CN116396396A - Gna-nkg2d融合蛋白及用于恶性肿瘤治疗的外泌体吸附剂 - Google Patents
Gna-nkg2d融合蛋白及用于恶性肿瘤治疗的外泌体吸附剂 Download PDFInfo
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Abstract
本发明属于生物领域,公开了GNA‑NKG2D融合蛋白及用于恶性肿瘤治疗的外泌体吸附剂。本发明利用不同的连接肽将雪花莲凝集素与NKG2D受体蛋白进行连接,第一方面能将两种功能的蛋白进行融合,方便表达多效性的蛋白;第二方面,采用柔性连接肽能使相邻的两分子GNA蛋白进行灵活的伸展和弯曲,便于形成聚集体;第三方面,采用刚性连接肽能够使相邻两种功能蛋白保持一定的间距,不相互影响,保证功能域的完整,使活性达到最佳;第四方面,本发明采用的刚性连接肽提高了蛋白的抗水解能力,提升GNA‑NKG2D融合蛋白的稳定性和产率。融合蛋白具有与天然GNA、NKG2D蛋白相似的结构,保留了GNA对外泌体的吸附能力和NKG2D蛋白对MICA、MICB的吸附能力,具有意料之外的效果。
Description
技术领域
本发明属于蛋白领域,涉及一种GNA-NKG2D融合蛋白及其免疫吸附剂,具体涉及外泌体的异常表达相关肿瘤的治疗。通过制备GNA-NKG2D融合蛋白,将其偶联至固相载体上,通过免疫吸附的方式去除肿瘤患者血浆中的外泌体,抑制正常组织的癌变,以及肿瘤的转移、增殖和免疫逃逸。
背景技术
肿瘤分为良性和恶性两大类,良性肿瘤生长缓慢,对人体影响小;恶性肿瘤一般称为癌症,往往增长迅速,并且有侵袭性(向周围组织浸润)及转移性,是继心血管疾病后,影响人类寿命的第二大威胁。癌症拥有死亡率高、预后差、治疗费用昂贵的特点,癌症患者通常需要承受巨大的生理痛苦,是目前最急需解决的人类医疗卫生问题之一。传统的外科治疗不适用于血液肿瘤和多处转移的实体瘤,放疗和化疗靶向性差,可造成极大的副作用。靶向药物可特异性靶向肿瘤组织,副作用小,但容易产生耐药性。免疫治疗不直接作用于肿瘤组织,具有效果好、不良反应小和防复发等优点,是目前抗肿瘤药物的开发热点。目前,肿瘤免疫疗法主要分为细胞因子类、抗体类、细胞免疫、肿瘤疫苗免疫、溶瘤病毒免疫,以上主要作用于免疫细胞,但对肿瘤细胞所释放的免疫抑制性物质并没有良好的处理方案。
血液净化近年来已被多数学者所接受,是指将患者血液引出体外,并通过净化装置除去某些致病物质,净化血液,从而治疗疾病。其中免疫吸附是血液净化清除溶质的重要原理之一,将具有高度特异性的抗原、抗体或具有特定理化亲和力的配基与载体结合,制成吸附柱,使其可以特异性地吸附体内相应的致病因子,达到治疗疾病的目的。在与药物的联合治疗中,免疫治疗可增强机体对药物治疗的敏感性,使药物疗效增加,副作用减少,缩短疗程,降低复发率。近年来,免疫吸附技术,如蛋白A免疫吸附、IgE免疫吸附技术在自身免疫性疾病的治疗方面具有良好的效果。随着肿瘤相关免疫抑制性物质的发现,利用血液净化治疗恶性肿瘤成为可能。
外泌体是直径为40~160nm的膜性囊泡状小体,通常被认为是经由多泡体与细胞膜融合而释放到细胞外的。外泌体中含有多种生物活性物质,包括DNA、RNA、蛋白、脂质等。外泌体可通过其表面的蛋白分子或脂质配体直接激活目标细胞表面的受体,或与受体细胞的质膜融合而进入细胞并释放内含物,进而调控细胞的功能及生物学行为。源自转移乳腺癌细胞的外泌体可通过转运miR-200促进未转移乳腺癌细胞的MET和转移的发生。胃癌细胞分泌的外泌体能通过运输EGFR到Kupffer细胞和肝星状细胞促进肝脏特异性转移。来自B淋巴细胞的CD20+外泌体可竞争性结合抗CD20抗体,从而降低抗CD20疗法的效果。因此,清除外泌体是一种潜在的有效抑制肿瘤发生、转移、免疫逃逸以及提高药物疗效的方法。
