CN114644706B - 基于DNA纳米技术的pMHC多聚体制备方法及其应用 - Google Patents
基于DNA纳米技术的pMHC多聚体制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了基于DNA纳米技术的pMHC多聚体制备方法及其应用。本发明利用DNA纳米结构作为支架，连接不同数量的pMHC，并控制其数量、位置和方向，使用原子力显微镜可以清晰地观察到不同数量的pMHC被捕获和展示在DNA纳米结构上。使用DNA‑pMHC来检测抗原特异性T细胞，由于pMHC在DNA纳米结构上的单一取向所介导的单向多价效应，pMHC数量从3～12不等的DNA‑pMHC多聚体在抗原特异性T细胞检测时的灵敏度是pMHC四聚体的1.9‑4.3倍。本发明基于DNA纳米技术的pMHC多聚体在抗原特异性T细胞的分析中具有广阔的应用前景，为探索纳米尺度下精确蛋白质排列的空间生物学效应提供有力工具。
Description
技术领域
本发明属于T细胞检测领域，具体地说，涉及一种基于DNA纳米技术的pMHC多聚体制备方法及其应用。
背景技术
CTL是机体介导细胞免疫的主要效应细胞，是适应性细胞免疫的核心。在机体清除细胞内病原体感染，抗肿瘤和排斥移植物等反应中发挥着重要的生物学作用。抗原特异CTL是一小群极重要的细胞，但由于它们在淋巴细胞中的百比非常低，因此使得这群有重要意义的细胞的检测非常困难。准确监测疾病进展和评估疫苗效力需要高效、特异性和定量检测抗原特异性T细胞。
合成的肽-主要组织相容性复合物(pMHC)可以与抗原特异性T细胞表面的T细胞受体(TCR)结合，这种特异性结合可用于通过流式细胞术检测抗原特异性T细胞。然而，单价pMHC与TCR之间的亲和力远低于抗原与抗体之间的亲和力。这种相对较弱的结合导致形成的pMHC单体和TCR复合物的半衰期短和快速解离。为了增强pMHC和TCR之间的亲和力，已经开发了各种pMHC多聚体，例如二聚体、四聚体、五聚体、八聚体和右旋聚体。由于结构和制备简单，1996年建立的基于pMHC四聚体的检测方法仍然是抗原特异性T细胞分析的金标准，为T细胞免疫治疗奠定了基础。
然而，pMHC四聚体技术仍然存在一些局限性。由于结合亲和力不足，pMHC四聚体在抗原特异性CD4+T细胞、低亲和力αβT细胞和稀有γδT细胞的检测中表现不尽如人意。pMHC四聚体的最大化合价仅限于四个MHC分子，但由于链霉亲和素作为支架，其分子的四重对称性，四聚体上只有三个pMHC可以同时与T细胞表面的TCR结合。提高多聚体上的pMHC数量是增强结合亲和力的有效方法。然而，除了显示在多聚体支架上的pMHC数量外，它们的空间排列和方向也是重要的参数。当pMHC单体面向统一方向时，pMHC多聚体的亲和力更高。因此，迫切需要一种能够实现四个以上pMHC单体精确空间和单向组装的多聚体支架，以有效检测抗原特异性T细胞。而DNA纳米技术为此提供了契机。
DNA纳米结构，作为展示分子的多功能平台具有巨大的潜力，引起了包括生物医学在内的许多研究领域极大的关注。DNA纳米结构由一条或多条模板链和许多短链组成，通过碱基互补配对自组装。DNA纳米结构的每一条短链都是独一无二的，这使得DNA纳米结构成为一种可编程的纳米结构。通过序列延伸和末端修饰，可以将DNA纳米结构的短链设计为捕获链，用于距离、数量和模式可控的蛋白质链接支架。
发明内容
本发明的目的是提供一种基于DNA纳米技术的pMHC多聚体的精确空间组装方法，以及将其用于检测抗原特异性T细胞的方法。
为了实现本发明目的，第一方面，本发明提供一种基于DNA纳米技术的pMHC多聚体制备方法，包括以下步骤：
A、带有特定捕获链的矩形DNA纳米结构的制备：将1条模板链和若干条短链通过碱基互补配对自组装形成带有特定捕获链的矩形DNA纳米结构；
B、利用点击化学反应将叠氮化物修饰的SpyTag与含有DBCO修饰的互补链偶联链接，得到SpyTag-互补链；
C、将SpyTag-互补链、PE修饰的互补链与矩形DNA纳米结构上的捕获链通过退火发生碱基互补配对链接，得到带有SpyTag的DNA纳米结构；
其中，步骤B和C中所述的互补链与步骤A中所述的捕获链互补配对；
D、构建pMHC与SpyCatcher融合蛋白(在pMHC的C端融合SpyCatcher)，与带有SpyTag的DNA纳米结构混合，DNA纳米结构上的SpyTag与SpyCatcher通过异肽键从而将pMHC单体链接在DNA纳米结构表面。
优选地，所述模板链的核苷酸序列如SEQ ID NO:1所示，所述短链共143条，核苷酸序列分别如SEQ ID NO:2-144所示。
优选地，SpyTag的氨基酸序列为SLLFLLFSL。
优选地，所述互补链包括与链接PE的捕获链互补的互补链以及与链接pMHC的捕获链互补的互补链，与链接PE的捕获链互补的互补链的核苷酸序列为PE-TTTTTTTAATTAATTTTTT，与链接pMHC的捕获链互补的互补链的核苷酸序列为N3-TTTTTGGTAGTGTAGGACTCCTAGC。
优选地，所述捕获链包括链接PE的捕获链以及链接pMHC的捕获链。
链接PE的捕获链的核苷酸序列如SEQ ID NO:145所示；其中，SEQ ID NO:145所示捕获链替换DNA纳米结构中SEQ ID NO:24所示的短链。
链接3个pMHC的捕获链的核苷酸序列如SEQ ID NO:146-148所示；其中，SEQ IDNO:146所示捕获链替换DNA纳米结构中SEQ ID NO:139所示的短链，SEQ ID NO:147所示捕获链替换DNA纳米结构中SEQ ID NO:80所示的短链，SEQ ID NO:148所示捕获链替换DNA纳米结构中SEQ ID NO:97所示的短链。
链接6个pMHC的捕获链的核苷酸序列如SEQ ID NO:149-154所示；其中，SEQ IDNO:149所示捕获链替换DNA纳米结构中SEQ ID NO:15所示的短链，SEQ ID NO:150所示捕获链替换DNA纳米结构中SEQ ID NO:139所示的短链，SEQ ID NO:151所示捕获链替换DNA纳米结构中SEQ ID NO:101所示的短链，SEQ ID NO:152所示捕获链替换DNA纳米结构中SEQ IDNO:80所示的短链，SEQ ID NO:153所示捕获链替换DNA纳米结构中SEQ ID NO:142所示的短链，SEQ ID NO:154所示捕获链替换DNA纳米结构中SEQ ID NO:97所示的短链。
链接9个pMHC的捕获链的核苷酸序列如SEQ ID NO:155-163所示；其中，SEQ IDNO:155所示捕获链替换DNA纳米结构中SEQ ID NO:15所示的短链，SEQ ID NO:156所示捕获链替换DNA纳米结构中SEQ ID NO:10所示的短链，SEQ ID NO:157所示捕获链替换DNA纳米结构中SEQ ID NO:139所示的短链，SEQ ID NO:158所示捕获链替换DNA纳米结构中SEQ IDNO:101所示的短链，SEQ ID NO:159所示捕获链替换DNA纳米结构中SEQ ID NO:66所示的短链，SEQ ID NO:160所示捕获链替换DNA纳米结构中SEQ ID NO:80所示的短链，SEQ ID NO:161所示捕获链替换DNA纳米结构中SEQ ID NO:142所示的短链，SEQ ID NO:162所示捕获链替换DNA纳米结构中SEQ ID NO:67所示的短链，SEQ ID NO:163所示捕获链替换DNA纳米结构中SEQ ID NO:97所示的短链。
链接12个pMHC的捕获链的核苷酸序列如SEQ ID NO:164-175所示；其中，SEQ IDNO:164所示捕获链替换DNA纳米结构中SEQ ID NO:79所示的短链，SEQ ID NO:165所示捕获链替换DNA纳米结构中SEQ ID NO:103所示的短链，SEQ ID NO:166所示捕获链替换DNA纳米结构中SEQ ID NO:5所示的短链，SEQ ID NO:167所示捕获链替换DNA纳米结构中SEQ IDNO:55所示的短链，SEQ ID NO:168所示捕获链替换DNA纳米结构中SEQ ID NO:65所示的短链，SEQ ID NO:169所示捕获链替换DNA纳米结构中SEQ ID NO:8所示的短链，SEQ ID NO:170所示捕获链替换DNA纳米结构中SEQ ID NO:25所示的短链，SEQ ID NO:171所示捕获链替换DNA纳米结构中SEQ ID NO:56所示的短链，SEQ ID NO:172所示捕获链替换DNA纳米结构中SEQ ID NO:13所示的短链，SEQ ID NO:173所示捕获链替换DNA纳米结构中SEQ ID NO:44所示的短链，SEQ ID NO:174所示捕获链替换DNA纳米结构中SEQ ID NO:108所示的短链，SEQ ID NO:175所示捕获链替换DNA纳米结构中SEQ ID NO:50所示的短链。
优选地，pMHC与SpyCatcher融合蛋白的氨基酸序列如SEQ ID NO:176-177所示(α链和β链)。
本发明中，蛋白质链接方式(步骤B、步骤C和步骤D中的链接)可以是叠氮化物(如N3)与DBCO以及SpyTag与SpyCatcher之间的反应，或其他共价和非共价结合反应。例如，小分子交联剂N-Succinimidyl 3-(2-pyridyldithio)-propionate(SPDP)和Sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(Sulfo-SMCC)(连接蛋白质表面的赖氨酸残基和DNA上的硫醇，生成复合物)，非天然氨基酸对氨基苯丙氨酸与苯二胺修饰的DNA通过氧化偶联反应，以及tag蛋白的引入O6-alkylguanine-DNA-alkyltransferase(hAGT，即SNAP-tag)和haloalkaneDehalogenase(即Halo-tag)等。
进一步地，所述DNA纳米结构还带有荧光分子，所述荧光分子为PE、APC或FITC等。
第二方面，本发明提供按照上述方法制备的pMHC多聚体。
第三方面，本发明提供所述基于DNA纳米技术的pMHC多聚体在制备抗原特异性T细胞检测试剂中的应用。
第四方面，本发明提供一种抗原特异性T细胞检测试剂盒，其有效成分为按照上述方法制备的pMHC多聚体。
第五方面，本发明提供所述基于DNA纳米技术的pMHC多聚体在进行抗原特异性T细胞检测中的应用。
在进行T细胞检测和分析时，检测的特异性T细胞可以是CD4+T细胞、低亲和力αβT细胞和稀有γδT，流感，新冠等病毒性T细胞，包括人和鼠的T细胞等。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明通过设计并合成DNA纳米结构，基于DNA纳米技术开发了一种新的肽-主要组织相容性复合体(DNA-pMHC)多聚体检测分析特异性T细胞。通过修饰DNA纳米结构的短链可以在特定位点精确控制pMHC单体数量、位置和方向。使用原子力显微镜，可以清晰地在DNA纳米结构上观察到不同数量的pMHC单体。利用开发的DNA-pMHC来检测抗原特异性T细胞，由于pMHC在DNA纳米结构上的单一取向所介导的单向多价效应(UME)，pMHC数量从3～12不等的DNA-pMHC多聚体在抗原特异性T细胞检测中表现出的灵敏度是pMHC四聚体的1.9-4.3倍。
本发明提供的基于DNA纳米技术控制pMHC单体的精确空间组装，可以精确地控制数量，位置和方向。使用原子力显微镜，可以清晰地在DNA纳米结构上观察到不同数量的pMHC单体，相较现有的蛋白质精准组装，更精确更清晰。
本发明提供的基于DNA纳米技术控制pMHC单体的精确空间组装，最大组装效率可以达到80％。相较现有的蛋白质组链接方案效率更高，更加节约成本。
附图说明
图1为本发明较佳实施例中DNA-pMHC多聚体的表征。其中，A-D分别为链接不同数量(DNA-pMHC-3、DNA-pMHC-6、DNA-pMHC-9和DNA-pMHC-12)的pMHC单体的DNA纳米结构的AFM图像。颜色条代表AFM图像中的高度比例。AFM图像比例尺为100nm。
图2为本发明较佳实施例中基于DNA纳米技术的pMHC组装效率检测。其中，A-D分别为DNA纳米结构链接3-12个pMHC的组装效率分别由AFM图像计算得出。
图3为本发明较佳实施例中基于DNA纳米技术的肽-主要组织相容性复合体(DNA-pMHC)多聚体检测特异性T细胞的效率。其中，A为OVA+细胞在对照和疫苗接种小鼠的CD3+CD8+T细胞中的百分比。pE：DNA纳米结构只连接PE，DNA纳米结构-3、6、9、12、16：DNA纳米结构上链接的不同数量的pMHC单体。B为经统计软件处理减去阴性对照和未免疫小鼠的空白对照后OVA+细胞在CD3+CD8+T细胞中的百分比(n＝5)。
具体实施方式
