CN114891091B - 一种抗nmdar脑炎重组抗原及应用 - Google Patents
一种抗nmdar脑炎重组抗原及应用 Download PDFInfo
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Abstract
本发明公开了一种抗NMDAR脑炎重组抗原及应用,抗原包括NR1或NR2A或NR2B或NR2C蛋白的N端结构域,或包含NR1、NR2A、NR2B、NR2C蛋白中的任意两种或多种的N端结构域,不包含其的跨膜片段、跨膜区域和C端结构域。还公开了编码抗NMDAR脑炎重组抗原的基因片段、含基因片段的重组蛋白表达载体、以及含抗NMDAR脑炎重组抗原的试剂盒。本发明设计的重组蛋白不需要重组表达完整的NMDAR蛋白,不但简化了蛋白重组表达的过程,而且大大降低了重组蛋白的纯化难度,同时有效提高了抗原的特异性。本发明提供的试剂盒临床检测准确度高,灵敏性好,能够满足不同人群不同场景的需求。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种抗NMDAR脑炎重组抗原及应用。
背景技术
自身免疫性脑炎(Autoimmune encephalitis,AE)是一类由自身免疫机制介导的脑炎,抗N-甲基-D-天冬氨酸受体(NMDAR)脑炎是AE的最主要类型,抗NMDAR脑炎患者占AE的比例为80%。N-甲基-D-天冬氨酸受体(NMDAR)是配体门控型的阳离子通道,与正常的神经网络形成、突触的传递和可塑性、学习和记忆等大脑功能密切相关。NMDAR的过度激活已被证明介导了急性神经元死亡和慢性神经变性,相反NMDAR的低激活与精神状态的发展有关。在抗NMDAR脑炎患者体内,抗NMDAR抗体与NMDAR蛋白交联并内化靶受体,从突触中消耗NMDAR蛋白,从而导致突触功能的缺失。
在2016年发表于柳叶刀的抗NMDAR脑炎诊断标准中,通过工作记忆缺陷的亚急性发作,中枢神经系统局灶性新发现,癫痫发作,脑脊液细胞增多,磁共振成像等症状可诊断为疑似抗NMDAR脑炎。只有通过脑脊液或血清中神经元抗体的检测才能最终确诊为抗NMDAR脑炎,换而言之,自身抗体的准确检测是抗NMDAR脑炎诊断过程中至关重要的一环。并且早期的诊断与及时的免疫治疗对抗NMDAR脑炎患者预后较好。
目前临床常用间接免疫荧光法(IIF)来检测AE相关抗体,其对操作者技术、经验要求比较高,在结果判断过程中主观性较强,且各实验室检测结果难以标准化。由于AE研究起步较晚,目前市场上抗NMDAR抗体体外检测试剂盒种类较少,技术也不够成熟。
现有技术通常是设计重组表达完整的NMDAR蛋白,但是由于其冗长的基因序列和复杂的跨膜结构,都提高了蛋白重组表达和纯化的难度。因此,有必要开发新型的抗NMDAR脑炎重组抗原。
发明内容
本发明要解决的技术问题是现有的抗NMDAR脑炎重组抗原均是设计重组表达完整的NMDAR蛋白,导致蛋白重组表达和纯化难度高,抗原特异性较差。目的在于提供一种抗NMDAR脑炎重组抗原及应用,以解决以上问题。
本发明通过下述技术方案实现:
本发明的第一个目的在于提供一种抗NMDAR脑炎重组抗原,包括NR1蛋白、NR2A蛋白、NR2B蛋白、NR2C蛋白中的任一种或几种的N端结构域,所述N端结构域不包含相应蛋白的跨膜片段、跨膜区域和C端结构域;
所述NR1蛋白的N端结构域基因包括编码NR1蛋白的如SEQ ID NO:1所示的19~559氨基酸序列;
NR2A蛋白的N端结构域基因包括编码NR2A蛋白的如SEQ ID NO:2所示的23~555氨基酸序列;
NR2B蛋白的N端结构域基因包括编码NR2B蛋白的如SEQ ID NO:3所示的27~557氨基酸序列;
NR2C蛋白的N端结构域基因包括编码NR2C蛋白的如SEQ ID NO:4所示的20~554氨基酸序列。
优选地,所述重组抗原还包括N端真核KOZAK序列、蜂毒信号肽,按照N端真核KOZAK序列、蜂毒信号肽、NR1、NR2A、NR2B、NR2C蛋白的顺序融合。
本发明的第二个目的在于提供一种编码上述抗NMDAR脑炎重组抗原的基因片段。
优选地,编码NR1蛋白的19~559氨基酸的核苷酸序列如SEQ ID NO:5所示;编码NR2A蛋白的23~555氨基酸的核苷酸序列如SEQ ID NO:6所示;编码NR2B蛋白的27~557氨基酸的核苷酸序列如SEQ ID NO:7所示;编码NR2C蛋白的20~554氨基酸的核苷酸序列如SEQ ID NO:8所示。
本发明的第三个目的在于提供一种含上述基因片段的重组蛋白表达载体。
本发明的第四个目的在于提供一种试剂盒,该试剂盒包含上述的抗NMDAR脑炎重组抗原。
可选地,试剂盒为包含如上述任一项所述的抗NMDAR脑炎重组抗原的胶体金免疫层析试剂盒、免疫印迹检测试剂盒、酶联免疫检测试剂盒、纳米磁微粒管式化学发光检测试剂盒。
本发明的第五个目的在于提供上述任一项所述的抗NMDAR脑炎重组抗原在制备用于检测抗NMDAR脑炎抗体试剂盒的应用。
本发明具有如下的优点和有益效果:
本发明的重组蛋白的重组部分主要是NR1亚基和NR2亚基的胞外N端结构域(NTDs),不需要重组表达完整的NMDAR蛋白,不但简化了蛋白重组表达的过程,而且大大降低了重组蛋白的纯化难度。同时通过选取编码NR1、NR2A、NR2B、NR2C蛋白的合适位置的氨基酸序列,设计NMDAR蛋白的N端结构域基因,仅利用NR1、NR2A、NR2B和NR2C蛋白的部分胞外结构,相较于现有完整的NMDAR蛋白,不仅在NMDAR蛋白重组及纯化上简便易得,同时能够有效提高抗原的特异性。本发明提供的试剂盒临床检测准确度高,灵敏性好。且四种试剂盒各具特点,能够满足不同人群不同场景的需求。
