455 460
Gly
465
<210> 37
<211> 381
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - EGFP
<400> 37
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Met Val
130 135 140
Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu
145 150 155 160
Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
165 170 175
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr
180 185 190
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr
195 200 205
Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His
210 215 220
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
225 230 235 240
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys
245 250 255
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp
260 265 270
Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr
275 280 285
Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile
290 295 300
Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln
305 310 315 320
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val
325 330 335
Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys
340 345 350
Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr
355 360 365
Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
370 375 380
<210> 38
<211> 402
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - 2xTEVsite - NLS - EGFP
<400> 38
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Glu Asn
130 135 140
Leu Tyr Phe Gln Ser Glu Asn Leu Tyr Phe Gln Ser Pro Pro Lys Lys
145 150 155 160
Lys Arg Lys Val Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val
165 170 175
Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
180 185 190
Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu
195 200 205
Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu
210 215 220
Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp
225 230 235 240
His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
245 250 255
Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr
260 265 270
Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
275 280 285
Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys
290 295 300
Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys
305 310 315 320
Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu
325 330 335
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
340 345 350
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln
355 360 365
Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
370 375 380
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
385 390 395 400
Tyr Lys
<210> 39
<211> 402
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - 2xTEVsite - EGFP - NLS
<400> 39
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Glu Asn
130 135 140
Leu Tyr Phe Gln Ser Glu Asn Leu Tyr Phe Gln Ser Val Ser Lys Gly
145 150 155 160
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
165 170 175
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
180 185 190
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys
195 200 205
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val
210 215 220
Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe
225 230 235 240
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
245 250 255
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly
260 265 270
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu
275 280 285
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His
290 295 300
Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn
305 310 315 320
Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp
325 330 335
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
340 345 350
Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn
355 360 365
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
370 375 380
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Pro Pro Lys Lys Lys Arg
385 390 395 400
Lys Val
<210> 40
<211> 324
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - 2x TEVsite - INK4C
<400> 40
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Glu Asn
130 135 140
Leu Tyr Phe Gln Ser Glu Asn Leu Tyr Phe Gln Ser Met Ala Glu Pro
145 150 155 160
Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly Asp Leu Glu Gln
165 170 175
Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn Ala Gln Asn Gly
180 185 190
Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly Asn Pro Glu Ile
195 200 205
Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp Leu Lys Asp Arg
210 215 220
Thr Gly Phe Ala Val Ile His Asp Ala Ala Arg Ala Gly Phe Leu Asp
225 230 235 240
Thr Leu Gln Ala Leu Pro Glu Phe Gln Ala Asp Val Asn Ile Glu Asp
245 250 255
Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys Glu Gly His Leu
260 265 270
Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser Asn Val Gly His
275 280 285
Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala Arg Leu Tyr Gly
290 295 300
Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly Ala Gly Gly Ala
305 310 315 320
Thr Asn Leu Gln
<210> 41
<211> 479
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - 2x TEVsite - ET1
<400> 41
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Glu Asn
130 135 140
Leu Tyr Phe Gln Ser Glu Asn Leu Tyr Phe Gln Ser Met Pro Arg Pro
145 150 155 160
Lys Leu Lys Ser Asp Asp Glu Val Leu Glu Ala Ala Thr Val Val Leu
165 170 175
Lys Arg Cys Gly Pro Ile Glu Phe Thr Leu Ser Gly Val Ala Lys Glu
180 185 190
Val Gly Leu Ser Arg Ala Ala Leu Ile Gln Arg Phe Thr Asn Arg Asp
195 200 205
Thr Leu Leu Val Arg Met Met Glu Arg Gly Val Glu Gln Val Arg His
210 215 220
Tyr Leu Asn Ala Ile Pro Ile Gly Ala Gly Pro Gln Gly Leu Trp Glu
225 230 235 240
Phe Leu Gln Val Leu Val Arg Ser Met Asn Thr Arg Asn Asp Phe Ser
245 250 255
Val Asn Tyr Leu Ile Ser Trp Tyr Glu Leu Gln Val Pro Glu Leu Arg
260 265 270
Thr Leu Ala Ile Gln Arg Asn Arg Ala Val Val Glu Gly Ile Arg Lys
275 280 285
Arg Leu Pro Pro Gly Ala Pro Ala Ala Ala Glu Leu Leu Leu His Ser
290 295 300
Val Ile Ala Gly Ala Thr Met Gln Trp Ala Val Asp Pro Asp Gly Glu
305 310 315 320
Leu Ala Asp His Val Leu Ala Gln Ile Ala Ala Ile Leu Cys Leu Met
325 330 335
Phe Pro Glu His Asp Asp Phe Gln Leu Leu Gln Ala His Ala Ser Ala
340 345 350
Tyr Ser Arg Ala Arg Thr Lys Asn Asn Tyr Gly Ser Thr Ile Glu Gly
355 360 365
Leu Leu Asp Leu Pro Asp Asp Asp Ala Pro Glu Glu Ala Gly Leu Ala
370 375 380
Ala Pro Arg Leu Ser Phe Leu Pro Ala Gly His Thr Arg Arg Leu Ser
385 390 395 400
Thr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His Leu Asp
405 410 415
Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp
420 425 430
Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro
435 440 445
His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe
450 455 460
Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly
465 470 475
<210> 42
<211> 380
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - TEV protease S219V
<400> 42
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Glu Ser Leu Phe
130 135 140
Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu
145 150 155 160
Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe
165 170 175
Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly
180 185 190
Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn Thr
195 200 205
Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile Ile
210 215 220
Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg
225 230 235 240
Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gln
245 250 255
Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro
260 265 270
Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly
275 280 285
Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly
290 295 300
Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser
305 310 315 320
Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln
325 330 335
Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly
340 345 350
His Lys Val Phe Met Val Lys Pro Glu Glu Pro Phe Gln Pro Val Lys