本发明通过设计并合成DNA纳米结构，开发了一种基于DNA纳米技术的新pMHC多聚体(DNA-pMHC)，其中pMHC单体的数量、位置和方向得到精确控制。通过结合点击化学反应和肽胶技术的“雕刻-印刷”策略，实现了多种pMHC在DNA纳米结构上的高效、快速的位置可控以及功能化，使用原子力显微镜，可以清晰地在DNA纳米结构上观察到不同数量的pMHC单体。然后，使用开发的DNA-pMHC来检测抗原特异性T细胞，由于pMHC在DNA纳米结构上的单一取向所介导的单向多价效应，pMHC数量从3～12不等的DNA-pMHC多聚体在抗原特异性T细胞检测中表现出的灵敏度是pMHC四聚体的1.9～4.3倍。实验结果表明，基于DNA纳米技术的pMHC多聚体在抗原特异性T细胞的分析中具有广阔的应用前景，并为探索纳米尺度下精确蛋白质排列的空间生物学效应提供有力工具。
本发明采用如下技术方案：
本发明的技术方案之一，提供一种基于DNA纳米技术的pMHC多聚体的精确空间组装的方法。
通过设计并合成DNA纳米结构，基于DNA纳米技术组装多肽-主要组织相容性复合体(pMHC)多聚体来检测分析抗原特异性T细胞。通过修饰DNA纳米结构的短链可以在特定位点精确控制pMHC单体数量、位置和方向。使用原子力显微镜，可以清晰地在DNA纳米结构上观察到不同数量的pMHC单体。然后，使用开发的多聚体来检测抗原特异性T细胞。与商品化四聚体相比，该结构表现出更高的灵敏度、更强的信号强度、更高的结合亲和力和更慢的解离速率。
所述精确的空间组装可以是pMHC的精确空间组装，也可以是应用到任何蛋白质的精确空间组装。
DNA纳米结构由模板链和许多短链通过碱基互补配对自组装，通过延长目的修饰位置DNA纳米结构的短链使其形成延长的捕获链，与捕获链互补的链称为互补链。使用点击化学反应将叠氮化物修饰的SpyTag与含有DBCO修饰的互补链偶联。SpyTag-互补链与PE修饰的互补链与矩形DNA纳米结构上相应的捕获链通过退火时碱基互补配对链接。通过将SpyCatcher表达为pMHC的融合蛋白，其与DNA纳米结构上的SpyTag可以通过形成异肽键从而将pMHC单体链接在DNA纳米结构表面。
延长的数目可以是6-30个与模板链和短链不同的任意碱基，GC含量不要超过60％。
N3与DBCO既可以修饰在互补链上，也可以修饰在SpyTag上。
采用的蛋白质链接方式可以是N3与DBCO以及SpyTag与SpyCatcher之间的反应，也可以是其他共价结合反应，例如，小分子交联剂N-Succinimidyl3-(2-pyridyldithio)-propionate(SPDP)和Sulfo succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(Sulfo-SMCC)(连接蛋白质表面的赖氨酸残基和DNA上的硫醇，生成复合物)，非天然氨基酸对氨基苯丙氨酸与苯二胺修饰的DNA通过氧化偶联反应，以及tag蛋白的引入O6-alkylguanine-DNA-alkyltransferase(hAGT，即SNAP-tag)和haloalkaneDehalogenase(即Halo-tag)等。
采用的蛋白质链接方式也可以是非共价结合的方法如生物素与链霉亲和素，抗原与抗体，适配体与相应的蛋白质，Ni-NTA与His-tag等。
如果反应策略之间没有干扰，多个蛋白质可以同时使用不同的方法被链接在DNA“折纸”上。
本发明的技术方案之二，提供一种基于DNA纳米技术的pMHC多聚体的精确空间组装来检测特异性T细胞。
MHC可以是人或者鼠的不同的MHC等位基因，同时相应的多肽可以是不同的多肽，如SLLFLLFSL等。
多肽链接的荧光分子可以是PE、APC、FITC等。
检测的特异性T细胞可以是CD4+T细胞、低亲和力αβT细胞和稀有γδT，流感，新冠等病毒相关特异性T细胞，包括人和鼠的T细胞等。
在组装与检测方面，其组装效率可以达到80％以上，检测灵敏度可以提高2-4倍。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1基于DNA纳米技术的pMHC多聚体制备方法
本实施例提供一种基于DNA纳米技术控制pMHC单体的精确空间组装方法以及最大组装效率检测。
首先，利用点击化学反应将叠氮化物(N3)修饰的SpyTag与含有DBCO的互补链偶联，并使用100KDa离心过滤器纯化产物。
其次，SpyTag-DNA互补链和pE修饰的互补链与矩形DNA纳米结构(具有相应的捕获链)在PCR仪中退火链接。并通过100KDa离心过滤器纯化，以去除多余的蛋白质。其中，带有特定捕获链的矩形DNA纳米结构的制备方法如下：将1条模板链和若干条短链通过碱基互补配对自组装形成带有特定捕获链的矩形DNA纳米结构。
最后，通过将SpyCatcher与pMHC一起表达为融合蛋白，然后就可以与SpyTag形成异肽键，从而链接在DNA纳米结构表面上。最后使用AFM进行表征。
本实施例中，模板链的核苷酸序列如SEQ ID NO:1所示，短链共143条，核苷酸序列分别如SEQ ID NO:2-144所示。
SpyTag的氨基酸序列为SLLFLLFSL。
其中，捕获链包括链接PE的捕获链以及链接pMHC的捕获链。
链接PE的捕获链的核苷酸序列如SEQ ID NO:145所示。
链接3个pMHC的捕获链的核苷酸序列如SEQ ID NO:146-148所示；其中，SEQ IDNO:146所示捕获链替换DNA纳米结构中SEQ ID NO:139所示的短链，SEQ ID NO:147所示捕获链替换DNA纳米结构中SEQ ID NO:80所示的短链，SEQ ID NO:148所示捕获链替换DNA纳米结构中SEQ ID NO:97所示的短链。
链接6个pMHC的捕获链的核苷酸序列如SEQ ID NO:149-154所示；其中，SEQ IDNO:149所示捕获链替换DNA纳米结构中SEQ ID NO:15所示的短链，SEQ ID NO:150所示捕获链替换DNA纳米结构中SEQ ID NO:139所示的短链，SEQ ID NO:151所示捕获链替换DNA纳米结构中SEQ ID NO:101所示的短链，SEQ ID NO:152所示捕获链替换DNA纳米结构中SEQ IDNO:80所示的短链，SEQ ID NO:153所示捕获链替换DNA纳米结构中SEQ ID NO:142所示的短链，SEQ ID NO:154所示捕获链替换DNA纳米结构中SEQ ID NO:97所示的短链。
链接9个pMHC的捕获链的核苷酸序列如SEQ ID NO:155-163所示；其中，SEQ IDNO:155所示捕获链替换DNA纳米结构中SEQ ID NO:15所示的短链，SEQ ID NO:156所示捕获链替换DNA纳米结构中SEQ ID NO:10所示的短链，SEQ ID NO:157所示捕获链替换DNA纳米结构中SEQ ID NO:139所示的短链，SEQ ID NO:158所示捕获链替换DNA纳米结构中SEQ IDNO:101所示的短链，SEQ ID NO:159所示捕获链替换DNA纳米结构中SEQ ID NO:66所示的短链，SEQ ID NO:160所示捕获链替换DNA纳米结构中SEQ ID NO:80所示的短链，SEQ ID NO:161所示捕获链替换DNA纳米结构中SEQ ID NO:142所示的短链，SEQ ID NO:162所示捕获链替换DNA纳米结构中SEQ ID NO:67所示的短链，SEQ ID NO:163所示捕获链替换DNA纳米结构中SEQ ID NO:97所示的短链。
链接12个pMHC的捕获链的核苷酸序列如SEQ ID NO:164-175所示；其中，SEQ IDNO:164所示捕获链替换DNA纳米结构中SEQ ID NO:79所示的短链，SEQ ID NO:165所示捕获链替换DNA纳米结构中SEQ ID NO:103所示的短链，SEQ ID NO:166所示捕获链替换DNA纳米结构中SEQ ID NO:5所示的短链，SEQ ID NO:167所示捕获链替换DNA纳米结构中SEQ IDNO:55所示的短链，SEQ ID NO:168所示捕获链替换DNA纳米结构中SEQ ID NO:65所示的短链，SEQ ID NO:169所示捕获链替换DNA纳米结构中SEQ ID NO:8所示的短链，SEQ ID NO:170所示捕获链替换DNA纳米结构中SEQ ID NO:25所示的短链，SEQ ID NO:171所示捕获链替换DNA纳米结构中SEQ ID NO:56所示的短链，SEQ ID NO:172所示捕获链替换DNA纳米结构中SEQ ID NO:13所示的短链，SEQ ID NO:173所示捕获链替换DNA纳米结构中SEQ ID NO:44所示的短链，SEQ ID NO:174所示捕获链替换DNA纳米结构中SEQ ID NO:108所示的短链，SEQ ID NO:175所示捕获链替换DNA纳米结构中SEQ ID NO:50所示的短链。
pMHC与SpyCatcher融合蛋白的氨基酸序列如SEQ ID NO:176-177所示(α链和β链)。
实验结果显示，使用原子力显微镜，可以清晰地在DNA纳米结构上观察到不同数量的pMHC单体，最大组装效率可以达到80％。
基于DNA纳米技术控制pMHC单体的精确空间组装见图1，基于DNA纳米技术的pMHC组装效率检测结果见图2。
实施例2基于DNA纳米技术的肽-主要组织相容性复合体(DNA-pMHC)多聚体检测分析特异性T细胞的效率评估
为了对本发明的特异性T细胞的检测效率进行测试，选择了C57BL/6小鼠脾脏细胞进行检测。
首先，用OVA 257-264(SIINFEKL)抗原肽和PolyIC佐剂(IC)质量比1:1的混合物皮下免疫C57BL/6小鼠，每3天免疫一次，共免疫3次。7天后，取出脾脏，制备脾脏单细胞悬液。
其次，使用H-2Kb-OVA 257-264(SIINFEKL)四聚体作为对照和DNA纳米结构多聚体分别用于染色H-2Kb限制的OVA257-264特异性TCR。
最后，使用流式细胞仪(NovoCyte 2060R，安捷伦)测量样品，并使用NovoExpress分析数据。
实验结果显示，使用DNA-pMHC来检测抗原特异性T细胞，pMHC数量从3～12不等的DNA-pMHC多聚体在抗原特异性T细胞检测中表现出的灵敏度是pMHC四聚体的1.9-4.3倍。
基于DNA纳米技术的肽-主要组织相容性复合体(DNA-pMHC)多聚体检测特异性T细胞的效率见图3。
本发明利用DNA纳米结构作为支架，连接不同数量的pMHC，并控制其数量、位置和方向，使用原子力显微镜(AFM)，可以清晰地观察到不同数量的pMHC被捕获和展示在DNA纳米结构上。使用开发的DNA-pMHC来检测抗原特异性T细胞，由于pMHC在DNA纳米结构上的单一取向所介导的单向多价效应(UME)，pMHC数量从3～12不等的DNA-pMHC多聚体在抗原特异性T细胞检测中表现出的灵敏度是pMHC四聚体的1.9-4.3倍。实验结果表明，基于DNA纳米技术的pMHC多聚体在抗原特异性T细胞的分析中具有广阔的应用前景，并为探索纳米尺度下精确蛋白质排列的空间生物学效应提供有力工具。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 国家纳米科学中心
<120> 基于DNA纳米技术的pMHC多聚体制备方法及其应用
<130> KHP221110665.4
<160> 177
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7249
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aatgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60
atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120
cgttcgcaga attgggaatc aactgttata tggaatgaaa cttccagaca ccgtacttta 180
gttgcatatt taaaacatgt tgagctacag cattatattc agcaattaag ctctaagcca 240
tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg 300
ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag 360
tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt 420
cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 480
tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540
aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt 600
ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt 660
aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg 720
atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780
tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840
caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt 900
ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960
aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc 1020
tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1080
gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140
caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200
caaagatgag tgttttagtg tattcttttg cctctttcgt tttaggttgg tgccttcgta 1260
gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320
caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380
cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1440
tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500
attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560
ttttggagat tttcaacgtg aaaaaattat tattcgcaat tcctttagtt gttcctttct 1620
attctcactc cgctgaaact gttgaaagtt gtttagcaaa atcccataca gaaaattcat 1680
ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat gagggctgtc 1740
tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga aactcagtgt tacggtacat 1800
gggttcctat tgggcttgct atccctgaaa atgagggtgg tggctctgag ggtggcggtt 1860
ctgagggtgg cggttctgag ggtggcggta ctaaacctcc tgagtacggt gatacaccta 1920
ttccgggcta tacttatatc aaccctctcg acggcactta tccgcctggt actgagcaaa 1980
accccgctaa tcctaatcct tctcttgagg agtctcagcc tcttaatact ttcatgtttc 2040
agaataatag gttccgaaat aggcaggggg cattaactgt ttatacgggc actgttactc 2100
aaggcactga ccccgttaaa acttattacc agtacactcc tgtatcatca aaagccatgt 2160
atgacgctta ctggaacggt aaattcagag actgcgcttt ccattctggc tttaatgagg 2220
atttatttgt ttgtgaatat caaggccaat cgtctgacct gcctcaacct cctgtcaatg 2280
ctggcggcgg ctctggtggt ggttctggtg gcggctctga gggtggtggc tctgagggtg 2340
gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct ggttccggtg 2400
attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg 2460
aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg 2520
ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg 2580
gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt 2640
taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt 2700
ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat 2760
tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt 2820
ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg gtattccgtt 2880
attattgcgt ttcctcggtt tccttctggt aactttgttc ggctatctgc ttacttttct 2940
taaaaagggc ttcggtaaga tagctattgc tatttcattg tttcttgctc ttattattgg 3000
gcttaactca attcttgtgg gttatctctc tgatattagc gctcaattac cctctgactt 3060
tgttcagggt gttcagttaa ttctcccgtc taatgcgctt ccctgttttt atgttattct 3120
ctctgtaaag gctgctattt tcatttttga cgttaaacaa aaaatcgttt cttatttgga 3180
ttgggataaa taatatggct gtttattttg taactggcaa attaggctct ggaaagacgc 3240
tcgttagcgt tggtaagatt caggataaaa ttgtagctgg gtgcaaaata gcaactaatc 3300
ttgatttaag gcttcaaaac ctcccgcaag tcgggaggtt cgctaaaacg cctcgcgttc 3360
ttagaatacc ggataagcct tctatatctg atttgcttgc tattgggcgc ggtaatgatt 3420
cctacgatga aaataaaaac ggcttgcttg ttctcgatga gtgcggtact tggtttaata 3480
cccgttcttg gaatgataag gaaagacagc cgattattga ttggtttcta catgctcgta 3540
aattaggatg ggatattatt tttcttgttc aggacttatc tattgttgat aaacaggcgc 3600
gttctgcatt agctgaacat gttgtttatt gtcgtcgtct ggacagaatt actttacctt 3660
ttgtcggtac tttatattct cttattactg gctcgaaaat gcctctgcct aaattacatg 3720
ttggcgttgt taaatatggc gattctcaat taagccctac tgttgagcgt tggctttata 3780
ctggtaagaa tttgtataac gcatatgata ctaaacaggc tttttctagt aattatgatt 3840
ccggtgttta ttcttattta acgccttatt tatcacacgg tcggtatttc aaaccattaa 3900
atttaggtca gaagatgaaa ttaactaaaa tatatttgaa aaagttttct cgcgttcttt 3960
gtcttgcgat tggatttgca tcagcattta catatagtta tataacccaa cctaagccgg 4020
aggttaaaaa ggtagtctct cagacctatg attttgataa attcactatt gactcttctc 4080
agcgtcttaa tctaagctat cgctatgttt tcaaggattc taagggaaaa ttaattaata 4140
gcgacgattt acagaagcaa ggttattcac tcacatatat tgatttatgt actgtttcca 4200
ttaaaaaagg taattcaaat gaaattgtta aatgtaatta attttgtttt cttgatgttt 4260
gtttcatcat cttcttttgc tcaggtaatt gaaatgaata attcgcctct gcgcgatttt 4320
gtaacttggt attcaaagca atcaggcgaa tccgttattg tttctcccga tgtaaaaggt 4380
actgttactg tatattcatc tgacgttaaa cctgaaaatc tacgcaattt ctttatttct 4440
gttttacgtg caaataattt tgatatggta ggttctaacc cttccattat tcagaagtat 4500
aatccaaaca atcaggatta tattgatgaa ttgccatcat ctgataatca ggaatatgat 4560
gataattccg ctccttctgg tggtttcttt gttccgcaaa atgataatgt tactcaaact 4620
tttaaaatta ataacgttcg ggcaaaggat ttaatacgag ttgtcgaatt gtttgtaaag 4680
tctaatactt ctaaatcctc aaatgtatta tctattgacg gctctaatct attagttgtt 4740
agtgctccta aagatatttt agataacctt cctcaattcc tttcaactgt tgatttgcca 4800
actgaccaga tattgattga gggtttgata tttgaggttc agcaaggtga tgctttagat 4860
ttttcatttg ctgctggctc tcagcgtggc actgttgcag gcggtgttaa tactgaccgc 4920
ctcacctctg ttttatcttc tgctggtggt tcgttcggta tttttaatgg cgatgtttta 4980
gggctatcag ttcgcgcatt aaagactaat agccattcaa aaatattgtc tgtgccacgt 5040
attcttacgc tttcaggtca gaagggttct atctctgttg gccagaatgt cccttttatt 5100
actggtcgtg tgactggtga atctgccaat gtaaataatc catttcagac gattgagcgt 5160
caaaatgtag gtatttccat gagcgttttt cctgttgcaa tggctggcgg taatattgtt 5220
ctggatatta ccagcaaggc cgatagtttg agttcttcta ctcaggcaag tgatgttatt 5280
actaatcaaa gaagtattgc tacaacggtt aatttgcgtg atggacagac tcttttactc 5340
ggtggcctca ctgattataa aaacacttct caggattctg gcgtaccgtt cctgtctaaa 5400
atccctttaa tcggcctcct gtttagctcc cgctctgatt ctaacgagga aagcacgtta 5460