附图说明
为了更清楚地说明本发明示例性实施方式的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以如这些附图获得其他相关的附图。在附图中:
图1为本发明实施例二的NR1亚基抗体磁微粒化学发光试剂盒校准品线性图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
在以下描述中,为了提供对本发明的透彻理解阐述了大量特定细节。然而,对于本领域普通技术人员显而易见的是:不必采用这些特定细节来实行本发明。在其他实施例中,为了避免混淆本发明,未具体描述公知的材料或方法。
一、本发明提供了一种抗NMDAR脑炎重组抗原,此重组抗原是重组蛋白。包括NR1蛋白的N端结构域,不包含其的跨膜片段、跨膜区域和C端结构域,NR1蛋白的N端结构域基因包括编码NR1蛋白的如SEQ ID NO:1所示的19~559氨基酸序列。将此重组蛋白定义为重组蛋白NR1。
研究发现,NMDAR是包含不同亚基的异质复合体,所包含的亚基有NR1、NR2、NR3。NMDAR还具有亚单位多样性。功能性NMDAR以四聚体的形式存在,最常见的组合形式是包含两个NR1亚基和两个NR2亚基。NMDAR的不同亚基都有着相似的跨膜拓扑结构,包括胞外的N端结构域(NTDs)、由3个跨膜片段和回环组成的跨膜区域、胞浆内的C端结构域。现有技术是设计重组表达完整的NMDAR蛋白,但是由于其冗长的基因序列和复杂的跨膜结构,都提高了蛋白重组表达和纯化的难度。本发明利用NMDAR的胞外N端结构域(NTDs)作为靶抗原,提高检测的灵敏度、特异性;同时重组抗原中去除NR1蛋白的N端结构域中的跨膜片段、跨膜区域和C端结构域,不但简化了蛋白重组表达的过程,而且大大降低了重组蛋白的纯化难度。同时发明人经过大量的实验和验证,NR1蛋白的N端结构域基因包括编码NR1蛋白(Gene ID:2902)的19~559氨基酸序列。不需要重组表达完整的NMDAR蛋白,且检测灵敏性和特异性明显提高。其中编码NR1蛋白(Gene ID:2902)的19~559氨基酸序列如SEQ ID NO:1所示。
本发明提供的抗NMDAR脑炎重组抗原还可以是包括NR2A、NR2B、NR2C蛋白中的任意一种的N端结构域,不包含各蛋白的跨膜片段、跨膜区域和C端结构域;
NR2A蛋白的N端结构域基因包括编码NR2A蛋白(Gene ID:2903)的如SEQ ID NO:2所示的23~555氨基酸序列。将此重组蛋白定义为重组蛋白NR2A。NR2B蛋白的N端结构域基因包括编码NR2B蛋白(GeneID:2904)的如SEQIDNO:3所示的27~557氨基酸序列。将此重组蛋白定义为重组蛋白NR2B。NR2C蛋白的N端结构域基因包括编码NR2C蛋白(GeneID:2905)的如SEQIDNO:4所示的20~554氨基酸序列。将此重组蛋白定义为重组蛋白NR2C。
重组的NR2A或NR2B或NR2C蛋白同样不需要重组表达完整的NMDAR蛋白,不但简化了蛋白重组表达的过程,而且大大降低了重组蛋白的纯化难度。同时发明人经过大量的实验和验证,重组的NR2A或NR2B或NR2C蛋白的检测灵敏性和特异性均明显提高。
同样地,本发明的抗NMDAR脑炎重组抗原还可以是包括NR1、NR2A、NR2B、NR2C蛋白中的至少任意两种的N端结构域,不包含各蛋白的跨膜片段、跨膜区域和C端结构域;
NR1、NR2A、NR2B、NR2C蛋白的N端结构域基因采用与上述相同的氨基酸序列。定义重组蛋白为重组蛋白NR1+NR2A,其他重组方式按照相同的方法定义。
进一步地,重组蛋白还包括N端真核KOZAK序列、蜂毒信号肽,按照N端真核KOZAK序列、蜂毒信号肽、NR1、NR2A、NR2B、NR2C蛋白的顺序融合。本领域技术人员应该知晓,此处的按照N端真核KOZAK序列、蜂毒信号肽、NR1、NR2A、NR2B、NR2C蛋白的顺序融合是指:若重组蛋白中没有NR1或NR2A或NR2B或NR2C蛋白,则跳过并按照上述的顺序进行融合。
通过选取编码NR1、NR2A、NR2B、NR2C蛋白的合适位置的氨基酸序列,设计NMDAR蛋白的N端结构域基因,设计抗NMDAR脑炎重组抗原,不需要重组表达完整的NMDAR蛋白,仅利用NR1、NR2A、NR2B和NR2C蛋白的部分胞外结构,相较于现有完整的NMDAR蛋白,胞外结构域不仅在NMDAR蛋白重组及纯化上简便易得,同时能够有效提高抗原的特异性。
抗NMDAR脑炎重组抗原的制备过程包括:(1)将NMDAR重组蛋白基因序列构建重组杆粒;(2)将重组杆粒转化至昆虫细胞,进行表达;(3)细胞培养,筛选纯化。其中NMDAR重组抗原包括如上所述的NR1或NR2A或NR2B或NR2C蛋白,或者包括上述四种蛋白的任意两种或多种。
二、本发明实施例还提供了编码上述抗NMDAR脑炎重组抗原的基因片段。
其中编码NR1蛋白(Gene ID:2902)的19~559氨基酸的核苷酸序列如SEQ ID NO:5所示。编码NR2A蛋白(Gene ID:2903)的23~555氨基酸的核苷酸序列如SEQ ID NO:6所示。编码NR2B蛋白(Gene ID:2904)的27~557氨基酸的核苷酸序列如SEQ ID NO:7所示。编码NR2C蛋白(Gene ID:2905)的20~554氨基酸的核苷酸序列如SEQ ID NO:8所示。
三、本发明实施例还提供了还提供一种含上述基因片段的重组蛋白表达载体。具体的重组蛋白表达载体的构建过程见下具体实施例。
四、本发明实施例利用上述得到的重组抗原制备检测试剂盒,该试剂盒包含上述的抗NMDAR脑炎重组抗原。此试剂盒可以为胶体金免疫层析试剂盒、免疫印迹检测试剂盒、酶联免疫检测试剂盒、纳米磁微粒管式化学发光检测试剂盒中的任一种。