355 360 365
Glu Ala Thr Gln Leu Met Asn Arg Arg Arg Arg Arg
370 375 380
<210> 43
<211> 332
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - 2x TEVsite - Flag - INK4C
<400> 43
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Glu Asn
130 135 140
Leu Tyr Phe Gln Ser Glu Asn Leu Tyr Phe Gln Ser Asp Tyr Lys Asp
145 150 155 160
Asp Asp Asp Lys Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala
165 170 175
Ala Ala Arg Gly Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn
180 185 190
Val Asn Val Asn Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val
195 200 205
Met Lys Leu Gly Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly
210 215 220
Ala Asn Pro Asp Leu Lys Asp Arg Thr Gly Phe Ala Val Ile His Asp
225 230 235 240
Ala Ala Arg Ala Gly Phe Leu Asp Thr Leu Gln Ala Leu Pro Glu Phe
245 250 255
Gln Ala Asp Val Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His
260 265 270
Leu Ala Ala Lys Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys
275 280 285
His Thr Ala Ser Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala
290 295 300
Cys Asp Leu Ala Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met
305 310 315 320
Gln Ala Asn Gly Ala Gly Gly Ala Thr Asn Leu Gln
325 330
<210> 44
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_285
<400> 44
cataccatgg gagtgagcaa gggcgag 27
<210> 45
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_286
<400> 45
ggaagatctt tacttgtaca gctcgtccat 30
<210> 46
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_287
<400> 46
cggggtacct caactaaatg accgtggtg 29
<210> 47
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_288
<400> 47
gttaaagctt ttcgaatcta gactcgagcg tggcgaactg gtc 43
<210> 48
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_292
<400> 48
cagtctcgag caaattctaa acaaaatact tccac 35
<210> 49
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_293
<400> 49
cagtttcgaa ttaatttgta ttgctttgac gg 32
<210> 50
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_296
<400> 50
cagtctcgag actaacataa cactatccac ccag 34
<210> 51
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_297
<400> 51
gttaaagctt tcaggaggca ttctgaag 28
<210> 52
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_299
<400> 52
cagtctcgag caggccatca agtgtgtg 28
<210> 53
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_300
<400> 53
cagtttcgaa tcattttctc ttcctcttct tca 33
<210> 54
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_301
<400> 54
cagtctcgag gctgccatcc ggaa 24
<210> 55
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_302
<400> 55
cagtttcgaa tcacaagaca aggcaccc 28
<210> 56
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_306
<400> 56
gttaaagctt ggaggcattc tgaagatact tatt 34
<210> 57
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_307
<400> 57
cagtctcgag caaatacaga gcttctatca ctcag 35
<210> 58
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_308
<400> 58
gttaaagctt tcaagatgtg attaatgaag aaatg 35
<210> 59
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_317
<400> 59
cagtttcgaa cccataaaaa agccctgtc 29
<210> 60
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_318
<400> 60
gttaaagctt ctactctatc atcaaacgat aaaatgg 37
<210> 61
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_324
<400> 61
cagtctcgag ttcactcaag aaacgcaaa 29
<210> 62
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_339
<400> 62
cagtttcgaa ttttctcttc ctcttcttca cg 32
<210> 63
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_341
<400> 63
cgtatctaga aaaatgatga aaatggagac tg 32
<210> 64
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_342
<400> 64
gttaaagctt ttagctggag acggtgac 28
<210> 65
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_346
<400> 65
cagtctcgag ttccagatcc cagagtttg 29
<210> 66
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_347
<400> 66
gttaaagctt tcactgggag gggg 24
<210> 67
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_351
<400> 67
cagtctcgag ctcgagttat ctactcatag aaactacttt tgcag 45
<210> 68
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_352
<400> 68
cgcggatcct cagtgtctct gcggcatta 29
<210> 69
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_353
<400> 69
catttattcc tcctagttag tcacagcaac tgctgctcct ttc 43
<210> 70
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_354
<400> 70
gaaaggagca gcagttgctg tgactaacta ggaggaataa atg 43
<210> 71
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_355
<400> 71
cgattcacgg attgctttct cattattccc tccaggtact a 41
<210> 72
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_356
<400> 72
tagtacctgg agggaataat gagaaagcaa tccgtgaatc g 41
<210> 73
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_357
<400> 73
cgtatctaga cggctttaag tgcgacattc 30
<210> 74
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_364
<400> 74
cgtatctaga ctaaagtatg aggagagaaa attgaa 36
<210> 75
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_365
<400> 75
gttaaagctt tcagcttgcc gtcgt 25
<210> 76
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_367
<400> 76
cgtatctaga gacccgttcc tggtgc 26
<210> 77
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_369
<400> 77
cgtatctaga ccccccaaga agaagc 26
<210> 78
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_373
<400> 78
gttaaagctt gctggagacg gtgacc 26
<210> 79
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_386
<400> 79
cgtatctaga tcaggacgct tcggaggtag 30
<210> 80
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_387
<400> 80
cgtatctaga atggactgtg aggtcaacaa 30
<210> 81
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_389
<400> 81
cgtatctaga ggcaaccgca gca 23
<210> 82
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_391
<400> 82
gttaaagctt tcagtccatc ccatttctg 29
<210> 83
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_403
<400> 83
cgtatctaga tctggaatat ccctggaca 29
<210> 84
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_406
<400> 84
gttaaagctt gtctgtctca atgccacagt 30
<210> 85
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_410
<400> 85
cagtctcgag atgtccgggg tggtg 25
<210> 86
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_413
<400> 86
cagtttcgaa tcactgcagc atgatgtc 28
<210> 87
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_417
<400> 87
cagtctcgag agtggtgttg atgatgacat g 31
<210> 88
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_420
<400> 88
cagtttcgaa ttagtgataa aaatagagtt cttttgtgag 40
<210> 89
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_423
<400> 89
cagtctcgag atgcacataa ctaatttggg att 33
<210> 90
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_424
<400> 90
cagtttcgaa ttatacaaat gacgaatacc cttt 34
<210> 91
<211> 52
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_425
<400> 91
gttaaagctt ttacaccttg cgcttcttct tgggcgggct ggagacggtg ac 52
<210> 92
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_428
<400> 92
cgtatctaga atggacttca acaggaactt t 31
<210> 93
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_429
<400> 93
cgtatctaga ggacatagtc caccagcg 28
<210> 94
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_430
<400> 94
gttaaagctt tcagttggat ccgaaaaac 29
<210> 95
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_433
<400> 95
cgtatctaga gaattaaaaa aaacactcat ccca 34
<210> 96
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_434
<400> 96
cgtatctaga ccaaaggcaa aagcaaaaa 29
<210> 97
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_435
<400> 97
gttaaagctt ttagctagcc atggcaagc 29
<210> 98
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_436
<400> 98
cgtatctaga atgccccgcc cc 22
<210> 99
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_437
<400> 99
gttaaagctt ctacccaccg tactcgtcaa t 31
<210> 100
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_438
<400> 100
cgtatctaga atgtctgaca cgtccagaga g 31
<210> 101
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_439
