CN111808170A - 多肽、hla-dr蛋白及其制备方法和应用 - Google Patents
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Abstract
本发明提供了一种多肽、HLA‑DR蛋白及其制备方法和应用。所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的8～48个氨基酸残基的多肽；该HLA‑DR蛋白结构组成包括α链、β链和多肽；α链的C端与多肽的N端相连接；多肽的C端与所述β链的N端相连接。该蛋白质是将构成HLA‑DR蛋白的α链和β链通过特定的由8～48个包含甘氨酸和丝氨酸以肽键相互连接构成的多肽连接。设计的HLA‑DR蛋白能够在哺乳动物细胞中串联表达，可以模拟sHLA‑DR表达，其与天然sHLA‑DR结构类似，与膜蛋白mHLA‑DR相比构象更稳定，能够使用真核表达系统进行可溶性高效表达，有望在医学生物学领域和生物医药开发中有很好的应用。
Description
技术领域
本发明属于生物免疫技术领域，涉及一种多肽、HLA-DR蛋白及其制备方法和应用。
背景技术
主要组织相容性复合体(major histocompatibility complex，MHC)是一个与机体免疫反应相关的基因群，该基因群有100多个基因位点，其编码产物包括MHCI类分子和MHCII类分子，是一些能够提呈抗原的分子，在调节免疫应答和免疫细胞发育中发挥作用。MHCII类分子是呈现在免疫系统抗原呈递细胞(APC)表面的由α链和β链非共价链相连组成的一组高度多态性的跨膜糖蛋白，可提呈经过加工的外来抗原给辅助性T细胞的抗原受体，导致辅助性T细胞激活和分化，从而在诱发免疫应答中起重要的作用。
HLAII类(HLA-II)分子或蛋白质呈递来自细胞外蛋白质的肽抗原，包括细胞外病原体的蛋白质，而HLAI类蛋白质呈递细胞内蛋白质或病原体的肽。细胞表面上加载的HLAII类蛋白与CD4+辅助T细胞相互作用。该相互作用通过激活B细胞导致吞噬细胞的募集，局部炎症和/或体液反应。人MHC抗原通常称为人类白细胞抗原(HLA)，HLAII有DR，DQ，DP等许多种，其中DR等位基因最多，含量最丰富。
脓毒血症已有明确的诊断标准但患者预后各异，可能与免疫状态尤其是免疫抑制密切相关。CD14细胞的人类白细胞抗原-DR(HLA-DR)表达率作为一个新的细菌感染指标，已经比较普遍地应用于脓毒血症的判断，很多研究者将HLA-DR表达率小于或等于30％作为重症脓毒血症的一个指标，HLA-DR表达率下降提示机体处于免疫抑制状态，导致感染加重。但是用流式检测膜蛋白表达率的方法操作复杂，成本较高，急需一种快速简便的方法检测。
除膜结合型HLA-DR外，外周血中还存在可溶性形式，最近的研究表明，可溶性HLA-DR水平的增加与移植相关并发症和自身免疫性疾病有关。后者的作用机制可能是认为细菌和部分病毒衍生的超抗原是诱导多克隆T细胞活化的致病剂，脓毒症的致病机制也是如此。而可溶性HLA-DR分子可以作为超抗原的配体发挥作用，因此，可能在超级抗原的“解毒”中发挥作用，可以比膜结合型HLA-DR更早的预警脓毒症。
编码HLA-DR的α链的基因缺乏多态性，而编码β链的基因则存在着许多亚型，目前己经发现的亚型有HLA-DRBP0101，0102，0401，0404，0405，0410，1001和1402等。由于存在类型繁多的HLA-DR分子，而每一种HLA-DR分子都能与多种肽片段结合，从而保证了T细胞能对所有的抗原发生免疫反应，这就为检测可溶性HLA-DR带来困难。所选抗体能否识别全部或者绝大多数HLA-DR分子，用来定量的校准品能否精确定量HLA-DR分子都存在困难。
相关文献报道在原核和真核蛋白表达系统中报道了克隆和表达MHC II类分子的各种尝试。但是由于重组杂二聚体再折叠困难，得率极低。
现有技术中，有采用膜蛋白提取法，此类方法具体为大量培养、收集经鉴定的HLA-DR阳性细胞(比如人B淋巴瘤细胞系Raji以及LG2细胞)，用裂解液裂解细胞，并用免疫亲和层析法进行HLA-DR纯化。此类方法存在如下不足之处：(1)细胞膜表面HLA-DR分子的含量很少，甚至无法通过Western杂交进行检测，因此大量培养细胞则成为纯化过程的限制点。(2)在纯化过程中，操作复杂且免疫亲和柱存在非特异吸附导致微量的杂蛋白混入，纯度不够；(3)从细胞系中纯化获得的HLA-DR分子，大部分抗原结合位点都有抗原肽结合，这与可溶性HLA-DR存在差异。
现有技术中，有采用亮氨酸拉链组装法，此类方法具体采用RT-PCR方法从721.221细胞中扩增DRα和DRβ的信号肽及胞外序列，运用重叠PCR将DRα和DRβ片段分别与Fos和Jun的亮氨酸拉链序列相连，形成DRα-Fos和DRβ-Jun结构，DRα和DRβ可凭借Fos和Jun的亮氨酸拉链结合形成DR1分子，再通过EcoRI酶切位点将DRα-Fos和人IgG1的Fc段相连，形成DRα-Fos-Fc重组序列，2个同源的DR1分子可通过IgG1的Fc段的二硫键结合形成二聚体。分别将DRα-Fos-Fc及DRβ-Jun插入杆状病毒表达载体pFastBacTMDual的2个多克隆位点处，构建出重组载体pFast BacTMDual+[DR1/Fc]，对构建的载体进行PCR及限制性内切酶酶切鉴定和测序。采用脂质体转染方法将表达载体转入昆虫细胞系Sf9中，通过双抗夹心ELISA及Westernblot检测HLA-DR1的表达。此类方法存在如下不足之处：(1)方法繁琐；(2)DRα和DRβ为体外组装，得率低，成本高；(3)所得产物为二聚体，与天然可溶性HLA-DR存在差异。
现有技术中，有采用呈递多肽组装法，此类方法具体为克隆单个α链胞外段和β链胞外段，并分别在大肠杆菌系统中表达。分别纯化包涵体蛋白。在来自人髓鞘碱性蛋白(b-MBP(83-102)Y)辅助下将原核表达的α链和β链体外组装，并用异二聚体特异性单克隆抗体(L243)纯化组装成功的产物。此类方法存在如下不足之处：(1)原核表达，缺乏复杂的N-糖基化O-糖基化等多种翻译后修饰，与天然蛋白构象差异很大；(2)体外重组，方法繁琐且不可控，产率低，方法可操作性差。
由于现有HLA-DR类分子的提取和制备方法存在诸多缺点如：操作复杂、原料来源较少、得率低、成本高以及潜在携带外源病毒及其他病原微生物的危险，很难实现产业化，迄今为止，未发现国内外关于可溶HLA-DR蛋白定量检测相关产品的报道。
发明内容
基于现有技术存在的缺陷，本发明的第一目的在于提供一种多肽；本发明的第二目的在于提供该多肽作为HLA-DR蛋白的α链和β链的连接肽(命名Linker)在构建HLA-DR蛋白中的应用；本发明的第三目的在于提供一种HLA-DR蛋白，该蛋白具备可溶性；本发明的第四目的在于提供一种编码该HLA-DR蛋白的全长多核苷酸；本发明的第五目的在于提供包含编码该HLA-DR蛋白全长多核苷酸的重组载体或重组细胞；本发明的第六目的在于提供该HLA-DR蛋白的制备方法；本发明的第七目的在于提供该HLA-DR蛋白在制备用于脓毒血症诊断试剂中的用途。本发明的第八目的在于提供包含该HLA-DR蛋白的定量检测sHLA-DR的试剂盒。
本发明的目的通过以下技术手段得以实现：
一方面，本发明提供一种多肽，所述多肽是由甘氨酸(G)和丝氨酸(S)以肽键相互连接构成的8～48个氨基酸残基的多肽(命名为Linker)。
上述的多肽中，优选地，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的16～32个氨基酸残基的多肽。
上述的多肽中，优选地，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的20个氨基酸残基的多肽。
上述的多肽中，优选地，所述多肽的氨基酸序列选自如下序列中的一种：
GGGSGGGSGGGSGGGSGGGS(SEQ ID NO：1)，
GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：2)，
GGSGGSGGSGGSGGSGGSGG(SEQ ID NO：3)，
GSGSGSGSGSGSGSGSGSGS(SEQ ID NO：4)，
GGSGGGSGGGSGGGSGGGSG(SEQ ID NO：5)，
GSGGGSGGGSGGGSGGGSGG(SEQ ID NO：6)，
SGGGSGGGSGGGSGGGSGGG(SEQ ID NO：7)。
另一方面，本发明提供上述多肽作为HLA-DR蛋白的α链和β链的连接肽在构建HLA-DR蛋白中的应用。
再一方面，本发明还提供一种HLA-DR蛋白，该HLA-DR蛋白结构组成包括α链、β链和上述多肽；所述α链的C端与所述多肽的N端相连接；所述多肽的C端与所述β链的N端相连接。
由于现有HLA-DR类分子的提取方法存在着操作复杂、原料来源较少、得率低、成本高以及潜在携带外源病毒及其他病原微生物的危险，很难实现产业化；基于此，本发明创造性的设计了一种可溶HLA-DR蛋白质，该蛋白质是将构成HLA-DR蛋白的α链和β链通过特定的由8～48个包含甘氨酸和丝氨酸的氨基酸组成的多肽连接，本发明设计的HLA-DR蛋白质能够在哺乳动物细胞中串联表达，可以模拟可溶性sHLA-DR表达。本发明的HLA-DR蛋白质与天然sHLA-DR结构类似，能够被L234、CR3/43等抗体识别，与膜蛋白mHLA-DR相比构象更稳定，本发明的HLA-DR蛋白质可以使用真核表达系统进行可溶性高效表达，有望在医学生物学领域和生物医药开发中有很好的应用。
上述的HLA-DR蛋白中，优选地，所述α链包括α1结构域多肽；所述α1结构域多肽的C端与所述多肽的N端相连接；
所述α1结构域多肽的氨基酸序列包括SEQ ID NO：8所示氨基酸序列或与SEQ IDNO：8所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与α1结构域多肽具有相同功能的氨基酸序列。
α1结构域多肽氨基酸序列SEQ ID NO：8如下：
IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMTKRSNYTPITN
与SEQ ID NO：8所示氨基酸序列中经过取代一个或几个氨基酸且与α1结构域多肽具有相同功能的氨基酸序列可以包括如下氨基酸序列：
SEQ ID NO：41：
IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIAAKRSNYTPITN
上述的HLA-DR蛋白中，优选地，所述α链还包括α2结构域多肽，所述α1结构域多肽的C端与所述α2结构域多肽的N端相连接，所述α2结构域多肽的C端与所述多肽的N端相连接；
所述α2结构域多肽的氨基酸序列包括SEQ ID NO：9所示氨基酸序列或与SEQ IDNO：9所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与α2结构域多肽具有相同功能的氨基酸序列，或为SEQ ID NO：9所示氨基酸序列的部分片段且具备α2结构域多肽部分功能的氨基酸序列(例如下述α2结构域多肽氨基酸序列中划线部分)。
α2结构域多肽氨基酸序列SEQ ID NO：9如下：
VPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPF LPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPETTE
与SEQ ID NO：9所示氨基酸序列中经过取代一个或几个氨基酸且与α2结构域多肽具有相同功能的氨基酸序列可以包括如下氨基酸序列：
SEQ ID NO：42：
VPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPAPLPETTE
SEQ ID NO：43：
VPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTAGVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPAPLPETTE
SEQ ID NO：44：
VPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPFLPSTEDVYACRVEHWGLDEPLLKHWEFDAPSPLPETTE