(1)基于NMDAR重组抗原的胶体金免疫层析检测试剂盒
本发明开发的胶体金试剂固相载体为硝酸纤维素膜,膜上依次排布有样品垫、结合垫、检测线、对照线、吸水垫。其中结合垫吸附有胶体金标记的鼠抗人IgG抗体,检测线上吸附有NMDAR重组抗原,对照线上吸附有羊抗鼠IgG抗体。
检测时将待检样品滴加至样品垫上,样品通过毛细作用向前移动,通过结合垫时与胶体金标记的鼠抗人IgG抗体结合,再层析至检测线时,胶体金标记的NMDAR抗体被检测线上的NMDAR重组抗原特异性捕获,聚集在检测带上,可通过肉眼观察到显色结果。而剩余的胶体金继续层析至对照线,与对照线上的羊抗鼠IgG抗体结合而被截留。因此只有检测线与对照线同时出现红色线条,表示样本中存在NMDAR抗体。
其中检测线上固定的NMDAR重组抗原可以是重组NR1蛋白,也可以是重组NR2A蛋白、重组NR2B蛋白、重组NR2C蛋白中的任一种,或者任意两种或多种的结合。
(2)、基于NMDAR重组抗原的免疫印迹法检测试剂盒
本发明开发的免疫印迹试剂盒包括反应膜条、酶结合物、显色底物、清洗液、样本稀释液和终止液。
在硝酸纤维素膜上包被特异性NMDAR重组抗原,加入稀释后的待测样本,特异性的NMDAR抗体与抗原结合,清洗后加入酶结合物,形成抗原-抗体-酶标二抗的免疫复合物,加入的显色底物再酶催化作用下发生显色反应,颜色的深浅和待测抗体浓度成正比。
其中反应膜上可以平行包被重组蛋白NR1,也可以平行包被重组蛋白NR2A、重组蛋白NR2B、重组蛋白NR2C中的任一种,或者任意两种或多种的结合。
(3)、基于NMDAR重组抗原的酶联免疫吸附法检测试剂盒
本发明开发的ELISA试剂盒包括微孔板、校准品、阳性对照、阴性对照、酶结合物、样本稀释液、浓缩清洗液、显色底物、终止液。
在聚苯乙烯微孔板上包被特异性NMDAR抗原,加入稀释后的待测样本,特异性的NMDAR抗体与抗原结合,清洗后加入酶结合物,形成抗原-抗体-酶标二抗的免疫复合物,加入的显色底物再酶催化作用下发生显色反应,显色底物最终的吸光度和待测抗体浓度成正比。
其中微孔板包被的NMDAR重组抗原可以是重组蛋白NR1,也可以是重组蛋白NR2A、重组蛋白NR2B、重组蛋白NR2C中的任一种,或者任意两种或多种的结合。
(4)、基于纳米磁微粒管式化学发光技术的NMDAR抗体检测试剂盒
本发明开发的化学发光试剂盒包括磁珠(M)试剂、R1试剂(稳定剂)、R2试剂以及配套的校准品、质控品、发光底物液。
在本试剂盒中,M试剂为表面包被有NMDAR抗原的磁珠试剂,检测时依次将待测样本(血清或脑脊液)、R1试剂、M试剂加入到反应杯中,样本中的NMDAR抗体与磁珠表面的抗原特异性结合形成免疫复合物,在外加磁场中产生聚沉,去上清液后清洗沉淀的复合物,加入R2试剂,形成抗原-抗体-酶标二抗(或者抗原-抗体-发光剂标记二抗)的夹心免疫复合物,再次清洗后加入发光底物(或者激发剂),催化化学发光反应产生的光子数和待测样本中的NMDAR抗体成正比。
其中M试剂中的磁微粒可以选用链霉亲和素磁珠、羧基磁珠、甲苯磺酰基磁珠中的一种或多种。
其中二抗标记酶可以选用碱性磷酸酶或者辣根过氧化物酶,对应的发光底物可以选用AMPPD和鲁米诺。
其中发光剂可以选用吖啶酯、异鲁米诺或者三联吡啶钌,对应的激发剂可以选用NaOH/H2O2、三丙胺。
其中纳米磁微粒上偶联的NMDAR重组抗原可以是重组蛋白NR1,也可以是重组蛋白NR2A、重组蛋白NR2B、重组蛋白NR2C中的任一种,或者任意两种或多种的结合。
上述的四种试剂盒均能够实现对NMDAR抗体的准确高效的检测。四种试剂盒各具特点,能够满足不同人群不同场景的需求。其中,胶体金免疫层析试剂盒方便快捷、操作简便、结果判读直接明了,适用于小范围的初步诊断。免疫印迹法检测试剂盒可同时检测样本对NMDAR蛋白不同亚基的反应性,适用于实验室或研究所对抗原抗体结合以及致病机理的进一步研究。酶联免疫试剂盒具有较高的灵敏度,可根据需求实现小批量或大批量的筛查。纳米磁微粒管式化学发光检测试剂盒具有快速安全、高精准、全定量、全自动等优点,能够实现对患者体内的抗体水平进行动态监测的效果。
上述的各种试剂盒通过对4种蛋白进行联合检测,来提高NMDAR抗体检测的灵敏度以及特异性。
以下通过具体实施例对本发明技术方案进行进一步地阐述。
实施例一:不同重组蛋白的制备
1、重组NR1、NR2A、NR2B、NR2C蛋白基因的全序列合成
(1)人工构建重组蛋白NR1的基因序列,包括N端真核KOZAK序列、蜂毒信号肽、NMDAR蛋白N端结构域基因,其中NMDAR蛋白N端结构域基因包括编码NR1蛋白(GeneID:2902)的19~559氨基酸序列;重组蛋白的基因序列前后分别包含BamHI和EcoRI酶切位点,最后由擎科生物公司进行全基因合成,并构建至常规质粒载体中,具体的全基因合成过程采用常规技术。
(2)人工构建重组蛋白NR2A的基因序列,包括N端真核KOZAK序列、蜂毒信号肽、NMDAR蛋白N端结构域基因,其中NMDAR蛋白N端结构域基因包括编码NR2A蛋白(GeneID:2903)的23~555氨基酸序列。其余与(1)中相同。
(3)人工构建重组蛋白NR2B的基因序列,包括N端真核KOZAK序列、蜂毒信号肽、NMDAR蛋白N端结构域基因,其中NMDAR蛋白N端结构域基因包括编码NR2B蛋白(GeneID:2904)的27~557氨基酸序列。其余与(1)中相同。
(4)人工构建重组蛋白NR2C的基因序列,包括N端真核KOZAK序列、蜂毒信号肽、NMDAR蛋白N端结构域基因,其中NMDAR蛋白N端结构域基因包括编码NR2C蛋白(GeneID:2905)的20~554氨基酸序列。其余与(1)中相同。
