CN110964119A

CN110964119A - 抗疟二聚体免疫粘附素、药物组合物和用途

Info

Publication number: CN110964119A
Application number: CN201911231257.7A
Authority: CN
Inventors: 傅文燕; 丁敏; 凌月娥; 胡适
Original assignee: Fengchao Medical Technology Shanghai Co ltd
Current assignee: Fengchao Medical Technology Shanghai Co ltd
Priority date: 2019-12-05
Filing date: 2019-12-05
Publication date: 2020-04-07
Also published as: WO2021109913A1

Abstract

本发明提供了抗疟二聚体免疫粘附素、药物组合物和用途。该二聚体免疫粘附素包含二聚化的两条多肽链，第一条多肽链结构通式为Z1‑Z2，第二条多肽链结构通式为Y1‑Y2。其中，Z1是第一细胞表面受体的细胞外结构域或其功能变体或片段，Z2是二聚化结构域或其功能变体或片段；Y1是第二细胞表面受体的细胞外结构域或其功能变体或片段，Y2是二聚化结构域或其功能变体或片段。第一细胞表面受体和第二细胞表面受体分别选自GYPA、GYPB、GYPC、CR1、BSG中的任一个。本发明提供的二聚体免疫粘附素能有效阻断疟原虫株入侵红细胞，入侵抑制率接近100％；提高免疫细胞的抗疟作用。此外，本发明中抗疟二聚体免疫粘附素体内稳定性优异，具备广阔的临床应用前景。

Description

抗疟二聚体免疫粘附素、药物组合物和用途

技术领域

本发明涉及生物医药工程技术领域，具体涉及一种二聚体免疫粘附素、以其作为活性组分的药物组合物和其医药用途，尤其是用于预防和治疗疟疾的用途。

背景技术

疟疾是经蚊虫叮咬或输入带疟原虫者的血液而感染疟原虫所引起的虫媒传染病。寄生于人体的疟原虫共有四种，即间日疟原虫，三日疟原虫，恶性疟原虫和卵形疟原虫。在我国主要是间日疟原虫和恶性疟原虫；其他两种少见，近年偶见国外输入的一些病例。不同的疟原虫分别引起间日疟、三日疟、恶性疟及卵圆疟。本病主要表现为周期性规律发作，全身发冷、发热、多汗，长期多次发作后，可引起贫血和脾肿大。

在抗体工程领域，通过使用融合蛋白使天然受体胞外结构域实现二聚化，可以增强这些可溶性受体的结合性质，使得它们变成相应配体治疗上有用的拮抗剂。这样的二聚融合体的代表是免疫粘附素(例如，Sledziewski et al,美国专利号5,155,027和5,567,584；Jacobs et al,美国专利号5,605,690；Wallner et al,美国专利号5,914,111；和Ashkenazi and Chamow,Curr.Opin.Immunol.9:195-200,1997)。

二聚化的免疫粘附素克服了可溶性受体胞外结构域的不稳定性和损失生物功能等劣势，具有较强的应用价值。疟原虫入侵红细胞需要红细胞表面受体蛋白的参与，然而目前尚未有报道利用红细胞表面受体蛋白制备免疫粘附素的研究，能否实现制备、制备之后的免疫粘附素的生物学活性是否还存在、制备免疫粘附素是否有抗病效果、选择哪种受体蛋白进行工程化、选择哪种工程化方式均是未知的，需要进一步证实的。

发明内容

本发明的目的在于，依托上述研究背景，研究可溶性二聚体免疫粘附素是否能够用于抗疟应用，并对二聚体免疫粘附素的具体结构、制备方法和用途进行了描述，即提供了二聚体免疫粘附素、其制备方法和用途。

本发明的第一方面，提供了可溶性抗疟二聚体免疫粘附素，包含二聚化的第一条和第二条多肽链，第一条多肽链结构通式为Z1-Z2，第二条多肽链结构通式为Y1-Y2。其中Z1是第一细胞表面受体的细胞外结构域或其功能变体或片段，Z2是二聚化结构域或其功能变体或片段；Y1是第二细胞表面受体的细胞外结构域或其功能变体或片段，Y2是二聚化结构域或其功能变体或片段。

第一种细胞表面受体和或所述第二种细胞表面受体各自选自包括：GYPA(UniProtKB:P02724)；GYPC(UniProtKB:P04921)；GYPB(UniProtKB:P06028)；CR1(UniProtKB:P17927)；BSG(UniProtKB:P35613)。

在Z1和Y1都是细胞表面受体的细胞外结构域或其功能变体或片段的情况下，第一种和第二种细胞表面受体可以是相同的或不同的。

Z2和Y2二聚化结构域包括免疫球蛋白重链恒定区。在具体的变化中，二聚化结构域Z2和Y2是IgG的Fc片段，诸如人免疫球蛋白γ1Fc片段。当Z1与Y1不同时，二聚化结构域Z2和Y2可以采用工程化的手段以增加特异性的异源二聚化形成，如Knob-in-hole、改变电荷极性的ART-Ig、BiMab等双特异性抗体恒定区构建方法工程方法(综述文献Brinkmann U,Kontermann R E.mAbs,2017,9(2):182-212.)。

此外，二聚化结构域Z2和Y2还可以包含有肽接头，肽接头由15-32个氨基酸残基组成，其中这些残基中的1-8个(例如，2个)是半胱氨酸残基。在具体变化中，Z2和Y2包含免疫球蛋白铰链区或其变体。例如，在一个具体实施方案中，Z2和Y2包含免疫球蛋白铰链变体(例如，人免疫球蛋白γ1铰链变体)，其中Fc片段的220相对应的半胱氨酸残基被丝氨酸替代。根据上述二聚化结构域Z2和Y2使用的特别合适的肽接头包括这样的肽接头：所述接头包含多个甘氨酸残基，且任选地包含至少一个丝氨酸残基。

在本发明的的某些实施方案中，二聚化结构域Z2和Y2可以是人免疫球蛋白Fc片段的活性变体，如采用IgG2、IgG3或IgG4的Fc结构域。在某些实施方案中，可以进一步采用Fc的突变体以降低免疫球蛋白诸如ADCC、补体结合等生物活性，如LALA-PG突变体、L235E；E318A；K320A；K322A突变体等。

在本发明的一些优选实施例中，Z1和Y1中的每一种是BSG的细胞外结构域或其功能变体或片段。Z1和Y1的氨基酸序列与SEQ ID NO.1所示的氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。

此时二聚体免疫粘附素包含SEQ ID NO.2所示的人BSG免疫粘附素氨基酸序列。

在本发明的一些优选实施例中，Z1是BSG的细胞外结构域或其功能变体或片段，Y1是GYPA的细胞外结构域或其功能变体或片段。Z1的氨基酸序列与SEQ ID NO.1所示的人BSG胞外结构域氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。Y1的氨基酸序列与SEQ ID NO.3所示的人GYPA胞外结构域氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。

