CN111936624A - Cdkl5表达变体和cdkl5融合蛋白 - Google Patents
Cdkl5表达变体和cdkl5融合蛋白 Download PDFInfo
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Abstract
提供了新颖的CDKL5酶变体、以及包含全长CDKL5多肽或CDKL5变体的融合蛋白。此类融合蛋白可以包括细胞穿透多肽和任选地包含前导信号多肽和/或标签。还提供了产生此类CDKL5变体和融合蛋白的方法、以及此类重组蛋白的药物组合物、治疗方法和用途。
Description
技术领域
本发明总体上涉及激酶缺陷障碍的治疗,特别是用于治疗涉及CDKL5缺陷的新颖重组蛋白。
背景技术
CDKL5是丝氨酸/苏氨酸激酶,之前被称为STK9。所述基因突变最近与许多神经障碍相关联,例如精神发育迟缓、沟通和运动技能丧失、婴儿痉挛和癫痫、非典型Rett综合征和X-连锁West综合征。X-连锁基因细胞周期蛋白依赖激酶样5(CDKL5)的突变或缺失已显示引起伴随早发型严重神经损害和难治性癫痫发作的癫痫性脑病。
目前,医学文献中描述的已知患有CDKL5缺陷的年龄最大的人已经达到41岁。其他许多人都是二十几岁或十几岁,但由于这种疾病是在过去15年才被发现的,大多数新确诊的是幼儿或婴儿。被诊断患有CDKL5缺陷的个体通常患有神经发育迟缓,并且癫痫发作风险高,发病年龄中位数为6周。一项针对111名参与者的研究发现,85.6%的个体患有癫痫发作每日出现的癫痫,且平均每日癫痫发作6次。
当前的治疗方法包括癫痫发作药物、生酮饮食、迷走神经刺激和手术。通常施用的抗癫痫药物包括氯巴占、丙戊酸和托吡酯,并且在许多情况下,同时使用两种或多种药物方案。个体似乎具有“蜜月期”,在开始一种新型的药物后一段时间,他们的癫痫不发作,但最终癫痫会复发。观察到的蜜月期的持续时间范围从2个月到7年,中位数为6个月。例如,所述研究发现111名参与者中有16人目前没有癫痫发作,其中一人从未出现癫痫发作。
致病表现的确切机制尚不清楚。一些实验数据表明,C-末端某些无义突变导致蛋白组成性地定位至细胞核,而其他错义突变在细胞质中具有高度代表性。核定位信号和核输出信号均在蛋白C-末端被鉴定。
一些突变酶变体导致磷酸化功能的部分或全部丧失,而其他突变和截短导致磷酸化能力的增加,这表明功能的丧失和获得都可能是致病的。由于酶活性丧失/功能增加和酶核定位以及酶在细胞质中的停留而引起的相互作用和致病作用尚不清楚。患有广泛CDKL5突变且呈现临床症状的患者的分析表明,导致临床症状的突变更容易在C-末端或激酶活性结构域中发现,这表明CDKL5的激酶活性和蛋白易位能力均可能影响症状的临床表现。
发明内容
因此,本发明的各个方面涉及新颖的CDKL5变体和CDKL5融合蛋白,其可用于治疗CDKL5-介导的神经障碍,例如CDKL5缺陷或由CDKL5突变或缺陷引起的非典型Rett综合征。本发明的其他方面涉及产生此类CDKL5变体和融合蛋白的方法,以及此类重组蛋白的药物组合物、治疗方法以及用途。
本发明的一个方面涉及如本文所述的CDKL5多肽。在一个或多个实施例中,所述CDKL5多肽包含与SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12具有至少98%的序列一致性的序列。在一个或多个实施例中,所述CDKL5多肽包含与SEQ IDNO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12具有至少99%的序列一致性的序列。在一个或多个实施例中,所述CDKL5多肽包含与SEQ ID NO:2、SEQ ID NO:3、SEQ IDNO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12具有100%的序列一致性的序列。
本发明的另一个方面涉及缺乏核输出信号(NES)的CDKL5多肽。在一个或多个实施例中,所述CDKL5多肽含有核定位信号(NLS)。
本发明的另一个方面涉及缺乏核定位信号(NLS)和含有核输出信号(NES)的CDKL5多肽。
本发明的另一个方面涉及包含如本文所述的CDKL5多肽和细胞穿透多肽的融合蛋白。在一个或多个实施例中,所述细胞穿透多肽与SEQ ID NO:13、SEQ ID NO:14、SEQ IDNO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有至少90%的序列一致性。在一个或多个实施例中,所述细胞穿透多肽与SEQ ID NO:13、SEQ ID NO:14、SEQID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有至少95%的序列一致性。在一个或多个实施例中,所述细胞穿透多肽与SEQ ID NO:13、SEQ ID NO:14、SEQID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有100%的序列一致性。在一个或多个实施例中,所述细胞穿透多肽与SEQ ID NO:13、SEQ ID NO:14、SEQID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18具有至少90%的序列一致性。在一个或多个实施例中,所述细胞穿透多肽与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQID NO:16、SEQ ID NO:17或SEQ ID NO:18具有至少95%的序列一致性。在一个或多个实施例中,所述细胞穿透多肽与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18具有100%的序列一致性。在一个或多个实施例中,所述细胞穿透多肽与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:17或SEQ ID NO:18具有至少90%的序列一致性。在一个或多个实施例中,所述细胞穿透多肽与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:17或SEQ ID NO:18具有至少95%的序列一致性。在一个或多个实施例中,所述细胞穿透多肽与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:17或SEQ ID NO:18具有100%的序列一致性。在各种实施例中,所述CDKL5多肽是全长CDKL5多肽(例如,如在SEQ ID NO.1或SEQ ID NO:47中示出的)。在其他实施例中,所述CDKL5多肽是如本文所述的变体(例如,如在SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQID NO:11或SEQ ID NO:12中示出的)。
本发明的另一个方面涉及包含如本文所述的CDKL5多肽或如本文所述的融合蛋白以及药学上可接受的载体的药物配制品。
本发明的另一个方面涉及治疗CDKL5-介导的神经障碍的方法,所述方法包括施用包含如本文所述的CDKL5多肽或如本文所述的融合蛋白;以及药学上可接受的载体的配制品。在一个或多个实施例中,所述配制品是鞘内施用。在一个或多个实施例中,所述配制品是静脉内施用。在一个或多个实施例中,所述配制品是脑池内施用。在一个或多个实施例中,所述配制品是脑室内(intracerebroventrically)施用。在一个或多个实施例中,所述配制品是实质内(intraparenchymally)施用。在一个或多个实施例中,所述CDKL5-介导的神经障碍是CDKL5缺陷或由CDKL5突变或缺陷引起的非典型Rett综合征中的一种或多种。