上述的HLA-DR蛋白中，优选地，所述β链包括β1结构域多肽和β2结构域多肽；所述多肽的C端与所述β1结构域多肽的N端相连接，所述β1结构域多肽的C端与所述β2结构域多肽的N端相连接；
所述β1结构域多肽的氨基酸序列包括SEQ ID NO：10所示氨基酸序列或与SEQ IDNO：10所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与β1结构域多肽具有相同功能的氨基酸序列。
β1结构域多肽氨基酸序列SEQ ID NO：10如下：
FLEQSKSECHFFNGTERVRFLDRYFYNQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVHPK
与SEQ ID NO：10所示氨基酸序列中经过取代一个或几个氨基酸且与β1结构域多肽具有相同功能的氨基酸序列可以包括如下氨基酸序列：
SEQ ID NO：45：
FLEQSKSECHFFNGTERVRFLDRYFYNQEEYVRFDSDVGEYRAVTELGRPDAEYWNAQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVHPK所述β2结构域多肽的氨基酸序列包括SEQ ID NO：11所示氨基酸序列或与SEQ ID NO：11所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与β2结构域多肽具有相同功能的氨基酸序列，或为SEQ ID NO：11所示氨基酸序列的部分片段且具备β2结构域多肽部分功能的氨基酸序列(例如下述β2结构域氨基酸序列中划线部分)。
β2结构域多肽氨基酸序列SEQ ID NO：11如下：
VTVYPAKTQPLQHHNLLVCSVSGFYPGSIEVRWFRNGQEEKAGVVSTGLIQNGDWTFQTLVMLETVPRS GEVYTCQVEHPSVTSPLTVEW
与SEQ ID NO：11所示氨基酸序列中经过取代一个或几个氨基酸且与β2结构域多肽具有相同功能的氨基酸序列可以包括如下氨基酸序列：
SEQ ID NO：46：
VTVYPAKTQPLQHHNLLVCAVSGFYPGSIEVRWFRNGQEEKAGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEW
SEQ ID NO：47：
VTVYPAKTQPLQHHNLLVCSVAGFYPGSIEVRWFRNGQEEKAGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEW
SEQ ID NO：48：
VTVYPAKTQPLQHHNLLVCSVSGFYPGSIEVRWFRNGQEEKAGVVSTGLIQNGDAAFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEW
上述的HLA-DR蛋白中，优选地，所述β链还包括连接肽；所述β2结构域多肽的C端与所述连接肽的N端相连接；
所述连接肽的氨基酸序列包括SEQ ID NO：12所示氨基酸序列或与SEQ ID NO：12所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与连接肽具有相同功能的氨基酸序列。
连接肽氨基酸序列SEQ ID NO：12如下：
RARSESAQSK
上述的HLA-DR蛋白中，优选地，该HLA-DR蛋白的氨基酸序列包括SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：29、SEQ IDNO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：35、SEQ ID NO：36、SEQ IDNO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40所示序列中的一种。
上述的HLA-DR蛋白中，优选地，该HLA-DR蛋白的氨基酸序列如SEQ ID NO：13所示：
IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPETTEGGSGGGSGGGSGGGSGGGSGFLEQSKSECHFFNGTERVRFLDRYFYNQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVHPKVTVYPAKTQPLQHHNLLVCSVSGFYPGSIEVRWFRNGQEEKAGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK
再一方面，本发明还提供一种编码上述HLA-DR蛋白的全长多核苷酸。
例如，本发明编码氨基酸序列为SEQ ID NO：13的HLA-DR蛋白的所述全长多核苷酸的核苷酸序列如SEQ ID NO：51所示。
再一方面，本发明还提供包含上述全长多核苷酸的重组质粒或重组细胞。
再一方面，本发明还提供一种HLA-DR蛋白制备方法，该方法包括：将上述全长多核苷酸克隆到质粒载体上，将质粒载体转染至表达细胞中培养，表达目标蛋白，然后进行蛋白纯化得到HLA-DR蛋白。
上述的制备方法中，优选地，所述表达细胞包括CHO细胞。
现有技术中，异源二聚体体外分别表达后自发或人为组装方法繁琐且产率极低，本发明采用α链和β链串联表达的方式，产量高出其他方法几十倍且纯化工艺简单易重复。
再一方面，本发明还提供上述HLA-DR蛋白在制备用于脓毒血症诊断试剂中的用途。
再一方面，本发明还提供一种可溶性人白细胞抗原-DR(sHLA-DR)的定量检测试剂盒，该试剂盒包括上述的HLA-DR蛋白。
本发明的HLA-DR蛋白可以用作工作校准品、校准品、质控品；工作校准品是企业最高溯源物质，用于给校准品/质控品溯源定值。标准品用于生成/标准主曲线，给常规样本定值(例如：脓毒血症血浆样本)；质控品用于验证分析仪器或试剂盒的性能。
上述的试剂盒中，优选地，所述试剂盒还包括生物素标记sHLA-DR鼠单抗、吖啶酯标记sHLA-DR鼠单抗、链霉亲和素标记的免疫磁性微珠和缓冲液。
本发明的有益效果：
(1)通过特定Linker长度和序列的选择，将HLA-DR的α链和β链串联表达，并形成正确的空间结构，抗原活性与天然蛋白相似度95％左右；
(2)选取不同亚型β链的公共序列表达，可同时模拟多种类型的HLA-DR分子；
(3)现有技术是将异源二聚体体外表达后的产物进行自发或人为组装，操作繁琐且产率极低，本发明采用α链和β链串联表达的方式，产量高出其他方法几十倍且工艺简单易重复；
(4)应用：可以用于脓毒症诊断中可溶性人类白细胞DR抗原(sHLA-DR)的定量检测，比细胞膜表面的HLA-DR更早预警脓毒症。
附图说明：
图1为本发明实施例1中获得的HLA-DR蛋白的电泳图；
图2为本发明实施例2中获得的sHLA-DR标准曲线。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。
实施例1 HLA-DR蛋白制备方法(以SEQ ID NO：13为例)
1、载体构建
1.1、α链氨基酸序列获取
从NCBI数据库中获得了人源的人类白细胞抗原-DR(HLA-DR)的α链氨基酸序列以及基因序列(UniProtKB-P01903(DRA_HUMAN)；Gene ID:3122,updated on 16-Feb-2020)；HLA-DRα链核苷酸序列如SEQ ID NO：49所示。
1.2、β链氨基酸序列获取
β链主要是DRB1，DRB3，DRB4，DRB5这几种，其中DRB1变异最多。根据uniprot数据库，筛选了50份DRB1，24份DRB3，19份DRB4，22份DRB5的蛋白序列；进一步对比筛选获得同源性较高的序列，设计获得了β链氨基酸序列，共231氨基酸。
根据β链氨基酸序列，设计β链核苷酸序列如SEQ ID NO：50所示。
1.3、目的序列合成
目的序列设计：酶切位点+(GCCACC)+IL2信号肽+目的基因+10xhis+TAA+酶切
位点
其中IL-2信号肽：MYRMQLLSCIALSLALVTNS；酶切位点：BamHI和XhoI
目的蛋白的全长核苷酸序列如SEQ ID NO：51所示。
将设计的表达单元送交基因合成公司合成了该序列，合成的表达单元插入在pVAX1载体中，获得HLA-DR-α-L20-β-pVAX1重组质粒以及相应甘油菌HLA-DR-α-L20-β-pVAX1-DH5α。
2、真核表达
2.1、表达的质粒制备
用质粒试剂盒提取含有目的基因的表达质粒HLA-DR-α-L20-β-pVAX1，乙醇沉淀DNA，在生物安全柜中自然风干后，用无菌水溶解DNA，用Nanodrop测定DNA浓度。
2.2、CHO细胞株准备
2.2.1、解冻ExpiCHO-STM细胞
从液氮中取出细胞管，在37℃的水浴中转动冻存管1～2分钟后将细胞全部转移至含有30mL的预热ExpiCHOTM表达培养基的通气式锥形摇瓶中。将细胞置于轨道摇床上(37℃培养箱含8％CO2，相对湿度≥80％的空气条件下)培养。
解冻三天后，测定活细胞密度和存活率百分比(细胞存活率应≥90％)。继续监测细胞密度和存活率，当细胞密度达到4×106～6×106个活细胞/mL时，进行细胞传代。
2.3、转染
使新鲜解冻的细胞复苏培养两代或更多代，方可进行转染。
(1)传代并扩增ExpiCHO-STM细胞，直至细胞密度达到约4×106～6×106个活细胞/mL。
(2)第-1天：分种细胞：转染前一天(第-1天)，分种第(1)步中的ExpiCHO-STM培养物，最终密度为3×106～4×106个活细胞/mL，使细胞过夜生长。
(3)第0天：转染细胞：次日(第0天)，测定活细胞密度和存活率百分比。细胞密度应达到约7×106～10×106个活细胞/mL。存活率应为95％～99％，方可继续转染。
(4)使用新鲜的预热至37℃的ExpiCHOTM表达培养基将第(2)步中的细胞稀释至最终密度为6×106个活细胞/mL。轻轻晃动培养瓶，混匀细胞。
(5)使用冷的试剂(4℃)配制ExpiFectamineTM CHO/质粒DNA复合物。络合过程中无需将试剂置于冰上。只需从冰箱中取出试剂，开始DNA络合即可。
(6)室温孵育ExpiFectamineTM CHO/质粒DNA复合物(第5d步)1～5分钟，然后将溶液慢慢转移至第(4)步的培养瓶中，在添加过程中轻轻晃动培养瓶。
(7)将细胞置于轨道摇床上(37℃培养箱含8％CO2的湿化空气条件下)培养。
3、蛋白纯化
将30mLExpiCHO-STM细胞培养液于4000g离心10分钟，上清经过0.22um过滤澄清去除细胞碎片。澄清的细胞培养液中加入1/25(v/v)500mM咪唑，使用镍柱分离纯化。镍柱使用20mM PB(500mM NaCl 20mM咪唑pH7.0)平衡5个柱体积，然后进样分离，分别以100mM，300mM和500mM咪唑阶段洗脱，收集目标咪唑的洗脱部分作为目标蛋白洗脱峰。即为目的蛋白HLA-DR-α-L20-β-pVAX1(B1)蛋白。蛋白纯化结果如图1所示，SDS-PAGE验证纯度95％以上。
图1中，M：蛋白Marker；1：10ug HLA-DR-α-L20-β-pVAX1(B1)蛋白；2：20ug HLA-DR-α-L20-β-pVAX1(B1)蛋白。
4、蛋白定量：
将上述样本置换透析缓冲液至PBS中，超滤浓缩后采用BCA蛋白浓度测定试剂盒测定sHLA-DR蛋白浓度为1.2mg/mL。
实施例2 sHLA-DR定量检测试剂盒原理及检测步骤
1、检验原理
定量检测试剂盒采用双抗体夹心法检测血浆中可溶性人白细胞抗原-DR(sHLA-DR)的含量。试剂盒采用针对sHLA-DR的一对单克隆抗体(常规市售购买获得)，其中一株抗体标记生物素，另一株抗体标记吖啶酯。标本/校准液/质控液与生物素标记的单克隆抗体、吖啶酯标记的单克隆抗体以及包被有链霉亲和素的免疫磁性微粒反应形成免疫复合物，此复合物的形成量与待测抗原的含量成正比。复合物中的吖啶酯在底物液的激发下释放光子，自动监测3秒钟内发出的相对光强度RLU，与sHLA-DR浓度成一定的比例关系，仪器自动拟合计算sHLA-DR浓度。
其中，本发明制备的蛋白作为标准品、校准品、质控品，其中标准品用于生成定量标准曲线。工作校准品作为企业最高溯源物质，用于检测常规样本，生成标准曲线，给常规样本的定值，质控品是已知的浓度含量，用仪器测量来鉴定该仪器测量结果是否准确。