tacgtgctcg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 5520
tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 5580
cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 5640
ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5700
tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5760
gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 5820
tatctcgggc tattcttttg atttataagg gattttgccg atttcggaac caccatcaaa 5880
caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc 5940
caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg 6000
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 6060
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 6120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 6180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct 6240
cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg 6300
ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 6360
atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 6420
agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc 6480
cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact 6540
ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca 6600
atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg 6660
atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt cctattggtt 6720
aaaaaatgag ctgatttaac aaaaatttaa tgcgaatttt aacaaaatat taacgtttac 6780
aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 6840
ggtacatatg attgacatgc tagttttacg attaccgttc atcgattctc ttgtttgctc 6900
cagactctca ggcaatgacc tgatagcctt tgtagatctc tcaaaaatag ctaccctctc 6960
cggcattaat ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc 7020
cggcctttct cacccttttg aatctttacc tacacattac tcaggcattg catttaaaat 7080
atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc ccgcaaaagt 7140
attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt 7200
gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgtt 7249
<210> 2
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgcggaactg gcaattcatc aatacgtcgc tattaattaa agagtcaa 48
<210> 3
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcgcgaaaaa tcataaggga accgttgggc ttgagatggt taaaacga 48
<210> 4
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccacagacgt tgaaaatctc caatcaccct cagcagcgaa ataataa 47
<210> 5
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aaaacaaacg tagattttca ggtttaaagc taaatcggtt gtac 44
<210> 6
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
caactgtttt acgccagctg gcgagccagt gccaagcttg ggtaccga 48
<210> 7
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atttaacgca tcaaaaagat taactttacc ctgactattg gcttaatt 48
<210> 8
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caacagagat gtgagcgagt aacacatcgt aaccgtgcat aaagcgaa 48
<210> 9
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agctttccag ggagcccccg atttcgtcaa agggcgaaaa acaattccac 50
<210> 10
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ttttagtttt atcaaaatca taggccttgc ttctgtaaat taatcctg 48
<210> 11
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aggtcattgg taatagtaaa atgtctatca taaccctcgt gaaaaatc 48
<210> 12
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
taattttaga ttatacttct gaatatatgt gagtgaataa tctgagag 48
<210> 13
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccggaaccct aaggcaccgc ttctggtggc tcatttt 37
<210> 14
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
acaaagaatt taacctccgg cttacagtac ataaatcaat aatggaag 48
<210> 15
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
taacaacgaa atcaaaaatc aggtgaggaa gcccgaaaga acatcaag 48
<210> 16
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tgctcattgc cagtaataag agaagcagag gcattttcga ctttaaac 48
<210> 17
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ggggtcagtg ccttgagtaa cagtggaggt tgaggcaggt gtcagact 48
<210> 18
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gagggtaagc cctttttaag aaaatcggtc gctgaggctt tcggttta 48
<210> 19
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
attagataat attttcattt gggggaaagg ccggagacag ctagctga 48
<210> 20
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ataacataat aataacggaa taccaaccga ttgagggagg g 41
<210> 21
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
acccacaaaa caatgaaata gcaacgcttt tgcgggatcg aaaaaagg 48
<210> 22
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tacgagcatt aaaccaagta ccgcaatctt accaacgcta tttatccc 48
<210> 23
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tgataatctg ccggagaggg tagcagattc aaaagggtga cgcgagct 48
<210> 24
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tcaccttgat attaccgcca gccaaactca aa 32
<210> 25
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
atcaaaatct ttcgaggtga atttcgcata accgatatat gtaagcag 48
<210> 26
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ccagagggtt tgcggatggc ttagacatgt tttaaatatg gtttgacc 48
<210> 27
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tagtgaataa tttcatcttc tgacaataaa caccggaatc tcctaatt 48
<210> 28
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
caacggctaa gaggcaaaag aatatatacc aa 32
<210> 29
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
agaggtgaac aactaataga ttaaattcga caactcgtac ctaccata 48
<210> 30
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ccagcagatc accagtcaca cgacgtggga acaaacggcg gatt 44
<210> 31
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
cgtaaaacat tagactttac aaacgagccg tcaatagata acgaacca 48
<210> 32
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tcaaaaatga ttaagactcc ttattttacc agcgccaaag atcaccgt 48