本发明的另一个方面涉及产生如本文所述的CDKL5多肽或如本文所述的融合蛋白的方法。在一个或多个实施例中,所述方法包括表达所述CDKL5多肽或所述融合蛋白;以及纯化所述CDKL5多肽或所述融合蛋白。在一个或多个实施例中,所述CDKL5多肽或所述融合蛋白表达于中国仓鼠卵巢(CHO)细胞、HeLa细胞、人胚肾(HEK)细胞或大肠杆菌细胞。
本发明的另一个方面涉及编码如本文所述的CDKL5多肽或如本文所述的融合蛋白的多核苷酸。本发明的另一个方面涉及包含此类多核苷酸的载体。
附图说明
图1A显示了CDKL5107的多肽图谱。所述图谱识别了多肽的重要特征,包括ATP结合位点、激酶结构域和激酶活性位点、两个核定位信号和核输出信号。
图1B和1C显示了描绘合成的CDKL5构建体变体的图(1B),并且图例描述了多肽的长度,以及相关氨基酸缺失信息,以描述如何合成这些构建体(1C)。
图2A-2AD显示了用于在细胞(例如CHO细胞或大肠杆菌细胞)内表达各种融合蛋白的示例性质粒。
具体实施方式
在描述本发明若干示例性实施例之前,应当理解的是,本发明不限于以下描述中列出的构建或工艺步骤的细节。本发明能够有其他的实施例,并且能够以不同的方式被实施或进行。
本发明的各个方面涉及新颖的CDKL5变体和CDKL5融合蛋白。本发明的其他方面涉及产生此类CDKL5变体和融合蛋白的方法,以及此类重组蛋白的药物组合物、治疗方法以及用途。
不希望受任何特定理论的束缚,据信与全长CDKL5多肽相比,保留功能活性的较短的CDKL5变体可以提供益处,尤其是当掺入包含CDKL5多肽的融合蛋白时。在一个或多个实施例中,这类益处可以包括蛋白产生期间来自宿主细胞的改善的分泌、改善的溶解性、增强的穿过血脑屏障(BBB)的能力和/或增强的穿透靶细胞的能力。
定义
如本文中所使用,“CDKL5介导的神经系统障碍”是指可以通过表达或过表达CDKL5蛋白来治疗的任何疾病或障碍。
如本文中所使用,“CDKL5缺陷”是指蛋白质的生物学功能的任何缺陷。所述缺陷可以产生自编码蛋白质的DNA或DNA相关调节区中的任何DNA突变,或由于表观遗传DNA修饰的任何变化导致的蛋白质功能的任何变化,包括但不限于DNA甲基化或组蛋白修饰、CDKL5蛋白的二级、三级或四级结构的任何变化,或CDKL5蛋白相比于野生型或正常受试者执行其生物学功能的能力的任何变化。这种缺陷还包括CDKL5蛋白的缺乏,例如无效突变或完全功能的蛋白的低表达。
如本文中所使用,“由CDKL5突变或缺陷引起的非典型Rett综合征”是指Rett综合征的非典型形式,其与Rett综合征具有类似的临床征象,但由CDKL5突变或缺陷引起。
CDKL5缺陷、Rett综合征、或非典型Rett综合征的症状或标记包括但不限于癫痫发作、认知失能、张力减退以及自调、睡眠和胃肠紊乱。
如本文中所使用,术语“载体”旨在指代与化合物一起施用的稀释剂、助剂、赋形剂、或运载体。适合的药物载体是本领域已知的,并且在至少一个实施例中,其描述于“Remington's Pharmaceutical Sciences[雷明顿药物科学]”,E.W.Martin,第18版,或其他版本。
如本文中所使用,术语“酶替代疗法”或“ERT”旨在指代将外源的、经纯化的酶引入具有这种酶缺乏的个体中。施用的蛋白质可以从自然来源或通过重组表达而获得。所述术语也指将经纯化的酶引入个体,所述个体在其他情况下需要或受益于施用的经纯化的酶。在至少一个实施例中,这样的个体患有酶缺乏症。所述引入的酶可以是在体外产生的经纯化的重组酶,或从离体组织或体液例如像胎盘或动物奶,或从植物纯化的蛋白质。
如本文中所使用,术语“受试者”或“患者”旨在是指人类或非人类动物。在至少一个实施例中,受试者是哺乳动物。在至少一个实施例中,受试者是人类。
如本文中所使用,“治疗有效剂量”和“有效量”旨在是指足够导致受试者治疗反应的重组蛋白(例如,CDKL5变体或融合蛋白)的量。治疗反应可以是使用者(例如,临床医生)将会识别为对治疗的有效响应的任何反应,包括本文所述和本领域已知的任何替代性临床标记物或症状。因此,在至少一个实施例中,治疗反应可以是CDKL5缺陷、Rett综合征、或非典型Rett综合征的一个或多个症状或标记(例如本领域已知的那些)的改善或抑制。
CDKL5蛋白的功能
人类CDKL5基因由24个外显子组成,其中前三个外显子(外显子1、1a和1b)是不翻译的。
最初发现的人类CDKL5变体是1030个氨基酸,分子量为115kDa(CDKL5115)。另一个显著的变体CDKL5107含有改变的C-末端区域,因为与CDKL5115变体中的相比,可替代的选择性剪接结合不同的外显子。CDKL5107(107kDa)较短,因为它具有外显子19的替代版本,并且不含有CDKL5115变体中存在的外显子20-21。已经发现在人脑中hCDKL5107mRNA的丰度是hCDKL5115转录物的37倍,并且已经发现在鼠脑中鼠CDKL5107的丰度是鼠CDKL5105变体的160倍。与鼠CDKL5115变体相比,人和鼠CDKL5107同种型均显示较长的半衰期和对降解的抵抗性。
使用Lox-Cre重组系统产生了CDKL5敲除小鼠模型,并且这些小鼠表现出自闭症样社会互动中的缺陷、运动控制障碍的损害和恐惧记忆丧失等症状(Wang等人,Proc NatlAcad Sci U.S.A[美国国家科学院院刊]109(52),21516-21521)。例如,敲除CDKL5的小鼠在反复暴露于刺激物时,其具有运动协调降低的症状并显示出受损的记忆和恐惧应答。这些变化使得科学家们假设CDKL5激酶活性的丧失会导致神经网络发育受损。先前数据已经提示,CDKL5磷酸化甲基-CpG结合蛋白2(MeCP2)和MeCP2中独立的失功能突变可导致Rett综合征表型。CDKL5的其他底物包括Netrin G1配体(NGL-1)、Shootin1(SHTN1)、Mindbomb 1(MIB1)、DNA(胞嘧啶-5)-甲基转移酶1(DNMT1)、双载蛋白1(AMPH1)、末端结合蛋白EB2、微管相关蛋白1S(MAP1S)和组蛋白脱乙酰基酶4(HDAC4)。虽然CDKL5的确切作用尚未被确定,但这些数据表明,CDKL5在下游靶点磷酸化中发挥作用,这些靶点对包括MeCP2在内的正确的神经元发育至关重要。在人类中,CDKL5中的突变与以下表型有关,所述表型与Rett综合征重叠而且另外与早发型癫痫发作一起出现。虽然CDKL5KO小鼠没有表现出任何早发型癫痫发作症状,但它们确实表现出运动缺陷、社交能力下降和学习和记忆受损(Chen等人,CDKL5,一种与Rett综合征相关的蛋白,经由Rac1信号传导调控神经元形态发生,JNeurosci[神经科学杂志]30:12777-12786)
在大鼠体内发现两种CDKL5同种型,一种标记为CDKL5a,并且另一种标记为CDKL5b(Chen等人)。一般来说,除了C-末端附近的最后100-150个氨基酸外,在人、大鼠和小鼠物种的CDKL5基因中存在高水平序列保守性。蛋白质印迹数据显示出这两种变体都存在于大鼠的发育过程中,但成年大鼠似乎主要表达单一变体。此外,CDKL5在脑、肝和肺中以可鉴定的数量存在。