2、NMDAR蛋白基因的质粒构建
将获得的4种重组蛋白基因的PCR片段分别与经过限制性内切酶NcoI和EcoRI双酶切的载体pFastBacTMHTA进行连接。连接产物转化于感受态大肠杆菌DH5α中,体积不超过感受态细胞的10%,轻轻旋转几次混匀内容物,冰浴30min,将管放入42℃水浴,定时60s热休克,快速将管转移到冰浴120s,使细胞冷却,每管加入500μLLB培养基,37℃缓摇60min,使细菌复苏并表达质粒编码的抗生素抗性标记基因,低速离心2min,去上清,留约150μL培养基在离心管内,重悬菌体,用玻璃铺菌器将菌液在含Amp抗生素的LB琼脂板上铺匀。将平板倒置于37℃恒温培养箱培养12小时后,挑取阳性克隆提取质粒进行双酶切验证。
3、重组杆粒的构建及重组蛋白的表达
分别将4种正确重组质粒转化大肠杆菌DH10Bac感受态细胞,复苏时间4h,摇床转速提高至200rpm,用玻璃铺菌器将50μL菌液在含有Kan+Gent+Tet的LB琼脂板上铺匀,将平板倒置培养于37℃恒温培养箱中。48h后可出现蓝白斑菌落,挑取阳性白斑单菌落接入5mLKan+Gent+Tet的LB培养液中,37℃培养16h,取菌液进行PCR鉴定,结果显示杆粒重组正确。
将重组杆粒经转染试剂转染昆虫细胞sf9,3天后离心收取细胞上清液,-80℃保存作为第一代杆状病毒。用第一代杆状病毒感染昆虫细胞sf9约48-96小时后,收获第二代病毒,用于重组蛋白的表达。使用第二代病毒1:100体积比感染密度>2x106/mL的昆虫细胞sf9,在SIMSF培养基继续培养72h后,8000rpm离心10min收集细胞培养上清。4种样品-20℃保存待用,取少量上清煮沸至少3min,分别用SDS-PAGE检测4种重组蛋白的表达。
4、NMDAR重组蛋白的纯化
分别用200mL预冷的裂解液(10mMHEPESpH7.5,10mMMgCl2,20mMKCl)重悬1L细胞培养物离心沉淀,用匀浆器在冰上进行匀浆,匀浆后用超速离心机离心45min,去掉上清。重复洗涤3次,再用高盐溶液重复洗涤三次,提取物用含甘油的裂解液溶解并液氮速冻,储存在-80℃冰箱。冰上融化解冻提取物,加入茶碱和碘乙酰胺分别至终浓度为4mM和2mg/mL,冰上放置30min后按照每升提取物加入100mL溶膜缓冲液比例加入溶膜缓冲液,并继续在冰上放置3h溶膜,用超速离心机在160000g离心力下离心40min。
弃沉淀,上清加至平衡好的Ni-NTARESIN孵育16h,弃上清,加入适量平衡缓冲液重悬填料,将填料转移到自流柱里。
冲洗缓冲液1(25mMTris-Hcl pH8.0;500mMNaCl)洗10个柱体积。
冲洗缓冲液2(25mMTris-Hcl pH8.0;500mMNaCl;20mM咪唑)洗10个柱体积。
冲洗缓冲液3(25mMTris-Hcl pH8.0;500mMNaCl;250mM咪唑)洗5个柱体积。
洗脱液洗脱目的蛋白,纯化得到的目的蛋白保存在-80℃。SDS-PAGE电泳分别检测4种表达蛋白纯度及浓度,4种目的蛋白的纯度均大于80%,浓度在0.5~1.0mg/mL之间不等。实施例二:含不同重组蛋白的磁微粒化学发光试剂盒的检测性能研究
1、磁微粒化学发光试剂盒配制(M、R2、R1、校准品)
在羧基磁珠表面分别包被4种NMDAR重组蛋白(重组NR1,重组NR2A,重组NR2B,重组NR2C蛋白),用PBS缓冲液稀释成0.2mg/mL的工作液作为M试剂;
在碱性磷酸酶(AP)表面标记抗人IgG抗体,用MES缓冲液稀释,以此作为R2试剂;
在PBS缓冲液中添加1%BSA和0.5%TritonX100作为稳定剂,以此作为R1试剂;
以四种NMDAR重组蛋白为免疫原筛选出高亲和力IgG单克隆抗体,并与人源IgG抗体偶联,以此作为校准品和质控品的原料;将校准质控品的原料按一定比例用柠檬酸缓冲液稀释成12.5、25、50、100、200、400RU/mL作为校准品。
2、试剂盒性能测试
对以上设计好的试剂盒分别进行临床比对分析(阳性符合率、阴性符合率)、准确度、线性、精密度和稳定性的考察。
(1)临床比对
用以上4种亚基的试剂盒对20例临床诊断为抗NMDAR脑炎的样本进行了联合检测(表1)。结果表明,抗NMDAR抗体联合检测的阳性符合率为100%(表2),说明本试剂盒临床阳性检出率高,灵敏度好。对100例随机健康人体检样本进行了测定。结果表明,抗NMDAR抗体检测试剂盒阴性符合率为99%,说明本试剂盒临床阴性符合率高,特异性好。
表1:4种亚基在NMDAR脑炎患者以及健康人中的检出率
表2磁微粒化学发光试剂盒临床样本比对
(2)准确度及线性
以重组NR1蛋白为免疫原筛选出高亲和力IgG单克隆抗体,并与人源IgG抗体偶联,以此作为校准品和质控品的原料;将校准质控品的原料按一定比例用柠檬酸缓冲液稀释成12.5、25、50、100、200、400RU/mL作为校准品,用检测试剂盒(M、R1、R2)检测,结果见表3,校准品检测结果CV均小于5%,实测浓度与理论浓度相关系数R2=0.997。
表3磁微粒化学发光试剂盒校准品检测结果分析
(3)精密度
用人源抗NMDAR抗体配制浓度值为20RU/mL和200RU/mL的精密度参考品,重复低值参考品(20RU/mL)和高值参考品(200RU/mL)检测10次,计算10次测试结果的平均值和标准差,根据公式计算变异系数。检测结果见表4,表4显示,低值变异系数2.24%,高值变异系数2.91%。
表4精密度检测结果
(4)稳定性
NR1亚基抗体检测试剂盒(M,R2)于4℃、37℃分别放置1、4、7天,测定高、中、低3种浓度质控品的信号保留率,其中NMDAR 13、NMDAR 03、NMDAR 08分别代表3例不同的临床样本。结果见表5,由表5可知,信号保留率均大于90%,表明试剂盒稳定性好,符合临床要求。