<210> 33
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gagcaagaga attgagttaa gcccaagagg acagatgaac ccctgacg 48
<210> 34
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gtctgtccat caatttacat tggcagatag ataaaac 37
<210> 35
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gctcgaattc gtaatcatgg tcatacgttg taaaacgacg aaggggga 48
<210> 36
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
agtaagcgtc atacatggca gaatggaaag cgcacaccaa tg 42
<210> 37
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
attgtttgaa agtttgagta acatagttga aaggaattga gccacgct 48
<210> 38
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
actggcatga aaatagcagc cttttaaatc aagattagtt tcatcgta 48
<210> 39
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
tcagatgaaa agaaaccacc agaatcaatc aatatctggt ctaaagca 48
<210> 40
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
ggaaaaacgc agcaaatgaa aaatcagttg gcaaatcaac tatcattt 48
<210> 41
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
caccgactca agtttgcctt tagccagacg attggccttg gttttaac 48
<210> 42
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
gcaaagaatt tacatcggga gaaagcgaat tattcatttc agcgaacc 48
<210> 43
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
acgctcaaca ccgcctgcaa cagtggaagg ttatctaaaa aacgttat 48
<210> 44
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
atcggaacaa taataatttt ttcacagccc tcatagttag caagccca 48
<210> 45
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ctatatcaaa gcggattaat ttcatttgaa ttatatttgc a 41
<210> 46
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
gagagtctgg agcacaatat ttttaaggat aaaaattttt caggcaag 48
<210> 47
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
aaccgcctgg agcctttaat tgtagcaggg agttaaaggc tagctatc 48
<210> 48
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
ttaccgaatt gagcgctaat atcagcatag gctggctgac aacaaagc 48
<210> 49
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
cacgtatagg attttagaca ggaacttgcc tgagtagaag ttgcaaca 48
<210> 50
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
ggaatcatct gtttatcaac aataatgcgt tatacaaatt atcgcaag 48
<210> 51
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
aacgggtatg tagaaaccaa tcaataaggc gttaaataag ctaaattt 48
<210> 52
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
aaatgaatac agtttcagcg gagtctaaag actttttcat aatgccac 48
<210> 53
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
actaccttcg cgagaaaact ttttgcctgt ttagtatcat gataagtc 48
<210> 54
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
tacgaaggtt tgtatcatcg cctgtgttac ttagccggaa tcattgtg 48
<210> 55
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
ggttagaatt aaatcctttg cccgtatctt taggagcact aggcggtc 48
<210> 56
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
gtagcgcgtc attaaaggtg aattacaaaa gggcgacatt ccaaaaga 48
<210> 57
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
attgttatcc gctcaccgtc tatcagccag ggttt 35
<210> 58
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
tattcattag caaactccaa caggacaaaa tcgcgcagag caataacg 48
<210> 59
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
gatgcaaatc cacttaccag tataaagcct aatgcag 37
<210> 60
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
gacagaattg agccatttgg gaattagaaa attcatatgg tacgcagt 48
<210> 61
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
ttaggaatca gtgaataagg cttgggtgta cagaccaggc gagagata 48
<210> 62
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
aaaccatcac cagtagcacc attaaccacg gaataagttt tacataaa 48
<210> 63
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
tcccagtcac gagctgtttc ctgtgtgaa 29
<210> 64
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
tgtgctgcgc gccattcgcc attcgttaaa attcgcatta accccggt 48
<210> 65
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
atgcctgata gtagtagcat taatgattgc tttgaacagt tgat 44
<210> 66
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
gagagccagc tcatggaaat acctattagt aataacatca cggtacgc 48
<210> 67
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
atgttagcta agaaacgatt ttttctacaa ttttatcctg actcatcg 48
<210> 68
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
agaccggaga atccccctca aatgaccaca ttcaactaat gttgagat 48
<210> 69
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
tgccatctca gagccgccac cagttcggaa cctattattc 40
<210> 70
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
gagaccgtaa tgggataggt cacgaagtgt agcggtcacg caaaatc 47
<210> 71
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
aagggaccca ttaaaaatac cgaatacatt tgagaaaaac att 43
<210> 72
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
acaacatacg agccggaagc atagaacgtg gactccaaag agcttg 46
<210> 73
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
aaggccgaca atgagaaacc gaggaaacgc aaaaacagg 39
<210> 74
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
aataggtgta tcaccgtact caggcctcag aaccgccacc acgttagt 48
<210> 75
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
taggtaatat ttttgagaga tctcatgtca atcatatgta atttttg 47
<210> 76
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
tacgttaatt aatttcaact ttaacgaggc gcagacggtc caaagtac 48
<210> 77
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
ttaaatcacc ggaaaccagg caaaaaggcg attaagttgg gtaa 44
<210> 78
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
acggggaacc tcaggaagat cgcaaaataa ttcgcgtctg acgtggca 48
<210> 79
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
aggccaccca cccgccgcgc ttaaaaaatc ctgtttgatg gctgcatt 48
<210> 80
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