CDKL5在细胞核中起作用,但也可在培养的神经元的树突中发现,这表明它可能在细胞质中起着替代作用。通过培养的皮层神经元中RNAi(RNA干扰)下调CDKL5的表达,抑制神经突生长和树突分枝(分支),而CDKL5的过表达则具有相反的作用(Chen等人)。为了表征CDKL5的核作用和胞质作用,在培养的皮层神经元RNAi模型中表达了具有核输出序列(NES)的CDKL5a的变体。NES-CDKL5a变体对使野生型基因表达沉默的RNAi具有抗性,并且因此当仅在细胞质中表达时,NES-CDKL5a被用于创建模型CDKL5a。使用GFP标签确认所述CDKL5变体仅存在于细胞质中后,可看出神经突长度和神经突分支数量均有所增加。NES-GFP-CDKL5a能够部分挽救RNAi用于敲低内源性CDKL5表达时观察到的疾病表型,说明CDKL5在细胞质中的表达是神经突发育和生长中的重要因素。
人CDKL5中的突变与类似于Rett综合征的表型有关,并且CDKL5突变的个体也表现为早发型癫痫发作。所述癫痫发作与典型的Rett综合征表型不同,典型的Rett综合征表型在Rett症状发作之前有一个早期的正常发展期。典型的Rett综合征(RTT)患者在6-18个月之前似乎发育正常,然后开始出现神经症状,包括丧失语言和运动能力。对RTT大脑的解剖显示,在运动皮层和额叶皮层中,神经元更小更致密,树突更短,这表明神经元发育受损。大多数典型的RTT病例是由于MECP2基因中的突变引起的,MECP2基因是一种编码核蛋白的X-连锁基因,所述核蛋白选择性地与哺乳动物基因组中的CpG二核苷酸结合,并通过复合物的募集调控转录。虽然在这方面还知之甚少,但一般认为MECP2中的突变导致的基因表达失调是Rett综合征的根本原因。约20%的典型的Rett综合征病例和60%-80%的其他Rett综合征变体在MECP2中没有突变,这表明了发病机制的替代的遗传原因。最近,在患有某些RTT的变体和其他严重脑病的患者中鉴定出了一些CDKL5突变,且CDKL5显示在体内和体外都与MeCP2相互作用。在MeCP2之外,CDKL5已经显示与许多下游靶点(包括NGL-1)相互作用并磷酸化许多下游靶点(包括NGL-1)。磷酸化后,NGL-1与PSD95相互作用,且NGL-1对树突棘和突触形成的正确发生和发育至关重要(Ricciardi S等人,“CDKL5ensures excitatorysynapse stability by reinforcing NGL-1-PSD95interaction in the postsynapticcompartment and is impaired in patient iPSC-derived neurons.[CDKL5通过加强突触后室(postsynaptic compartment)NGL-1-PSD95的相互作用,确保了兴奋性突触的稳定性,并损害了患者的iPSC来源的神经元。]”Nat Cell Biol[自然细胞生物学]14(9):911–923)。
CDKL5还已显示磷酸化蛋白质DNA甲基转移酶1(DNMT1)(Kameshita I等人,“Cyclin-dependent kinase-like 5binds and phosphorylates DNAmethyltransferase1.[细胞周期蛋白依赖激酶样5结合并磷酸化DNA甲基转移酶1。]”BiochemBiophys Res Commun[生物化学与生物物理学研究通讯]377:1162-1167)。这种磷酸化导致DNMT1的活化,DNMT1是一种维持型甲基化蛋白,优先甲基化半甲基化的DNA。这个过程对于DNA复制过程中DNA甲基化模式的维持是有用的,这样新合成的子DNA链就能够维持它所取代的亲本链的甲基化模式。由于DNA甲基化通常被认为是一种使基因表达沉默的表观遗传机制,DNMT1的这种维持功能在跨细胞代保存基因表达模式中至关重要。
当前模型表明CDKL5激酶结构域磷酸化GSK-3β,以及表明GSK-3β的磷酸化导致其失活。缺乏CDKL5活性的个体因此似乎表现出增加的GSK-3β活性。以前的研究已经显示GSK-3β调节海马神经发生,以及显示增加的GSK-3β的活性严重损害新生海马神经元的树突形态。此外,GSK-3β似乎作为关键发育事件(例如神经元生存和成熟)的负调节物。使用CDKL5KO小鼠进行的研究表明,用GSK-3β抑制剂治疗几乎可以完全挽救缺乏CDKL5活性的小鼠中的海马发育和行为缺陷(Fuchs等人,“Inhibition of GSK3βRescues HippocampalDevelopment and Learning in a Mouse Model of CDKL5Disorder.[CDKL5障碍的小鼠模型中GSK3β的抑制挽救海马发育和学习。]”Neurobiology of Disease[疾病的神经生物学]82:298–310.)。这种发育挽救似乎也在治疗之外持续存在。
CDKL5107多肽构建体
图1A显示了CDKL5107的多肽图谱。SEQ ID NO:1中提供了野生型全长人CDKL5107同种型的氨基酸序列。CDKL5107蛋白由960个氨基酸组成,且其激酶结构域包含于约前300个氨基酸中。960个氨基酸中的残基42是一个关键的赖氨酸残基,其位于激酶结构域中,在磷酸化反应中参与ATP结合,而这个残基的突变通常会导致激酶活性的丧失(“激酶死亡”)。此外,还存在两个核定位信号跨残基312-315(NLS1)核定位信号和跨残基784-789(NLS2)核定位信号,以及存在一个跨残基836-845核输出信号(NES)。在C-末端跨残基905到960的氨基酸是CDKL5107所特有的,且在CDKL5115中不存在。CDKL5115和CDKL5107之间的氨基酸残基1-904相同。SEQ ID:47中提供了野生型全长人CDKL5115同种型的氨基酸序列。
本发明的各种实施例提供了新颖的CDKL5变体。图1B和1C显示了全长人CDKL5107同种型(构建体1)和新颖的CDKL5构建体(指定为构建体2-12)的多肽。这些CDKL5构建体通常分为两类:C-末端缺失一定数量的氨基酸(构建体2-7)的那些和多肽链中间缺失一定数量的氨基酸(构建体8-12)的那些。此外,在这些构建体中,CDKL5被C-末端融合到另外的N-末端氨基酸序列,CDKL5的初始蛋氨酸被除去。在这些构建体中,CDKL5多肽从第二个氨基酸赖氨酸开始。构建体1包含全长人CDKL5107同种型的全部960个氨基酸。构建体2,其含有全部960个氨基酸的链的前851个氨基酸,构建体2代表一个缩短的CDKL5多肽,除去CDKL5107和CDKL5115之间不同的尾巴序列但激酶结构域、核定位信号(NLS1和NLS2)和核输出信号(NES)保持不变。进一步缩短构建体3,其中另外将核定位信号(NLS2)和核输出信号(NES)除去。构建体4-7被进一步缩短,如图1B和1C所示。构建体2-7均包含活性激酶结构域,而构建体3-7不包含NLS2或NES序列。构建体7进一步缩短为NLS1序列。剩余的构建体(构建体8-12)均在多肽链的中部具有缺失,同时保留CDKL5107特有的C-末端氨基酸。在这些构建体中,构建体12缺少NES和NLS2序列。在SEQ ID NO:1-12中分别提供了构建体1-12的氨基酸序列。