表5稳定性检测结果
实施例三:含不同重组蛋白的胶体金免疫层析试剂盒的检测性能研究
1、胶体金免疫层析试剂盒配制
取0.01%的氯金酸溶液煮沸后加入0.03%的柠檬酸三钠溶液后制得胶体金颗粒,在胶体金颗粒表面包被鼠抗人IgG抗体得到胶体金-鼠抗人IgG抗体结合物。
胶体金免疫层析测试条有吸水纤维、硝酸纤维素膜、吸水滤纸和含双面胶的塑料板组成。在吸水纤维上加胶体金-鼠抗人IgG抗体结合物;用NMDAR重组抗原(分别为NR1,NR2A,NR2B,NR2C)和羊抗鼠IgG抗体在硝酸纤维素膜上分别划线包被成两条宽约1mm的线段,分别作为检测线和对照线。干燥后用含1%BSA的PBS溶液封闭。干燥后依次粘在塑料板上,并切成宽约5mm的小条。
2、试剂盒性能测试
对以上4种亚基的免疫层析测试条进行临床比对分析(阳性符合率、阴性符合率)。共选取20例临床诊断为抗NMDAR脑炎和100例健康人样本进行联合检测。对检测结果的分析具体如下:
3、结果
将以上4种亚基的胶体金免疫层析试剂盒对20例临床诊断为抗NMDAR脑炎的样本进行联合检测(表6),结果表明,4种亚基联合检测的阳性符合率为90%(表7),说明本试剂盒临床阳性检出率较高,灵敏度较好。对100例随机健康人体检样本进行了测定,结果表明,胶体金免疫层析试剂盒阴性符合率为98%,说明本试剂盒临床阴性符合率较高,特异性较好。
表6:4种亚基在NMDAR脑炎患者以及健康人中的检出率
表7抗NMDAR抗体胶体金免疫层析测试条临床样本比对
实施例四:免疫印迹检测试剂盒的检测性能研究
1、免疫印迹检测试剂盒的配制
用PBS溶液将NMDAR蛋白的4种重组蛋白(NR1、NR2A、NR2B和NR2C)稀释至点样浓度,通过多通道加样器按照点样矩阵点在硝酸纤维素膜上。烘干之后用BSA溶液充分浸泡,后用双蒸水清洗3次。充分晾干后,沿边框裁成条状。
2、试剂盒性能测试
对以上免疫印迹测试条进行临床比对分析。共选取20例临床诊断为抗NMDAR脑炎和100例健康人样本进行检测。
3、结果
对检测结果的分析具体如下:
不同亚基反应性:在20例抗NMDAR脑炎患者样本中,有17例样本中检测出NR1亚基抗体,4例检测出NR2A亚基抗体,另有1例检测出NR2B亚基抗体(表8)。在本实施例的NMDAR抗体阳性样本测试中,表明NMDAR抗体的主要表位集中在NR1亚基上,但其它亚基上也有少量分布。
阳性符合率:4种亚基试剂盒对20例临床诊断为抗NMDAR脑炎的样本进行了联合检测。结果表明,抗NMDAR抗体检测试剂盒阳性符合率为90%(表9),说明本试剂盒临床阳性检出率较高,灵敏度较好。
阴性符合率:本发明试剂盒对100例随机健康人体检样本进行了测定。结果表明,抗NMDAR抗体检测试剂盒阴性符合率为97%,说明本试剂盒临床阴性符合率较高,特异性较好。
表8:4种亚基在NMDAR脑炎患者以及健康人中的检出率
表9抗NMDAR抗体免疫印迹测试条临床样本比对
实施例五:酶联免疫检测试剂盒测试性能研究
1、酶联免疫检测试剂盒配制
用CBS溶液将NMDAR重组抗原(包括重组NR1蛋白,重组NR2A蛋白,重组NR2B蛋白,重组NR2C蛋白)稀释至1μg/mL,将其包被到聚苯乙烯微孔中,清洗后用BSA溶液封闭3小时。
其中配套的校准品的制备:其原料的制备是以重组NMDAR蛋白为免疫原筛选出高亲和力IgG单克隆抗体,并进行人源化改造,使之能模拟患者血清中NMDARIgG抗体和试剂盒中NMDAR重组抗原结合;得到的NMDAR蛋白人源IgG单克隆抗体,用稀释液按一定比例稀释配制成校准品。
2、性能测试
对以上4种亚基的ELISA试剂盒进行临床比对分析(阳性符合率、阴性符合率),共选取20例临床诊断为抗NMDAR脑炎和100例健康人样本进行检测。
3、结果
本实施例的试剂盒对20例临床诊断为抗NMDAR脑炎的样本进行联合检测(表10)。结果表明,ELISA试剂盒阳性符合率为100%(表11),说明本试剂盒临床阳性检出率高,灵敏度好。本发明试剂盒对100例随机健康人体检样本进行了测定。结果表明,抗NMDAR抗体检测试剂盒阴性符合率为98%,说明本试剂盒临床阴性符合率较高,特异性较好。
表10:4种亚基在NMDAR脑炎患者以及健康人中的检出率
表11抗NMDAR抗体ELISA试剂盒临床样本比对
综上,含本发明的重组蛋白NR1、重组蛋白NR2A、重组蛋白NR2B、重组蛋白NR2C的四种试剂盒的临床检测准确度高,灵敏性好,具有十分良好的应用前景。
本发明各实施例中未提及的方法、检测过程等均是采用已知技术进行,且未提及的仪器、设备、试剂等均是可以通过外购或其他的方式得到,均是现有技术,在此不进行赘述。
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 四川携光生物技术有限公司
<120> 一种抗NMDAR脑炎重组抗原及应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 541
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile Gly Ala Val Leu Ser
1 5 10 15
Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala Val Asn Gln Ala Asn
20 25 30
Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn Ala Thr Ser Val Thr