<400> 101
gttaaagctt tcatcttctt cgcaggaaaa ag 32
<210> 102
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_445
<400> 102
cgcggatcct tatgggttct cacagcaaaa 30
<210> 103
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_446
<400> 103
catttattcc tcctagttag tcaaggcaac agccaatcaa gag 43
<210> 104
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_447
<400> 104
ctcttgattg gctgttgcct tgactaacta ggaggaataa atg 43
<210> 105
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_448
<400> 105
ttgattgcag tgacatggtg cattattccc tccaggtact a 41
<210> 106
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_449
<400> 106
tagtacctgg agggaataat gcaccatgtc actgcaatca a 41
<210> 107
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_450
<400> 107
cgtatctaga tagccgcaga tgttggtatg 30
<210> 108
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_451
<400> 108
cgtatctaga gatcaagtcc aactggtgg 29
<210> 109
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_463
<400> 109
cagtctcgag gaaagcttgt ttaaggggc 29
<210> 110
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_464
<400> 110
cagtttcgaa ttagcgacgg cgacg 25
<210> 111
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_476
<400> 111
gttaaagctt ttacttgtac agctcgtcca t 31
<210> 112
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_477
<400> 112
cgtatctaga gtgagcaagg gcgag 25
<210> 113
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_478
<400> 113
cagtctcgag atggaagatt ataccaaaat agagaaa 37
<210> 114
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_479
<400> 114
gttaaagctt ctacatcttc ttaatctgat tgtcca 36
<210> 115
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_482
<400> 115
cgtatctaga atggcgctgc agct 24
<210> 116
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_483
<400> 116
gttaaagctt tcagtcattg acaggaattt tg 32
<210> 117
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_486
<400> 117
cgtatctaga atggagccgg cggcg 25
<210> 118
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_487
<400> 118
gttaaagctt tcaatcgggg atgtctg 27
<210> 119
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_492
<400> 119
cgtatctaga atgcgcgagg agaacaaggg 30
<210> 120
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_493
<400> 120
gttaaagctt tcagtcccct gtggctgtgc 30
<210> 121
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_494
<400> 121
cgtatctaga atggccgagc cttg 24
<210> 122
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_495
<400> 122
gttaaagctt ttattgaaga tttgtggctc c 31
<210> 123
<211> 64
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_504
<400> 123
cgtatctaga gaaaatctgt attttcaaag tgaaaatctg tattttcaaa gtatgccccg 60
cccc 64
<210> 124
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_505
<400> 124
gttaaagctt cccaccgtac tcgtcaattc 30
<210> 125
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_508
<400> 125
cgtatctaga gaaaatctgt attttcaaag tgaaaatctg tattttcaaa gtatggccga 60
gccttg 66
<210> 126
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_509
<400> 126
gttaaagctt ttgaagattt gtggctccc 29
<210> 127
<211> 67
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_511
<400> 127
cgtatctaga gaaaatctgt attttcaaag tgaaaatctg tattttcaaa gtgtgagcaa 60
gggcgag 67
<210> 128
<211> 91
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_512
<400> 128
cgtatctaga gaaaatctgt attttcaaag tgaaaatctg tattttcaaa gtccgccgaa 60
aaaaaaacgt aaagttgtga gcaagggcga g 91
<210> 129
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_513
<400> 129
gttaaagctt ttaaacttta cgtttttttt tcggcggctt gtacagctcg tccat 55
<210> 130
<211> 90
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_515
<400> 130
cgtatctaga gaaaatctgt attttcaaag tgaaaatctg tattttcaaa gtgattataa 60
agatgatgat gataaaatgg ccgagccttg 90
<210> 131
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_558
<400> 131
cgtatctaga atgaccagtt ttgaagatgc 30
<210> 132
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_559
<400> 132
gttaaagctt tcatgactca ttttcatcca t 31
<210> 133
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_561
<400> 133
cgtatctaga atgagtctct taaactgtga gaacag 36
<210> 134
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_562
<400> 134
gttaaagctt ctacaccccc gcatca 26
<210> 135
<211> 405
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - SopE - MycHis
<400> 135
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Val Thr Asn Ile
130 135 140
Thr Leu Ser Thr Gln His Tyr Arg Ile His Arg Ser Asp Val Glu Pro
145 150 155 160
Val Lys Glu Lys Thr Thr Glu Lys Asp Ile Phe Ala Lys Ser Ile Thr
165 170 175
Ala Val Arg Asn Ser Phe Ile Ser Leu Ser Thr Ser Leu Ser Asp Arg
180 185 190
Phe Ser Leu His Gln Gln Thr Asp Ile Pro Thr Thr His Phe His Arg
195 200 205
Gly Asn Ala Ser Glu Gly Arg Ala Val Leu Thr Ser Lys Thr Val Lys
210 215 220
Asp Phe Met Leu Gln Lys Leu Asn Ser Leu Asp Ile Lys Gly Asn Ala
225 230 235 240
Ser Lys Asp Pro Ala Tyr Ala Arg Gln Thr Cys Glu Ala Ile Leu Ser
245 250 255
Ala Val Tyr Ser Asn Asn Lys Asp Gln Cys Cys Lys Leu Leu Ile Ser
260 265 270
Lys Gly Val Ser Ile Thr Pro Phe Leu Lys Glu Ile Gly Glu Ala Ala
275 280 285
Gln Asn Ala Gly Leu Pro Gly Glu Ile Lys Asn Gly Val Phe Thr Pro
290 295 300
Gly Gly Ala Gly Ala Asn Pro Phe Val Val Pro Leu Ile Ala Ser Ala
305 310 315 320
Ser Ile Lys Tyr Pro His Met Phe Ile Asn His Asn Gln Gln Val Ser
325 330 335
Phe Lys Ala Tyr Ala Glu Lys Ile Val Met Lys Glu Val Thr Pro Leu
340 345 350
Phe Asn Lys Gly Thr Met Pro Thr Pro Gln Gln Phe Gln Leu Thr Ile
355 360 365
Glu Asn Ile Ala Asn Lys Tyr Leu Gln Asn Ala Ser Lys Leu Gly Pro
370 375 380
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His
385 390 395 400
His His His His His
405
<210> 136
<211> 435
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - BepG 715-end
<400> 136
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Phe Thr Gln Glu
130 135 140
Thr Gln Lys Met Leu Ile Glu Lys Glu Ile Ile Pro Pro Leu Ser Tyr
145 150 155 160
Val Asp Val Ala Ser Lys Ile Arg Glu Ser Glu Val Val Lys Ser Ser
165 170 175
Met Gln Lys Ile Lys Thr Leu Cys Gly Val Val Tyr Gly Asn Pro Asp
180 185 190
Ile Leu Glu Gly Lys Met Pro Lys Met Gly Ile Pro Val Thr Asn Lys
195 200 205
Asn Val Glu Glu Leu Glu Lys Phe Ala Arg Gln Val Gly Asn Phe Pro
210 215 220
Ser Ser Cys Gly Lys Ile Val Gly Phe Ser Phe Leu Gly Ile Lys Ser
225 230 235 240
Glu Ala Arg Ala His Ala Glu Glu Asn Phe Leu Pro Leu Ser His Ala
245 250 255
Ile Phe Ser Tyr Ala His Asn Val Lys Gln Ala Glu Lys Asp Ile Leu
260 265 270
Glu Ala Tyr Phe Lys Glu Gln Glu Arg Cys Ala Gln Ser Val Glu Thr
275 280 285
Pro Ser Glu Glu Ile Thr Asn Leu Leu Ser Phe Thr Gln Glu Gln Gln
290 295 300
Lys Glu Ile Leu Ser Asn Ser Pro Lys Leu Arg Thr Gln Val Lys Ala
305 310 315 320
Tyr Ser Gln Lys Leu His Asn Arg Leu Ser Pro Asn Asp Leu Gln Ala
325 330 335
Ile Ser Glu Arg Ser His Thr Lys Leu Ala Glu Ser Leu Gly Thr Ser
340 345 350
Val Asn Gln Ala Glu Lys Ile Ala Gln Ile Leu Thr Gln Thr Lys Asp
355 360 365
Val Val Gln Ile Leu Gln Gln Gln Glu Lys Leu Gly Leu Tyr Gln Ser
370 375 380
Ile Met Lys Gly Asp Gly Arg Glu Thr Ala Lys Val Asn Met Ser Ala
385 390 395 400
Ile Lys Ala Thr Gln Met Thr Thr Lys Val Thr Ser Leu Lys Ala Val
405 410 415
Glu Gln Ile Val Arg Pro Pro Lys Val Glu Thr Ala Lys Val Val Ser
420 425 430
Met Ser Arg
435
<210> 137
<211> 354
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Rac1 Q61E - MycHis
<400> 137
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Gln Ala Ile Lys
130 135 140
Cys Val Val Val Gly Asp Gly Ala Val Gly Lys Thr Cys Leu Leu Ile
145 150 155 160
Ser Tyr Thr Thr Asn Ala Phe Pro Gly Glu Tyr Ile Pro Thr Val Phe
165 170 175
Asp Asn Tyr Ser Ala Asn Val Met Val Asp Gly Lys Pro Val Asn Leu
180 185 190
Gly Leu Trp Asp Thr Ala Gly Glu Glu Asp Tyr Asp Arg Leu Arg Pro
195 200 205
Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Ile Cys Phe Ser Leu Val
210 215 220