ttaaccagta atcgtaaaac tagacaaagg ctatcaggtt taaatgca 48
<210> 81
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
cttctgactt aatgcgcgaa ctgatacttt tgcgggagaa ataaagcc 48
<210> 82
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
gagaatcgtc cagacgacga caagaaggct tatccggtat tc 42
<210> 83
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
ccttataaac ccgctttcca gtcgggaaac ctgtcgtg 38
<210> 84
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 84
gcaaaatcga tagcagcacc gtaaaaatcc tcattaaagc cttttgat 48
<210> 85
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 85
agttcagatt aattcgagct tcaaaattac ctgagcaaaa tacagtaa 48
<210> 86
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 86
aggagcggag atagggttga gtgtgctaac tcacattaat tgcg 44
<210> 87
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 87
tcagcttgca ccggaaccag agcccctcag aaccgccacc ttaagagg 48
<210> 88
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 88
cgatttagaa gtagaaataa agaaattgat taattac 37
<210> 89
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 89
agaacaagga aaaataatat cccaataatt actagaaaaa caaatata 48
<210> 90
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 90
taaattaaag aaaagcccca aaaataaacg ttaatatttt aggctgcg 48
<210> 91
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 91
agggatagcg taacgatcta aagtaactaa aggaattgcg gagggtag 48
<210> 92
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 92
gatacaggag tgtactggta ataaatattc acaaacaaat tcagtagc 48
<210> 93
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 93
ccagtggttc cgaaatcggc tgcgcgtaac caccagagta aaa 43
<210> 94
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 94
ctccaaaata caaactacaa cgcctgagtt tcgtcaccag aggattag 48
<210> 95
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 95
gattcgccca tccaataaat cataagaacc ctcatatatt cattgcct 48
<210> 96
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 96
gaaaaggtcg aacgagtaga tttacaacta aagtacggtg ttgataag 48
<210> 97
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 97
aactttgatt tgacccccag cgatcactaa aacactcatc aagacagc 48
<210> 98
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 98
cggggttttg ctcagtacca ggcgccatgt accgtaacac tgtagcatt 49
<210> 99
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 99
aagcctgggg tgcctaatga gtgatgttcc agtttggaac gagaaagg 48
<210> 100
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 100
aacgcgctac cgcgcccaat agcggaggtt ttgaagccta cagagaga 48
<210> 101
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 101
atgaccctgt aatagcccta aaacatcgat tctggc 36
<210> 102
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 102
cgctggcttg gtgtagatgg gcgacccgtc ggattctccc agtaataa 48
<210> 103
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 103
ttacgaggaa caacattatt acagcagaac gagtagtaaa aactgacc 48
<210> 104
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 104
cagaatccgc gtactatggt tgctaagcgg tccacgctgg gaggcggt 48
<210> 105
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 105
agtatcggag ccggcgaacg tggcaagagt ccactattaa aaagtgta 48
<210> 106
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 106
aaaggaactt tgtcgtcttt ccagctcaga gccaccaccc tagcccgg 48
<210> 107
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 107
aattacctgt caggacgttg ggaattacca gacgacgata aagttttg 48
<210> 108
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 108
taccggatat tcaaaggtaa agtaattctg ccatatt 37
<210> 109
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 109
aacggagaca ccaacctaaa acgaacagag gctttgagga gagaatag 48
<210> 110
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 110
tcccaattct gggcatcaat tctactaagt aatgtg 36
<210> 111
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 111
agtattaatc gtctgaaatg gattcgcaaa ttaaccgttg aatcagtg 48
<210> 112
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 112
atgttcagca acgctcaaca gtagatagtc agaaggtaaa tgct 44
<210> 113
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 113
tgctcctttc tggaagtttc attccatata ataccaa 37
<210> 114
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 114
ggcgttttag cgattagacg ggagaattac aaagtta 37
<210> 115
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 115
tcagagcata acgtcagatg aatagaagat gatgaaacaa cttcaaat 48
<210> 116
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 116
cagtaccttt agcaaaatta agcagccttt atttcaacgc gaatggct 48
<210> 117
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 117
aatgaatcgg ccaacgcgcg gggatttgcc ccagcaggcg tgcgccgc 48
<210> 118
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 118
actaacggca tagtaagagc aacattagac tggatagcgt cctttaat 48
<210> 119
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 119
aatggtttga ttaagacgct gagattttcc cttagaatcc cctgatta 48
<210> 120
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 120
tgaaacatga aagtactcag agccaccacc cttttcata 39
<210> 121
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 121
gaagcgcaac ctcccgactt gcgaagcaaa tcagatatat aaacaac 47
<210> 122
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 122
aagggaagct gccagtttga ggggctttca tcaacattaa atagaacc 48
<210> 123
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 123
aatccaaaaa acgtagaaaa tacaattttg tcacaatcaa tagagcca 48
<210> 124
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 124
ttgcgtattg ggcgccaggg tggtctgaga gagttgcagc ttgacgag 48
<210> 125
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 125
cagaaacaag agaatcgatg aacgatagga acgccatcaa ctccagcc 48
<210> 126