2、主要组成成分
试剂盒主要成分如下表1所示：
表1：
3、检测步骤
3.1、前期准备
仪器准备：反应杯、微量移液器和吸头、科斯迈全自动磁微粒化学发光仪SMART6500/500S。
试剂准备：准备预激发液、激发液和洗涤液(含磷酸盐缓冲液、表面活性剂)，并连同试剂盒组分一起安装到仪器上。
3.2、检测步骤
3.2.1、基本操作步骤如表2所示：
表2：
3.2.2、全自动化学发光仪操作步骤：开机准备→仪器自检→试剂安装→校准→质控→样本加载→仪器运行测定→结果审核→报告。
4、标准曲线如图2所示，样本浓度计算：仪器软件会自动将样本发光值根据标曲计算出浓度。
实施例3 Linker长度变化对融合蛋白活性影响
本实施例设计了6组不同长度的Linker，如下表3所示：
表3：
表4：
表4中发光值，是将表3不同设计方案所获取的目的蛋白稀释成不同浓度(10000ng/mL、5000ng/mL等)作为样本在定量检测体系里的发光值；其中膜蛋白为对照组。等浓度样本发光值与膜蛋白越相近，蛋白活性越好；蛋白活性是通过计算等浓度样本发光值与膜蛋白发光值比值的平均值来评价。
由上述实验数据可知：Linker长度在8～48个氨基酸时，蛋白活性占天然蛋白活性的20％以上，可以作为检测体系校准品；更优为16～32个氨基酸；尤其当Linker的长度为20个氨基酸时，蛋白活性占天然蛋白活性的75％，HLA-DR蛋白表达量高达5mg/30mL细胞。蛋白活性越高，越接近天然蛋白，检测样本时越能反应实际情况。
实施例4 Linker氨基酸组成的变化对融合蛋白活性影响
根据实施例3可以看出，20个氨基酸长度的Linker效果最佳，在此前提下，基于实施例3中L4组实验，本实施例针对20个氨基酸长度的Linker中甘氨酸(G)和丝氨酸(S)的组成对HLA-DR蛋白活性的影响，设计了6组实验，如下表5所示：
表5：
表6：
由上述实验数据可知：Linker中氨基酸由甘氨酸和丝氨酸混合组成相比单一甘氨酸或丝氨酸组成具备更优的抗原活性，且5个GGGS循环和4个GGGGS循环获得的目的蛋白抗原活性最好，尤其是5个GGGS循环最优，抗原活性占天然蛋白活性的76％，HLA-DR蛋白表达量高达4.5mg/30mL细胞。
实施例5 Linker为5个GGGS循环时，甘氨酸和丝氨酸的组成顺序变化对融合蛋白活性影响
根据实施例4可以看出，5个GGGS循环构成的Linker效果最佳，在此前提下，基于实施例4中L4组实验，本实施例针对GGGS中甘氨酸和丝氨酸的组成顺序变化对HLA-DR蛋白活性的影响，设计了4组实验，如下表7所示：
表7：
| 编号 | 蛋白活性 | 表达方式 | 表达量(30mL细胞) |
| L4(SEQ ID NO：17) | 78％ | 80％胞外分泌 | 5mg |
| B1(SEQ ID NO：13) | 98％ | 80％胞外分泌 | 4mg |
| B2(SEQ ID NO：25) | 90％ | 80％胞外分泌 | 3mg |
| B3(SEQ ID NO：26) | 110％ | 80％胞外分泌 | 2.5mg |
表8：
由上述实验数据可知：20个氨基酸的Linker组成中，5个GGSG循环获得的目的蛋白抗原活性最好，抗原活性占天然蛋白活性的98％，HLA-DR蛋白表达量高达4mg/30mL细胞。
实施例6 α链序列对融合蛋白活性影响
根据实施例3～5确定Linker最佳为5个GGSG循环(即：SEQ ID NO：5)的前提下，本实施例针对α链序列的变化对HLA-DR蛋白活性的影响，设计了6组实验，如下表9所示：
表9：
| 编号 | 蛋白活性 | 表达方式 | 表达量(30mL细胞) |
| B1(SEQ ID NO：13) | 96％ | 80％胞外分泌 | 5mg |
| C1(SEQ ID NO：27) | 0％ | 70％胞外分泌 | 1.5mg |
| C2(SEQ ID NO：28) | 0％ | 80％胞外分泌 | 2mg |
| C3(SEQ ID NO：29) | 36％ | 80％胞外分泌 | 3mg |
| C4(SEQ ID NO：30) | 48％ | 80％胞外分泌 | 1.2mg |
| C5(SEQ ID NO：31) | 76％ | 80％胞外分泌 | 1.8mg |
表10：
由上述实验数据可知：α1功能区域是必要的，α2区域有助于抗原构象生成。α1结构域+α2结构域胞外段全长肽段构建获得的HLA-DR蛋白抗原活性最好，抗原活性占天然蛋白活性的96％，HLA-DR蛋白表达量高达5mg/30mL细胞。
实施例7 β链序列对融合蛋白活性影响
根据实施例3～5确定Linker最佳为5个GGSG循环(即：SEQ ID NO：5)，根据实施例5确定α链为α1结构域多肽(即：SEQ ID NO：8)+α2结构域多肽(即：SEQ ID NO：9)胞外段全长肽段的前提下，本实施设计了4组实验考察β链序列变化对HLA-DR蛋白活性的影响，如下表11所示：
表11：
| 编号 | 蛋白活性 | 表达方式 | 表达量(30mL细胞) |
| B1(SEQ ID NO：13) | 96％ | 80％胞外分泌 | 5mg |
| D1(SEQ ID NO：32) | 71％ | 70％胞外分泌 | 2mg |
| D2(SEQ ID NO：33) | 37％ | 80％胞外分泌 | 2.6mg |
| D3(SEQ ID NO：34) | 0％ | 80％胞外分泌 | 1.3mg |
表12：
由上述实验数据可知：β1结构域多肽+β2结构域多肽、β1结构域多肽+β2结构域多肽+连接肽公共区全长肽段构建获得的HLA-DR蛋白抗原活性最好，尤其是包含公共区全长肽段的β链构建的蛋白抗原活性占天然蛋白活性的96％，HLA-DR蛋白表达量高达5mg/30mL细胞。
实施例8 α链和β链中部分氨基酸突变对融合蛋白活性影响
根据实施例3～7确定了最佳的Linker、α链和β链，由此构建获得的HLA-DR蛋白(即：B1组)，其氨基酸序列如SEQ ID NO：13所示。设计了7组实验考察α链和β链中氨基酸突变对融合蛋白活性的影响，如下表13所示：
表13：
表14：
注：相同实验组，在不同批操作时，实验结果不会完全一致，会有些许误差，一般产品重复性CV<10％均符合要求，这里默认误差在±10％均是合理的。
由上述实验数据可知：α链和β链部分氨基酸突变(抗原表位除外)对HLA-DR蛋白活性影响较小。
实施例9 本发明与现有方法比较
1、表达量对比实验如下表15和表16所示：
表15：
| 表达方案 | 产量 |
| 天然膜蛋白 | 74pg/1.8×10<sup>8</sup>个细胞 |
| 本发明 | 4mg/1.8×10<sup>8</sup>个细胞 |
表16：
| 表达方案 | 产量 |
| 体外重组 | 12ug/30mL细胞 |
| 本发明 | 4mg/30mL细胞 |
由表15和表16所示：相比之下，本发明的方案产量是天然膜蛋白产量的107倍；是在DRα和DRβ通过亮氨酸拉链体外组装方案的300多倍。
参考文献：
1.HLA-DR分子的分离纯化，贾帅争，孙红琰，刘晓达，杜芝燕，王全立，(军事医学科学院野战输血研究所，北京100850)军事医学科学院院刊2001年3月，第25卷第1期。
2.Kalandadze A,Galleno M,Foncerrada L,et al.Expression of recombinantHLA-DR2 molecules[J].Journal of Biological Chemistry,1996,271(33):20156-20162。
2、稳定性对比实验：
比较本发明B1组蛋白和天然蛋白的热加速稳定性。
方案：
实验组：将相同浓度(均为1ug/mL)的B1蛋白和天然蛋白放在37℃温箱中热加速破坏3天；
对照组是1ug/mL的B1组蛋白和天然蛋白4度冰箱放置3天。
用实施例2中的定量检测试剂盒验证两组蛋白浓度变化；根据图2标准曲线计算蛋白浓度。
回收率＝实验组浓度/对照组浓度(回收率越接近100％说明稳定性越好)
结果如下表17所示：
表17：
由表17可以看出：实验数据计算获得的B1组回收率＝94％；对照组膜蛋白回收率＝37％。由此可见，本发明B1组HLA-DR蛋白的稳定性显著性优于天然膜蛋白的稳定性。
序列表
<110> 江苏为真生物医药技术股份有限公司
<120> 多肽、HLA-DR蛋白及其制备方法和应用
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1 5 10 15
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Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
1 5 10 15
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20
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Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
1 5 10 15
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20
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<213> Artificial Sequence
<220>
<223> 用作HLA-DR蛋白的α和β链之间的连接肽
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Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
1 5 10 15
Ser Gly Gly Gly
20
<210> 8
<211> 84
<212> PRT
<213> Artificial Sequence
<220>
<223> α1结构域多肽
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Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn
<210> 9
<211> 107
<212> PRT
<213> Artificial Sequence
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<223> α2结构域多肽
<400> 9
Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro Val Glu Leu Arg
1 5 10 15
Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pro Pro Val
20 25 30
Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Thr Gly Val
35 40 45
Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe
50 55 60
His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg
65 70 75 80
Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe
85 90 95
Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu
100 105
<210> 10
<211> 92
<212> PRT
<213> Artificial Sequence
<220>
<223> β1结构域多肽
<400> 10
Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn Gly Thr Glu
1 5 10 15
Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu Glu Tyr Val
20 25 30
Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly
35 40 45
Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln
50 55 60
Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val Gly
65 70 75 80
Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys
85 90
<210> 11
<211> 90
<212> PRT
<213> Artificial Sequence
<220>
<223> β2结构域多肽
<400> 11
Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu
1 5 10 15
Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg
20 25 30
Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly
35 40 45
Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu
50 55 60
Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro
65 70 75 80
Ser Val Thr Ser Pro Leu Thr Val Glu Trp
85 90
<210> 12
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 连接肽
<400> 12
Arg Ala Arg Ser Glu Ser Ala Gln Ser Lys
1 5 10
<210> 13
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 13
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 14
<211> 387
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 14
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Gly Ser Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
195 200 205
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
210 215 220
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
225 230 235 240
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
245 250 255
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
260 265 270
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
275 280 285
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
290 295 300
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
305 310 315 320
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
325 330 335
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
340 345 350
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
355 360 365
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
370 375 380
Gln Ser Lys
385
<210> 15
<211> 391
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 15
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Gly Ser Gly Gly Gly Ser Phe Leu Glu Gln Ser Lys Ser Glu Cys
195 200 205
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
210 215 220
Tyr Asn Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr
225 230 235 240
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser
245 250 255
Gln Lys Asp Leu Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys
260 265 270
Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val
275 280 285
His Pro Lys Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His
290 295 300
His Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile
305 310 315 320
Glu Val Arg Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val
325 330 335
Ser Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val
340 345 350
Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val
355 360 365
Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg
370 375 380
Ser Glu Ser Ala Gln Ser Lys
385 390
<210> 16
<211> 399
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 16
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Phe
195 200 205
Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn Gly Thr Glu Arg
210 215 220
Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu Glu Tyr Val Arg
225 230 235 240
Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg
245 250 255
Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln Arg
260 265 270
Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val Gly Glu
275 280 285
Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val Thr Val Tyr Pro
290 295 300
Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu Val Cys Ser Val
305 310 315 320
Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp Phe Arg Asn Gly
325 330 335
Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu Ile Gln Asn Gly
340 345 350
Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr Val Pro Arg Ser
355 360 365
Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser Val Thr Ser Pro
370 375 380
Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala Gln Ser Lys
385 390 395
<210> 17
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 17
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
195 200 205
Gly Gly Ser Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 18
<211> 415
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 18
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
195 200 205
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Phe
210 215 220
Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn Gly Thr Glu Arg
225 230 235 240
Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu Glu Tyr Val Arg
245 250 255
Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg
260 265 270
Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln Arg
275 280 285
Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val Gly Glu
290 295 300
Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val Thr Val Tyr Pro
305 310 315 320
Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu Val Cys Ser Val
325 330 335
Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp Phe Arg Asn Gly
340 345 350
Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu Ile Gln Asn Gly
355 360 365
Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr Val Pro Arg Ser
370 375 380
Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser Val Thr Ser Pro
385 390 395 400
Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala Gln Ser Lys
405 410 415
<210> 19
<211> 431
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 19
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
195 200 205
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
210 215 220
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Phe
225 230 235 240
Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn Gly Thr Glu Arg
245 250 255
Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu Glu Tyr Val Arg
260 265 270
Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg
275 280 285
Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln Arg
290 295 300
Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val Gly Glu
305 310 315 320
Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val Thr Val Tyr Pro
325 330 335
Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu Val Cys Ser Val
340 345 350
Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp Phe Arg Asn Gly
355 360 365
Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu Ile Gln Asn Gly
370 375 380
Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr Val Pro Arg Ser
385 390 395 400
Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser Val Thr Ser Pro
405 410 415
Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala Gln Ser Lys
420 425 430
<210> 20
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 20
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
195 200 205
Gly Gly Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 21
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 21
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Ser
180 185 190
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
195 200 205
Ser Ser Ser Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 22
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 22
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Gly Ser Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 23
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 23
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
195 200 205
Ser Gly Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 24
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 24
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
195 200 205
Ser Gly Ser Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 25
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 25
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
195 200 205
Ser Gly Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 26
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 26
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Ser
180 185 190
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
195 200 205
Gly Gly Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 27
<211> 233
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 27
Asn Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu
1 5 10 15
Ile Phe His Val Asp Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
20 25 30
Gly Gly Gly Ser Gly Gly Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser
35 40 45
Glu Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg
50 55 60
Tyr Phe Tyr Asn Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly
65 70 75 80
Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp
85 90 95
Asn Ser Gln Lys Asp Leu Leu Glu Gln Arg Arg Ala Ala Val Asp Thr
100 105 110
Tyr Cys Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg
115 120 125
Arg Val His Pro Lys Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu
130 135 140
Gln His His Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly
145 150 155 160
Ser Ile Glu Val Arg Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly
165 170 175
Val Val Ser Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr
180 185 190
Leu Val Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys
195 200 205
Gln Val Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg
210 215 220
Ala Arg Ser Glu Ser Ala Gln Ser Lys
225 230
<210> 28
<211> 281
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 28
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
65 70 75 80
Gly Gly Gly Ser Gly Gly Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser
85 90 95
Glu Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg
100 105 110
Tyr Phe Tyr Asn Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly
115 120 125
Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp
130 135 140
Asn Ser Gln Lys Asp Leu Leu Glu Gln Arg Arg Ala Ala Val Asp Thr
145 150 155 160
Tyr Cys Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg
165 170 175
Arg Val His Pro Lys Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu
180 185 190
Gln His His Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly
195 200 205
Ser Ile Glu Val Arg Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly
210 215 220
Val Val Ser Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr
225 230 235 240
Leu Val Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys
245 250 255
Gln Val Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg
260 265 270
Ala Arg Ser Glu Ser Ala Gln Ser Lys
275 280
<210> 29
<211> 296
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 29
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
85 90 95
Gly Gly Ser Gly Gly Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu
100 105 110
Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr
115 120 125
Phe Tyr Asn Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu
130 135 140
Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn
145 150 155 160
Ser Gln Lys Asp Leu Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr
165 170 175
Cys Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg
180 185 190
Val His Pro Lys Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln
195 200 205
His His Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser
210 215 220
Ile Glu Val Arg Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val
225 230 235 240
Val Ser Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu
245 250 255
Val Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln
260 265 270
Val Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala
275 280 285
Arg Ser Glu Ser Ala Gln Ser Lys
290 295
<210> 30
<211> 376
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 30
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
165 170 175
Gly Gly Ser Gly Gly Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu
180 185 190
Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr
195 200 205
Phe Tyr Asn Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu
210 215 220
Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn
225 230 235 240
Ser Gln Lys Asp Leu Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr
245 250 255
Cys Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg
260 265 270
Val His Pro Lys Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln
275 280 285
His His Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser
290 295 300
Ile Glu Val Arg Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val
305 310 315 320
Val Ser Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu
325 330 335
Val Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln
340 345 350
Val Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala
355 360 365
Arg Ser Glu Ser Ala Gln Ser Lys
370 375
<210> 31
<211> 390
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 31
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
180 185 190
Ser Gly Gly Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His
195 200 205
Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr
210 215 220
Asn Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg
225 230 235 240
Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln
245 250 255
Lys Asp Leu Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg
260 265 270
His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His
275 280 285
Pro Lys Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His
290 295 300
Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu
305 310 315 320
Val Arg Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser
325 330 335
Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met
340 345 350
Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu
355 360 365
His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser
370 375 380
Glu Ser Ala Gln Ser Lys
385 390
<210> 32
<211> 393
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 32
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp
385 390
<210> 33
<211> 377
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 33
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr
370 375
<210> 34
<211> 303
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 34
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys
290 295 300
<210> 35
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 35
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ala Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 36
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 36
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Ala Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ala Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 37
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 37
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ala Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 38
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 38
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ala Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ala Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 39
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 39
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Ala Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ala Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 40
<211> 403
<212> PRT
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 40
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Ala Ala Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro
85 90 95
Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe
100 105 110
Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val
115 120 125
Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu
130 135 140
Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val
145 150 155 160
Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys
165 170 175
His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Gly
180 185 190
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn
210 215 220
Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu
225 230 235 240
Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
245 250 255
Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu
260 265 270
Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr
275 280 285
Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys Val
290 295 300
Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu
305 310 315 320
Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp
325 330 335
Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu
340 345 350
Ile Gln Asn Gly Asp Ala Ala Phe Gln Thr Leu Val Met Leu Glu Thr
355 360 365
Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser
370 375 380
Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala
385 390 395 400
Gln Ser Lys
<210> 41
<211> 84
<212> PRT
<213> Artificial Sequence
<220>
<223> α1结构域多肽
<400> 41
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
1 5 10 15
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
20 25 30
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
35 40 45
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
50 55 60
Val Asp Lys Ala Asn Leu Glu Ile Ala Ala Lys Arg Ser Asn Tyr Thr
65 70 75 80
Pro Ile Thr Asn
<210> 42
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> α2结构域多肽
<400> 42
Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro Val Glu Leu Arg
1 5 10 15
Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pro Pro Val
20 25 30
Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Thr Gly Val
35 40 45
Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe
50 55 60
His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg
65 70 75 80
Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe
85 90 95
Asp Ala Pro Ala Pro Leu Pro Glu Thr Thr Glu
100 105
<210> 43
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> α2结构域多肽
<400> 43
Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro Val Glu Leu Arg
1 5 10 15
Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pro Pro Val
20 25 30
Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Ala Gly Val
35 40 45
Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe
50 55 60
His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg
65 70 75 80
Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe
85 90 95
Asp Ala Pro Ala Pro Leu Pro Glu Thr Thr Glu
100 105
<210> 44
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> α2结构域多肽
<400> 44
Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro Val Glu Leu Arg
1 5 10 15
Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pro Pro Val
20 25 30
Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Thr Gly Val
35 40 45
Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe
50 55 60
His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Ala Cys Arg
65 70 75 80
Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe
85 90 95
Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu
100 105
<210> 45
<211> 92
<212> PRT
<213> Artificial Sequence
<220>
<223> β1结构域多肽
<400> 45
Phe Leu Glu Gln Ser Lys Ser Glu Cys His Phe Phe Asn Gly Thr Glu
1 5 10 15
Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu Glu Tyr Val
20 25 30
Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly
35 40 45
Arg Pro Asp Ala Glu Tyr Trp Asn Ala Gln Lys Asp Leu Leu Glu Gln
50 55 60
Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val Gly
65 70 75 80
Glu Ser Phe Thr Val Gln Arg Arg Val His Pro Lys
85 90
<210> 46
<211> 90
<212> PRT
<213> Artificial Sequence
<220>
<223> β2结构域多肽
<400> 46
Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu
1 5 10 15
Leu Val Cys Ala Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg
20 25 30
Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly
35 40 45
Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu
50 55 60
Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro
65 70 75 80
Ser Val Thr Ser Pro Leu Thr Val Glu Trp
85 90
<210> 47
<211> 90
<212> PRT
<213> Artificial Sequence
<220>
<223> β2结构域多肽
<400> 47
Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu
1 5 10 15
Leu Val Cys Ser Val Ala Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg
20 25 30
Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly
35 40 45
Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu
50 55 60
Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro
65 70 75 80
Ser Val Thr Ser Pro Leu Thr Val Glu Trp
85 90
<210> 48
<211> 90
<212> PRT
<213> Artificial Sequence
<220>
<223> β2结构域多肽
<400> 48
Val Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu
1 5 10 15
Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg
20 25 30
Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Val Ser Thr Gly
35 40 45
Leu Ile Gln Asn Gly Asp Ala Ala Phe Gln Thr Leu Val Met Leu Glu
50 55 60
Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro
65 70 75 80
Ser Val Thr Ser Pro Leu Thr Val Glu Trp
85 90
<210> 49
<211> 573
<212> DNA
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白α链
<400> 49
atcaaagaag aacatgtgat catccaggcc gagttctatc tgaatcctga ccaatcaggc 60
gagtttatgt ttgactttga tggtgatgag attttccatg tggatatggc aaagaaggag 120
acggtctggc ggcttgaaga atttggacga tttgccagct ttgaggctca aggtgcattg 180
gccaacatag ctgtggacaa agccaacctg gaaatcatga caaagcgctc caactatact 240
ccgatcacca atgtacctcc agaggtaact gtgctcacaa acagccctgt ggaactgaga 300
gagcccaacg tcctcatctg tttcatagac aagttcaccc caccagtggt caatgtcacg 360
tggcttcgaa atggaaaacc tgtcaccaca ggagtgtcag agacagtctt cctgcccagg 420
gaagaccacc ttttccgcaa gttccactat ctccccttcc tgccctcaac tgaggacgtt 480