Ser Pro Ala Ser Phe Glu Asn Val Arg Ala Lys Trp Tyr Pro Glu Val
225 230 235 240
Arg His His Cys Pro Asn Thr Pro Ile Ile Leu Val Gly Thr Lys Leu
245 250 255
Asp Leu Arg Asp Asp Lys Asp Thr Ile Glu Lys Leu Lys Glu Lys Lys
260 265 270
Leu Thr Pro Ile Thr Tyr Pro Gln Gly Leu Ala Met Ala Lys Glu Ile
275 280 285
Gly Ala Val Lys Tyr Leu Glu Cys Ser Ala Leu Thr Gln Arg Gly Leu
290 295 300
Lys Thr Val Phe Asp Glu Ala Ile Arg Ala Val Leu Cys Pro Pro Pro
305 310 315 320
Val Lys Lys Arg Lys Arg Lys Phe Glu Lys Leu Gly Pro Glu Gln Lys
325 330 335
Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His
340 345 350
His His
<210> 138
<211> 163
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Y. enterocolitica codon optimized murine BID BH3 part
<400> 138
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Ser Arg Phe Glu
130 135 140
Glu Ile Ile His Asn Ile Ala Arg His Leu Ala Gln Ile Gly Asp Glu
145 150 155 160
Met Asp His
<210> 139
<211> 156
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138 - Y. enterocolitica codon optimized murine Bax BH3 part
<400> 139
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Lys Lys Leu Ser
130 135 140
Glu Cys Leu Arg Arg Ile Gly Asp Glu Leu Asp Ser
145 150 155
<210> 140
<211> 20
<212> PRT
<213> 肠炎沙门氏菌(Salmonella enterica)
<220>
<223> SteA1-20
<400> 140
Met Pro Tyr Thr Ser Val Ser Thr Tyr Ala Arg Ala Leu Ser Gly Asn
1 5 10 15
Lys Leu Pro His
20
<210> 141
<211> 210
<212> PRT
<213> 肠炎沙门氏菌(Salmonella enterica)
<220>
<223> SteA
<400> 141
Met Pro Tyr Thr Ser Val Ser Thr Tyr Ala Arg Ala Leu Ser Gly Asn
1 5 10 15
Lys Leu Pro His Val Ala Ala Gly Asp Tyr Glu Asn Lys Leu Ser Thr
20 25 30
Lys Ile Met Lys Gly Ile Leu Tyr Val Leu Thr Ala Gly Leu Ala Tyr
35 40 45
Gly Phe Thr Arg Val Ile Glu His Tyr Cys Asn Val Thr Pro Lys Val
50 55 60
Ala Glu Phe Cys Ala Asn Ala Gly Asn Ile His Asn His Leu Ala Asp
65 70 75 80
Ala Val Arg Asp Gly Leu Phe Thr Ile Asp Val Glu Leu Ser Asp Gly
85 90 95
Arg Met Leu Thr Phe Glu Gln Leu Ser Leu Ile Ala Glu Gly Lys Pro
100 105 110
Ile Val Arg Ile Ser Asp Gly Glu His Thr Val Glu Val Glu Gly Thr
115 120 125
Phe Glu Glu Ile Cys Met Arg Leu Glu Glu Gly Phe Phe Glu Ala Pro
130 135 140
Ala Tyr Tyr Asp Tyr Asp Ile Asp Glu Lys Tyr Lys Thr Val Arg Glu
145 150 155 160
Arg Met Ala Ala Tyr Asn Ala Leu Pro Gln Ala Leu Gly Ala Ile Pro
165 170 175
Cys Leu Glu Tyr Tyr Ile Ala Arg Ala Ser Asn Met Gln Glu Ala Lys
180 185 190
Ala Gln Trp Ala Ala Asp Ile Lys Ala Arg Tyr His Asn Tyr Leu Asp
195 200 205
Asn Tyr
210
<210> 142
<211> 81
<212> PRT
<213> 肠炎沙门氏菌(Salmonella enterica)
<220>
<223> SopE1-81
<400> 142
Val Thr Lys Ile Thr Leu Ser Pro Gln Asn Phe Arg Ile Gln Lys Gln
1 5 10 15
Glu Thr Thr Leu Leu Lys Glu Lys Ser Thr Glu Lys Asn Ser Leu Ala
20 25 30
Lys Ser Ile Leu Ala Val Lys Asn His Phe Ile Glu Leu Arg Ser Lys
35 40 45
Leu Ser Glu Arg Phe Ile Ser His Lys Asn Thr Glu Ser Ser Ala Thr
50 55 60
His Phe His Arg Gly Ser Ala Ser Glu Gly Arg Ala Val Leu Thr Asn
65 70 75 80
Lys
<210> 143
<211> 105
<212> PRT
<213> 肠炎沙门氏菌(Salmonella enterica)
<220>
<223> SopE1-105
<400> 143
Val Thr Lys Ile Thr Leu Ser Pro Gln Asn Phe Arg Ile Gln Lys Gln
1 5 10 15
Glu Thr Thr Leu Leu Lys Glu Lys Ser Thr Glu Lys Asn Ser Leu Ala
20 25 30
Lys Ser Ile Leu Ala Val Lys Asn His Phe Ile Glu Leu Arg Ser Lys
35 40 45
Leu Ser Glu Arg Phe Ile Ser His Lys Asn Thr Glu Ser Ser Ala Thr
50 55 60
His Phe His Arg Gly Ser Ala Ser Glu Gly Arg Ala Val Leu Thr Asn
65 70 75 80
Lys Val Val Lys Asp Phe Met Leu Gln Thr Leu Asn Asp Ile Asp Ile
85 90 95
Arg Gly Ser Ala Ser Lys Asp Pro Ala
100 105
<210> 144
<211> 158
<212> PRT
<213> 人工序列
<220>
<223> SteA1-20 - S. enterica codon optimized murine t-BID
<400> 144
Met Pro Tyr Thr Ser Val Ser Thr Tyr Ala Arg Ala Leu Ser Gly Asn
1 5 10 15
Lys Leu Pro His Gly Thr Gly Ser Gln Ala Ser Arg Ser Phe Asn Gln
20 25 30
Gly Arg Ile Glu Pro Asp Ser Glu Ser Gln Glu Glu Ile Ile His Asn
35 40 45
Ile Ala Arg His Leu Ala Gln Ile Gly Asp Glu Met Asp His Asn Ile
50 55 60
Gln Pro Thr Leu Val Arg Gln Leu Ala Ala Gln Phe Met Asn Gly Ser
65 70 75 80
Leu Ser Glu Glu Asp Lys Arg Asn Cys Leu Ala Lys Ala Leu Asp Glu
85 90 95
Val Lys Thr Ala Phe Pro Arg Asp Met Glu Asn Asp Lys Ala Met Leu
100 105 110
Ile Met Thr Met Leu Leu Ala Lys Lys Val Ala Ser His Ala Pro Ser
115 120 125
Leu Leu Arg Asp Val Phe His Thr Thr Val Asn Phe Ile Asn Gln Asn
130 135 140
Leu Phe Ser Tyr Val Arg Asn Leu Val Arg Asn Glu Met Asp
145 150 155
<210> 145
<211> 348
<212> PRT
<213> 人工序列
<220>
<223> SteA - S. enterica codon optimized murine t-BID
<400> 145
Met Pro Tyr Thr Ser Val Ser Thr Tyr Ala Arg Ala Leu Ser Gly Asn
1 5 10 15
Lys Leu Pro His Val Ala Ala Gly Asp Tyr Glu Asn Lys Leu Ser Thr
20 25 30
Lys Ile Met Lys Gly Ile Leu Tyr Val Leu Thr Ala Gly Leu Ala Tyr
35 40 45
Gly Phe Thr Arg Val Ile Glu His Tyr Cys Asn Val Thr Pro Lys Val
50 55 60
Ala Glu Phe Cys Ala Asn Ala Gly Asn Ile His Asn His Leu Ala Asp
65 70 75 80
Ala Val Arg Asp Gly Leu Phe Thr Ile Asp Val Glu Leu Ser Asp Gly
85 90 95
Arg Met Leu Thr Phe Glu Gln Leu Ser Leu Ile Ala Glu Gly Lys Pro
100 105 110
Ile Val Arg Ile Ser Asp Gly Glu His Thr Val Glu Val Glu Gly Thr
115 120 125
Phe Glu Glu Ile Cys Met Arg Leu Glu Glu Gly Phe Phe Glu Ala Pro
130 135 140
Ala Tyr Tyr Asp Tyr Asp Ile Asp Glu Lys Tyr Lys Thr Val Arg Glu
145 150 155 160
Arg Met Ala Ala Tyr Asn Ala Leu Pro Gln Ala Leu Gly Ala Ile Pro
165 170 175
Cys Leu Glu Tyr Tyr Ile Ala Arg Ala Ser Asn Met Gln Glu Ala Lys
180 185 190
Ala Gln Trp Ala Ala Asp Ile Lys Ala Arg Tyr His Asn Tyr Leu Asp
195 200 205
Asn Tyr Gly Thr Gly Ser Gln Ala Ser Arg Ser Phe Asn Gln Gly Arg
210 215 220
Ile Glu Pro Asp Ser Glu Ser Gln Glu Glu Ile Ile His Asn Ile Ala
225 230 235 240
Arg His Leu Ala Gln Ile Gly Asp Glu Met Asp His Asn Ile Gln Pro
245 250 255
Thr Leu Val Arg Gln Leu Ala Ala Gln Phe Met Asn Gly Ser Leu Ser
260 265 270
Glu Glu Asp Lys Arg Asn Cys Leu Ala Lys Ala Leu Asp Glu Val Lys
275 280 285
Thr Ala Phe Pro Arg Asp Met Glu Asn Asp Lys Ala Met Leu Ile Met
290 295 300
Thr Met Leu Leu Ala Lys Lys Val Ala Ser His Ala Pro Ser Leu Leu
305 310 315 320
Arg Asp Val Phe His Thr Thr Val Asn Phe Ile Asn Gln Asn Leu Phe
325 330 335
Ser Tyr Val Arg Asn Leu Val Arg Asn Glu Met Asp
340 345
<210> 146
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> SopE1-81 - S. enterica codon optimized murine t-BID
<400> 146
Val Thr Lys Ile Thr Leu Ser Pro Gln Asn Phe Arg Ile Gln Lys Gln
1 5 10 15
Glu Thr Thr Leu Leu Lys Glu Lys Ser Thr Glu Lys Asn Ser Leu Ala
20 25 30
Lys Ser Ile Leu Ala Val Lys Asn His Phe Ile Glu Leu Arg Ser Lys
35 40 45
Leu Ser Glu Arg Phe Ile Ser His Lys Asn Thr Glu Ser Ser Ala Thr
50 55 60
His Phe His Arg Gly Ser Ala Ser Glu Gly Arg Ala Val Leu Thr Asn
65 70 75 80
Lys Gly Thr Gly Ser Gln Ala Ser Arg Ser Phe Asn Gln Gly Arg Ile
85 90 95
Glu Pro Asp Ser Glu Ser Gln Glu Glu Ile Ile His Asn Ile Ala Arg
100 105 110
His Leu Ala Gln Ile Gly Asp Glu Met Asp His Asn Ile Gln Pro Thr
115 120 125
Leu Val Arg Gln Leu Ala Ala Gln Phe Met Asn Gly Ser Leu Ser Glu
130 135 140
Glu Asp Lys Arg Asn Cys Leu Ala Lys Ala Leu Asp Glu Val Lys Thr
145 150 155 160
Ala Phe Pro Arg Asp Met Glu Asn Asp Lys Ala Met Leu Ile Met Thr
165 170 175
Met Leu Leu Ala Lys Lys Val Ala Ser His Ala Pro Ser Leu Leu Arg
180 185 190
Asp Val Phe His Thr Thr Val Asn Phe Ile Asn Gln Asn Leu Phe Ser
195 200 205
Tyr Val Arg Asn Leu Val Arg Asn Glu Met Asp
210 215
<210> 147
<211> 243
<212> PRT
<213> 人工序列
<220>
<223> SopE1-105 - S. enterica codon optimized murine t-BID
<400> 147
Val Thr Lys Ile Thr Leu Ser Pro Gln Asn Phe Arg Ile Gln Lys Gln
1 5 10 15
Glu Thr Thr Leu Leu Lys Glu Lys Ser Thr Glu Lys Asn Ser Leu Ala
20 25 30
Lys Ser Ile Leu Ala Val Lys Asn His Phe Ile Glu Leu Arg Ser Lys
35 40 45
Leu Ser Glu Arg Phe Ile Ser His Lys Asn Thr Glu Ser Ser Ala Thr
50 55 60
His Phe His Arg Gly Ser Ala Ser Glu Gly Arg Ala Val Leu Thr Asn
65 70 75 80
Lys Val Val Lys Asp Phe Met Leu Gln Thr Leu Asn Asp Ile Asp Ile