可溶性二聚体免疫粘附素的两条多肽链包含选自下述的氨基酸序列：(a)Z1-Z2多肽链包括SEQ ID NO.4所示的人BSG免疫粘附素Hole突变体氨基酸序列，和(b)Y1-Y2多肽链包括SEQ ID NO.5所示的人GYPA胞外结构域Knob突变体氨基酸序列。

本发明的一些优选实施例中，Z1是BSG的细胞外结构域或其功能变体或片段，Y1是GYPB的细胞外结构域或其功能变体或片段。Z1的氨基酸序列与SEQ ID NO.1所示的人BSG胞外结构域氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。Y1的氨基酸序列与SEQ ID NO.6所示的人GYPB胞外结构域氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。

可溶性二聚体免疫粘附素的两条多肽链包含选自下述的氨基酸序列：(a)Z1-Z2多肽链具有SEQ ID NO:4所示人BSG免疫粘附素Hole突变体的氨基酸序列，和(b)Y1-Y2多肽链具有SEQ ID NO:7所示的人GYPB胞外结构域Knob突变体氨基酸序列。

本发明的一些优选实施例中，Z1是BSG的细胞外结构域或其功能变体或片段。Y1是GYPC的细胞外结构域或其功能变体或片段。Z1的氨基酸序列与SEQ ID NO.1所示的氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。Y1的氨基酸序列与SEQ ID NO.8所示的人GYPC胞外结构域氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。

可溶性二聚体免疫粘附素的两条多肽链包含选自下述的氨基酸序列：(a)Z1-Z2多肽链包括SEQ ID NO:4所示的氨基酸序列，和(b)Y1-Y2多肽链包括SEQ ID NO:9所示的人GYPC胞外结构域Knob突变体氨基酸序列。

本发明的一些优选实施例中，Z1是BSG的细胞外结构域或其功能变体或片段。Y1是CR1的细胞外结构域或其功能变体或片段。Z1的氨基酸序列与SEQ ID NO.1所示的氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。Y1的氨基酸序列与SEQ ID NO.10所示的人CR1胞外结构域氨基酸序列具有至少60％、优选至少65％、优选至少70％、更优选至少75％、更优选至少80％、更优选至少85％、甚至更优选至少90％、甚至更优选至少95％和最优选至少99％同一性。

可溶性二聚体免疫粘附素的两条多肽链包含选自下述的氨基酸序列：(a)Z1-Z2多肽链包括SEQ ID NO:4所示的氨基酸序列，和(b)Y1-Y2多肽链包括SEQ ID NO:11所示的人CR1胞外结构域Knob突变体氨基酸序列。

本发明的第二方面，提供了编码上述抗疟二聚体免疫粘附素的多核苷酸、运载该核苷酸的载体以及包含这种载体的细胞。

本发明提供的表达载体包含下述可操作地连接的元件：转录启动子、编码上述二聚体免疫融合蛋白的DNA区和转录终止子。

通过培养包含载体的细胞，用于生产如上公开的多肽或二聚蛋白，包括：(i)培养包含如上公开的表达载体的细胞，其中细胞表达由所述DNA区段编码的二聚体免疫融合蛋白，并生产编码的二聚体免疫融合蛋白；(ii)回收可溶性二聚体免疫融合蛋白。

类似地，在制备二聚蛋白的方法的某些变化中，方法包括：(i)培养包含如上公开的表达载体的细胞，其中细胞表达由所述DNA区段编码的二聚体免疫融合蛋白，并生产编码的二聚体免疫融合蛋白作为二聚蛋白；和(ii)回收二聚蛋白。

本发明的第三方面，提供了一种药物组合物，包含上述可溶性抗疟二聚体免疫粘附素和至少一种药学上可接受的载体。该药物组合物以可溶性抗疟二聚体免疫粘附素为主要或唯一活性成分，辅料可以保证本发明公开的TIGIT免疫粘附素氨基酸核心序列的构像完整性，同时还要保护蛋白质的多官能团，防止其降解(包括但不限于凝聚、脱氨或氧化)，从而更稳定地发挥疗效。

在药物形式上，可为制药领域常用的混悬、水针、冻干等制剂，优选水针或冻干制剂。液体制剂可以在2℃-8℃条件下保存至少稳定一年，冻干制剂在30℃至少六个月保持稳定。

对于本发明公开的上述二聚体免疫粘附素的水针或冻干制剂，药学上可以接受的辅料包括表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合。其中，表面活性剂包括非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)；poloxamer(如poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸；Pluronics；MONAQUATTM等，其加入量应使双功能双特异性抗体蛋白的颗粒化趋势最小；溶液稳定剂可以为糖类，包括还原性糖和非还原性糖，氨基酸类包括谷氨酸单钠或组氨酸，醇类包括三元醇、高级糖醇、丙二醇、聚乙二醇之一或其组合，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态；等渗调节剂可以为氯化钠、甘露醇之一；缓冲液可以为TRIS、组氨酸缓冲液、磷酸盐缓冲液之一。

本发明的第四方面，提供了本发明的抗疟二聚体免疫粘附素、药物组合物、多核苷酸、载体或宿主细胞在治疗或预防疟疾中的用途，尤其是：1)在阻断疟原虫入侵红细胞作用，如实施例3-5；以及介导免疫细胞的抗疟作用，如实施例6。

本发明的有益保障及效果：

通过细胞实验以及动物模型实验，本发明提供的二聚体免疫粘附素能够有效阻断四种疟原虫株入侵红细胞，入侵抑制率接近100％；此外，通过体内稳定性实验，本发明中的抗疟二聚体免疫粘附素的体内半衰期与市面上抗疟抗体西妥昔单抗半衰期接近，体内稳定性优异。因此，单独应用或与其他相关病症药物联用，能有效地预防和治疗疟疾，具备广阔的临床应用前景。

附图说明

图1为本发明抗疟二聚体免疫粘附素的结构示意图；

图2为抗疟二聚体免疫粘附素体外抗疟原虫入侵红细胞的实验结果；

图3为抗疟二聚体免疫粘附素体内感染治疗实验结果；

图4为抗疟二聚体免疫粘附素体内预防感染实验结果。

具体实施方式

以下实施例、实验例对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质拉的方法，将编码蛋白的基因插入到这样的载体和质拉的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2^ndedition，Cold spring Harbor Laboratory Press。