tacgactgca gggtggagca ctggggcttg gatgagcctc ttctcaagca ctgggagttt 540
gatgctccaa gccctctccc agagactaca gag 573
<210> 50
<211> 576
<212> DNA
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白β链
<400> 50
tttctggaac agtccaaaag cgagtgccac ttcttcaacg gtaccgaacg cgttcgtttt 60
ctggatcgct acttctacaa ccaggaagaa tatgtccgct ttgattctga cgttggcgaa 120
tatcgcgcag ttaccgaact gggtcgtccg gacgcagaat attggaacag ccagaaagac 180
ctgctggaac aacgtcgcgc agcagttgat acctattgtc gtcataacta cggcgttggc 240
gaaagtttca ccgttcaacg tcgcgttcat ccgaaagtta ccgtttatcc ggcgaaaacc 300
caaccgctgc aacatcataa cctgctggtt tgcagcgtaa gcggctttta tccgggcagc 360
attgaagtac gctggtttcg taacggtcag gaagaaaaag cgggcgttgt tagtaccggt 420
ctgattcaga acggcgattg gacctttcaa accctggtta tgctggaaac cgttccgcgt 480
tctggcgaag tttatacctg ccaggtcgaa catccgtctg ttaccagtcc gctgaccgtt 540
gaatggcgtg cacgttctga atctgcacag agcaaa 576
<210> 51
<211> 1320
<212> DNA
<213> Artificial Sequence
<220>
<223> HLA-DR蛋白
<400> 51
ggatccgcca ccatgtacag gatgcaactc ctgtcttgca ttgcactaag tcttgcactt 60
gtcacgaatt cgatcaaaga agaacatgtg atcatccagg ccgagttcta tctgaatcct 120
gaccaatcag gcgagtttat gtttgacttt gatggtgatg agattttcca tgtggatatg 180
gcaaagaagg agacggtctg gcggcttgaa gaatttggac gatttgccag ctttgaggct 240
caaggtgcat tggccaacat agctgtggac aaagccaacc tggaaatcat gacaaagcgc 300
tccaactata ctccgatcac caatgtacct ccagaggtaa ctgtgctcac aaacagccct 360
gtggaactga gagagcccaa cgtcctcatc tgtttcatag acaagttcac cccaccagtg 420
gtcaatgtca cgtggcttcg aaatggaaaa cctgtcacca caggagtgtc agagacagtc 480
ttcctgccca gggaagacca ccttttccgc aagttccact atctcccctt cctgccctca 540
actgaggacg tttacgactg cagggtggag cactggggct tggatgagcc tcttctcaag 600
cactgggagt ttgatgctcc aagccctctc ccagagacta cagagggcgg tggtagcggc 660
ggtggcagcg gcggtggtag cggcggtggc agcggcggtg gtagctttct ggaacagtcc 720
aaaagcgagt gccacttctt caacggtacc gaacgcgttc gttttctgga tcgctacttc 780
tacaaccagg aagaatatgt ccgctttgat tctgacgttg gcgaatatcg cgcagttacc 840
gaactgggtc gtccggacgc agaatattgg aacagccaga aagacctgct ggaacaacgt 900
cgcgcagcag ttgataccta ttgtcgtcat aactacggcg ttggcgaaag tttcaccgtt 960
caacgtcgcg ttcatccgaa agttaccgtt tatccggcga aaacccaacc gctgcaacat 1020
cataacctgc tggtttgcag cgtaagcggc ttttatccgg gcagcattga agtacgctgg 1080
tttcgtaacg gtcaggaaga aaaagcgggc gttgttagta ccggtctgat tcagaacggc 1140
gattggacct ttcaaaccct ggttatgctg gaaaccgttc cgcgttctgg cgaagtttat 1200
acctgccagg tcgaacatcc gtctgttacc agtccgctga ccgttgaatg gcgtgcacgt 1260
tctgaatctg cacagagcaa acatcaccat caccaccatc atcaccatca ctaactcgag 1320
Claims (12)
1.一种多肽，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的8～48个氨基酸残基的多肽。
2.根据权利要求1所述的多肽，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的16～32个氨基酸残基的多肽；
优选地，所述多肽是由甘氨酸和丝氨酸以肽键相互连接构成的20个氨基酸残基的多肽；
优选地，所述多肽的氨基酸序列选自如下序列中的一种：
GGGSGGGSGGGSGGGSGGGS(SEQ ID NO：1)，
GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：2)，
GGSGGSGGSGGSGGSGGSGG(SEQ ID NO：3)，
GSGSGSGSGSGSGSGSGSGS(SEQ ID NO：4)，
GGSGGGSGGGSGGGSGGGSG(SEQ ID NO：5)，
GSGGGSGGGSGGGSGGGSGG(SEQ ID NO：6)，
SGGGSGGGSGGGSGGGSGGG(SEQ ID NO：7)。
3.权利要求1所述多肽作为HLA-DR蛋白的α链和β链的连接肽在构建HLA-DR蛋白中的应用。
4.一种HLA-DR蛋白，该HLA-DR蛋白结构组成包括α链、β链和权利要求1或2所述多肽；所述α链的C端与所述多肽的N端相连接；所述多肽的C端与所述β链的N端相连接。
5.根据权利要求4所述的HLA-DR蛋白，其中，所述α链包括α1结构域多肽；所述α1结构域多肽的C端与所述多肽的N端相连接；
所述α1结构域多肽的氨基酸序列包括SEQ ID NO：8所示氨基酸序列或与SEQ ID NO：8所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与α1结构域多肽具有相同功能的氨基酸序列；
优选地，所述α链还包括α2结构域多肽，所述α1结构域多肽的C端与所述α2结构域多肽的N端相连接，所述α2结构域多肽的C端与所述多肽的N端相连接；
所述α2结构域多肽的氨基酸序列包括SEQ ID NO：9所示氨基酸序列或与SEQ ID NO：9所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与α2结构域多肽具有相同功能的氨基酸序列，或为SEQ ID NO：9所示氨基酸序列的部分片段且具备α2结构域多肽部分功能的氨基酸序列。
6.根据权利要求4所述的HLA-DR蛋白，其中，所述β链包括β1结构域多肽和β2结构域多肽；所述多肽的C端与所述β1结构域多肽的N端相连接，所述β1结构域多肽的C端与所述β2结构域多肽的N端相连接；
所述β1结构域多肽的氨基酸序列包括SEQ ID NO：10所示氨基酸序列或与SEQ ID NO：10所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与β1结构域多肽具有相同功能的氨基酸序列；
所述β2结构域多肽的氨基酸序列包括SEQ ID NO：11所示氨基酸序列或与SEQ ID NO：11所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与β2结构域多肽具有相同功能的氨基酸序列，或为SEQ ID NO：11所示氨基酸序列的部分片段且具备β2结构域多肽部分功能的氨基酸序列；
优选地，所述β链还包括连接肽；所述β2结构域多肽的C端与所述连接肽的N端相连接；
所述连接肽的氨基酸序列包括SEQ ID NO：12所示氨基酸序列或与SEQ ID NO：12所示氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且与连接肽具有相同功能的氨基酸序列。
7.根据权利要求4所述的HLA-DR蛋白，其中，该HLA-DR蛋白的氨基酸序列包括SEQ IDNO：13、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ IDNO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：29、SEQ IDNO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：35、SEQ ID NO：36、SEQ IDNO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40所示序列中的一种；
优选地，该HLA-DR蛋白的氨基酸序列包括SEQ ID NO：13所示的序列。
8.一种编码权利要求4～7任一项所述HLA-DR蛋白的全长多核苷酸；
优选地，编码氨基酸序列为SEQ ID NO：13的HLA-DR蛋白的全长多核苷酸的核苷酸序列如SEQ ID NO：51所示。
9.一种包含权利要求8所述全长多核苷酸的重组载体或重组细胞。
10.一种HLA-DR蛋白的制备方法，该方法包括：将权利要求8所述全长多核苷酸克隆到质粒载体上，将质粒载体转染至表达细胞中培养，表达目标蛋白，然后进行蛋白纯化得到HLA-DR蛋白；
优选地，所述表达细胞包括CHO细胞。
11.权利要求4～7任一项所述HLA-DR蛋白在制备用于脓毒血症诊断试剂中的用途。
12.一种定量检测sHLA-DR的试剂盒，该试剂盒包括权利要求4～7任一项所述HLA-DR蛋白；
优选地，所述试剂盒还包括生物素标记sHLA-DR鼠单抗、吖啶酯标记sHLA-DR鼠单抗、链霉亲和素标记的免疫磁性微珠和缓冲液。
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| CN103852407A (zh) * | 2014-03-18 | 2014-06-11 | 浙江大学 | 通过检测hla-dr表达量评估cd14阳性细胞抗原呈递能力的方法 |
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| CN103852407A (zh) * | 2014-03-18 | 2014-06-11 | 浙江大学 | 通过检测hla-dr表达量评估cd14阳性细胞抗原呈递能力的方法 |
| CN111278864A (zh) * | 2017-09-07 | 2020-06-12 | 库尔生物制药有限公司 | 抗原呈递多肽及其使用方法 |
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