在一个或多个实施例中,所述CDKL5多肽与SEQ ID NO:2、SEQ ID NO:3、SEQ IDNO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12具有至少98%、至少98.5%、至少99%或至少99.5%的序列一致性。所述CDKL5多肽相对于SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12可以包含缺失、取代和/或插入,如相对于由SEQ ID NO:2、SEQ ID NO:3、SEQID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ IDNO:10、SEQ ID NO:11或SEQ ID NO:12描述的氨基酸序列具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15或更多个缺失、取代和/或插入。
在一个或多个实施例中,所述CDKL5多肽与SEQ ID NO:1或SEQ ID NO:47具有至少98%、至少98.5%、至少99%或至少99.5%的序列一致性。所述CDKL5多肽相对于SEQ IDNO:1或SEQ ID NO:47可以包含缺失、取代和/或插入,如相对于由SEQ ID NO:1或SEQ IDNO:47述的氨基酸序列,具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15或更多个缺失、取代和/或插入。
可以使用不同比对算法和/或程序来计算两个序列之间的一致性,包括可用作GCG序列分析包(威斯康辛大学,麦迪逊市,威斯康辛州)的一部分的FASTA或BLAST,并且可以与例如,默认设置一起使用。例如,考虑与本文所述的特定多肽具有至少98%、98.5%、99%或99.5%一致性,并且优选地表现出基本相同功能的多肽,连同编码此类多肽的多核苷酸。除非另有说明,相似性分数将基于BLOSUM62的使用。当使用BLASTP时,相似性百分比是基于BLASTP阳性得分,并且序列一致性百分比是基于BLASTP一致性得分。BLASTP“一致性”示出相同的高分序列对中总残基的数量和分数;并且BLASTP“阳性”示出具有正值的比对分数并且彼此相似的残基的数量和分数。本披露考虑和涵盖具有与本文披露的氨基酸序列具有这些程度的一致性或相似性或任何中间程度的一致性或相似性的氨基酸序列。使用遗传密码推导出的相似多肽的多核苷酸序列,并且可以通过常规手段(具体地通过使用遗传密码来逆转录其氨基酸序列)获得。
本领域技术人员可以很容易地推导出编码特定多肽序列的多核苷酸序列。此类多核苷酸序列可以使用可商购的产品,例如使用OptimumGeneTM密码子优化工具(金斯瑞公司(Genscript),皮斯卡塔韦,新泽西州),针对靶细胞中的表达进行密码子优化。
细胞穿透肽(CPP)
多种病毒和细胞蛋白具有介导跨细胞膜易位的碱性多肽序列。跨细胞膜易位的能力已成为一种跨膜递送高分子量多肽的重要工具。短语“蛋白质转导结构域”(PTD)和“细胞穿透肽”(CPP)通常用于指短肽(<30个氨基酸),这些短肽可以穿过许多哺乳动物细胞(如果不是全部的话)的质膜。在鉴定了允许其共同地跨质膜的结构域特性的研究后,研究人员发现这些结构域含有大量的碱性氨基酸残基,如赖氨酸和精氨酸。因此,细胞穿透肽可分为两类:第一类由含有赖氨酸残基的两亲性螺旋肽组成,这些赖氨酸残基导致正电荷;而第二类包括富含精氨酸的肽。这些多肽如果与其他难以递送到细胞内靶标的蛋白质结合使用,可能具有治疗潜力。PTD最常见的实验用途是TAT、触角足素(Antennapedia,Antp)和其他多精氨酸肽。
到目前为止,TAT已经成为最具特征性的PTD,并已被用于成功地将小型载物,例如短肽和寡核苷酸,递送到细胞内靶标。HIV-TAT(HIV反式激活的激活因子)是涉及人免疫缺陷病毒1型(HIV-1)的复制的86个氨基酸蛋白质,并且许多研究表明,TAT能够通过质膜易位并到达细胞核,从而激活病毒基因组的转录。研究还表明,TAT在与几种不同的蛋白质连接时仍能保持其穿透性质。为了了解TAT蛋白的哪些区域对易位特性至关重要,我们进行了实验,合成了不同长度的TAT的肽片段,并对其穿透能力进行了评估。(Lebleu等人,“ATruncated HIV-1TAT Protein Basic Domain Rapidly Translocates through thePlasma Membrane and Accumulates in the Cell Nucleus.[截短的HIV-1TAT蛋白质的碱性结构域迅速通过质膜易位并在细胞核内积累。]”J.Biol.Chem.[生物化学杂志]1997,272:16010-16017)。碱性氨基酸的区域被认为是保持这种穿透性的TAT的方面,并且在没有这种碱性氨基酸簇的TAT蛋白质的实验中,无法穿透细胞质膜。在一些情况下,较短的序列细胞穿透肽已经被修饰,以防止在分泌过程中被内切蛋白酶(如弗林蛋白酶(furin))裂解。这些修饰将缩短的细胞穿透TAT氨基酸序列由YGRKKRRQRRR改变至YARKAARQARA,并且这种短肽被称为TATκ。
TAT能够跨质膜易位的确切机制仍不确定。最近的研究已经探索了一种特殊类型的内吞作用与TAT摄取有关的可能性,并且已经鉴定出一些似乎对TAT穿透有抗性的细胞系。通过TAT递送的特定载物也可能对递送的有效性发挥作用。先前的研究数据表明,TAT融合蛋白在变性条件下制备时具有更好的细胞摄取能力,因为由于结构限制,正确折叠的蛋白载物可能需要更多的能量(δG)以穿过质膜。
细胞内蛋白伴侣对TAT货物进行再折叠的能力可能会根据将要再折叠的蛋白载物的身份和大小而有所不同。在一些情况下,TAT-融合蛋白当置于水性环境中时沉淀,因此不能以变性的方式制备,也不能以天然构象长时间保持稳定性。TAT-融合蛋白的设计还必须针对要递送的特定载物进行定制。如果载物蛋白紧密结合在N-末端,且TAT结构域也在N-末端发现,则TAT易位结构域可能埋藏在载物蛋白中,且转导可能较差。
许多TAT-载物变体已经成功地递送到各种细胞类型,包括初级培养细胞、转化的细胞和小鼠组织中存在的细胞。在培养过程中,TAT-融合蛋白通常很容易扩散进入细胞内或扩散至细胞外,导致浓度非常迅速地达到一致。
许多药物制剂,例如酶、抗体、其他蛋白质,或甚至载药载体颗粒,都需要在细胞内递送,以在细胞质、细胞核或其他特定细胞器内发挥其治疗作用。因此,这些不同类型的大分子的递送代表了生物制剂发展中的一个重大挑战。目前的数据表明TAT能够通过多于一种的机制穿过质膜。
TAT转导结构域也已经与超氧化物歧化酶(SOD)融合。(Torchilin,“Intracellular delivery of protein and peptide therapeutics.[细胞内递送蛋白质和肽疗法]”Protein Therapeutics.[蛋白质疗法]2008.5(2-3):e95-e103)。所述融合蛋白用于证明它可以跨细胞膜易位以递送SOD酶至细胞内环境,并且因此在这里融合蛋白在治疗酶缺乏症障碍方面具有治疗潜力,可导致活性氧类的更高积累和对宿主细胞的氧化应激。
TAT融合蛋白也已被证明可以通过血脑屏障进行转导。与神经保护性蛋白Bcl-xL融合的TAT结构域在培养过程中能够快速穿透细胞,并且当向患有脑缺血的小鼠给药时,融合蛋白在1-2小时内转导脑细胞。转导后,脑梗死灶的大小以剂量依赖性的方式减小(Cao,G.等人,“In Vivo Delivery of a Bcl-xL Fusion Protein Containing the TATProtein Transduction Domain Protects against Ischemic Brain Injury andNeuronal Apoptosis.[在体内递送含有TAT蛋白质转导结构域的Bcl-xL融合蛋白防止缺血性脑损伤和神经元细胞凋亡]”J.Neurosci.[神经科学杂志]22,5423,2002.)