外泌体表面含有大量的糖蛋白,越来越多关于外泌体糖基化分析的论文在近年发表。Bianca S. Batista等用凝集素微阵列技术分析了来自人黑色素瘤细胞、人结肠癌细胞、人T淋巴细胞、人T淋巴白血病细胞以及人乳汁来源的外泌体,发现外泌体富集高甘露糖、聚乳糖胺、α-2,6唾液酸和复杂的N-连聚糖,其中雪花莲凝集素、菜豆凝集素、鲎凝集素等可区分正常和恶性的外泌体。利用这一性质,有研究者利用凝集素捕获各种生物样本(如血浆、尿液)中的外泌体。US9707333以抗MHC I/II抗体、雪花莲凝集素(GNA)、抗HIV单克隆抗体、抗Fas抗体等为配基,分别偶联到不同的载体上,通过膜分离结合免疫吸附技术清除病人血液中的外泌体。CN111257073A将以小扁豆凝集素为配基,以sephrose 4B为固相载体制备吸附剂,该吸附剂能分离细胞培养上清、血浆、尿液、脑脊液中的外泌体,粒径为30-150nm。
NKG2D是一种同源二聚体II型跨膜C类凝集素受体,其表达于所有NK细胞、大部分CD8+T细胞、NKT细胞和γδT细胞的表面。免疫效应细胞可通过NKG2D受体与其配体的结合,识别并清除异常的体细胞。NKG2D配体种类繁多,包括MICA、MICB和六个不同的ULBP蛋白。NKG2D配体通常不表达,或者在健康人的组织里低水平表达,但通常在被感染细胞和癌细胞高水平表达 (Miller and Lanier 2019)。但是,NKG2D配体可从肿瘤细胞表面释放,形成可溶性NKG2D配体,包含游离的蛋白和外泌体形式的蛋白。Veronika Groh等研究表明,sMICA能诱导T细胞表面NKG2D的内化和降解。Ashiru等研究发现,MICA*08能以外泌体的形式存在,含MICA*08的外泌体可以显著下调NK细胞表面的NKG2D表达量,同时显著抑制NK细胞对靶细胞的毒性。Fernández-Messina等发现,可溶性ULBP2和ULBP3都能下调NK细胞表面NKG2D受体的表达,外泌体的ULBP3蛋白比可溶性的ULBP2蛋白能更有效地下调NKG2D受体,而且与不含ULBP3的外泌体相比,含ULBP3的外泌体能显著降低NK细胞对MICA表达细胞的溶解活性。免疫吸附是一种潜在的有效清除血液中可溶性NKG2D配体的方案。Weil等将抗MICA抗体共价偶联到微球上,然后制备成sMICA免疫吸附柱并连接到血浆分离器中,以100μg/L向恒河猴注射sMICA*004蛋白,经过三倍血浆体积的交换后,血浆中的sMICA几乎被完全清除。CN113683710A以NKG2D受体蛋白为活性配基制备了一种免疫吸附剂,该吸附剂对多种MICA、MICB都有良好的吸附效果。
将具有不同吸附能力的蛋白融合,理由上可以得到对多种组分具有吸附能力的融合蛋白,但是多数情况下,融合蛋白的吸附能力往往因为融合蛋白的结构不合理,导致其吸附能力较弱,稳定性差,难以满足实际应用的需要。开发一种对外泌体和MICA、MICB均具有良好吸附能力的GNA-NKG2D融合蛋白,具有非常的难度。
发明内容
本发明的目的在于克服现有技术的至少一个不足,提供一种GNA-NKG2D融合蛋白及用于恶性肿瘤治疗的外泌体吸附剂。
本发明利用不同的连接肽将雪花莲凝集素与NKG2D受体蛋白进行连接,第一方面能将两种功能的蛋白进行融合,方便表达多效性的蛋白;第二方面,采用柔性连接肽能使相邻的两分子GNA蛋白进行灵活的伸展和弯曲,便于形成聚集体;第三方面,采用刚性连接肽能够使相邻两种功能蛋白保持一定的间距,不相互影响,保证功能域的完整,使活性达到最佳;第四方面,本发明采用的刚性连接肽提高了蛋白的抗水解能力,提升GNA-NKG2D融合蛋白的稳定性和产率。
本发明所采取的技术方案是:
本发明的第一个方面,提供:
一种GNA-NKG2D融合蛋白,其结构式为(GNA)m-(NKG2D)n或(NKG2D)n-(GNA)m,m=2或4,n=1或2,其中,GNA的氨基酸序列如SEQ ID NO.:3所示,NKG2D的氨基酸序列如SEQ IDNO.:4所示。
在一些GNA-NKG2D融合蛋白的实例中,具有如下至少一个特征:
所述GNA-NKG2D融合蛋白的至少一端带有标签序列;
所述GNA-NKG2D融合蛋白的N端带Fc段;
GNA和GNA间、GNA和NKG2D间独立通过柔性连接肽或刚性连接肽连接。
在一些GNA-NKG2D融合蛋白的实例中,GNA和GNA间、GNA和NKG2D间通过柔性连接肽连接。不同功能肽片段间的连接肽相同或者不同。
在一些GNA-NKG2D融合蛋白的实例中,具有如下至少一个特征:
所述标签序列为His标签序列;
所述柔性连接肽由氨基酸G/S组成,其氨基酸序列优选为(GS)x、(GGS)x、(GGGS)x或(GGGGS)x,x=1~5;