85 90 95
Arg Gly Ser Ala Ser Lys Asp Pro Ala Gly Thr Gly Ser Gln Ala Ser
100 105 110
Arg Ser Phe Asn Gln Gly Arg Ile Glu Pro Asp Ser Glu Ser Gln Glu
115 120 125
Glu Ile Ile His Asn Ile Ala Arg His Leu Ala Gln Ile Gly Asp Glu
130 135 140
Met Asp His Asn Ile Gln Pro Thr Leu Val Arg Gln Leu Ala Ala Gln
145 150 155 160
Phe Met Asn Gly Ser Leu Ser Glu Glu Asp Lys Arg Asn Cys Leu Ala
165 170 175
Lys Ala Leu Asp Glu Val Lys Thr Ala Phe Pro Arg Asp Met Glu Asn
180 185 190
Asp Lys Ala Met Leu Ile Met Thr Met Leu Leu Ala Lys Lys Val Ala
195 200 205
Ser His Ala Pro Ser Leu Leu Arg Asp Val Phe His Thr Thr Val Asn
210 215 220
Phe Ile Asn Gln Asn Leu Phe Ser Tyr Val Arg Asn Leu Val Arg Asn
225 230 235 240
Glu Met Asp
<210> 148
<211> 82
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_677
<400> 148
ttactattcg aagaaattat tcataatatt gcccgccatc tggcccaaat tggtgatgaa 60
atggatcatt aagcttggag ta 82
<210> 149
<211> 82
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_678
<400> 149
tactccaagc ttaatgatcc atttcatcac caatttgggc cagatggcgg gcaatattat 60
gaataatttc ttcgaatagt aa 82
<210> 150
<211> 73
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_682
<400> 150
ttactactcg agaaaaaact gagcgaatgt ctgcgccgca ttggtgatga actggatagc 60
taagcttgga gta 73
<210> 151
<211> 73
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_683
<400> 151
tactccaagc ttagctatcc agttcatcac caatgcggcg cagacattcg ctcagttttt 60
tctcgagtag taa 73
<210> 152
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_580
<400> 152
catgccatgg atttatggtc atagatatga cctc 34
<210> 153
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_612
<400> 153
cggggtacca tgaggtagct tatttcctga taaag 35
<210> 154
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_613
<400> 154
cggggtacca taattgtcca aatagttatg gtagc 35
<210> 155
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_614
<400> 155
catgccatgg cggcaaggct cctc 24
<210> 156
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_615
<400> 156
cggggtacct ttatttgtca acactgccc 29
<210> 157
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_616
<400> 157
cggggtacct gcggggtctt tactcg 26
<210> 158
<211> 164
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138-Y. enterocolitica codon optimized Ink4A 84-103
<400> 158
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Gly Ala Ile Asp
130 135 140
Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val Leu His Arg
145 150 155 160
Ala Gly Ala Arg
<210> 159
<211> 164
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138-Y. enterocolitica codon optimized p107/RBL1 657-662
(AAA02489.1)
<400> 159
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Gly Ala Ile Asp
130 135 140
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Pro Val Lys Arg
145 150 155 160
Arg Leu Phe Gly
<210> 160
<211> 164
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138-Y. enterocolitica codon optimized p21 141-160
(AAH13967.1)
<400> 160
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Gly Ala Ile Asp
130 135 140
Lys Arg Arg Gln Thr Ser Met Thr Ala Phe Tyr His Ser Lys Arg Arg
145 150 155 160
Leu Ile Phe Ser
<210> 161
<211> 160
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138-Y. enterocolitica codon optimized p21 145-160
(AAH13967.1)
<400> 161
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Gly Ala Ile Asp
130 135 140
Thr Ser Met Thr Ala Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser
145 150 155 160
<210> 162
<211> 161
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138-Y. enterocolitica codon optimized p21 17-33
(AAH13967.1)
<400> 162
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Gly Ala Ile Asp
130 135 140
Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser Arg
145 150 155 160
Asp
<210> 163
<211> 153
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138-Y. enterocolitica codon optimized cyclin D2 139-147 (
CAA48493.1)
<400> 163
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Gly Ala Ile Asp
130 135 140
Trp Glu Leu Val Val Leu Gly Lys Leu
145 150
<210> 164
<211> 397
<212> PRT
<213> 人工序列
<220>
<223> SteA-Ink4a-MycHis
<400> 164
Met Pro Tyr Thr Ser Val Ser Thr Tyr Ala Arg Ala Leu Ser Gly Asn
1 5 10 15
Lys Leu Pro His Val Ala Ala Gly Asp Tyr Glu Asn Lys Leu Ser Thr
20 25 30
Lys Ile Met Lys Gly Ile Leu Tyr Val Leu Thr Ala Gly Leu Ala Tyr
35 40 45
Gly Phe Thr Arg Val Ile Glu His Tyr Cys Asn Val Thr Pro Lys Val
50 55 60
Ala Glu Phe Cys Ala Asn Ala Gly Asn Ile His Asn His Leu Ala Asp
65 70 75 80
Ala Val Arg Asp Gly Leu Phe Thr Ile Asp Val Glu Leu Ser Asp Gly
85 90 95
Arg Met Leu Thr Phe Glu Gln Leu Ser Leu Ile Ala Glu Gly Lys Pro
100 105 110
Ile Val Arg Ile Ser Asp Gly Glu His Thr Val Glu Val Glu Gly Thr
115 120 125
Phe Glu Glu Ile Cys Met Arg Leu Glu Glu Gly Phe Phe Glu Ala Pro
130 135 140
Ala Tyr Tyr Asp Tyr Asp Ile Asp Glu Lys Tyr Lys Thr Val Arg Glu
145 150 155 160
Arg Met Ala Ala Tyr Asn Ala Leu Pro Gln Ala Leu Gly Ala Ile Pro
165 170 175
Cys Leu Glu Tyr Tyr Ile Ala Arg Ala Ser Asn Met Gln Glu Ala Lys
180 185 190
Ala Gln Trp Ala Ala Asp Ile Lys Ala Arg Tyr His Asn Tyr Leu Asp
195 200 205
Asn Tyr Gly Thr Ile Trp Glu Phe Met Glu Pro Ala Ala Gly Ser Ser
210 215 220
Met Glu Pro Ser Ala Asp Trp Leu Ala Thr Ala Ala Ala Arg Gly Arg
225 230 235 240
Val Glu Glu Val Arg Ala Leu Leu Glu Ala Gly Ala Leu Pro Asn Ala
245 250 255
Pro Asn Ser Tyr Gly Arg Arg Pro Ile Gln Val Met Met Met Gly Ser
260 265 270
Ala Arg Val Ala Glu Leu Leu Leu Leu His Gly Ala Glu Pro Asn Cys
275 280 285
Ala Asp Pro Ala Thr Leu Thr Arg Pro Val His Asp Ala Ala Arg Glu
290 295 300
Gly Phe Leu Asp Thr Leu Val Val Leu His Arg Ala Gly Ala Arg Leu
305 310 315 320
Asp Val Arg Asp Ala Trp Gly Arg Leu Pro Val Asp Leu Ala Glu Glu
325 330 335
Leu Gly His Arg Asp Val Ala Arg Tyr Leu Arg Ala Ala Ala Gly Gly
340 345 350
Thr Arg Gly Ser Asn His Ala Arg Ile Asp Ala Ala Glu Gly Pro Ser
355 360 365
Asp Ile Pro Asp Lys Leu Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu
370 375 380
Asp Leu Asn Ser Ala Val Asp His His His His His His
385 390 395
<210> 165
<211> 292
<212> PRT
<213> 人工序列
<220>
<223> SopE1-105-Ink4a-MycHis
<400> 165
Val Thr Lys Ile Thr Leu Ser Pro Gln Asn Phe Arg Ile Gln Lys Gln
1 5 10 15
Glu Thr Thr Leu Leu Lys Glu Lys Ser Thr Glu Lys Asn Ser Leu Ala
20 25 30
Lys Ser Ile Leu Ala Val Lys Asn His Phe Ile Glu Leu Arg Ser Lys
35 40 45
Leu Ser Glu Arg Phe Ile Ser His Lys Asn Thr Glu Ser Ser Ala Thr
50 55 60
His Phe His Arg Gly Ser Ala Ser Glu Gly Arg Ala Val Leu Thr Asn
65 70 75 80
Lys Val Val Lys Asp Phe Met Leu Gln Thr Leu Asn Asp Ile Asp Ile
85 90 95
Arg Gly Ser Ala Ser Lys Asp Pro Ala Gly Thr Ile Trp Glu Phe Met
100 105 110
Glu Pro Ala Ala Gly Ser Ser Met Glu Pro Ser Ala Asp Trp Leu Ala
115 120 125
Thr Ala Ala Ala Arg Gly Arg Val Glu Glu Val Arg Ala Leu Leu Glu
130 135 140
Ala Gly Ala Leu Pro Asn Ala Pro Asn Ser Tyr Gly Arg Arg Pro Ile
145 150 155 160
Gln Val Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu Leu
165 170 175
His Gly Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg Pro
180 185 190
Val His Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val Leu
195 200 205
His Arg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg Leu
210 215 220
Pro Val Asp Leu Ala Glu Glu Leu Gly His Arg Asp Val Ala Arg Tyr
225 230 235 240
Leu Arg Ala Ala Ala Gly Gly Thr Arg Gly Ser Asn His Ala Arg Ile
245 250 255
Asp Ala Ala Glu Gly Pro Ser Asp Ile Pro Asp Lys Leu Gly Pro Glu
260 265 270
Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His
275 280 285
His His His His
290
<210> 166
<211> 409
<212> PRT
<213> 人工序列
<220>
<223> SteA-Ink4c-MycHis
<400> 166
Met Pro Tyr Thr Ser Val Ser Thr Tyr Ala Arg Ala Leu Ser Gly Asn
1 5 10 15
Lys Leu Pro His Val Ala Ala Gly Asp Tyr Glu Asn Lys Leu Ser Thr
20 25 30
Lys Ile Met Lys Gly Ile Leu Tyr Val Leu Thr Ala Gly Leu Ala Tyr
35 40 45
Gly Phe Thr Arg Val Ile Glu His Tyr Cys Asn Val Thr Pro Lys Val