本文所述的CDKL5变体可操作地连接至CPP,例如TAT、修饰的TAT(TATκ)、转运素、触角足素或P97。如在此所使用的,TAT可以指具有11个氨基酸的原始TAT肽(指定TAT11),也可以指具有另外的16个N-末端氨基酸的TAT肽(指定为TAT28),这些N-末端氨基酸来源于用于克隆的质粒的多接头。类似地,TATκ可以是指TAT11的修饰版本(指定TATκ11)或TAT28的修饰版本(指定TATκ28)。CPP TAT28、TATκ28、TAT11、TATκ11、转运素、触角足素和P97的氨基酸序列分别提供于SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ IDNO:17、SEQ ID NO:18和SEQ ID NO:50。
在一些实施例中,所述CPP与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ IDNO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有至少90%的序列一致性。在一些实施例中,所述CPP与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ IDNO:17、SEQ ID NO:18或SEQ ID NO:50具有至少95%的序列一致性。在一些实施例中,所述CPP与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ IDNO:18或SEQ ID NO:50具有100%的序列一致性。在一些实施例中,所述CPP与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18具有至少90%的序列一致性。在一些实施例中,所述CPP与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18具有至少95%的序列一致性。在一些实施例中,所述CPP与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18具有100%的序列一致性。在各种实施例中,所述CPP不具有SEQ ID NO:16的序列。
在各种实施例中,CPP可以添加N-末端甘氨酸。例如,TATκ28和TAT28另外具有N-末端天冬氨酸残基,其具有较低的稳定性。向序列中添加N-末端甘氨酸可以经由N-末端规则增加蛋白质的稳定性。因此,在一些实施例中,任何具有前导信号多肽的融合蛋白都可以具有在前导信号多肽的C-末端添加甘氨酸,这样当前导信号多肽的裂解后,融合蛋白的新N-末端将以甘氨酸开始。以类似的方式,那些缺乏前导信号多肽的融合蛋白也可以具有在N-末端蛋氨酸和融合蛋白的其余部分之间添加的甘氨酸。也以类似的方式,那些具有CPP而不是TAT28或TATκ28的融合蛋白也可以具有在前导信号多肽和CPP之间添加的甘氨酸。
包含CDKL5变体的融合蛋白
如上所述,CDKL5变体可以用于融合蛋白,例如也包含CPP的蛋白。其他多肽也可以被掺入到此类融合蛋白中,例如用于增强蛋白分泌的前导信号多肽,或用于检测和/或纯化融合蛋白的标签,以及用于连接功能性多肽的接头多肽。
前导信号多肽的实例包括但不限于,人免疫球蛋白重链结合蛋白的修饰的片段(修饰的BiP,例如SEQ ID NO:48、SEQ ID NO:51、SEQ ID NO:52或SEQ ID NO:53)或鼠Igκ链前导多肽(SEQ ID NO:49,例如pSecTag2,来自赛默飞世尔公司(ThermoFisher)载体)。修饰的BiP信号多肽的实例包括美国专利号9,279,007中描述的那些,其全部内容通过引用结合在此。
配制品、治疗方法与用途
可以根据常规程序将所述重组蛋白(例如CDKL5变体或融合蛋白)配制为适合向人类施用的药物组合物。例如,在一个或多个实施例中,用于静脉内施用的组合物是在无菌等渗水缓冲液中的溶液。必要时,所述组合物还可以包括增溶剂和局部麻醉剂以缓解注射部位的疼痛。通常,成分以单位剂量形式分开或混合在一起提供,例如,作为在气密容器中的干燥冻干粉或无水浓缩物,气密容器如指明活性剂的量的安瓿或小药囊。在通过输注施用组合物时,可以用包含无菌药用级的水、盐水或右旋糖/水的输液瓶进行分配。在通过注射施用组合物时,可以提供无菌注射用水或盐水的安瓿,这样使得可以在施用前将这些成分混合。
通过适当的途径施用重组蛋白(例如,CDKL5变体或融合蛋白)(或包含重组蛋白的组合物或药物)。在一个或多个实施例中,所述重组蛋白是静脉内施用。在其他实施例中,通过直接施用至靶组织(如至心脏或骨骼肌(例如,肌内;心室内))或神经系统(例如,直接注射到脑中;鞘内)来施用重组蛋白。如果需要,可以同时使用多于一个途径。
将所述重组蛋白(例如,CDKL5变体或融合蛋白)(或包含重组蛋白的组合物或药物)以治疗有效量施用(例如,当以规律间隔施用时,足以治疗疾病的剂量,例如通过减轻与与疾病相关的症状,预防或延迟疾病的发作,和/或减轻疾病的症状的严重性或频率实现的)。在治疗疾病中治疗有效的量将取决于疾病影响的性质和程度。另外,可以任选地采用体外或体内测定来帮助鉴定最佳剂量范围。待采用的确切剂量还取决于施用途径和疾病的严重程度,并且应根据从业者的判断和每个患者的情况来决定。可以从源自体外或动物模型测试系统的剂量-反应曲线外推有效剂量。
以规律的间隔施用的重组蛋白(例如,CDKL5变体或融合蛋白)(或包含重组蛋白的组合物或药物)的治疗有效量取决于疾病影响的性质和程度,和/或持续进行的基础。如本文中所使用,以“规律的间隔”施用表示,将治疗有效量定期地施用(如区别于一次性剂量)。单个个体的施用间隔不需要是固定的间隔,但是可以是随时间变化的,其取决于个体的需要。
可以制备所述重组蛋白(例如,CDKL5变体或融合蛋白)用于以后使用,例如在单位剂量小瓶或注射器中,或在用于静脉施用的瓶或袋中。包含重组蛋白(例如,CDKL5变体或融合蛋白)连同任选的赋形剂或其他活性成分例如其他药物的试剂盒,可以封装在包装材料中,并且附有用于复水、稀释或给药的说明书,用于治疗有需要的受试者,如患有CDKL5缺陷、Rett综合征或Rett综合征变体的患者。
产生方法
重组蛋白(例如CDKL5变体或融合蛋白)可以使用适当载体在宿主细胞中表达和从宿主细胞中分泌。例如,可以使用哺乳动物细胞(例如CHO细胞或HEK细胞)或细菌细胞(例如大肠杆菌或假交替单胞菌(P.haloplanktis)TAC 125细胞)。示例性质粒描述于下面的实例中,并示于图2A-2AD中。本领域普通技术人员可以选择适合于转化、转染或转导细胞的替代载体,从而产生本文所述的CDKL5变体和融合蛋白。
表达和分泌后,使用标准技术从周围细胞培养基中回收和纯化重组蛋白。可替代地,重组蛋白可以直接从细胞中分离和纯化,而不是从培养基中。
实例
实例1–CDKL5融合蛋白
图2A-2AD示出了合适细胞中表达融合蛋白的质粒,例如哺乳动物细胞(例如CHO细胞)或细菌细胞(例如大肠杆菌细胞)。这些蛋白质具有SEQ ID NO:19-46中所示的氨基酸序列。缺失或截短的编号与全长CDKL5107多肽(1-960)有关。在这些构建体中,CDKL5被C-末端融合到另外的N-末端氨基酸序列,CDKL5的初始蛋氨酸(氨基酸1)被除去。在这些构建体中,CDKL5多肽从第二个氨基酸赖氨酸开始。图2A-2AD和SEQ ID NO:19-46和54-55中使用的缩写汇总于下表1中:
表1
图2A显示了在CHO细胞中用于表达SEQ ID NO:19的融合蛋白的示例性质粒。所述融合蛋白包含经修饰的BiP前导信号多肽、TATκ28和全长人CDKL5107同种型。
图2B显示了在CHO细胞中用于表达SEQ ID NO:20的融合蛋白的示例性质粒。所述融合蛋白包含鼠Igκ链前导多肽、TATκ28和全长人CDKL5107同种型。
图2C显示了在CHO细胞中用于表达SEQ ID NO:21的融合蛋白的示例性质粒。所述融合蛋白包含经修饰的BiP前导信号多肽、TATκ28和全长人CDKL5115同种型。
图2D显示了在CHO细胞中用于表达SEQ ID NO:22的融合蛋白的示例性质粒。所述融合蛋白包含鼠Igκ链前导多肽、TATκ28和全长人CDKL5115同种型。
图2E显示了在CHO细胞中用于表达SEQ ID NO:23的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和全长人CDKL5107同种型。
图2F显示了在大肠杆菌细胞中用于表达SEQ ID NO:24的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和全长人CDKL5107同种型。
图2G显示了在大肠杆菌细胞中用于表达SEQ ID NO:25的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体2的CDKL5107变体。
图2H显示了在大肠杆菌细胞中用于表达SEQ ID NO:26的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体3的CDKL5107变体。
图2I显示了在大肠杆菌细胞中用于表达SEQ ID NO:27的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体4的CDKL5107变体。
图2J显示了在大肠杆菌细胞中用于表达SEQ ID NO:28的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体5的CDKL5107变体。
图2K显示了在大肠杆菌细胞中用于表达SEQ ID NO:29的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体6的CDKL5107变体。
图2L显示了在大肠杆菌细胞中用于表达SEQ ID NO:30的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体7的CDKL5107变体。
图2M显示了在大肠杆菌细胞中用于表达SEQ ID NO:31的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体8的CDKL5107变体。
图2N显示了在大肠杆菌细胞中用于表达SEQ ID NO:32的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体9的CDKL5107变体。