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 126
ctatcggcag gaggccgatt aaagacgtgc tt 32
<210> 127
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 127
ggtggcaaat aaacagccat attaacgagc gtctttccag attccaag 48
<210> 128
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 128
tcaaaatcct tttttaatgg aaaggttggg ttatataa 38
<210> 129
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 129
accgacaatt acccaaatca acgtcttcat caagagtaat caaagtca 48
<210> 130
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 130
aaacagttaa tgccccctgc ctataaccac caccagagcc cggtcata 48
<210> 131
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 131
ttgcgctcac tgtcaaaaga atagcccggc gctaggg 37
<210> 132
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 132
ctgaacaaca agccgttttt atttgctatt ttgcacccag gtttaacg 48
<210> 133
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 133
tcctcgttcc ttcaccgcct ggccttttct tttcaccagt ga 42
<210> 134
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 134
ataggaacga taagtgccgt cgagagggtt gatataagta tcattttc 48
<210> 135
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 135
tatttttcat cggcattttg ccgccagcat tgacagcccg tat 43
<210> 136
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 136
tacagggctg agaagtgttt ttattagcaa tacttctttg acattttg 48
<210> 137
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 137
atcgcgttaa acgagaatga ccatccaaca tgtaatttag tataaagt 48
<210> 138
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 138
agaaacacgt agaaagattc atcagcagat acataacgcc gtcataaa 48
<210> 139
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 139
gtttcaggat tagagagtac caatactgcg gaatcaaaag gaa 43
<210> 140
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 140
ctgagactcc tcaagagaag gattccctca gagccgccac accaccgg 48
<210> 141
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 141
ccagaagcaa caaccatcgc ccacttaaac agcttgataa ttagcgtt 48
<210> 142
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 142
atagccgaaa ctgaacaccc tgaacttgac aagaaagaac gcga 44
<210> 143
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 143
gcccccttcc gatagttgcg taaatattga cggaaat 37
<210> 144
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 144
attagtctct gaaagcgtaa gaatgccttc ctgtagccag acgacgac 48
<210> 145
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 145
tcaccttgat attaccgcca gccaaactca aaaaaaaatt aattaa 46
<210> 146
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 146
gtttcaggat tagagagtac caatactgcg gaatcaaaag gaagctagga gtcctacact 60
acc 63
<210> 147
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 147
ttaaccagta atcgtaaaac tagacaaagg ctatcaggtt taaatgcagc taggagtcct 60
acactacc 68
<210> 148
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 148
aactttgatt tgacccccag cgatcactaa aacactcatc aagacagcgc taggagtcct 60
acactacc 68
<210> 149
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 149
taacaacgaa atcaaaaatc aggtgaggaa gcccgaaaga acatcaagcg taggagtcct 60
acactacc 68
<210> 150
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 150
gtttcaggat tagagagtac caatactgcg gaatcaaaag gaagctagga gtcctacact 60
acc 63
<210> 151
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 151
atgaccctgt aatagcccta aaacatcgat tctggcgcta ggagtcctac actacc 56
<210> 152
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 152
ttaaccagta atcgtaaaac tagacaaagg ctatcaggtt taaatgcagc taggagtcct 60
acactacc 68
<210> 153
<211> 64
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 153
atagccgaaa ctgaacaccc tgaacttgac aagaaagaac gcgagctagg agtcctacac 60
tacc 64
<210> 154
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 154
aactttgatt tgacccccag cgatcactaa aacactcatc aagacagcgc taggagtcct 60
acactacc 68
<210> 155
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 155
taacaacgaa atcaaaaatc aggtgaggaa gcccgaaaga acatcaagcg taggagtcct 60
acactacc 68
<210> 156
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 156
ttttagtttt atcaaaatca taggccttgc ttctgtaaat taatcctggc taggagtcct 60
acactacc 68
<210> 157
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 157
gtttcaggat tagagagtac caatactgcg gaatcaaaag gaagctagga gtcctacact 60
acc 63
<210> 158
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 158
atgaccctgt aatagcccta aaacatcgat tctggcgcta ggagtcctac actacc 56
<210> 159
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 159
gagagccagc tcatggaaat acctattagt aataacatca cggtacgcgc taggagtcct 60
acactacc 68
<210> 160
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 160
ttaaccagta atcgtaaaac tagacaaagg ctatcaggtt taaatgcagc taggagtcct 60
acactacc 68
<210> 161
<211> 64
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 161
atagccgaaa ctgaacaccc tgaacttgac aagaaagaac gcgagctagg agtcctacac 60
tacc 64
<210> 162
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 162
atgttagcta agaaacgatt ttttctacaa ttttatcctg actcatcggc taggagtcct 60
acactacc 68
<210> 163
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 163
aactttgatt tgacccccag cgatcactaa aacactcatc aagacagcgc taggagtcct 60
acactacc 68
<210> 164
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 164
aggccaccca cccgccgcgc ttaaaaaatc ctgtttgatg gctgcattcg taggagtcct 60
acactacc 68
<210> 165
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 165
ttacgaggaa caacattatt acagcagaac gagtagtaaa aactgaccgc taggagtcct 60
acactacc 68
<210> 166
<211> 64
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 166
aaaacaaacg tagattttca ggtttaaagc taaatcggtt gtacgctagg agtcctacac 60
tacc 64
<210> 167
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 167