50 55 60
Ala Glu Phe Cys Ala Asn Ala Gly Asn Ile His Asn His Leu Ala Asp
65 70 75 80
Ala Val Arg Asp Gly Leu Phe Thr Ile Asp Val Glu Leu Ser Asp Gly
85 90 95
Arg Met Leu Thr Phe Glu Gln Leu Ser Leu Ile Ala Glu Gly Lys Pro
100 105 110
Ile Val Arg Ile Ser Asp Gly Glu His Thr Val Glu Val Glu Gly Thr
115 120 125
Phe Glu Glu Ile Cys Met Arg Leu Glu Glu Gly Phe Phe Glu Ala Pro
130 135 140
Ala Tyr Tyr Asp Tyr Asp Ile Asp Glu Lys Tyr Lys Thr Val Arg Glu
145 150 155 160
Arg Met Ala Ala Tyr Asn Ala Leu Pro Gln Ala Leu Gly Ala Ile Pro
165 170 175
Cys Leu Glu Tyr Tyr Ile Ala Arg Ala Ser Asn Met Gln Glu Ala Lys
180 185 190
Ala Gln Trp Ala Ala Asp Ile Lys Ala Arg Tyr His Asn Tyr Leu Asp
195 200 205
Asn Tyr Gly Thr Ile Trp Glu Phe Met Ala Glu Pro Trp Gly Asn Glu
210 215 220
Leu Ala Ser Ala Ala Ala Arg Gly Asp Leu Glu Gln Leu Thr Ser Leu
225 230 235 240
Leu Gln Asn Asn Val Asn Val Asn Ala Gln Asn Gly Phe Gly Arg Thr
245 250 255
Ala Leu Gln Val Met Lys Leu Gly Asn Pro Glu Ile Ala Arg Arg Leu
260 265 270
Leu Leu Arg Gly Ala Asn Pro Asp Leu Lys Asp Arg Thr Gly Phe Ala
275 280 285
Val Ile His Asp Ala Ala Arg Ala Gly Phe Leu Asp Thr Leu Gln Thr
290 295 300
Leu Leu Glu Phe Gln Ala Asp Val Asn Ile Glu Asp Asn Glu Gly Asn
305 310 315 320
Leu Pro Leu His Leu Ala Ala Lys Glu Gly His Leu Arg Val Val Glu
325 330 335
Phe Leu Val Lys His Thr Ala Ser Asn Val Gly His Arg Asn His Lys
340 345 350
Gly Asp Thr Ala Cys Asp Leu Ala Arg Leu Tyr Gly Arg Asn Glu Val
355 360 365
Val Ser Leu Met Gln Ala Asn Gly Ala Gly Gly Ala Thr Asn Leu Gln
370 375 380
Lys Leu Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser
385 390 395 400
Ala Val Asp His His His His His His
405
<210> 167
<211> 304
<212> PRT
<213> 人工序列
<220>
<223> SopE1-105-Ink4c-MycHis
<400> 167
Val Thr Lys Ile Thr Leu Ser Pro Gln Asn Phe Arg Ile Gln Lys Gln
1 5 10 15
Glu Thr Thr Leu Leu Lys Glu Lys Ser Thr Glu Lys Asn Ser Leu Ala
20 25 30
Lys Ser Ile Leu Ala Val Lys Asn His Phe Ile Glu Leu Arg Ser Lys
35 40 45
Leu Ser Glu Arg Phe Ile Ser His Lys Asn Thr Glu Ser Ser Ala Thr
50 55 60
His Phe His Arg Gly Ser Ala Ser Glu Gly Arg Ala Val Leu Thr Asn
65 70 75 80
Lys Val Val Lys Asp Phe Met Leu Gln Thr Leu Asn Asp Ile Asp Ile
85 90 95
Arg Gly Ser Ala Ser Lys Asp Pro Ala Gly Thr Ile Trp Glu Phe Met
100 105 110
Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly Asp
115 120 125
Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn Ala
130 135 140
Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly Asn
145 150 155 160
Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp Leu
165 170 175
Lys Asp Arg Thr Gly Phe Ala Val Ile His Asp Ala Ala Arg Ala Gly
180 185 190
Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val Asn
195 200 205
Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys Glu
210 215 220
Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser Asn
225 230 235 240
Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala Arg
245 250 255
Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly Ala
260 265 270
Gly Gly Ala Thr Asn Leu Gln Lys Leu Gly Pro Glu Gln Lys Leu Ile
275 280 285
Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His His
290 295 300
<210> 168
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> SteA-Mad2-MycHis
<400> 168
Met Pro Tyr Thr Ser Val Ser Thr Tyr Ala Arg Ala Leu Ser Gly Asn
1 5 10 15
Lys Leu Pro His Val Ala Ala Gly Asp Tyr Glu Asn Lys Leu Ser Thr
20 25 30
Lys Ile Met Lys Gly Ile Leu Tyr Val Leu Thr Ala Gly Leu Ala Tyr
35 40 45
Gly Phe Thr Arg Val Ile Glu His Tyr Cys Asn Val Thr Pro Lys Val
50 55 60
Ala Glu Phe Cys Ala Asn Ala Gly Asn Ile His Asn His Leu Ala Asp
65 70 75 80
Ala Val Arg Asp Gly Leu Phe Thr Ile Asp Val Glu Leu Ser Asp Gly
85 90 95
Arg Met Leu Thr Phe Glu Gln Leu Ser Leu Ile Ala Glu Gly Lys Pro
100 105 110
Ile Val Arg Ile Ser Asp Gly Glu His Thr Val Glu Val Glu Gly Thr
115 120 125
Phe Glu Glu Ile Cys Met Arg Leu Glu Glu Gly Phe Phe Glu Ala Pro
130 135 140
Ala Tyr Tyr Asp Tyr Asp Ile Asp Glu Lys Tyr Lys Thr Val Arg Glu
145 150 155 160
Arg Met Ala Ala Tyr Asn Ala Leu Pro Gln Ala Leu Gly Ala Ile Pro
165 170 175
Cys Leu Glu Tyr Tyr Ile Ala Arg Ala Ser Asn Met Gln Glu Ala Lys
180 185 190
Ala Gln Trp Ala Ala Asp Ile Lys Ala Arg Tyr His Asn Tyr Leu Asp
195 200 205
Asn Tyr Gly Thr Ile Trp Glu Phe Met Ala Leu Gln Leu Ser Arg Glu
210 215 220
Gln Gly Ile Thr Leu Arg Gly Ser Ala Glu Ile Val Ala Glu Phe Phe
225 230 235 240
Ser Phe Gly Ile Asn Ser Ile Leu Tyr Gln Arg Gly Ile Tyr Pro Ser
245 250 255
Glu Thr Phe Thr Arg Val Gln Lys Tyr Gly Leu Thr Leu Leu Val Thr
260 265 270
Thr Asp Leu Glu Leu Ile Lys Tyr Leu Asn Asn Val Val Glu Gln Leu
275 280 285
Lys Asp Trp Leu Tyr Lys Cys Ser Val Gln Lys Leu Val Val Val Ile
290 295 300
Ser Asn Ile Glu Ser Gly Glu Val Leu Glu Arg Trp Gln Phe Asp Ile
305 310 315 320
Glu Cys Asp Lys Thr Ala Lys Asp Asp Ser Ala Pro Arg Glu Lys Ser
325 330 335
Gln Lys Ala Ile Gln Asp Glu Ile Arg Ser Val Ile Arg Gln Ile Thr
340 345 350
Ala Thr Val Thr Phe Leu Pro Leu Leu Glu Val Ser Cys Ser Phe Asp
355 360 365
Leu Leu Ile Tyr Thr Asp Lys Asp Leu Val Val Pro Glu Lys Trp Glu
370 375 380
Glu Ser Gly Pro Gln Phe Ile Thr Asn Ser Glu Glu Val Arg Leu Arg
385 390 395 400
Ser Phe Thr Thr Thr Ile His Lys Val Asn Ser Met Val Ala Tyr Lys
405 410 415
Ile Pro Val Asn Asp Lys Leu Gly Pro Glu Gln Lys Leu Ile Ser Glu
420 425 430
Glu Asp Leu Asn Ser Ala Val Asp His His His His His His
435 440 445
<210> 169
<211> 341
<212> PRT
<213> 人工序列
<220>
<223> SopE1-105-Mad2-MycHis
<400> 169
Val Thr Lys Ile Thr Leu Ser Pro Gln Asn Phe Arg Ile Gln Lys Gln
1 5 10 15
Glu Thr Thr Leu Leu Lys Glu Lys Ser Thr Glu Lys Asn Ser Leu Ala
20 25 30
Lys Ser Ile Leu Ala Val Lys Asn His Phe Ile Glu Leu Arg Ser Lys
35 40 45
Leu Ser Glu Arg Phe Ile Ser His Lys Asn Thr Glu Ser Ser Ala Thr
50 55 60
His Phe His Arg Gly Ser Ala Ser Glu Gly Arg Ala Val Leu Thr Asn
65 70 75 80
Lys Val Val Lys Asp Phe Met Leu Gln Thr Leu Asn Asp Ile Asp Ile
85 90 95
Arg Gly Ser Ala Ser Lys Asp Pro Ala Gly Thr Ile Trp Glu Phe Met
100 105 110
Ala Leu Gln Leu Ser Arg Glu Gln Gly Ile Thr Leu Arg Gly Ser Ala
115 120 125
Glu Ile Val Ala Glu Phe Phe Ser Phe Gly Ile Asn Ser Ile Leu Tyr
130 135 140
Gln Arg Gly Ile Tyr Pro Ser Glu Thr Phe Thr Arg Val Gln Lys Tyr
145 150 155 160
Gly Leu Thr Leu Leu Val Thr Thr Asp Leu Glu Leu Ile Lys Tyr Leu
165 170 175
Asn Asn Val Val Glu Gln Leu Lys Asp Trp Leu Tyr Lys Cys Ser Val
180 185 190
Gln Lys Leu Val Val Val Ile Ser Asn Ile Glu Ser Gly Glu Val Leu
195 200 205
Glu Arg Trp Gln Phe Asp Ile Glu Cys Asp Lys Thr Ala Lys Asp Asp
210 215 220
Ser Ala Pro Arg Glu Lys Ser Gln Lys Ala Ile Gln Asp Glu Ile Arg
225 230 235 240
Ser Val Ile Arg Gln Ile Thr Ala Thr Val Thr Phe Leu Pro Leu Leu
245 250 255
Glu Val Ser Cys Ser Phe Asp Leu Leu Ile Tyr Thr Asp Lys Asp Leu
260 265 270
Val Val Pro Glu Lys Trp Glu Glu Ser Gly Pro Gln Phe Ile Thr Asn
275 280 285
Ser Glu Glu Val Arg Leu Arg Ser Phe Thr Thr Thr Ile His Lys Val
290 295 300
Asn Ser Met Val Ala Tyr Lys Ile Pro Val Asn Asp Lys Leu Gly Pro
305 310 315 320
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His
325 330 335
His His His His His
340
<210> 170
<211> 538
<212> PRT
<213> 人工序列
<220>
<223> SteA-Cdk1-MycHis
<400> 170
Met Pro Tyr Thr Ser Val Ser Thr Tyr Ala Arg Ala Leu Ser Gly Asn
1 5 10 15
Lys Leu Pro His Val Ala Ala Gly Asp Tyr Glu Asn Lys Leu Ser Thr
20 25 30
Lys Ile Met Lys Gly Ile Leu Tyr Val Leu Thr Ala Gly Leu Ala Tyr
35 40 45
Gly Phe Thr Arg Val Ile Glu His Tyr Cys Asn Val Thr Pro Lys Val
50 55 60
Ala Glu Phe Cys Ala Asn Ala Gly Asn Ile His Asn His Leu Ala Asp
65 70 75 80
Ala Val Arg Asp Gly Leu Phe Thr Ile Asp Val Glu Leu Ser Asp Gly
85 90 95
Arg Met Leu Thr Phe Glu Gln Leu Ser Leu Ile Ala Glu Gly Lys Pro
100 105 110
Ile Val Arg Ile Ser Asp Gly Glu His Thr Val Glu Val Glu Gly Thr
115 120 125
Phe Glu Glu Ile Cys Met Arg Leu Glu Glu Gly Phe Phe Glu Ala Pro