实施例1.可溶性二聚免疫粘附素的构建及表达

如图1所示，可溶性抗疟二聚体免疫粘附素是一种带有抗体IgGFc的二聚体，二聚体免疫粘附素本身的构建和表达的方法为领域内的常规实验技术，简单描述如下：

(1)全基因合成可溶性二聚免疫粘附素BSG-Fc(包含两条多肽链，每条多肽链的氨基酸序列和核苷酸序列如SEQ ID NO.2和SEQ ID NO.12所示)；BSG/GYPA-Fc(包含两条多肽链，第一条多肽链的氨基酸序列和核苷酸序列如SEQ ID NO.4和SEQ ID NO.13所示，第二条多肽链的氨基酸序列和核苷酸序列如SEQ ID NO.5和SEQ ID NO.14所示)；BSG/GYPB-Fc(包含两条多肽链，第一条多肽链的氨基酸序列和核苷酸序列如SEQ ID NO.4和SEQ ID NO.13所示，第二条多肽链的氨基酸序列和核苷酸序列如SEQ ID NO.7和SEQ ID NO.15所示)。BSG/GYPC-Fc(包含两条多肽链，第一条多肽链的氨基酸序列和核苷酸序列如SEQ ID NO.4和SEQ ID NO.13所示，第二条多肽链的氨基酸序列和核苷酸序列如SEQ ID NO.9和SEQ IDNO.16所示)。

(2)融合蛋白的表达纯化

按照文献(Finck B K.Science,265.；Mihara M et al..Journal of ClinicalInvestigation.2000；106:91-101；Yu X,et al.Nature Immunology.2009；10:48-57.LiuS,et al.Clin Immunol.2019 Jun；203:72-80.)方法进行可溶性二聚免疫粘附素的表达。

实施例2.可溶性二聚免疫粘附素的体内稳定性试验

利用文献(Hu S,et al.Science translational medicine,2017,9(380):eaag0339.)方法在NOG小鼠中评估抗疟二聚免疫粘附素的半衰期。结果显示，BSG-Fc、BSG/GYPA-Fc、BSG/GYPB-Fc、BSG/GYPC-Fc的体内半衰期分别为8.9天、8.1天、7.5天和8.4天；阳性对照西妥昔单抗为8.6天；而三种BSG抗原肽(氨基酸序列TNINTLENSDHTCFAR；SNPYFIVGSR；ENYYNSDIAGPAR)和的半衰期太短未能测出，BSG胞外段蛋白半衰期半衰期太短未能测出。

实施例3.可溶性二聚免疫粘附素体外入侵实验

利用O型人红细胞进行疟原虫入侵实验，试验方法同文献(Zhang M Y,etal.blood,2018,131(10):1111-1121.)。利用四种疟原虫珠Dd2、3D7、FCC1、Nf54进行本实验，健康人IgG作为对照组，分别给药BSG-Fc、BSG/GYPA-Fc、BSG/GYPB-Fc、BSG/GYPC-Fc和对照IgG，给药剂量为10μg/ml。入侵抑制率计算方式也同文献，结果如图2所示：显示BSG-Fc、BSG/GYPA-Fc、BSG/GYPB-Fc、BSG/GYPC-Fc具有很强的抑制入侵能力，入侵抑制率接近100％。

实施例4.可溶性二聚免疫粘附素体内感染治疗实验

利用疟原虫感染NOG小鼠模型进行本实验，模型构建方法同文献(Zhang M Y,etal.blood,2018,131(10):1111-1121.)。将小鼠分为BSG-Fc、BSG/GYPA-Fc、BSG/GYPB-Fc、BSG/GYPC-Fc、对照IgG和空白组。空白组给与等体积PBS，其余各组给药10mg/kg，于感染第2天一次给药，各组N＝8。感染7天后计算小鼠感染率，检测方法和计算方法同文献，结果如图3所示：BSG-Fc、BSG/GYPA-Fc、BSG/GYPB-Fc、BSG/GYPC-Fc具有很强体内的抑制入侵能力，BSG/GYPA-Fc组的感染率为个位数，其余三组的感染率均为零。

实施例5.可溶性二聚免疫粘附素体内预防感染实验

利用疟原虫感染NOG小鼠模型进行本实验模型构建方法同文献(Zhang M Y,etal.blood,2018,131(10):1111-1121.)。采用预防性的给药方式，在小鼠感染疟原虫之前一天将小鼠分为BSG-Fc、BSG/GYPA-Fc、BSG/GYPB-Fc、BSG/GYPC-Fc、对照IgG和空白组。空白组给与等体积PBS，其余各组给药10mg/kg.各组N＝8。感染第7天再次给药，感染15天后计算小鼠感染率，检测方法和计算方法同文献。结果如图4所示，BSG-Fc、BSG/GYPA-Fc、BSG/GYPB-Fc、BSG/GYPC-Fc具有很强的预防感染能力，给药组小鼠无一感染疟原虫。

实施例6.可溶性二聚免疫粘附素介导NK细胞抗疟原虫作用

健康志愿者外周单个核细胞中的NK细胞获取、感染疟原虫珠Dd2的人红细胞(iRBC)建立、血红蛋白释放试验均按照文献(Arora G,et al.Elife,2018,7:e36806.)。简述如下：将iRBC细胞核NK细胞分别用RPMI 1640离心清洗两次，按照NK细胞：iRBC细胞5:1的比例混合并培养与不含有血清的RPMI 1640培养基中；然后分组，分为为BSG-Fc、BSG/GYPA-Fc、BSG/GYPB-Fc、BSG/GYPC-Fc、对照IgG和空白组，每组3个复孔，给药浓度为10μg/ml。37℃孵育4小时后，收集孔板上清，血红蛋白用QuantiChrom血红蛋白分析试剂盒(BioAssay生产)，使用96孔读板仪(Enspire，Perkin Elmer)。血红蛋白的释放以1％Triton-X-100中溶解的iRBC作为标准品。在4小时结束时间，以标准品和空白孔计算各组的血红蛋白释放量。结果如表1：

表1血红蛋白释放量

结果可以看出，可溶性二聚免疫粘附素有效介导NK细胞裂解感染疟原虫的红细胞，有效抗疟疾。

综上，在疟疾模型中，二聚体免疫粘附素对于疟疾具有良好的预防治疗效果，有利于后续的临床试验的开展。

序列表

<110> 沣潮医药科技（上海）有限公司

<120> 抗疟二聚体免疫粘附素、药物组合物和用途

<130> 权利要求书、说明书

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Asp Ala Leu Pro Gly Gln Lys Thr Glu Phe Lys Val Asp Ser Asp Asp

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Asp Ala Leu Pro Gly Gln Lys Thr Glu Phe Lys Val Asp Ser Asp Asp

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Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

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Asp Ala Leu Pro Gly Gln Lys Thr Glu Phe Lys Val Asp Ser Asp Asp

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Gln Trp Gly Glu Tyr Ser Cys Val Phe Leu Pro Glu Pro Met Gly Thr

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Ala Asn Ile Gln Leu His Gly Pro Pro Arg Val Lys Ala Val Lys Ser

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Ser Glu His Ile Asn Glu Gly Glu Thr Ala Met Leu Val Cys Lys Ser

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Ser Glu Asp Lys Ala Leu Met Asn Gly Ser Glu Ser Arg Phe Phe Val

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Ser Ser Ser Gln Gly Arg Ser Glu Leu His Ile Glu Asn Leu Asn Met