ggttagaatt aaatcctttg cccgtatctt taggagcact aggcggtcgc taggagtcct 60
acactacc 68
<210> 168
<211> 64
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 168
atgcctgata gtagtagcat taatgattgc tttgaacagt tgatgctagg agtcctacac 60
tacc 64
<210> 169
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 169
caacagagat gtgagcgagt aacacatcgt aaccgtgcat aaagcgaagc taggagtcct 60
acactacc 68
<210> 170
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 170
atcaaaatct ttcgaggtga atttcgcata accgatatat gtaagcaggc taggagtcct 60
acactacc 68
<210> 171
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 171
gtagcgcgtc attaaaggtg aattacaaaa gggcgacatt ccaaaagagc taggagtcct 60
acactacc 68
<210> 172
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 172
ccggaaccct aaggcaccgc ttctggtggc tcattttgct aggagtccta cactacc 57
<210> 173
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 173
atcggaacaa taataatttt ttcacagccc tcatagttag caagcccagc taggagtcct 60
acactacc 68
<210> 174
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 174
taccggatat tcaaaggtaa agtaattctg ccatattgct aggagtccta cactacc 57
<210> 175
<211> 68
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 175
ggaatcatct gtttatcaac aataatgcgt tatacaaatt atcgcaaggc taggagtcct 60
acactacc 68
<210> 176
<211> 419
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 176
Met Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro
1 5 10 15
Gly Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr Val Asp Asp Thr
20 25 30
Glu Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Tyr Glu Pro
35 40 45
Arg Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg Glu
50 55 60
Thr Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg Val Asp Leu Arg
65 70 75 80
Thr Leu Leu Gly Tyr Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Ile
85 90 95
Gln Val Ile Ser Gly Cys Glu Val Gly Ser Asp Gly Arg Leu Leu Arg
100 105 110
Gly Tyr Gln Gln Tyr Ala Tyr Asp Gly Cys Asp Tyr Ile Ala Leu Asn
115 120 125
Glu Asp Leu Lys Thr Trp Thr Ala Ala Asp Met Ala Ala Leu Ile Thr
130 135 140
Lys His Lys Trp Glu Gln Ala Gly Glu Ala Glu Arg Leu Arg Ala Tyr
145 150 155 160
Leu Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Lys Asn Gly
165 170 175
Asn Ala Thr Leu Leu Arg Thr Asp Ser Pro Lys Ala His Val Thr His
180 185 190
His Ser Arg Pro Glu Asp Lys Val Thr Leu Arg Cys Trp Ala Leu Gly
195 200 205
Phe Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Glu
210 215 220
Leu Ile Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly
225 230 235 240
Thr Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu Gly Lys Glu Gln
245 250 255
Tyr Tyr Thr Cys His Val Tyr His Gln Gly Leu Pro Glu Pro Leu Thr
260 265 270
Leu Arg Trp Glu Pro Pro Pro Ser Thr Val Ser Asn Met Gly Gly Gly
275 280 285
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Val Thr Thr
290 295 300
Leu Ser Gly Leu Ser Gly Glu Gln Gly Pro Ser Gly Asp Met Thr Thr
305 310 315 320
Glu Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp
325 330 335
Gly Arg Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly
340 345 350
Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly His Val Lys Asp Phe Tyr
355 360 365
Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly
370 375 380
Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln
385 390 395 400
Val Thr Val Asn Gly Glu Ala Thr Lys Gly Asp Ala His Thr Gly Ser
405 410 415
Ser Gly Ser
<210> 177
<211> 100
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 177
Met Ile Gln Lys Thr Pro Gln Ile Gln Val Tyr Ser Arg His Pro Pro
1 5 10 15
Glu Asn Gly Lys Pro Asn Ile Leu Asn Cys Tyr Val Thr Gln Phe His
20 25 30
Pro Pro His Ile Glu Ile Gln Met Leu Lys Asn Gly Lys Lys Ile Pro
35 40 45
Lys Val Glu Met Ser Asp Met Ser Phe Ser Lys Asp Trp Ser Phe Tyr
50 55 60
Ile Leu Ala His Thr Glu Phe Thr Pro Thr Glu Thr Asp Thr Tyr Ala
65 70 75 80
Cys Arg Val Lys His Ala Ser Met Ala Glu Pro Lys Thr Val Tyr Trp
85 90 95
Asp Arg Asp Met
100
Claims (8)
1.基于DNA纳米技术的pMHC多聚体制备方法，其特征在于，包括以下步骤：
A、带有特定捕获链的矩形DNA纳米结构的制备：将1条模板链和若干条短链通过碱基互补配对自组装形成带有特定捕获链的矩形DNA纳米结构；
B、利用点击化学反应将叠氮化物修饰的SpyTag与含有DBCO修饰的互补链偶联链接，得到SpyTag-互补链；
C、将SpyTag-互补链、PE修饰的互补链与矩形DNA纳米结构上的捕获链通过退火发生碱基互补配对链接，得到带有SpyTag的DNA纳米结构；
其中，步骤B和C中所述的互补链与步骤A中所述的捕获链互补配对；
D、构建pMHC与SpyCatcher融合蛋白，在pMHC的C端融合SpyCatcher，与带有SpyTag的DNA纳米结构混合，DNA纳米结构上的SpyTag与SpyCatcher通过异肽键从而将pMHC单体链接在DNA纳米结构表面；
所述模板链的核苷酸序列如SEQ ID NO:1所示，所述短链共143条，核苷酸序列分别如SEQ ID NO:2-144所示。
2.根据权利要求1所述的方法，其特征在于，SpyTag的氨基酸序列为SLLFLLFSL。
3.根据权利要求1所述的方法，其特征在于，所述捕获链包括链接PE的捕获链以及链接pMHC的捕获链；
链接PE的捕获链的核苷酸序列如SEQ ID NO:145所示；
链接3个pMHC的捕获链的核苷酸序列如SEQ ID NO:146-148所示；
链接6个pMHC的捕获链的核苷酸序列如SEQ ID NO:149-154所示；
链接9个pMHC的捕获链的核苷酸序列如SEQ ID NO:155-163所示；
链接12个pMHC的捕获链的核苷酸序列如SEQ ID NO:164-175所示；
所述互补链包括与链接PE的捕获链互补的互补链以及与链接pMHC的捕获链互补的互补链，与链接PE的捕获链互补的互补链的核苷酸序列为PE-TTTTTTTAATTAATTTTTT，与链接pMHC的捕获链互补的互补链的核苷酸序列为N3-TTTTTGGTAGTGTAGGACTCCTAGC。
4.根据权利要求1所述的方法，其特征在于，pMHC与SpyCatcher融合蛋白的氨基酸序列如SEQ ID NO:176-177所示。
5.根据权利要求1所述的方法，其特征在于，蛋白质链接方式为叠氮化物与DBCO以及SpyTag与SpyCatcher之间的反应，或其他共价和非共价结合反应。
6.根据权利要求1所述的方法，其特征在于，蛋白质链接方式为非共价结合和共价结合。
7.按照权利要求1-6任一项所述方法制备的pMHC多聚体。
8.权利要求7所述pMHC多聚体在制备抗原特异性T细胞检测试剂或试剂盒中的应用。
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| CN110507817A (zh) * | 2019-09-09 | 2019-11-29 | 国家纳米科学中心 | 一种dna纳米疫苗及其制备方法和应用 |
| CN111234002A (zh) * | 2020-01-15 | 2020-06-05 | 华东师范大学 | 肽-主要组织相容性复合物多聚体DNA-pMHC及其制备方法和应用 |
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| WO2021262872A2 (en) * | 2020-06-24 | 2021-12-30 | Repertoire Immune Medicines, Inc. | Mhc multimer expression constructs and uses thereof |
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