130 135 140
Ala Tyr Tyr Asp Tyr Asp Ile Asp Glu Lys Tyr Lys Thr Val Arg Glu
145 150 155 160
Arg Met Ala Ala Tyr Asn Ala Leu Pro Gln Ala Leu Gly Ala Ile Pro
165 170 175
Cys Leu Glu Tyr Tyr Ile Ala Arg Ala Ser Asn Met Gln Glu Ala Lys
180 185 190
Ala Gln Trp Ala Ala Asp Ile Lys Ala Arg Tyr His Asn Tyr Leu Asp
195 200 205
Asn Tyr Gly Thr Ile Trp Glu Phe Met Glu Asp Tyr Thr Lys Ile Glu
210 215 220
Lys Ile Gly Glu Gly Thr Tyr Gly Val Val Tyr Lys Gly Arg His Lys
225 230 235 240
Thr Thr Gly Gln Val Val Ala Met Lys Lys Ile Arg Leu Glu Ser Glu
245 250 255
Glu Glu Gly Val Pro Ser Thr Ala Ile Arg Glu Ile Ser Leu Leu Lys
260 265 270
Glu Leu Arg His Pro Asn Ile Val Ser Leu Gln Asp Val Leu Met Gln
275 280 285
Asp Ser Arg Leu Tyr Leu Ile Phe Glu Phe Leu Ser Met Asp Leu Lys
290 295 300
Lys Tyr Leu Asp Ser Ile Pro Pro Gly Gln Tyr Met Asp Ser Ser Leu
305 310 315 320
Val Lys Ser Tyr Leu Tyr Gln Ile Leu Gln Gly Ile Val Phe Cys His
325 330 335
Ser Arg Arg Val Leu His Arg Asp Leu Lys Pro Gln Asn Leu Leu Ile
340 345 350
Asp Asp Lys Gly Thr Ile Lys Leu Ala Asp Phe Gly Leu Ala Arg Ala
355 360 365
Phe Gly Ile Pro Ile Arg Val Tyr Thr His Glu Val Val Thr Leu Trp
370 375 380
Tyr Arg Ser Pro Glu Val Leu Leu Gly Ser Ala Arg Tyr Ser Thr Pro
385 390 395 400
Val Asp Ile Trp Ser Ile Gly Thr Ile Phe Ala Glu Leu Ala Thr Lys
405 410 415
Lys Pro Leu Phe His Gly Asp Ser Glu Ile Asp Gln Leu Phe Arg Ile
420 425 430
Phe Arg Ala Leu Gly Thr Pro Asn Asn Glu Val Trp Pro Glu Val Glu
435 440 445
Ser Leu Gln Asp Tyr Lys Asn Thr Phe Pro Lys Trp Lys Pro Gly Ser
450 455 460
Leu Ala Ser His Val Lys Asn Leu Asp Glu Asn Gly Leu Asp Leu Leu
465 470 475 480
Ser Lys Met Leu Ile Tyr Asp Pro Ala Lys Arg Ile Ser Gly Lys Met
485 490 495
Ala Leu Asn His Pro Tyr Phe Asn Asp Leu Asp Asn Gln Ile Lys Lys
500 505 510
Met Lys Leu Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn
515 520 525
Ser Ala Val Asp His His His His His His
530 535
<210> 171
<211> 433
<212> PRT
<213> 人工序列
<220>
<223> SopE1-105-Cdk1-MycHis
<400> 171
Val Thr Lys Ile Thr Leu Ser Pro Gln Asn Phe Arg Ile Gln Lys Gln
1 5 10 15
Glu Thr Thr Leu Leu Lys Glu Lys Ser Thr Glu Lys Asn Ser Leu Ala
20 25 30
Lys Ser Ile Leu Ala Val Lys Asn His Phe Ile Glu Leu Arg Ser Lys
35 40 45
Leu Ser Glu Arg Phe Ile Ser His Lys Asn Thr Glu Ser Ser Ala Thr
50 55 60
His Phe His Arg Gly Ser Ala Ser Glu Gly Arg Ala Val Leu Thr Asn
65 70 75 80
Lys Val Val Lys Asp Phe Met Leu Gln Thr Leu Asn Asp Ile Asp Ile
85 90 95
Arg Gly Ser Ala Ser Lys Asp Pro Ala Gly Thr Ile Trp Glu Phe Met
100 105 110
Glu Asp Tyr Thr Lys Ile Glu Lys Ile Gly Glu Gly Thr Tyr Gly Val
115 120 125
Val Tyr Lys Gly Arg His Lys Thr Thr Gly Gln Val Val Ala Met Lys
130 135 140
Lys Ile Arg Leu Glu Ser Glu Glu Glu Gly Val Pro Ser Thr Ala Ile
145 150 155 160
Arg Glu Ile Ser Leu Leu Lys Glu Leu Arg His Pro Asn Ile Val Ser
165 170 175
Leu Gln Asp Val Leu Met Gln Asp Ser Arg Leu Tyr Leu Ile Phe Glu
180 185 190
Phe Leu Ser Met Asp Leu Lys Lys Tyr Leu Asp Ser Ile Pro Pro Gly
195 200 205
Gln Tyr Met Asp Ser Ser Leu Val Lys Ser Tyr Leu Tyr Gln Ile Leu
210 215 220
Gln Gly Ile Val Phe Cys His Ser Arg Arg Val Leu His Arg Asp Leu
225 230 235 240
Lys Pro Gln Asn Leu Leu Ile Asp Asp Lys Gly Thr Ile Lys Leu Ala
245 250 255
Asp Phe Gly Leu Ala Arg Ala Phe Gly Ile Pro Ile Arg Val Tyr Thr
260 265 270
His Glu Val Val Thr Leu Trp Tyr Arg Ser Pro Glu Val Leu Leu Gly
275 280 285
Ser Ala Arg Tyr Ser Thr Pro Val Asp Ile Trp Ser Ile Gly Thr Ile
290 295 300
Phe Ala Glu Leu Ala Thr Lys Lys Pro Leu Phe His Gly Asp Ser Glu
305 310 315 320
Ile Asp Gln Leu Phe Arg Ile Phe Arg Ala Leu Gly Thr Pro Asn Asn
325 330 335
Glu Val Trp Pro Glu Val Glu Ser Leu Gln Asp Tyr Lys Asn Thr Phe
340 345 350
Pro Lys Trp Lys Pro Gly Ser Leu Ala Ser His Val Lys Asn Leu Asp
355 360 365
Glu Asn Gly Leu Asp Leu Leu Ser Lys Met Leu Ile Tyr Asp Pro Ala
370 375 380
Lys Arg Ile Ser Gly Lys Met Ala Leu Asn His Pro Tyr Phe Asn Asp
385 390 395 400
Leu Asp Asn Gln Ile Lys Lys Met Lys Leu Gly Pro Glu Gln Lys Leu
405 410 415
Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His
420 425 430
His
<210> 172
<211> 95
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_745
<400> 172
catgctcgag ggtgccatcg atgatgccgc ccgcgaaggt tttctggata ccctggtggt 60
gctgcatcgc gccggtgccc gctaattcga acatg 95
<210> 173
<211> 95
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_746
<400> 173
catgttcgaa ttagcgggca ccggcgcgat gcagcaccac cagggtatcc agaaaacctt 60
cgcgggcggc atcatcgatg gcaccctcga gcatg 95
<210> 174
<211> 95
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_747
<400> 174
catgctcgag ggtgccatcg attatggtcg caaaaaacgc cgccaacgcc gccgcggtcc 60
ggtgaaacgc cgcctgtttg gttaattcga acatg 95
<210> 175
<211> 95
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_748
<400> 175
catgttcgaa ttaaccaaac aggcggcgtt tcaccggacc gcggcggcgt tggcggcgtt 60
ttttgcgacc ataatcgatg gcaccctcga gcatg 95
<210> 176
<211> 95
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_749
<400> 176
catgctcgag ggtgccatcg ataaacgccg ccaaaccagc atgaccgcct tttatcatag 60
caaacgccgc ctgattttta gctaattcga acatg 95
<210> 177
<211> 95
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_750
<400> 177
catgttcgaa ttagctaaaa atcaggcggc gtttgctatg ataaaaggcg gtcatgctgg 60
tttggcggcg tttatcgatg gcaccctcga gcatg 95
<210> 178
<211> 83
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_753
<400> 178
catgctcgag ggtgccatcg ataccagcat gaccgccttt tatcatagca aacgccgcct 60
gatttttagc taattcgaac atg 83
<210> 179
<211> 83
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_754
<400> 179
catgttcgaa ttagctaaaa atcaggcggc gtttgctatg ataaaaggcg gtcatgctgg 60
tatcgatggc accctcgagc atg 83
<210> 180
<211> 86
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_755
<400> 180
catgctcgag ggtgccatcg atgcctgtcg ccgcctgttt ggtccggtgg atagcgaaca 60
actgagccgc gattaattcg aacatg 86
<210> 181
<211> 86
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_756
<400> 181
catgttcgaa ttaatcgcgg ctcagttgtt cgctatccac cggaccaaac aggcggcgac 60
aggcatcgat ggcaccctcg agcatg 86
<210> 182
<211> 62
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_757
<400> 182
catgctcgag ggtgccatcg attgggaact ggtggtgctg ggtaaactgt aattcgaaca 60
tg 62
<210> 183
<211> 62
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_758
<400> 183
catgttcgaa ttacagttta cccagcacca ccagttccca atcgatggca ccctcgagca 60
tg 62
<210> 184
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_703
<400> 184
gacatggaat tcatggagcc ggcggcg 27
<210> 185
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_704
<400> 185
catgaagctt atcggggatg tctgaggg 28
<210> 186
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_705
<400> 186
gacatggaat tcatggccga gccttgggg 29
<210> 187
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_706
<400> 187
gttaacatca gcttgaaact ccagcaaagt ctgtaaagtg tccaggaaac c 51
<210> 188
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_707
<400> 188
ggtttcctgg acactttaca gactttgctg gagtttcaag ctgatgttaa c 51
<210> 189
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_708
<400> 189
catgaagctt ttgaagattt gtggctcccc 30
<210> 190
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_709
<400> 190
gacatggaat tcatggcgct gcagctctcc 30
<210> 191
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_710
<400> 191
catgaagctt gtcattgaca ggaattttgt agg 33
<210> 192
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_711
<400> 192
gacatggaat tcatggaaga ttataccaaa atagagaa 38
<210> 193
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_712
<400> 193
catgaagctt catcttctta atctgattgt ccaa 34
<210> 194
<211> 276
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138-Y. enterocolitica codon optimized murine tBid
<400> 194
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Gly Ser Gln Ala
130 135 140
Ser Arg Ser Phe Asn Gln Gly Arg Ile Glu Pro Asp Ser Glu Ser Gln
145 150 155 160