所述刚性连接肽的结构式为(PX)n P,n=2~10的整数,X选自丙氨酸、组氨酸、亮氨酸、脯氨酸、苏氨酸、谷氨酰胺、精氨酸或缬氨酸;优选的刚性连接肽的结构式为 (PT)n P,n=4~7的整数。
在一些GNA-NKG2D融合蛋白的实例中,所述GNA-NKG2D融合蛋白的氨基酸序列如SEQ ID NO.:1、SEQ ID NO.:2、SEQ ID NO.:5或SEQ ID NO.:6所示。
本发明的第二个方面,提供:
一种GNA-NKG2D免疫吸附剂,包括固相载体,所述固相载体上共价偶联有本发明第一个方面所述的GNA-NKG2D融合蛋白。
在一些GNA-NKG2D免疫吸附剂的实例中,所述固相载体选自壳聚糖、琼脂糖、纤维素、葡聚糖、树脂、改性树脂中的至少一种和/或所述固相载体为超大孔微球介质。
在一些GNA-NKG2D免疫吸附剂的实例中,所述固相载体为Sephacryl S-1000SF葡聚糖、Giga AP琼脂糖改性聚苯乙烯超大孔微球介质或Giga AP琼脂糖改性聚苯乙烯超大孔微球介质;和/或
所述微球介质的孔径范围为100-1200nm和/或对球蛋白分离范围为105-108Da。
在一些GNA-NKG2D免疫吸附剂的实例中,所述微球介质的孔径范围为200-800nm和/或对球蛋白分离范围为107-108Da。
在一些GNA-NKG2D免疫吸附剂的实例中,其可吸附血液中的外泌体、可溶性NKG2D配体。
在一些GNA-NKG2D免疫吸附剂的实例中,用于净化血液,从而解除肿瘤相关外泌体异常升高引起的正常组织的癌变,以及肿瘤的转移、增殖和免疫逃逸和/或解除NKG2D配体异常升高引起的肿瘤免疫抑制作用。
本发明的有益效果是:
本发明的一些实例的GNA-NKG2D融合蛋白,具有与天然GNA、NKG2D蛋白相似的结构,保留了GNA对外泌体的吸附能力和NKG2D蛋白对MICA、MICB的吸附能力,具有意料之外的效果。
本发明的一些实例的GNA-NKG2D融合蛋白,有助于其形成GNA四聚体和NKG2D二聚体结构,更接近天然的活性结构,具有更好的吸附活性。
本发明一些实例的GNA-NKG2D融合蛋白制备的吸附剂可以有效吸附外泌体、MICA、MICB,以抑制恶性肿瘤的增殖、转移和免疫逃逸,具有治疗恶性肿瘤的潜力。
附图说明
图1是构建得到的质粒DNA电泳图,其中泳道1和2分别是SEQ ID NO.:1和SEQ IDNO.:2所示氨基酸序列对应的质粒,M为marker。
具体实施方式
本发明的第一个方面,提供:
一种GNA-NKG2D融合蛋白,其结构式为(GNA)m-(NKG2D)n或(NKG2D)n-(GNA)m,m=2或4,n=1或2,其中,GNA的氨基酸序列如SEQ ID NO.:3所示,NKG2D的氨基酸序列如SEQ IDNO.:4所示。
在一些GNA-NKG2D融合蛋白的实例中,所述GNA-NKG2D融合蛋白的至少一端带有标签序列。标签序列的引入,方便对融合蛋白进行纯化。标签序列本身没有特别的要求,可以是本领域常用的标签序列。
在一些GNA-NKG2D融合蛋白的实例中,所述GNA-NKG2D融合蛋白的N端带Fc段。Fc段的存在,使得融合蛋白可以自发形成二聚体,得到更接近天然活性结构的融合蛋白,发挥更好的效果。
在一些GNA-NKG2D融合蛋白的实例中,GNA和GNA间、GNA和NKG2D间独立通过柔性连接肽或刚性连接肽连接。
采用柔性连接肽能使相邻的两分子GNA蛋白进行灵活的伸展和弯曲,便于形成聚集体。柔性连接肽的种类没有特别要求,只要不影响GNA和NKG2D活性即可。
采用刚性连接肽能够使相邻两种功能蛋白保持一定的间距,不相互影响,保证功能域的完整,使活性达到最佳,刚性连接肽提高了蛋白的抗水解能力,提升GNA-NKG2D融合蛋白的稳定性和产率。刚性连接肽可以是本领域常用的刚性连接肽。
在一些GNA-NKG2D融合蛋白的实例中,GNA和GNA间、GNA和NKG2D间通过柔性连接肽连接。不同功能肽片段间的连接肽相同或者不同。
在一些GNA-NKG2D融合蛋白的实例中,GNA和GNA间、GNA和NKG2D间通过刚性连接肽连接。不同功能肽片段间的连接肽相同或者不同。
在一些GNA-NKG2D融合蛋白的实例中,GNA和GNA间、GNA和NKG2D间同时具有刚性连接肽和柔性连接肽。
His标签序列易于引入,同时具有良好的分离效果。在一些GNA-NKG2D融合蛋白的实例中,所述标签序列为His标签序列。