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Glu Ala Asp Pro Gly Gln Tyr Arg Cys Asn Gly Thr Ser Ser Lys Gly

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Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

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Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

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Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

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Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

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Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

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Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

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Met Trp Ser Thr Arg Ser Pro Asn Ser Thr Ala Trp Pro Leu Ser Leu

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Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu

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Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys

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Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn

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Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys Arg

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Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly Ile

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Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val Ile

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Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe

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Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg Asp Lys

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Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu Pro Gly

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Trp Ser Pro Ala Ala Pro Thr Cys Glu Val Lys Ser Cys Asp Asp Phe

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Leu Gly Ala Lys Val Asp Phe Val Cys Asp Glu Gly Phe Gln Leu Lys

340 345 350

Gly Ser Ser Ala Ser Tyr Cys Val Leu Ala Gly Met Glu Ser Leu Trp

355 360 365

Asn Ser Ser Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser Pro Pro

370 375 380

Val Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val Phe Pro

385 390 395 400

Phe Gly Lys Thr Val Asn Tyr Thr Cys Asp Pro His Pro Asp Arg Gly

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Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr Ser Asp

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Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys Gly Ile

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Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys Leu Lys

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Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu Lys Tyr

465 470 475 480

Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr Cys Leu

485 490 495

Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg Lys Ser

500 505 510

Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Thr

515 520 525

Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr Gly His

530 535 540

Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly Asn Ala

545 550 555 560

Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg Ile Pro Cys Gly

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Leu Pro Pro Thr Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu

580 585 590

Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser

595 600 605

Gly Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys

610 615 620

Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln

625 630 635 640

Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn Gly Ile

645 650 655

Leu Val Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val Val Glu

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Phe Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg Val Lys

675 680 685

Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys Ser Arg

690 695 700

Val Cys Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg

705 710 715 720

Asp Lys Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu

725 730 735

Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Met Arg Cys Thr Pro Gln

740 745 750

Gly Asp Trp Ser Pro Ala Ala Pro Thr Cys Glu Val Lys Ser Cys Asp

755 760 765

Asp Phe Met Gly Gln Leu Leu Asn Gly Arg Val Leu Phe Pro Val Asn

770 775 780

Leu Gln Leu Gly Ala Lys Val Asp Phe Val Cys Asp Glu Gly Phe Gln

785 790 795 800

Leu Lys Gly Ser Ser Ala Ser Tyr Cys Val Leu Ala Gly Met Glu Ser

805 810 815

Leu Trp Asn Ser Ser Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser

820 825 830

Pro Pro Val Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val

835 840 845

Phe Pro Phe Gly Lys Ala Val Asn Tyr Thr Cys Asp Pro His Pro Asp

850 855 860

Arg Gly Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr

865 870 875 880

Ser Asp Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys

885 890 895

Gly Ile Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys

900 905 910

Leu Lys Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu

915 920 925

Lys Tyr Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr

930 935 940

Cys Leu Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg

945 950 955 960

Lys Ser Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val

965 970 975

Ile Thr Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr

980 985 990

Gly His Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly

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Asn Thr Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg Ile Pro

1010 1015 1020

Cys Gly Leu Pro Pro Thr Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn

1025 1030 1035 1040

Arg Glu Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Leu

1045 1050 1055

Gly Ser Arg Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile

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Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala

1075 1080 1085

Pro Gln Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn

1090 1095 1100

Gly Ile Leu Val Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val

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Val Glu Phe Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg

1125 1130 1135

Val Lys Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys

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Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu His Gly Glu His Thr