图2O显示了在大肠杆菌细胞中用于表达SEQ ID NO:33的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体10的CDKL5107变体。
图2P显示了在大肠杆菌细胞中用于表达SEQ ID NO:34的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体11的CDKL5107变体。
图2Q显示了在大肠杆菌细胞中用于表达SEQ ID NO:35的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和构建体12的CDKL5107变体。
图2R显示了在大肠杆菌细胞中用于表达SEQ ID NO:36的融合蛋白的示例性质粒。所述融合蛋白包含TAT28和全长人CDKL5107同种型。
图2S显示了在大肠杆菌细胞中用于表达SEQ ID NO:37的融合蛋白的示例性质粒。所述融合蛋白包含TATκ28和增强的绿色荧光蛋白(eGFP)。
图2T显示了在大肠杆菌细胞中用于表达SEQ ID NO:38的融合蛋白的示例性质粒。所述融合蛋白包含不含CPP的eGFP。
图2U显示了在大肠杆菌细胞中用于表达SEQ ID NO:39的融合蛋白的示例性质粒。所述融合蛋白包含人双载蛋白1(AMPH1)。
图2V显示了在CHO细胞中用于表达SEQ ID NO:40的融合蛋白的示例性质粒。所述融合蛋白包含人双载蛋白1(AMPH1)。
图2W显示了在CHO细胞中用于表达SEQ ID NO:41的融合蛋白的示例性质粒。所述融合蛋白包含经修饰的BiP前导信号多肽、TATκ11和全长人CDKL5107同种型。
图2X显示了在CHO细胞中用于表达SEQ ID NO:42的融合蛋白的示例性质粒。所述融合蛋白包含鼠Igκ链前导多肽、TATκ11和全长人CDKL5107同种型。
图2Y显示了在CHO细胞中用于表达SEQ ID NO:43的融合蛋白的示例性质粒。所述融合蛋白包含TATκ11和不含前导信号多肽的全长人CDKL5107同种型。
图2Z显示了在大肠杆菌细胞中用于表达SEQ ID NO:44的融合蛋白的示例性质粒。所述融合蛋白包含TATκ11和不含前导信号多肽的全长人CDKL5107同种型。
图2AA显示了在大肠杆菌细胞中用于表达SEQ ID NO:45的融合蛋白的示例性质粒。所述融合蛋白包含TAT11和不含前导信号多肽的全长人CDKL5107同种型。
图2AB显示了在CHO细胞中用于表达SEQ ID NO:46的融合蛋白的示例性质粒。所述融合蛋白包含TAT11和不含前导信号多肽的全长人CDKL5107同种型。
图2AC显示了在CHO细胞中用于表达SEQ ID NO:54的融合蛋白的示例性质粒。所述融合蛋白包含触角足素CPP和不含前导信号多肽的全长人CDKL5107同种型。
图2AD显示了在CHO细胞中用于表达SEQ ID NO:55的融合蛋白的示例性质粒。所述融合蛋白包含转运素CPP和不含前导信号多肽的全长人CDKL5107同种型。
分别使用图2A-2R和2W-2AB的质粒表达SEQ ID NO:19-36和41-46的CDKL5融合蛋白并评价其活性。人双载蛋白1(AMPH1)将作为CDKL5激酶测定的底物。图2U和2V的质粒将用于表达用于CDKL5激酶测定的亲和力标签化的AMPH1(SEQ ID NO:39和40)。亲和力标签化的eGFP(SEQ ID NO.38)单独以及亲和力标签化的TATk28-eGFP(SEQ ID NO.37)作为CDKL5融合蛋白的对照,其分别使用图2S和2T的质粒表达。
各种CDKL5融合蛋白在CHO和HEK细胞中表达,以及用HeLa细胞裂解物使用体外转录/翻译进行表达。。简言之,使用Maxcyte STX和如下八种质粒电穿孔CHO-S细胞(20x10^6个细胞):(1)pOptiVec空载体;2)TATk28-CDKL5-107-3xFlagHis;3)TATk11-CDKL5-107-3xFlagHis;4)TAT11-CDKL5-107-3xFlagHis;5)TAT28-CDKL5-107-3xFlagHis;6)ANTP-CDKL5-107-3xFlagHis;7)TRANSP-CDKL5-107-3xFlagHis和8)MBiP-TATK28-CDKL5-107-3xFlagHis(编码序列是经CHO密码子优化的)。在培养基中回收细胞,并培养1天。收集细胞并裂解。对于每个转染,将20μg的裂解物经受4%-12%BisTris SDS-PAGE,并使用iBlot2系统转移到硝酸纤维素印迹。在1xTBS-T的5%牛奶中,阻断印迹。通过用1:2000稀释的兔抗-His抗体孵育过夜,将印迹进行蛋白质印迹。经一系列洗涤后,用1:10000抗兔IgG DyaLight680二抗孵育印迹。进行了另外的洗涤。在Licor Odyssey扫描仪上成像印迹。印迹证实CDKL5融合蛋白的表达。
使用FuGeneHD(24μl FuGeneHD:8μg DNA比率)和如下7种质粒转染HEK293F细胞(8x10^6个细胞):1)空pOptiVec;2)TATk11-CDKL5_107-3xFlagHis;3)TAT11-CDKL5_1-FH;4)TAT28-CDKL5_1-FH;5)ANTP-CDKL5_107-3xFlagHis;6)TRANSP-CDKL5_107-3xFlagHis和7)TATk28-CDKL5_107-3xFlagHis(编码序列是经人密码子优化的)。孵育细胞并在转染后2天收获细胞。裂解细胞,并且将20μg的裂解物经受4%-12%BisTris SDS-PAGE,并使用iBlot2系统转移到硝酸纤维素印迹。在1xTBS-T的5%牛奶中,阻断印迹。通过用1:2000稀释的兔抗-His抗体孵育过夜,将印迹进行蛋白质印迹。经一系列洗涤后,用1:10000抗兔IgGDyaLight 680二抗孵育印迹。进行了另外的洗涤。在Licor Odyssey扫描仪上成像印迹。印迹证实CDKL5融合蛋白的表达。
在整篇说明书中对“一个实施例”、“某些实施例”、“各种实施例”、“一个或多个实施例”、或“实施例”的引用意指与实施例相联系地描述的具体的特征、结构、材料、或特性是包括在本披露的至少一个实施例中的。因此,在整篇说明书中多个位置中出现的短语如“在一个或多个实施例中”、“在某些实施例中”、“在各种实施例中”、“在一个实施例中”、或“在实施例中”不一定是指本披露中的相同实施例。此外,在一个或多个实施例中,具体的特征、结构、材料、或特性可以是以任何适合的方式进行组合。
尽管此处的披露已参照具体实施例提供了说明,应理解这些实施例对于本披露的原理和应用仅仅是说明性的。对本领域的普通技术人员而言将明显地是,在不偏离本披露的范围和精神的情况下,可以对本披露进行多种修改和变化。因此,意图是本披露包括在所附的权利要求书及其等效物的范围内的修改和变化。
SEQ ID NO: 1 CDKL5107同种型多肽1-960 (全长)
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETAL
SEQ ID NO: 2 CDKL5107 变体 ?853-960
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHP
SEQ ID NO: 3 CDKL5107 变体 ?745-960
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESS
SEQ ID NO: 4 CDKL5107 变体 ?637-960
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMA
SEQ ID NO: 5 CDKL5107 变体 ?529-960
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPT
SEQ ID NO: 6 CDKL5107 变体 ?421-960
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFN
SEQ ID NO: 7 CDKL5107 变体 ?315-960
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRK
SEQ ID NO: 8 CDKL5107 变体 ?315-420
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETAL
SEQ ID NO: 9 CDKL5107 变体 ?315-528
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETAL
SEQ ID NO: 10 CDKL5107 变体 ?315-636
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETAL
SEQ ID NO: 11 CDKL5107 变体 ?315-744
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETAL
SEQ ID NO: 12 CDKL5107 变体 ?315-852
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETAL
SEQ ID NO: 13 TAT28 CPP
DAAQPARRARRTKLAAYGRKKRRQRRR
SEQ ID NO: 14 TAT?28 CPP
DAAQPARRARRTKLAAYARKAARQARA
SEQ ID NO: 15 TAT11 CPP
YGRKKRRQRRR
SEQ ID NO: 16 TAT?11 CPP
YARKAARQARA
SEQ ID NO: 17 转运素 CPP
AGYLLGK于LKALAALAKKIL
SEQ ID NO: 18 触角足素 CPP
RQIKIWFQNRRMKWKK
SEQ ID NO: 19 >MBip_Tk28p_107_3xFlagHis_cho-opt 于 pOptiVec中
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 20 >IgK_Tk28p_107_3xFlagHis_cho-opt 于 pOptiVec中
METDTLLLWVLLLWVPGSTGGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 21 >MBiP_Tk28p_115_3xFlagHis_cho-opt 于 pOptiVec中
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPDGGCDGRRQRHHSGPQDRRFMLRTTEQQGEYFCCGDPKKPHTPCVPNRALHRPISSPAPYPVLQVRGTSMCPTLQVRGTDAFSCPTQQSGFSFFVRHVMREALIHRAQVNQAALLTYHENAALTGKGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 22 >IgK_Tk28p_115_3xFlagHis_cho-opt 于 pOptiVec中
METDTLLLWVLLLWVPGSTGGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPDGGCDGRRQRHHSGPQDRRFMLRTTEQQGEYFCCGDPKKPHTPCVPNRALHRPISSPAPYPVLQVRGTSMCPTLQVRGTDAFSCPTQQSGFSFFVRHVMREALIHRAQVNQAALLTYHENAALTGKGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 23 >Tk28p_107_3xFlagHis_cho-opt 于 pOptiVec中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 24 >Tk28p_107_3xFlagHis_ecoli-opt 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 25 >?853-960 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 26 >?745-960 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 27 >?637-960 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 28 >?529-960 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 29 >?421-960 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 30 >?315-960 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 31 >?315-420 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 32 >?315-528 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 33 >?315-636 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 34 >?315-744 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 35 >?315-852 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 36 >Tt28p_107_3xFlagHis_ecoli-opt 于 pEX-1中
MGDAAQPARRARRTKLAAYGRKKRRQRRRGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 37 >Tk28p_eGFP_ecoli-opt_3xFlagHis 于 pEX-1中
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 38 >eGFP_3xFlagHis_ecoli-opt 于 pEX-1中
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 39 >AMPH1-3xFlagHis 于 pEX-1中 (ecoli-opt)
MADIKTGIFAKNVQKRLNRAQEKVLQKLGKADETKDEQFEEYVQNFKRQEAEGTRLQRELRGYLAAIKGMQEASMKLTESLHEVYEPDWYGREDVKMVGEKCDVLWEDFHQKLVDGSLLTLDTYLGQFPDIKNRIAKRSRKLVDYDSARHHLEALQSSKRKDESRISKAEEEFQKAQKVFEEFNVDLQEELPSLWSRRVGFYVNTFKNVSSLEAKFHKEIAVLCHKLYEVMTKLGDQHADKAFTIQGAPSDSGPLRIAKTPSPPEEPSPLPSPTASPNHTLAPASPAPARPRSPSQTRKGPPVPPLPKVTPTKELQQENIISFFEDNFVPEISVTTPSQNEVPEVKKEETLLDLDFDPFKPEVTPAGSAGVTHSPMSQTLPWDLWTTSTDLVQPASGGSFNGFTQPQDTSLFTMQTDQSMICNLAESEQAPPTEPKAEEPLAAVTPAVGLDLGMDTRAEEPVEEAVIIPGADADAAVGTLVSAAEGAPGEEAEAEKATVPAGEGVSLEEAKIGTETTEGAESAQPEAEELEATVPQEKVIPSVVIEPASNHEEEGENEITIGAEPKETTEDAAPPGPTSETPELATEQKPIQDPQPTPSAPAMGAADQLASAREASQELPPGFLYKVETLHDFEAANSDELTLQRGDVVLVVPSDSEADQDAGWLVGVKESDWLQYRDLATYKGLFPENFTRRLDENLYFQGGGGGSDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 40 >AMPH1-3xFlagHis cho-opt 于 pOptiVec中
MADIKTGIFAKNVQKRLNRAQEKVLQKLGKADETKDEQFEEYVQNFKRQEAEGTRLQRELRGYLAAIKGMQEASMKLTESLHEVYEPDWYGREDVKMVGEKCDVLWEDFHQKLVDGSLLTLDTYLGQFPDIKNRIAKRSRKLVDYDSARHHLEALQSSKRKDESRISKAEEEFQKAQKVFEEFNVDLQEELPSLWSRRVGFYVNTFKNVSSLEAKFHKEIAVLCHKLYEVMTKLGDQHADKAFTIQGAPSDSGPLRIAKTPSPPEEPSPLPSPTASPNHTLAPASPAPARPRSPSQTRKGPPVPPLPKVTPTKELQQENIISFFEDNFVPEISVTTPSQNEVPEVKKEETLLDLDFDPFKPEVTPAGSAGVTHSPMSQTLPWDLWTTSTDLVQPASGGSFNGFTQPQDTSLFTMQTDQSMICNLAESEQAPPTEPKAEEPLAAVTPAVGLDLGMDTRAEEPVEEAVIIPGADADAAVGTLVSAAEGAPGEEAEAEKATVPAGEGVSLEEAKIGTETTEGAESAQPEAEELEATVPQEKVIPSVVIEPASNHEEEGENEITIGAEPKETTEDAAPPGPTSETPELATEQKPIQDPQPTPSAPAMGAADQLASAREASQELPPGFLYKVETLHDFEAANSDELTLQRGDVVLVVPSDSEADQDAGWLVGVKESDWLQYRDLATYKGLFPENFTRRLDENLYFQGGGGGSDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 41 >MBip_Tatk11_107_3xFlagHis_cho-opt 于 pOptiVec中
MKLSLVAAMLLLLSLVAAMLLLLSAARAGYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 42 >IgK_Tatk11_107_3xFlagHis_cho-opt 于 pOptiVec中
METDTLLLWVLLLWVPGSTGGYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 43 >Tatk11_107_3xFlagHis_cho-opt 于 pOptiVec中 (无前导多肽)
MGYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 44 >Tatk11_107_3xFlagHis_ecoli-opt 于 pEX-1中
MGYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 45 >Tat11_107_3xFlagHis_ecoli-opt 于 pEX-1中
MGYGRKKRRQRRRGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 46 >Tat11_107_3xFlagHis_cho-opt 于 pOptiVec中 (无前导多肽)
MGYGRKKRRQRRRGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH
SEQ ID NO: 47 CDKL5115同种型多肽1-1030 (全长)
MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPDGGCDGRRQRHHSGPQDRRFMLRTTEQQGEYFCCGDPKKPHTPCVPNRALHRPISSPAPYPVLQVRGTSMCPTLQVRGTDAFSCPTQQSGFSFFVRHVMREALIHRAQVNQAALLTYHENAALTGK
SEQ ID NO: 48 MBiP
MKLSLVAAMLLLLSLVAAMLLLLSAARA
SEQ ID NO: 49 鼠Ig?