在一些GNA-NKG2D融合蛋白的实例中,所述柔性连接肽由氨基酸G/S组成。
在一些GNA-NKG2D融合蛋白的实例中,所述柔性连接肽的氨基酸序列为(GS)x、(GGS)x、(GGGS)x或(GGGGS)x,x=1~5。
在一些GNA-NKG2D融合蛋白的实例中,所述柔性连接肽的氨基酸序列为(GGGS)2、(GGGS)3。
在一些GNA-NKG2D融合蛋白的实例中,所述刚性连接肽的结构式为(PX)n P,n=2~10的整数,X选自丙氨酸、组氨酸、亮氨酸、脯氨酸、苏氨酸、谷氨酰胺、精氨酸或缬氨酸。
在一些GNA-NKG2D融合蛋白的实例中,刚性连接肽的氨基酸序列为 (PT)n P,n=4~7的整数。
在一些GNA-NKG2D融合蛋白的实例中,刚性连接肽的氨基酸序列为(PT)5 P。
上述特征在不互斥的情况下,可以自由组合。
在一些GNA-NKG2D融合蛋白的实例中,所述GNA-NKG2D融合蛋白的氨基酸序列如SEQ ID NO.:1、SEQ ID NO.:2、SEQ ID NO.:5或SEQ ID NO.:6所示。
本发明的第二个方面,提供:
一种GNA-NKG2D免疫吸附剂,包括固相载体,所述固相载体上共价偶联有本发明第一个方面所述的GNA-NKG2D融合蛋白。
在一些GNA-NKG2D免疫吸附剂的实例中,所述固相载体选自壳聚糖、琼脂糖、纤维素、葡聚糖、树脂、改性树脂中的至少一种和/或所述固相载体为超大孔微球介质。超大孔微球介质为本领域常用的固相载体。
在一些GNA-NKG2D免疫吸附剂的实例中,所述固相载体为Sephacryl S-1000SF葡聚糖、Giga AP琼脂糖改性聚苯乙烯超大孔微球介质或Giga AP琼脂糖改性聚苯乙烯超大孔微球介质。
在一些GNA-NKG2D免疫吸附剂的实例中,所述微球介质的孔径范围为100-1200nm和/或对球蛋白分离范围为105-108Da。
在一些GNA-NKG2D免疫吸附剂的实例中,所述微球介质的孔径范围为200-800nm和/或对球蛋白分离范围为107-108Da。
在一些GNA-NKG2D免疫吸附剂的实例中,其可吸附血液中的外泌体、可溶性NKG2D配体。
在一些GNA-NKG2D免疫吸附剂的实例中,用于净化血液,从而解除肿瘤相关外泌体异常升高引起的正常组织的癌变,以及肿瘤的转移、增殖和免疫逃逸和/或解除NKG2D配体异常升高引起的肿瘤免疫抑制作用。
下面结合实例,进一步说明本发明的技术方案。
实施例1 GNA-NKG2D载体构建及瞬时转染
分别使用不同的连接肽或不使用连接肽,设计得到不同的GNA-NKG2D融合蛋白,其中:
Fc-(GNA)2-NKG2D-1,同时含有柔性连接肽和刚性连接肽连接,氨基酸序列如SEQID NO.:1所示;
Fc-(GNA)2-NKG2D-2,由柔性连接肽GGGGS简单连接,氨基酸序列如SEQ ID NO.:5所示;
Fc-(GNA)2-NKG2D-3,不含连接肽,氨基酸序列如SEQ ID NO.:7所示;
(GNA)4-(NKG2D)2-His-1,同时含有柔性连接肽和刚性连接肽连接,氨基酸序列如SEQ ID NO.:2所示;
(GNA)4-(NKG2D)2-His-2,由柔性连接肽GGGGS简单连接,氨基酸序列如SEQ IDNO.:6所示;
(GNA)4-(NKG2D)2-His-3,不含连接肽,氨基酸序列如SEQ ID NO.:8所示。
选择pcDNA3.1为真核细胞表达载体,用生信软件将上述氨基酸序列转换成核苷酸序列,并进行序列优化。然后,分别设计限制性酶切位点并在C端加上终止密码子,委外进行全基因合成。获得含质粒的转化菌后,用LB液体培养基发酵,用QIAGEN EndoFree PlasmidKit处理菌液并提取质粒。电泳结果 (图1)表明质粒构建成功。
取处于对数生长期的且存活率≥90%的293F细胞,用无血清培养基将细胞稀释至4×106细胞/mL,置锥形培养瓶中备用。以生理盐水为介质,以120μL/mL加入Polyjet(SignaGen)配制成A液,以40μg/mL加入质粒DNA配制成B液,在室温静置5min,将A、B液等比例混合后静置10min,再将转染复合物以1:20加入到配制好的100mL 293F细胞悬液中,置二氧化碳振荡培养箱中以130rpm进行悬浮培养。转染72h后,离心,收集细胞培养上清。