1155 1160 1165

Pro Ser His Gln Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser

1170 1175 1180

Cys Glu Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Leu His Cys Thr

1185 1190 1195 1200

Pro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg Cys Ala Val Lys Ser

1205 1210 1215

Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly Arg Val Leu Phe Pro

1220 1225 1230

Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe Val Cys Asp Glu Gly

1235 1240 1245

Phe Arg Leu Lys Gly Ser Ser Val Ser His Cys Val Leu Val Gly Met

1250 1255 1260

Arg Ser Leu Trp Asn Asn Ser Val Pro Val Cys Glu His Ile Phe Cys

1265 1270 1275 1280

Pro Asn Pro Pro Ala Ile Leu Asn Gly Arg His Thr Gly Thr Pro Ser

1285 1290 1295

Gly Asp Ile Pro Tyr Gly Lys Glu Ile Ser Tyr Thr Cys Asp Pro His

1300 1305 1310

Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly Glu Ser Thr Ile Arg

1315 1320 1325

Cys Thr Ser Asp Pro His Gly Asn Gly Val Trp Ser Ser Pro Ala Pro

1330 1335 1340

Arg Cys Glu Leu Ser Val Arg Ala Gly His Cys Lys Thr Pro Glu Gln

1345 1350 1355 1360

Phe Pro Phe Ala Ser Pro Thr Ile Pro Ile Asn Asp Phe Glu Phe Pro

1365 1370 1375

Val Gly Thr Ser Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Phe Gly Lys

1380 1385 1390

Met Phe Ser Ile Ser Cys Leu Glu Asn Leu Val Trp Ser Ser Val Glu

1395 1400 1405

Asp Asn Cys Arg Arg Lys Ser Cys Gly Pro Pro Pro Glu Pro Phe Asn

1410 1415 1420

Gly Met Val His Ile Asn Thr Asp Thr Gln Phe Gly Ser Thr Val Asn

1425 1430 1435 1440

Tyr Ser Cys Asn Glu Gly Phe Arg Leu Ile Gly Ser Pro Ser Thr Thr

1445 1450 1455

Cys Leu Val Ser Gly Asn Asn Val Thr Trp Asp Lys Lys Ala Pro Ile

1460 1465 1470

Cys Glu Ile Ile Ser Cys Glu Pro Pro Pro Thr Ile Ser Asn Gly Asp

1475 1480 1485

Phe Tyr Ser Asn Asn Arg Thr Ser Phe His Asn Gly Thr Val Val Thr

1490 1495 1500

Tyr Gln Cys His Thr Gly Pro Asp Gly Glu Gln Leu Phe Glu Leu Val

1505 1510 1515 1520

Gly Glu Arg Ser Ile Tyr Cys Thr Ser Lys Asp Asp Gln Val Gly Val

1525 1530 1535

Trp Ser Ser Pro Pro Pro Arg Cys Ile Ser Thr Asn Lys Cys Thr Ala

1540 1545 1550

Pro Glu Val Glu Asn Ala Ile Arg Val Pro Gly Asn Arg Ser Phe Phe

1555 1560 1565

Ser Leu Thr Glu Ile Ile Arg Phe Arg Cys Gln Pro Gly Phe Val Met

1570 1575 1580

Val Gly Ser His Thr Val Gln Cys Gln Thr Asn Gly Arg Trp Gly Pro

1585 1590 1595 1600

Lys Leu Pro His Cys Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu

1605 1610 1615

His Gly Glu His Thr Leu Ser His Gln Asp Asn Phe Ser Pro Gly Gln

1620 1625 1630

Glu Val Phe Tyr Ser Cys Glu Pro Ser Tyr Asp Leu Arg Gly Ala Ala

1635 1640 1645

Ser Leu His Cys Thr Pro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg

1650 1655 1660

Cys Thr Val Lys Ser Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly

1665 1670 1675 1680

Arg Val Leu Leu Pro Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe

1685 1690 1695

Val Cys Asp Glu Gly Phe Arg Leu Lys Gly Arg Ser Ala Ser His Cys

1700 1705 1710

Val Leu Ala Gly Met Lys Ala Leu Trp Asn Ser Ser Val Pro Val Cys

1715 1720 1725

Glu Gln Ile Phe Cys Pro Asn Pro Pro Ala Ile Leu Asn Gly Arg His

1730 1735 1740

Thr Gly Thr Pro Phe Gly Asp Ile Pro Tyr Gly Lys Glu Ile Ser Tyr

1745 1750 1755 1760

Ala Cys Asp Thr His Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly

1765 1770 1775

Glu Ser Ser Ile Arg Cys Thr Ser Asp Pro Gln Gly Asn Gly Val Trp

1780 1785 1790

Ser Ser Pro Ala Pro Arg Cys Glu Leu Ser Val Pro Ala Ala Cys Pro

1795 1800 1805

His Pro Pro Lys Ile Gln Asn Gly His Tyr Ile Gly Gly His Val Ser

1810 1815 1820

Leu Tyr Leu Pro Gly Met Thr Ile Ser Tyr Ile Cys Asp Pro Gly Tyr

1825 1830 1835 1840

Leu Leu Val Gly Lys Gly Phe Ile Phe Cys Thr Asp Gln Gly Ile Trp

1845 1850 1855

Ser Gln Leu Asp His Tyr Cys Lys Glu Val Asn Cys Ser Phe Pro Leu

1860 1865 1870

Phe Met Asn Gly Ile Ser Lys Glu Leu Glu Met Lys Lys Val Tyr His

1875 1880 1885

Tyr Gly Asp Tyr Val Thr Leu Lys Cys Glu Asp Gly Tyr Thr Leu Glu

1890 1895 1900

Gly Ser Pro Trp Ser Gln Cys Gln Ala Asp Asp Arg Trp Asp Pro Pro

1905 1910 1915 1920

Leu Ala Lys Cys Thr Ser Arg Thr His Asp

1925 1930

<210> 11

<211> 2162

<212> PRT

<213> 人工序列(Artificial sequence )