35 40 45
His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser Val Cys Glu Asp Leu
50 55 60
Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser His Pro Pro Thr Pro
65 70 75 80
Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr Thr Ala Gly Phe Tyr
85 90 95
Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met Ser Ile Tyr Ser Asp
100 105 110
Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val Pro Pro Tyr Ser His
115 120 125
Gln Ser Ser Val Trp Phe Glu Met Met Arg Val Tyr Ser Trp Asn His
130 135 140
Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala Gln Lys
145 150 155 160
Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu Lys Val
165 170 175
Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu Met Glu
180 185 190
Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser Glu Asp
195 200 205
Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met Thr Gly
210 215 220
Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly Asn Ala
225 230 235 240
Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu Gln Leu Ile Asn Gly
245 250 255
Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val Gly Val Val Ala Gln
260 265 270
Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile Thr Asp Pro Pro Arg
275 280 285
Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr Gly Pro Leu Phe Lys
290 295 300
Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly Val Thr Gly Arg Val
305 310 315 320
Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala Asn Tyr Ser Ile Met
325 330 335
Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly Ile Tyr Asn Gly Thr
340 345 350
His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp Pro Gly Gly Glu Thr
355 360 365
Glu Lys Pro Arg Gly Tyr Gln Met Ser Thr Arg Leu Lys Ile Val Thr
370 375 380
Ile His Gln Glu Pro Phe Val Tyr Val Lys Pro Thr Leu Ser Asp Gly
385 390 395 400
Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp Pro Val Lys Lys Val
405 410 415
Ile Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly Ser Pro Arg His Thr
420 425 430
Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp Leu Leu Ile Lys Leu
435 440 445
Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His Leu Val Ala Asp Gly
450 455 460
Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser Asn Lys Lys Glu Trp
465 470 475 480
Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln Ala Asp Met Ile Val
485 490 495
Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln Tyr Ile Glu Phe Ser
500 505 510
Lys Pro Phe Lys Tyr Gln Gly Leu Thr Ile Leu Val Lys Lys Glu Ile
515 520 525
Pro Arg Ser Thr Leu Asp Ser Phe Met Gln Pro Phe Gln
530 535 540
<210> 2
<211> 533
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Pro Ser Ala Ala Ala Glu Lys Gly Pro Pro Ala Leu Asn Ile Ala Val
1 5 10 15
Met Leu Gly His Ser His Asp Val Thr Glu Arg Glu Leu Arg Thr Leu
20 25 30
Trp Gly Pro Glu Gln Ala Ala Gly Leu Pro Leu Asp Val Asn Val Val
35 40 45
Ala Leu Leu Met Asn Arg Thr Asp Pro Lys Ser Leu Ile Thr His Val
50 55 60
Cys Asp Leu Met Ser Gly Ala Arg Ile His Gly Leu Val Phe Gly Asp
65 70 75 80
Asp Thr Asp Gln Glu Ala Val Ala Gln Met Leu Asp Phe Ile Ser Ser
85 90 95
His Thr Phe Val Pro Ile Leu Gly Ile His Gly Gly Ala Ser Met Ile
100 105 110
Met Ala Asp Lys Asp Pro Thr Ser Thr Phe Phe Gln Phe Gly Ala Ser
115 120 125
Ile Gln Gln Gln Ala Thr Val Met Leu Lys Ile Met Gln Asp Tyr Asp
130 135 140
Trp His Val Phe Ser Leu Val Thr Thr Ile Phe Pro Gly Tyr Arg Glu
145 150 155 160
Phe Ile Ser Phe Val Lys Thr Thr Val Asp Asn Ser Phe Val Gly Trp
165 170 175
Asp Met Gln Asn Val Ile Thr Leu Asp Thr Ser Phe Glu Asp Ala Lys
180 185 190
Thr Gln Val Gln Leu Lys Lys Ile His Ser Ser Val Ile Leu Leu Tyr
195 200 205
Cys Ser Lys Asp Glu Ala Val Leu Ile Leu Ser Glu Ala Arg Ser Leu
210 215 220
Gly Leu Thr Gly Tyr Asp Phe Phe Trp Ile Val Pro Ser Leu Val Ser
225 230 235 240
Gly Asn Thr Glu Leu Ile Pro Lys Glu Phe Pro Ser Gly Leu Ile Ser
245 250 255
Val Ser Tyr Asp Asp Trp Asp Tyr Ser Leu Glu Ala Arg Val Arg Asp
260 265 270
Gly Ile Gly Ile Leu Thr Thr Ala Ala Ser Ser Met Leu Glu Lys Phe
275 280 285
Ser Tyr Ile Pro Glu Ala Lys Ala Ser Cys Tyr Gly Gln Met Glu Arg
290 295 300
Pro Glu Val Pro Met His Thr Leu His Pro Phe Met Val Asn Val Thr
305 310 315 320
Trp Asp Gly Lys Asp Leu Ser Phe Thr Glu Glu Gly Tyr Gln Val His
325 330 335
Pro Arg Leu Val Val Ile Val Leu Asn Lys Asp Arg Glu Trp Glu Lys
340 345 350
Val Gly Lys Trp Glu Asn His Thr Leu Ser Leu Arg His Ala Val Trp
355 360 365
Pro Arg Tyr Lys Ser Phe Ser Asp Cys Glu Pro Asp Asp Asn His Leu
370 375 380
Ser Ile Val Thr Leu Glu Glu Ala Pro Phe Val Ile Val Glu Asp Ile
385 390 395 400
Asp Pro Leu Thr Glu Thr Cys Val Arg Asn Thr Val Pro Cys Arg Lys
405 410 415
Phe Val Lys Ile Asn Asn Ser Thr Asn Glu Gly Met Asn Val Lys Lys
420 425 430
Cys Cys Lys Gly Phe Cys Ile Asp Ile Leu Lys Lys Leu Ser Arg Thr
435 440 445