实施例2 融合蛋白的纯化及鉴定
带Fc段的GNA-NKG2D融合蛋白的纯化:取1mL的蛋白A亲和层析填料,用纯化水清洗5倍柱体积,再用1X PBS平衡5倍柱体积,将收集的细胞培养液用孔径为0.45μm的滤膜过滤后上样至吸附柱,上样完毕后再用1X PBS平衡5倍柱体积,最后用pH=2.8, 100mM甘氨酸-盐酸缓冲液进行洗脱,再用pH=8.8的Tris-盐酸缓冲液中和,透析成PBS体系,用BCA法测蛋白浓度,计算产率,用HPLC测蛋白纯度,结果见表1。
带His标签的GNA-NKG2D融合蛋白的纯化:取1mL的钴离子亲和层析填料,用纯化水清洗5倍柱体积,再用1X PBS平衡5倍柱体积,将收集的细胞培养液用孔径为0.45μm的滤膜过滤后上样至吸附柱,上样完毕后再用1X PBS平衡5倍柱体积,最后用pH=7.4,500mM的咪唑-PBS溶液进行洗脱,透析成PBS体系,用BCA法测蛋白浓度,计算产率,用HPLC测蛋白纯度,结果见表1。
表1、不同融合蛋白的产率及纯度
由表1的结果可知,连接肽的加入会使融合蛋白的产率和纯度降低,但是通过优化连接肽的序列,可以减少连接肽对融合蛋白的产率和蛋白纯度的影响。对比SEQ ID NO.:1和SEQ ID NO.:5可知,通过优化连接肽,可以显著提高融合蛋白的产率和纯度。对比SEQ IDNO.:2和SEQ ID NO.:6可以得出类似的结论。SEQ ID NO.:1和SEQ ID NO.:2使用的连接肽,具有显著更佳的效果。
实施例3 GNA-NKG2D免疫吸附剂的制备
取5mL的Giga AP微球,加入10mL 0.02M的氢氧化钠,加入3.0mL的环氧氯丙烷溶液,在37℃以120rpm反应2h后,用纯化水冲洗填料。然后加入5mL的1X PBS溶液,加入3.0mL的乙二胺溶液,在37℃以120rpm反应3h后,用纯化水冲洗填料。再加入5mL的1X PBS溶液,加入5mL的戊二醛溶液,在37℃以120rpm反应3h后,用纯化水冲洗填料。
实施例2制备的GNA-NKG2D融合蛋白缓冲液置换成pH=8.3的碳酸盐缓冲液,按照投料量10mg/mL加入填料中,在37℃以120rpm反应3h。然后加入10mL的1M的乙醇胺,在25℃以120rpm反应2h后,用纯化水冲洗填料,得到GNA-NKG2D免疫吸附剂,保存于20%乙醇中备用。
实施例4 外泌体的分离纯化
取贴壁型宫颈癌细胞Hela(MICA*008/008),用含10%胎牛血清的DMEM培养基培养至细胞达70%汇合度后,弃去培养上清,用1X PBS清洗贴壁细胞1~2次后,用无血清的DMEM培养基培养24h。收集培养上清,以2000xg离心30分钟,弃去细胞及其碎片。取上清,用截留分子量为500kD的超滤管进行超滤浓缩并去除杂蛋白,浓缩倍数为20倍。加入0.5倍体积的总外泌体分离试剂(ThermoFisher,4478359),混匀,在4℃放置过夜。随后在4℃以10,000xg离心1小时,弃上清,用1X PBS重悬沉淀,即为外泌体。用纳米颗粒示踪分析进行定量和纯度鉴定,并保存于4℃备用。
从-80℃取出冻存的胰腺癌、乳腺癌、头颈癌病人的血浆样品,以37℃水浴融解样品,以2000xg在室温离心20分钟,除去细胞及其碎片。取上清至新的离心管,以10,000xg在室温离心20分钟,进一步除去碎片。取上清,用截留分子量为500kD的超滤管进行超滤浓缩并去除杂蛋白,浓缩倍数为5倍。将浓缩后的样品转移到新的离心管中,并加入0.5倍体积的1X PBS,混匀。再加入0.2倍总溶液体积的外泌体沉淀试剂(ThermoFisher,4484450),混匀后在室温孵育10分钟,再以10,000xg在室温离心5分钟。弃上清,以10,000xg在室温离心30秒,弃去残留试剂。用1X PBS重悬沉淀,即为外泌体。用纳米颗粒示踪分析进行定量和纯度鉴定,并保存于4℃备用。
实施例5 免疫吸附剂对外泌体的吸附性能评价
取实施例4中从Hela培养上清提取的外泌体和胰腺癌病人血浆中提取的外泌体,每种外泌体溶液的浓度为2×1010个/mL,各取2mL外泌体溶液,与1mL实施例3合成的GNA-NKG2D免疫吸附剂混合,在室温以90rpm反应1h。取反应前后的溶液,分别用纳米颗粒示踪分析法检测吸附前后上清液中的外泌体浓度,计算清除率,并以未偶联蛋白的Giga AP微球为空白对照。实验结果如表2所示。