<400> 11

Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu

1 5 10 15

Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys

20 25 30

Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn

35 40 45

Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys Arg

50 55 60

Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly Ile

65 70 75 80

Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu

85 90 95

Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val Ile

100 105 110

Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro

115 120 125

Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe

130 135 140

His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly

145 150 155 160

Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser

165 170 175

Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys Ile

180 185 190

Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn Gly Ile Leu Val

195 200 205

Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val Val Glu Phe Arg

210 215 220

Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg Val Lys Cys Gln

225 230 235 240

Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys Ser Arg Val Cys

245 250 255

Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg Asp Lys

260 265 270

Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu Pro Gly

275 280 285

Tyr Asp Leu Arg Gly Ala Ala Ser Met Arg Cys Thr Pro Gln Gly Asp

290 295 300

Trp Ser Pro Ala Ala Pro Thr Cys Glu Val Lys Ser Cys Asp Asp Phe

305 310 315 320

Met Gly Gln Leu Leu Asn Gly Arg Val Leu Phe Pro Val Asn Leu Gln

325 330 335

Leu Gly Ala Lys Val Asp Phe Val Cys Asp Glu Gly Phe Gln Leu Lys

340 345 350

Gly Ser Ser Ala Ser Tyr Cys Val Leu Ala Gly Met Glu Ser Leu Trp

355 360 365

Asn Ser Ser Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser Pro Pro

370 375 380

Val Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val Phe Pro

385 390 395 400

Phe Gly Lys Thr Val Asn Tyr Thr Cys Asp Pro His Pro Asp Arg Gly

405 410 415

Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr Ser Asp

420 425 430

Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys Gly Ile

435 440 445

Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys Leu Lys

450 455 460

Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu Lys Tyr

465 470 475 480

Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr Cys Leu

485 490 495

Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg Lys Ser

500 505 510

Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Thr

515 520 525

Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr Gly His

530 535 540

Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly Asn Ala

545 550 555 560

Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg Ile Pro Cys Gly

565 570 575

Leu Pro Pro Thr Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu

580 585 590

Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser

595 600 605

Gly Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys

610 615 620

Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln

625 630 635 640

Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn Gly Ile

645 650 655

Leu Val Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val Val Glu

660 665 670

Phe Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg Val Lys

675 680 685

Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys Ser Arg

690 695 700

Val Cys Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg

705 710 715 720

Asp Lys Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu

725 730 735

Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Met Arg Cys Thr Pro Gln

740 745 750

Gly Asp Trp Ser Pro Ala Ala Pro Thr Cys Glu Val Lys Ser Cys Asp

755 760 765

Asp Phe Met Gly Gln Leu Leu Asn Gly Arg Val Leu Phe Pro Val Asn

770 775 780

Leu Gln Leu Gly Ala Lys Val Asp Phe Val Cys Asp Glu Gly Phe Gln

785 790 795 800

Leu Lys Gly Ser Ser Ala Ser Tyr Cys Val Leu Ala Gly Met Glu Ser

805 810 815

Leu Trp Asn Ser Ser Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser

820 825 830

Pro Pro Val Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val

835 840 845

Phe Pro Phe Gly Lys Ala Val Asn Tyr Thr Cys Asp Pro His Pro Asp

850 855 860

Arg Gly Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr

865 870 875 880

Ser Asp Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys

885 890 895

Gly Ile Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys

900 905 910

Leu Lys Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu

915 920 925

Lys Tyr Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr

930 935 940

Cys Leu Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg

945 950 955 960

Lys Ser Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val

965 970 975

Ile Thr Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr

980 985 990

Gly His Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly

995 1000 1005

Asn Thr Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg Ile Pro

1010 1015 1020

Cys Gly Leu Pro Pro Thr Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn

1025 1030 1035 1040

Arg Glu Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Leu

1045 1050 1055

Gly Ser Arg Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile

1060 1065 1070

Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala

1075 1080 1085

Pro Gln Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn

1090 1095 1100

Gly Ile Leu Val Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val

1105 1110 1115 1120

Val Glu Phe Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg

1125 1130 1135

Val Lys Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys

1140 1145 1150

Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu His Gly Glu His Thr

1155 1160 1165

Pro Ser His Gln Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser

1170 1175 1180

Cys Glu Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Leu His Cys Thr

1185 1190 1195 1200

Pro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg Cys Ala Val Lys Ser

1205 1210 1215

Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly Arg Val Leu Phe Pro

1220 1225 1230

Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe Val Cys Asp Glu Gly

1235 1240 1245

Phe Arg Leu Lys Gly Ser Ser Val Ser His Cys Val Leu Val Gly Met

1250 1255 1260

Arg Ser Leu Trp Asn Asn Ser Val Pro Val Cys Glu His Ile Phe Cys

1265 1270 1275 1280

Pro Asn Pro Pro Ala Ile Leu Asn Gly Arg His Thr Gly Thr Pro Ser

1285 1290 1295

Gly Asp Ile Pro Tyr Gly Lys Glu Ile Ser Tyr Thr Cys Asp Pro His

1300 1305 1310

Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly Glu Ser Thr Ile Arg

1315 1320 1325

Cys Thr Ser Asp Pro His Gly Asn Gly Val Trp Ser Ser Pro Ala Pro

1330 1335 1340

Arg Cys Glu Leu Ser Val Arg Ala Gly His Cys Lys Thr Pro Glu Gln

1345 1350 1355 1360

Phe Pro Phe Ala Ser Pro Thr Ile Pro Ile Asn Asp Phe Glu Phe Pro

1365 1370 1375

Val Gly Thr Ser Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Phe Gly Lys

1380 1385 1390

Met Phe Ser Ile Ser Cys Leu Glu Asn Leu Val Trp Ser Ser Val Glu

1395 1400 1405

Asp Asn Cys Arg Arg Lys Ser Cys Gly Pro Pro Pro Glu Pro Phe Asn

1410 1415 1420

Gly Met Val His Ile Asn Thr Asp Thr Gln Phe Gly Ser Thr Val Asn

1425 1430 1435 1440

Tyr Ser Cys Asn Glu Gly Phe Arg Leu Ile Gly Ser Pro Ser Thr Thr

1445 1450 1455

Cys Leu Val Ser Gly Asn Asn Val Thr Trp Asp Lys Lys Ala Pro Ile

1460 1465 1470

Cys Glu Ile Ile Ser Cys Glu Pro Pro Pro Thr Ile Ser Asn Gly Asp

1475 1480 1485

Phe Tyr Ser Asn Asn Arg Thr Ser Phe His Asn Gly Thr Val Val Thr

1490 1495 1500

Tyr Gln Cys His Thr Gly Pro Asp Gly Glu Gln Leu Phe Glu Leu Val

1505 1510 1515 1520

Gly Glu Arg Ser Ile Tyr Cys Thr Ser Lys Asp Asp Gln Val Gly Val

1525 1530 1535

Trp Ser Ser Pro Pro Pro Arg Cys Ile Ser Thr Asn Lys Cys Thr Ala

1540 1545 1550

Pro Glu Val Glu Asn Ala Ile Arg Val Pro Gly Asn Arg Ser Phe Phe

1555 1560 1565

Ser Leu Thr Glu Ile Ile Arg Phe Arg Cys Gln Pro Gly Phe Val Met

1570 1575 1580

Val Gly Ser His Thr Val Gln Cys Gln Thr Asn Gly Arg Trp Gly Pro

1585 1590 1595 1600

Lys Leu Pro His Cys Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu

1605 1610 1615

His Gly Glu His Thr Leu Ser His Gln Asp Asn Phe Ser Pro Gly Gln

1620 1625 1630

Glu Val Phe Tyr Ser Cys Glu Pro Ser Tyr Asp Leu Arg Gly Ala Ala

1635 1640 1645

Ser Leu His Cys Thr Pro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg

1650 1655 1660

Cys Thr Val Lys Ser Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly

1665 1670 1675 1680

Arg Val Leu Leu Pro Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe

1685 1690 1695

Val Cys Asp Glu Gly Phe Arg Leu Lys Gly Arg Ser Ala Ser His Cys

1700 1705 1710

Val Leu Ala Gly Met Lys Ala Leu Trp Asn Ser Ser Val Pro Val Cys

1715 1720 1725

Glu Gln Ile Phe Cys Pro Asn Pro Pro Ala Ile Leu Asn Gly Arg His

1730 1735 1740

Thr Gly Thr Pro Phe Gly Asp Ile Pro Tyr Gly Lys Glu Ile Ser Tyr

1745 1750 1755 1760

Ala Cys Asp Thr His Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly

1765 1770 1775

Glu Ser Ser Ile Arg Cys Thr Ser Asp Pro Gln Gly Asn Gly Val Trp

1780 1785 1790

Ser Ser Pro Ala Pro Arg Cys Glu Leu Ser Val Pro Ala Ala Cys Pro

1795 1800 1805

His Pro Pro Lys Ile Gln Asn Gly His Tyr Ile Gly Gly His Val Ser

1810 1815 1820

Leu Tyr Leu Pro Gly Met Thr Ile Ser Tyr Ile Cys Asp Pro Gly Tyr

1825 1830 1835 1840

Leu Leu Val Gly Lys Gly Phe Ile Phe Cys Thr Asp Gln Gly Ile Trp

1845 1850 1855

Ser Gln Leu Asp His Tyr Cys Lys Glu Val Asn Cys Ser Phe Pro Leu

1860 1865 1870

Phe Met Asn Gly Ile Ser Lys Glu Leu Glu Met Lys Lys Val Tyr His

1875 1880 1885

Tyr Gly Asp Tyr Val Thr Leu Lys Cys Glu Asp Gly Tyr Thr Leu Glu

1890 1895 1900

Gly Ser Pro Trp Ser Gln Cys Gln Ala Asp Asp Arg Trp Asp Pro Pro

1905 1910 1915 1920

Leu Ala Lys Cys Thr Ser Arg Thr His Asp Glu Pro Lys Ser Cys Asp

1925 1930 1935

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

1940 1945 1950

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

1955 1960 1965

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

1970 1975 1980

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

1985 1990 1995 2000

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

2005 2010 2015

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

2020 2025 2030

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

2035 2040 2045

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

2050 2055 2060

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

2065 2070 2075 2080

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

2085 2090 2095

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

2100 2105 2110

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

2115 2120 2125

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

2130 2135 2140

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

2145 2150 2155 2160

Gly Lys

<210> 12

<211> 1311

<212> DNA

<213> 人工序列(Artificial sequence )