Glu Glu Ile Ile His Asn Ile Ala Arg His Leu Ala Gln Ile Gly Asp
165 170 175
Glu Met Asp His Asn Ile Gln Pro Thr Leu Val Arg Gln Leu Ala Ala
180 185 190
Gln Phe Met Asn Gly Ser Leu Ser Glu Glu Asp Lys Arg Asn Cys Leu
195 200 205
Ala Lys Ala Leu Asp Glu Val Lys Thr Ala Phe Pro Arg Asp Met Glu
210 215 220
Asn Asp Lys Ala Met Leu Ile Met Thr Met Leu Leu Ala Lys Lys Val
225 230 235 240
Ala Ser His Ala Pro Ser Leu Leu Arg Asp Val Phe His Thr Thr Val
245 250 255
Asn Phe Ile Asn Gln Asn Leu Phe Ser Tyr Val Arg Asn Leu Val Arg
260 265 270
Asn Glu Met Asp
275
<210> 195
<211> 245
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138-Ubiquitin
<400> 195
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Met Gln Ile Phe
130 135 140
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser
145 150 155 160
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile
165 170 175
Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp
180 185 190
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His
195 200 205
Leu Val Leu Arg Leu Arg Gly Gly Phe Glu Ala Ser Lys Leu Gly Pro
210 215 220
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His
225 230 235 240
His His His His His
245
<210> 196
<211> 419
<212> PRT
<213> 人工序列
<220>
<223> YopE1-138-Ubiquitin-Flag-INK4C-MycHis
<400> 196
Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Ala Ser
1 5 10 15
Val Ser Gly Ser Ser Ser Val Gly Glu Met Ser Gly Arg Ser Val Ser
20 25 30
Gln Gln Lys Ser Asp Gln Tyr Ala Asn Asn Leu Ala Gly Arg Thr Glu
35 40 45
Ser Pro Gln Gly Ser Ser Leu Ala Ser Arg Ile Ile Glu Arg Leu Ser
50 55 60
Ser Met Ala His Ser Val Ile Gly Phe Ile Gln Arg Met Phe Ser Glu
65 70 75 80
Gly Ser His Lys Pro Val Val Thr Pro Ala Leu Thr Pro Ala Gln Met
85 90 95
Pro Ser Pro Thr Ser Phe Ser Asp Ser Ile Lys Gln Leu Ala Ala Glu
100 105 110
Thr Leu Pro Lys Tyr Met Gln Gln Leu Ser Ser Leu Asp Ala Glu Thr
115 120 125
Leu Gln Lys Asn His Asp Gln Phe Ala Thr Leu Glu Met Gln Ile Phe
130 135 140
Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser
145 150 155 160
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile
165 170 175
Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp
180 185 190
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His
195 200 205
Leu Val Leu Arg Leu Arg Gly Gly Phe Glu Asp Tyr Lys Asp Asp Asp
210 215 220
Asp Lys Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala
225 230 235 240
Arg Gly Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn
245 250 255
Val Asn Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys
260 265 270
Leu Gly Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn
275 280 285
Pro Asp Leu Lys Asp Arg Thr Gly Phe Ala Val Ile His Asp Ala Ala
290 295 300
Arg Ala Gly Phe Leu Asp Thr Leu Gln Ala Leu Pro Glu Phe Gln Ala
305 310 315 320
Asp Val Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala
325 330 335
Ala Lys Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Thr
340 345 350
Ala Ser Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp
355 360 365
Leu Ala Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala
370 375 380
Asn Gly Ala Gly Gly Ala Thr Asn Leu Gln Lys Leu Gly Pro Glu Gln
385 390 395 400
Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His
405 410 415
His His His
<210> 197
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_585
<400> 197
cagtctcgag atgcagatct tcgtcaagac 30
<210> 198
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_586
<400> 198
gttaaagctt gctagcttcg aaaccaccac gtagacgtaa gac 43
<210> 199
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 引物No. : Si_588
<400> 199
cagtttcgaa gattataaag atgatgatga taaaatggcc gagccttg 48

Claims (34)

1.一种选自耶尔森氏菌属(Yersinia)、埃希氏菌属(Escherichia)、沙门氏菌属(Salmonella)和假单胞菌属(Pseudomonas)的重组革兰氏阴性细菌菌株，其中所述革兰氏阴性细菌菌株用载体转化，所述载体在5'至3'方向包含：
启动子；
编码细菌T3SS效应蛋白的递送信号的第一DNA序列，可操作地连接到所述启动子；
框内融合到所述第一DNA序列的3'末端的编码异源蛋白质的第二DNA序列，其中所述异源蛋白质选自参与凋亡或凋亡调节的蛋白质。
2.权利要求1的重组革兰氏阴性细菌菌株，其中所述载体包含编码蛋白酶切割位点的第三DNA序列，其中所述第三DNA序列位于所述第一DNA序列的3'端和所述第二DNA序列的5'端之间。
3.权利要求1或2的重组革兰氏阴性细菌菌株，其中所述重组革兰氏阴性细菌菌株对于至少一种T3SS效应蛋白的产生是缺陷的。
4.根据权利要求1-3中任一项所述的重组革兰氏阴性细菌菌株，其中所述重组革兰氏阴性细菌菌株选自耶尔森氏菌属和沙门氏菌属。
5.根据权利要求1-4中任一项所述的重组革兰氏阴性细菌菌株，其中所述重组革兰氏阴性细菌菌株是耶尔森氏菌菌株且由所述第一DNA序列编码的细菌T3SS效应蛋白的递送信号包含YopE效应蛋白或其N末端片段，或者其中所述重组革兰氏阴性细菌菌株是沙门氏菌菌株且由所述第一DNA序列编码的细菌T3SS效应蛋白的递送信号包含SopE或SteA效应子蛋白或其N末端片段。
6.根据权利要求1-4中任一项所述的重组革兰氏阴性细菌菌株，其中所述重组革兰氏阴性细菌菌株是耶尔森氏菌菌株，并且其中所述耶尔森氏菌菌株是野生型或对于至少一种T3SS效应蛋白的产生是缺陷的且来自细菌T3SS效应蛋白的递送信号包含小肠结肠炎耶尔森氏菌(Y.enterocolitica)YopE效应蛋白的N-末端138个氨基酸，或者其中所述重组革兰氏阴性细菌菌株是沙门氏菌菌株且其中所述沙门氏菌菌株是野生型或对于至少一种T3SS效应蛋白的产生是缺陷的且其中来自细菌T3SS效应蛋白的递送信号包含肠炎沙门氏菌(S.entrica)SteA效应蛋白或肠炎沙门氏菌SopE效应蛋白的N-末端81或105个氨基酸。
7.权利要求1-6中任一项的重组革兰氏阴性细菌菌株，其中另外的DNA序列融合到所述第二DNA序列的5'端或3'端，其中所述另外的DNA序列编码标记分子或标记分子的受体位点。
8.权利要求1-7中任一项的重组革兰氏阴性细菌菌株，其中另外的DNA序列融合到所述第二DNA序列的5'端或3'端，其中所述另外的DNA序列编码核定位信号。
9.根据权利要求1-8中任一项所述的重组革兰氏阴性细菌菌株，其中所述革兰氏阴性细菌菌株对于产生生长必需的氨基酸是缺陷的。
10.根据权利要求1-9中任一项所述的重组革兰氏阴性细菌菌株，其中所述革兰氏阴性细菌菌株对于产生结合真核细胞表面或细胞外基质的粘附蛋白是缺陷的。
11.根据权利要求1-10中任一项所述的重组革兰氏阴性细菌菌株，其中所述表达载体在所述第一DNA的3'端包含多克隆位点。
12.一种载体，其在5'至3'方向上包含：
启动子；
编码来自细菌T3SS效应蛋白的递送信号的第一DNA序列，可操作地连接到所述启动子；
框内融合到所述第一DNA序列的3'末端的编码异源蛋白质的第二DNA序列，其中所述异源蛋白质参与凋亡或凋亡调节；和作为选择的
编码蛋白酶切割位点的第三DNA序列，其中所述第三DNA序列位于所述第一DNA序列的3'端和所述第二DNA序列的5'端之间。
13.权利要求1-4和7-11中任一项的重组革兰氏阴性细菌菌株或权利要求12的载体，其中所述细菌T3SS效应蛋白选自SoPE、SopE2、SptP、SteA、ExoS、SipA、SipB、SipD、SopA、SopB、SopD、IpgB1、IpgD、SipC、SifA、SseJ、Sse、SrfH、SspH1、YopJ、AvrA、AvrBsT、YopT、YopH、YpkA、Tir、EspF、TccP2、IpgB2、OspF、Map、OspG、OspI、VirA、IpaA、IpaH、SspH1、VopF、ExoS、ExoT、HopAB2、XopD、AvrRpt2、HopAo1、HopPtoD2、HopU1、GALA蛋白家族、AvrBs2、AvrD1、AvrBS3、YopO、YopP、YopE、YopT、EspG、EspH、EspZ、IpaA、IpaB、IpaC、VirA、IcsB、OspC1、OspE2、IpaH9.8、IpaH7.8、AvrB、AvrD、AvrPphB、AvrPphC、AvrPphEPto、AvrPpiBPto、AvrPto、AvrPto、AvrPtoB、VirPphA、AvrRpm1、AvrRpt2、AvrRpt2、HopPtoD2、HopPtoE、HopPtoF、HopPtoN、PopB、PopP2、AvrBs3、XopD和AvrXv3。
14.权利要求1-4和7-11中任一项的重组革兰氏阴性细菌菌株或权利要求12的载体，其中所述细菌T3SS效应蛋白选自SopE、SptP、SteA、SifB、SopB、IpgB1、IpgD、YopJ、YopH、EspF、OspF、Exo S、YopO、YopP、YopE、YopT。
15.权利要求1-4和7-11中任一项的重组革兰氏阴性细菌菌株或权利要求12的载体，其中所述细菌T3SS效应蛋白选自IpgB1、SopE、SopB、SptP、SteA、SifB、OspF、IpgD、YopH、YopO、YopP、YopE和YopT。
16.根据权利要求1-4和7-11中任一项所述的重组革兰氏阴性细菌菌株或权利要求12所述的载体，其中所述细菌T3SS效应蛋白选自SopE，SteA和YopE。
17.权利要求1-4和7-11中任一项的重组革兰氏阴性细菌菌株或权利要求12的载体，其中所述细菌T3SS效应蛋白是SteA或YopE。
18.权利要求15-17中任一项所述的重组革兰氏阴性细菌菌株或载体，其中所述异源蛋白质选自参与凋亡或凋亡调节的蛋白质、细胞周期调节剂、锚蛋白重复蛋白、细胞信号传导蛋白、报告蛋白、转录因子、蛋白酶、小GTP酶、GPCR相关蛋白、纳米抗体融合构建体和纳米抗体、细菌T3SS效应子、细菌T4SS效应子和病毒蛋白。
19.权利要求13-17中任一项所述的重组革兰氏阴性细菌菌株或载体，其中所述异源蛋白质选自参与凋亡或凋亡调节的蛋白质、细胞周期调节剂、锚蛋白重复蛋白、报告蛋白、小GTPases、GPCR相关蛋白、纳米抗体融合构建体、细菌T3SS效应子、细菌T4SS效应子和病毒蛋白。
20.权利要求13-17中任一项的重组革兰氏阴性细菌菌株或载体，其中所述异源蛋白选自参与凋亡或凋亡调节、细胞周期调节剂和锚蛋白重复蛋白的蛋白质。
21.根据权利要求1-11中任一项所述的重组革兰氏阴性细菌菌株或权利要求12所述的载体，其中参与细胞凋亡或凋亡调节的蛋白质选自唯BH3蛋白、胱天蛋白酶和凋亡的死亡受体控制的细胞内信号传导蛋白。
22.根据权利要求1-21中任一项所述的重组革兰氏阴性细菌菌株或载体，其中所述载体包含相互独立地框内融合到所述第一DNA序列的3'端的编码相同的或两个不同的异源蛋白的两个第二DNA序列。
23.如权利要求22所述的重组革兰氏阴性细菌菌株或载体，其中所述异源蛋白质是参与凋亡或凋亡调节的蛋白质，并且其中一种蛋白质是促凋亡蛋白质，另一种蛋白质是凋亡预防途径的抑制剂或者其中一种蛋白是促凋亡蛋白，而另一种蛋白是促生存信号传导或通路的抑制剂。
24.根据权利要求1-23中任一项所述的重组革兰氏阴性细菌菌株或载体，其中由所述第一DNA序列编码的细菌T3SS效应蛋白的递送信号包含细菌T3SS效应蛋白或其N末端片段，其中所述N末端片段包括细菌T3SS效应蛋白的至少前10个氨基酸。
25.根据权利要求1-23中任一项所述的重组革兰氏阴性细菌菌株或载体，其中由所述第一DNA序列编码的细菌T3SS效应蛋白的递送信号包含细菌T3SS效应蛋白或其N末端片段，其中所述细菌T3SS效应子蛋白或其N末端片段包含伴侣结合位点。
26.权利要求1-23中任一项的重组革兰氏阴性细菌菌株或载体，其中所述启动子在所述革兰氏阴性细菌菌株中是有功能的。
27.一种将异源蛋白质递送到真核细胞中的方法，包括以下步骤：i)培养权利要求1-11或13-26中任一项所述的革兰氏阴性细菌菌株；和ii)使真核细胞与i)的革兰氏阴性细菌菌株接触，其中包含细菌T3SS效应蛋白的递送信号和异源蛋白的融合蛋白由革兰氏阴性细菌菌株表达并且被转运(translocated)进入真核细胞。
28.权利要求27所述的方法，还包括iii)切割所述融合蛋白，使得所述异源蛋白与所述细菌T3SS效应蛋白的递送信号中切割开来。
29.权利要求27或28的方法，其中融合蛋白在体外或体内转移到真核细胞中。
30.根据权利要求27-29中任一项所述的方法，其中包含细菌T3SS效应蛋白的递送信号和异源蛋白的至少两种融合蛋白，由革兰氏阴性细菌菌株表达，并且被转运至真核细胞。
31.一种纯化异源蛋白的方法，包括培养权利要求1-11或13-26任一项的革兰氏阴性细菌菌株，使得包含来自细菌T3SS效应蛋白的递送信号和异源蛋白的融合蛋白被表达并分泌到培养物的上清液中。
32.根据权利要求1-11或13-26中任一项所述的重组革兰氏阴性菌株，其用于将异源蛋白质作为药物或作为疫苗递送至受试者。
33.权利要求1-11或13-26中任一项的重组革兰氏阴性菌株用于高通量筛选由递送的异源蛋白质引发的细胞途径或事件的抑制剂的用途。
34.根据权利要求1-11或13-26中任一项所述的革兰氏阴性细菌菌株的文库，其中由所述革兰氏阴性细菌菌株的表达载体的第二DNA序列编码的异源蛋白质是人或鼠蛋白质，其中由革兰氏阴性细菌菌株表达的每种人或鼠蛋白质在氨基酸序列上不同。
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