表2 不同免疫吸附剂对外泌体的吸附效果
由表2可知,不同的GNA-NKG2D融合蛋白均具有一定的吸附能力,但其吸附性能存在显著的差异。根据吸附性能评价的结果,以Fc-(GNA)2-NKG2D蛋白(SEQ ID NO.:1)和(GNA)4-(NKG2D)2-His蛋白(SEQ ID NO.:2)为配基制备的吸附剂对各种类型的外泌体都具有良好的吸附效果,吸附率在80%以上,为优选配基。
Fc-(GNA)2-NKG2D-1氨基酸序列:
METDTLLLWVLLLWVPGSTG- THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK- PTPTPTPTPTPTPTP- SCLSDNILYSGETLSTGEFLNYGSFVFIMQED CNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWA TG -GGGGSGGGGS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQT DGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG - PTPTPTPTPGGGGS-IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV(SEQ ID NO.:1)。
Fc-(GNA)2-NKG2D-2氨基酸序列:
METDTLLLWVLLLWVPGSTG- THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK- GGGGSGGGGSGGGGS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQED CNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWA TG- GGGGSGGGGS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQT DGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG - GGGGSGGGGSGGGGS-IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV(SEQ ID NO.:5)。
Fc-(GNA)2-NKG2D-3氨基酸序列:
METDTLLLWVLLLWVPGSTG- THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNT GGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG - SCLSDNILYSGETLS TGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCI LQKDRNVVIYGTDRWATG -IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV(SEQ ID NO.:7)。
上述氨基酸序列中,单纯下划线标记的部分为信号肽,单纯斜体标记的部分为Fc标签序列,粗体为连接肽序列,下划线+斜体标记部分为雪花莲凝集素(GNA)部分,最后一部分是NKG2D序列。
(GNA)4-(NKG2D)2-His-1氨基酸序列:
MYRMQLLSCIALSLALVTNS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWAT NTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG -GGGGSGGGGS- SC LSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWA SNTGGQNGNYVCILQKDRNVVIYGTDRWATG -GGGGSGGGGS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDC NLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG -GGGGSGGGGS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGN LVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG -PTPTPTPTPGGGGS-IWSAVFLNSLFNQEVQ IPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNG SWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV-GGGGSGGGGS-IWSAVFLNSLFN QEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHI PTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV-HHHHHH(SEQ ID NO.:2)。
(GNA)4-(NKG2D)2-His-2氨基酸序列:
MYRMQLLSCIALSLALVTNS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWAT NTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG -GGGGSGGGGS- SC LSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWA SNTGGQNGNYVCILQKDRNVVIYGTDRWATG -GGGGSGGGGS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDC NLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG -GGGGSGGGGS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGN LVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG -GGGGSGGGGSGGGGS-IWSAVFLNSLFNQEV QIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTN GSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV-GGGGSGGGGS-IWSAVFLNSLF NQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVH IPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV-HHHHHH(SEQ ID NO.:6)。
(GNA)4-(NKG2D)2-His-3氨基酸序列:
MYRMQLLSCIALSLALVTNS- SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWAT NTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG - SCLSDNILYSGE TLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNY VCILQKDRNVVIYGTDRWATG - SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLS RSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG - SCLSDNILYSGETLSTGEF LNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKD RNVVIYGTDRWATG -IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNAS LLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTY ICMQRTV-IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSK EDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV-HHHHHH(SEQ ID NO.:8)。