<400> 12

atggcagccg cattgtttgt gctgttggga tttgcattgc tcggaaccca cggagcttct 60

ggagccgctg gaactgtgtt caccacggtg gaggatctcg ggagtaaaat tctgttgacc 120

tgttccttga atgattccgc caccgaggta accggacacc gatggttgaa agggggtgtt 180

gtgctgaagg aggatgccct gccagggcag aaaactgagt tcaaggttga ttccgacgac 240

cagtgggggg agtactcttg cgtgtttctt cctgaaccaa tgggcaccgc taatatccaa 300

cttcatgggc ctcccagagt taaagcagtg aagtcaagcg agcacattaa tgaaggagaa 360

actgctatgc tggtgtgcaa gtctgagagc gtgccaccag tgacagattg ggcttggtac 420

aagattacgg atagcgagga caaagcactg atgaatggtt cagagagcag gttcttcgtc 480

tcctctagtc agggccgctc agagctccac attgaaaatc tgaacatgga ggctgatcct 540

gggcagtatc gctgcaacgg tacctctagc aagggcagcg accaggcaat cattaccctg 600

agagtgagat cccatgaacc taagtcttgc gacaaaaccc acacatgccc tccttgtccg 660

gcacctgaac tgctgggagg gccttctgtg tttctgttcc cacctaagcc taaggatacc 720

ctgatgatct cccgcactcc tgaggtgaca tgtgtcgttg tcgacgtgtc ccatgaggac 780

ccagaagtca aattcaattg gtacgtggac ggcgtggaag ttcataacgc caagacaaag 840

ccaagagagg agcaatacaa ctctacctat cgggttgtgt ccgtgctgac tgtcctgcac 900

caagattggc ttaatggtaa ggaatacaag tgcaaggtct ccaacaaagc tctgccagct 960

ccaatcgaaa aaactatttc caaggctaag gggcagccga gagagccaca ggtgtatacc 1020

ctgccaccaa gtagagacga gcttactaag aatcaggtct cacttacctg tttggtgaag 1080

ggcttttacc caagtgatat agcagtcgaa tgggaatcta atgggcaacc agagaataac 1140

tataagacca ctccccccgt gctggactca gatgggtcct tttttctgta ctcaaagttg 1200

acagtggaca aatctcggtg gcagcagggc aacgtgttca gctgctccgt gatgcatgaa 1260

gcactgcaca accattacac acagaaatcc ctctccctgt cacccggtaa g 1311

<210> 13

<211> 1311

<212> DNA

<213> 人工序列(Artificial sequence )

<400> 13

atggctgccg ctttgtttgt acttttggga tttgccctgc tcggtactca tggtgcatcc 60

ggtgctgcgg gaaccgtctt cacaactgtc gaggacctcg gcagcaaaat cctcctgaca 120

tgttcactga acgactccgc cacagaagtg acagggcacc gctggctcaa aggtggggtc 180

gttctgaaag aggatgccct gccgggccag aaaaccgaat ttaaggttga cagtgacgat 240

cagtgggggg aatatagttg cgtgttcttg ccagaaccga tggggaccgc aaatatccag 300

cttcatgggc ctccaagagt gaaggcggtg aagtcctcag aacacattaa tgagggcgag 360

actgccatgc tcgtgtgcaa gagtgaatcc gtgccgcctg ttaccgattg ggcctggtat 420

aagatcaccg atagcgagga taaggcactt atgaatggga gtgaaagtcg ctttttcgtt 480

tcctctagtc agggtaggtc tgagctgcac attgagaacc tgaatatgga agcggaccct 540

ggacagtacc gatgcaatgg cactagctcc aaaggaagcg accaggctat tattactctg 600

agagtgcgat ctcatgagcc caaatcatgc gataagacac acacgtgtcc cccctgtcca 660

gctcctgaac ttcttggtgg cccatcagtt tttctgtttc cacccaagcc caaggatacc 720

ctgatgataa gcaggacacc agaggtgact tgtgtcgttg tcgacgtctc acatgaagac 780

ccggaagtta aatttaactg gtacgtggac ggggtagaag ttcacaatgc caagacaaag 840

cccagagaag agcagtacaa cagtacgtat cgagtggtca gtgttctcac agtactgcac 900

caagactggc ttaacggtaa ggagtacaaa tgcaaagtgt ctaataaagc ccttccagcc 960

cctatcgaga aaaccatttc taaagctaag ggtcaaccta gggaaccgca agtatgcaca 1020

ctgcccccga gtcgagatga actgaccaag aatcaggttt ctctgtcatg cgctgtgaag 1080

ggtttctacc cctccgatat agcagttgag tgggaaagta atgggcagcc ggaaaataat 1140

tacaaaacaa caccgcctgt gcttgactct gacgggtcct tctttctcgt gtccaaactt 1200

acagtggaca aatcaaggtg gcaacaggga aatgtctttt cttgcagcgt gatgcacgag 1260

gcgctccaca atcattatac acagaaaagc ctgagcctta gtcccggtaa g 1311

<210> 14

<211> 912

<212> DNA

<213> 人工序列(Artificial sequence )

<400> 14

tcaagcacta caggcgttgc tatgcatact tccacgagca gctctgtgac aaaaagctac 60

atatccagcc agacgaacga tacccataag agagacacat acgcggctac tccacgggca 120

cacgaagtgt cagagatcag tgtacggacg gtgtatcctc cagaagaaga gacaggtgag 180

agagtccaac ttgcacatca cttctctgag cctgaagagc caaaatcatg cgacaaaact 240

cacacttgtc caccatgtcc cgcacctgag ttgctgggag gtccttccgt cttccttttc 300

ccaccgaaac cgaaggacac gctgatgatt tcccgcactc ccgaagtaac atgcgtggtg 360

gtggacgtct cacacgagga tccagaggtg aaatttaact ggtacgttga cggcgtagag 420

gtgcacaacg ccaagacaaa accgagggaa gaacagtaca acagcaccta tagggtggtt 480

agtgttctta ccgttcttca tcaggactgg ctcaacggta aagagtataa gtgtaaggtg 540

tccaataagg ctttgcccgc accgatcgaa aaaacaatat ccaaagcaaa gggacagcca 600

agagaacccc aggtgtacac attgccacct tgccgcgacg aactgactaa gaaccaggta 660

tccctgtggt gtctggttaa aggcttctac ccaagcgata tcgccgtgga gtgggaatct 720

aatggccagc ccgagaataa ttataagaca acaccacctg tgctggatag cgatggttct 780

ttcttcctgt actccaagtt gacggtcgac aagtcccggt ggcaacaggg caatgtgttt 840

tcatgctcag taatgcatga ggccctccac aatcactaca cacagaaaag cctttccctg 900

tccccgggaa aa 912

<210> 15

<211> 816

<212> DNA

<213> 人工序列(Artificial sequence )

<400> 15

ctgagcacta ctgaagtcgc catgcacact tccacatcct caagcgtcac caaatcttac 60

atcagttctc aaaccaacgg agagacagga cagttggtcc ataggttcac agttccggcc 120

gagccaaagt cttgcgacaa aacgcatact tgtccacctt gccccgcccc tgaactgttg 180

gggggcccat cagtattcct cttcccgccc aagcccaaag acacccttat gatcagtcgg 240

actcccgaag tcacatgcgt ggtggtggac gtatcccacg aggatcccga ggtgaaattt 300

aattggtacg tcgacggggt cgaggtacat aacgccaaga caaagccaag ggaggagcag 360

tacaacagca cttatcgcgt cgtgtcagtt ctgaccgtac tgcaccagga ttggctgaat 420

gggaaggaat acaaatgcaa ggtctcaaat aaggccctgc ctgcccccat tgaaaagaca 480

attagcaagg cgaaaggtca gcctagggag ccccaggtgt ataccctgcc tccctgcaga 540

gacgagctca caaagaatca ggtgtccctg tggtgtctgg tgaagggctt ctacccatct 600

gatatcgctg tcgagtggga aagcaacgga cagccagaaa ataattataa gactactccc 660

cctgtccttg acagcgatgg aagttttttt ctgtactcca agctgaccgt ggacaaatca 720

agatggcagc agggcaatgt gttctcttgt tccgtcatgc acgaggcctt gcataatcac 780

tatacacaga agagtctttc tcttagccct ggaaaa 816

<210> 16

<211> 867

<212> DNA

<213> 人工序列(Artificial sequence )