METDTLLLWVLLLWVPGSTG
SEQ ID NO: 50 P97
DSSHAFTLDELR
SEQ ID NO: 51 MBiP2
MKLSLVAAMLLLLWVALLLLSAARA
SEQ ID NO: 52 MBiP3
MKLSLVAAMLLLLSLVALLLLSAARA
SEQ ID NO: 53 MBiP4
MKLSLVAAMLLLLALVALLLLSAARA
SEQ ID NO: 54 >ANTP_107_3xFlagHis_cho-opt 于 pOptiVec中
MGRQIKIWFQNRRMKWKKGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
SEQ ID NO: 55 >TRANSP_107_3xFlagHis_cho-opt 于 pOptiVec中
MGAGYLLGK于LKALAALAKKILGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETALGGGGSENLYFQGDYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
Claims (44)
1.一种CDKL5多肽,其中所述CDKL5多肽包含与SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12具有至少98%的序列一致性的序列。
2.如权利要求1所述的CDKL5多肽,其中所述CDKL5多肽包含与SEQ ID NO:2、SEQ IDNO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12具有至少99%的序列一致性的序列。
3.如权利要求1所述的CDKL5多肽,其中所述CDKL5多肽包含与SEQ ID NO:2、SEQ IDNO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12具有100%的序列一致性的序列。
4.一种缺乏核输出信号(NES)的CDKL5多肽。
5.如权利要求4所述的CDKL5多肽,其中所述CDKL5多肽含有核定位信号(NLS)。
6.如权利要求4所述的CDKL5多肽,其中所述CDKL5多肽不含有核定位信号(NLS)。
7.一种缺乏核定位信号(NLS)且含有核输出信号(NES)的CDKL5多肽。
8.一种融合蛋白,所述融合蛋白包含如权利要求1-7中任一项所述的CDKL5多肽和可操作地连接至所述CDKL5多肽的前导信号多肽。
9.一种融合蛋白,所述融合蛋白包含如权利要求1-7中任一项所述的CDKL5多肽和可操作地连接至所述CDKL5多肽的细胞穿透多肽。
10.如权利要求9所述的融合蛋白,其中所述细胞穿透多肽包含与SEQ ID NO:13、SEQID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有至少90%的序列一致性的序列。
11.如权利要求9所述的融合蛋白,其中所述细胞穿透多肽包含与SEQ ID NO:13、SEQID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有100%的序列一致性的序列。
12.如权利要求9-11中任一项所述的融合蛋白,其进一步包含可操作地连接至如权利要求9-11中任一项所述的融合蛋白的前导信号多肽。
13.如权利要求8或12所述的融合蛋白,其中所述前导信号多肽包含与SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:52或SEQ ID NO:53具有至少90%的序列一致性的序列。
14.如权利要求8或12所述的融合蛋白,其中所述前导信号多肽包含与SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:52或SEQ ID NO:53具有100%的序列一致性的序列。
15.一种药物配制品,所述药物配制品包含:
如权利要求1-7中任一项所述的CDKL5多肽或如权利要求8-14中任一项所述的融合蛋白;以及
药学上可接受的载体。
16.一种治疗CDKL5介导的神经障碍的方法,所述方法包括将如权利要求15所述的配制品施用至有需要的患者。
17.如权利要求16所述的方法,其中所述配制品是鞘内、静脉内、脑池内、脑室内或实质内施用。
18.如权利要求16或17所述的方法,其中所述配制品是鞘内或静脉内施用。
19.如权利要求16-18中任一项所述的方法,其中所述CDKL5介导的神经障碍是CDKL5缺陷或由CDKL5突变或缺陷引起的非典型Rett综合征中的一种或多种。
20.一种产生如权利要求1-7中任一项所述的CDKL5多肽或如权利要求8-14中任一项所述的融合蛋白的方法,所述方法包括:
表达所述CDKL5多肽或所述融合蛋白;以及
纯化所述CDKL5多肽或所述融合蛋白。
21.如权利要求20所述的方法,其中所述CDKL5多肽或所述融合蛋白在中国仓鼠卵巢(CHO)细胞、HeLa细胞、人胚肾(HEK)细胞或大肠杆菌细胞中表达。
22.一种多核苷酸,所述多核苷酸编码如权利要求1-7中任一项所述的CDKL5多肽或如权利要求8-14中任一项所述的融合蛋白。
23.一种载体,所述载体包含如权利要求22所述的多核苷酸。
24.一种融合蛋白,所述融合蛋白包含CDKL5多肽和可操作地连接在一起的细胞穿透多肽,其中所述CDKL5多肽包含与SEQ ID NO:1或SEQ ID NO:47具有至少98%的序列一致性的序列且所述细胞穿透多肽包含与SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有至少90%的序列一致性的序列。
25.如权利要求24所述的融合蛋白,其中所述细胞穿透多肽包含与SEQ ID NO:13、SEQID NO:14、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有至少95%的序列一致性的序列。
26.如权利要求25所述的融合蛋白,其中所述细胞穿透多肽包含与SEQ ID NO:13、SEQID NO:14、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有100%的序列一致性的序列。
27.如权利要求24-26中任一项所述的融合蛋白,所述融合蛋白进一步包含前导信号多肽。
28.如权利要求27所述的融合蛋白,其中所述前导信号多肽包含与SEQ ID NO:48、SEQID NO:49、SEQ ID NO:51、SEQ ID NO:52或SEQ ID NO:53具有至少90%的序列一致性的序列。
29.如权利要求28所述的融合蛋白,其中所述前导信号多肽包含与SEQ ID NO:48、SEQID NO:49、SEQ ID NO:51、SEQ ID NO:52或SEQ ID NO:53具有100%的序列一致性的序列。
30.一种融合蛋白,所述融合蛋白包含CDKL5多肽和可操作地连接在一起的前导信号多肽,其中所述CDKL5多肽包含与SEQ ID NO:1或SEQ ID NO:47具有至少98%的序列一致性的序列且所述前导信号多肽包含与SEQ ID NO:48、SEQ ID NO:51、SEQ ID NO:52或SEQ IDNO:53具有至少90%的序列一致性的序列。
31.如权利要求30所述的融合蛋白,其中所述前导信号多肽包含与SEQ ID NO:48、SEQID NO:51、SEQ ID NO:52或SEQ ID NO:53具有100%的序列一致性的序列。
32.如权利要求30或31所述的融合蛋白,所述融合蛋白进一步包含细胞穿透多肽。
33.如权利要求32所述的融合蛋白,其中所述细胞穿透多肽包含与SEQ ID NO:13、SEQID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有至少90%的序列一致性的序列。
34.如权利要求33所述的融合蛋白,其中所述细胞穿透多肽包含与SEQ ID NO:13、SEQID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有至少95%的序列一致性的序列。
35.如权利要求34所述的融合蛋白,其中所述细胞穿透多肽包含与SEQ ID NO:13、SEQID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:50具有100%的序列一致性的序列。
36.一种药物配制品,所述药物配制品包含如权利要求24-35中任一项所述的融合蛋白;以及药学上可接受的载体。
37.一种治疗CDKL5介导的神经障碍的方法,所述方法包括将如权利要求36所述的配制品施用至有需要的患者。
38.如权利要求37所述的方法,其中所述配制品是鞘内、静脉内、脑池内、脑室内或实质内施用。
39.如权利要求37或38所述的方法,其中所述配制品是鞘内或静脉内施用。
40.如权利要求37-39中任一项所述的方法,其中所述CDKL5介导的神经障碍是CDKL5缺陷或由CDKL5突变或缺陷引起的非典型Rett综合征中的一种或多种。
41.一种产生如权利要求24-35中任一项所述的融合蛋白的方法,所述方法包括:
表达所述融合蛋白;以及
纯化所述融合蛋白。
42.如权利要求41所述的方法,其中所述融合蛋白在中国仓鼠卵巢(CHO)细胞、HeLa细胞、人胚肾(HEK)细胞或大肠杆菌细胞中表达。
43.一种多核苷酸,所述多核苷酸编码如权利要求24-35中任一项所述的融合蛋白。
44.一种载体,所述载体包含如权利要求43所述的多核苷酸。
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WO2022119890A1 (en) | 2020-12-01 | 2022-06-09 | The Trustees Of The University Of Pennsylvania | Compositions and uses thereof for treatment of angelman syndrome |
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