上述氨基酸序列中,单纯下划线标记部分为信号肽,下划线+斜体标记部分为雪花莲凝集素(GNA)部分,粗体为连接肽序列,单纯斜体标记部分为NKG2D序列,最后为His标签序列。
GNA氨基酸序列:
SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG (SEQ ID NO.:3) 。
NKG2D氨基酸序列:
IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV(SEQ ID NO.:4) 。
以上是对本发明所作的进一步详细说明,不可视为对本发明的具体实施的局限。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的简单推演或替换,都在本发明的保护范围之内。
Claims (10)
1.一种GNA-NKG2D融合蛋白,其结构式为(GNA)m-(NKG2D)n或(NKG2D)n-(GNA)m,m=2或4,n=1或2,其中,GNA的氨基酸序列如SEQ ID NO.:3所示,NKG2D的氨基酸序列如SEQ IDNO.:4所示。
2.根据权利要求1所述的GNA-NKG2D融合蛋白,其特征在于,具有如下至少一个特征:
所述GNA-NKG2D融合蛋白的至少一端带有标签序列;
所述GNA-NKG2D融合蛋白的N端带Fc段;
GNA和GNA间、GNA和NKG2D间独立通过柔性连接肽和/或刚性连接肽连接。
3.根据权利要求2所述的GNA-NKG2D融合蛋白,其特征在于,具有如下至少一个特征:
所述标签序列为His标签序列;
所述柔性连接肽由氨基酸G/S组成;
所述刚性连接肽的结构式为(PX)n P,n=2~10的整数,X选自丙氨酸、组氨酸、亮氨酸、脯氨酸、苏氨酸、谷氨酰胺、精氨酸或缬氨酸;
所述GNA-NKG2D融合蛋白的氨基酸序列如SEQ ID NO.:1、SEQ ID NO.:2、SEQ ID NO.:5或SEQ ID NO.:6所示。
4.根据权利要求3所述的GNA-NKG2D融合蛋白,其特征在于,所述柔性连接肽的氨基酸序列为(GS)x、(GGS)x、(GGGS)x或(GGGGS)x,x=1~5;和/或
所述刚性连接肽的氨基酸序列为(PT)n P,n=4~7的整数。
5.一种GNA-NKG2D免疫吸附剂,包括固相载体,其特征在于,所述固相载体上共价偶联有权利要求1~4任一项所述的GNA-NKG2D融合蛋白。
6.根据权利要求5所述的GNA-NKG2D免疫吸附剂,其特征在于,所述固相载体选自壳聚糖、琼脂糖、纤维素、葡聚糖、树脂、改性树脂中的至少一种和/或所述固相载体为超大孔微球介质。
7.根据权利要求6所述的GNA-NKG2D免疫吸附剂,其特征在于,所述固相载体为Sephacryl S-1000SF葡聚糖、Giga AP琼脂糖改性聚苯乙烯超大孔微球介质或Giga AP琼脂糖改性聚苯乙烯超大孔微球介质;和/或
所述微球介质的孔径范围为100-1200nm和/或对球蛋白分离范围为105-108Da。
8.根据权利要求6或7所述的GNA-NKG2D免疫吸附剂,其特征在于,所述微球介质的孔径范围为200-800nm和/或对球蛋白分离范围为107-108Da。
9.根据权利要求5~7任一项所述的GNA-NKG2D免疫吸附剂,其特征在于,其可吸附血液中的外泌体、可溶性NKG2D配体。
10.根据权利要求5~7任一项所述的GNA-NKG2D免疫吸附剂,其特征在于,用于净化血液,从而解除肿瘤相关外泌体异常升高引起的正常组织的癌变,以及肿瘤的转移、增殖和免疫逃逸和/或解除NKG2D配体异常升高引起的肿瘤免疫抑制作用。
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