<400> 16

atgtggtcca cgcggagtcc aaattctact gcatggccat tgagcctgga gcctgaccca 60

ggcatggcct ctgctagcac cactatgcac acaacaacaa tcgcagaacc ggaccctggt 120

atgtcaggat ggccagatgg ccggatggag actagcaccc cgacaataat ggaaccgaag 180

agttgtgaca aaactcacac ttgtccaccg tgtccagccc ccgaattgct gggcggtcca 240

tctgttttcc tcttcccccc caagcccaag gatacactca tgatctcccg aacccctgaa 300

gtaacttgcg tcgtcgttga cgtgagccac gaggacccgg aagtaaagtt caattggtac 360

gtcgacggcg tggaggtgca caatgccaaa actaagccca gagaggaaca gtataacagt 420

acgtacagag tcgtcagtgt gcttactgtt ctgcatcagg actggctcaa cgggaaggag 480

tataagtgca aggtgagcaa taaggcgctg cctgcaccaa tcgagaaaac gatttctaaa 540

gctaaaggtc agccaagaga accacaggtg tatacacttc ctccttgtag agacgagctt 600

accaagaacc aggtttcatt gtggtgcttg gtgaagggat tctacccatc tgatatagct 660

gtggagtggg aatccaacgg acaacccgag aataattaca aaacgacacc acctgtgctg 720

gacagcgatg gatccttctt tttgtactca aaactcactg tggacaagag tcgctggcag 780

cagggaaacg tatttagttg ctctgtcatg cacgaagcac tgcataacca ttacacacaa 840

aagagtctga gcctctcacc cggtaaa 867

Claims

1.一种抗疟二聚体免疫粘附素，其特征在于，包含二聚化的第一条多肽链和第二条多肽链，所述第一条多肽链的结构通式为Z1-Z2，所述第二条多肽链的结构通式为Y1-Y2，

其中，Z1是第一细胞表面受体的细胞外结构域或其功能变体或片段，Z2是二聚化结构域或其功能变体或片段；Y1是第二细胞表面受体的细胞外结构域或其功能变体或片段，Y2是二聚化结构域或其功能变体或片段，

所述第一细胞表面受体和所述第二细胞表面受体分别选自：GYPA、GYPB、GYPC、CR1、BSG中的任一个。

2.根据权利要求1所述的抗疟二聚体免疫粘附素，其特征在于：

其中，Z2和Y2为IgG的Fc片段或改变其生物活性的Fc突变体，或利用Knob-in-hole技术、改变电荷极性的ART-Ig技术或BiMab技术构建的异源二聚IgG-Fc片段。

3.根据权利要求1所述的抗疟二聚体免疫粘附素，其特征在于：

其中，二聚化结构域Z2和Y2还包含有肽接头，所述肽接头由15-32个氨基酸残基组成，该氨基酸残基包含多个甘氨酸残基、一至八个半胱氨酸残基以及至少一个丝氨酸残基。

4.根据权利要求1所述的抗疟二聚体免疫粘附素，其特征在于，

其中，Z1和Y1来源相同，均为BSG的细胞外结构域或其功能变体或片段，Z1和Y1的氨基酸序列与SEQ ID NO.1所示的氨基酸序列具有至少95％的同一性；

所述抗疟二聚体免疫粘附素具有SEQ ID NO.2所示的人BSG免疫粘附素氨基酸序列。

5.根据权利要求4所述的抗疟二聚体免疫粘附素，其特征在于：

其中，Z1和Y1来源不同，为如下任一所述情况：

(1)Z1是BSG的细胞外结构域或其功能变体或片段，Y1是GYPA的细胞外结构域或其功能变体或片段；Z1氨基酸序列与SEQ ID NO.1所示的氨基酸序列具有至少95％的同一性，Y1氨基酸序列与SEQ ID NO.3所示的氨基酸序列具有至少95％的同一性；Z1-Z2多肽链序列如SEQ ID NO.4所示，Y1-Y2多肽链序列如SEQ ID NO.5所示；

(2)Z1是BSG的细胞外结构域或其功能变体或片段，Y1是GYPB的细胞外结构域或其功能变体或片段；Z1氨基酸序列与SEQ ID NO.1所示的氨基酸序列具有至少95％的同一性，Y1氨基酸序列与SEQ ID NO.6所示的氨基酸序列具有至少95％的同一性；Z1-Z2多肽链序列如SEQ ID NO.4所示，Y1-Y2多肽链序列如SEQ ID NO.7所示；

(3)Z1是BSG的细胞外结构域或其功能变体或片段，Y1是GYPC的细胞外结构域或其功能变体或片段；Z1氨基酸序列与SEQ ID NO.1所示的氨基酸序列具有至少95％的同一性，Y1氨基酸序列与SEQ ID NO.8所示的氨基酸序列具有至少95％的同一性；Z1-Z2多肽链序列如SEQ ID NO.4所示，Y1-Y2多肽链序列如SEQ ID NO.9所示；

Z1是BSG的细胞外结构域或其功能变体或片段，Y1是CR1的细胞外结构域或其功能变体或片段；Z1氨基酸序列与SEQ ID NO.1所示的氨基酸序列具有至少95％的同一性，Y1氨基酸序列与SEQ ID NO.10所示的氨基酸序列具有至少95％的同一性；Z1-Z2多肽链序列如SEQID NO.4所示，Y1-Y2多肽链序列如SEQ ID NO.11所示。

6.一种药物组合物，其特征在于，包括活性成分以及括药学上可接受的药物载体，所述活性成分包括权利要求1～5任一项所述的抗疟二聚体免疫粘附素、编码该抗疟二聚体免疫粘附素的核苷酸或运载所述抗疟二聚体免疫粘附素或所述核苷酸的载体。

7.权利要求1～5任一项所述的抗疟二聚体免疫粘附素在制备抗疟药物中的用途。

8.根据权利要求7所述的抗疟二聚体免疫粘附素在制备抗疟药物中的用途，其特征在于：

其中，所述抗疟药物为阻断疟原虫入侵红细胞的药物或介导免疫细胞裂解染疟红细胞的药物。

9.根据权利要求8所述的抗疟二聚体免疫粘附素在制备抗疟药物中的用途，其特征在于：

其中，所述介导免疫细胞裂解染疟原虫红细胞的药物为介导NK细胞裂解染疟红细胞的药物。