Val Lys Phe Thr Tyr Asp Leu Tyr Leu Val Thr Asn Gly Lys His Gly
450 455 460
Lys Lys Val Asn Asn Val Trp Asn Gly Met Ile Gly Glu Val Val Tyr
465 470 475 480
Gln Arg Ala Val Met Ala Val Gly Ser Leu Thr Ile Asn Glu Glu Arg
485 490 495
Ser Glu Val Val Asp Phe Ser Val Pro Phe Val Glu Thr Gly Ile Ser
500 505 510
Val Met Val Ser Arg Ser Asn Gly Thr Val Ser Pro Ser Ala Phe Leu
515 520 525
Glu Pro Phe Ser Ala
530
<210> 3
<211> 531
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Arg Ser Gln Lys Ser Pro Pro Ser Ile Gly Ile Ala Val Ile Leu Val
1 5 10 15
Gly Thr Ser Asp Glu Val Ala Ile Lys Asp Ala His Glu Lys Asp Asp
20 25 30
Phe His His Leu Ser Val Val Pro Arg Val Glu Leu Val Ala Met Asn
35 40 45
Glu Thr Asp Pro Lys Ser Ile Ile Thr Arg Ile Cys Asp Leu Met Ser
50 55 60
Asp Arg Lys Ile Gln Gly Val Val Phe Ala Asp Asp Thr Asp Gln Glu
65 70 75 80
Ala Ile Ala Gln Ile Leu Asp Phe Ile Ser Ala Gln Thr Leu Thr Pro
85 90 95
Ile Leu Gly Ile His Gly Gly Ser Ser Met Ile Met Ala Asp Lys Asp
100 105 110
Glu Ser Ser Met Phe Phe Gln Phe Gly Pro Ser Ile Glu Gln Gln Ala
115 120 125
Ser Val Met Leu Asn Ile Met Glu Glu Tyr Asp Trp Tyr Ile Phe Ser
130 135 140
Ile Val Thr Thr Tyr Phe Pro Gly Tyr Gln Asp Phe Val Asn Lys Ile
145 150 155 160
Arg Ser Thr Ile Glu Asn Ser Phe Val Gly Trp Glu Leu Glu Glu Val
165 170 175
Leu Leu Leu Asp Met Ser Leu Asp Asp Gly Asp Ser Lys Ile Gln Asn
180 185 190
Gln Leu Lys Lys Leu Gln Ser Pro Ile Ile Leu Leu Tyr Cys Thr Lys
195 200 205
Glu Glu Ala Thr Tyr Ile Phe Glu Val Ala Asn Ser Val Gly Leu Thr
210 215 220
Gly Tyr Gly Tyr Thr Trp Ile Val Pro Ser Leu Val Ala Gly Asp Thr
225 230 235 240
Asp Thr Val Pro Ala Glu Phe Pro Thr Gly Leu Ile Ser Val Ser Tyr
245 250 255
Asp Glu Trp Asp Tyr Gly Leu Pro Ala Arg Val Arg Asp Gly Ile Ala
260 265 270
Ile Ile Thr Thr Ala Ala Ser Asp Met Leu Ser Glu His Ser Phe Ile
275 280 285
Pro Glu Pro Lys Ser Ser Cys Tyr Asn Thr His Glu Lys Arg Ile Tyr
290 295 300
Gln Ser Asn Met Leu Asn Arg Tyr Leu Ile Asn Val Thr Phe Glu Gly
305 310 315 320
Arg Asn Leu Ser Phe Ser Glu Asp Gly Tyr Gln Met His Pro Lys Leu
325 330 335
Val Ile Ile Leu Leu Asn Lys Glu Arg Lys Trp Glu Arg Val Gly Lys
340 345 350
Trp Lys Asp Lys Ser Leu Gln Met Lys Tyr Tyr Val Trp Pro Arg Met
355 360 365
Cys Pro Glu Thr Glu Glu Gln Glu Asp Asp His Leu Ser Ile Val Thr
370 375 380
Leu Glu Glu Ala Pro Phe Val Ile Val Glu Ser Val Asp Pro Leu Ser
385 390 395 400
Gly Thr Cys Met Arg Asn Thr Val Pro Cys Gln Lys Arg Ile Val Thr
405 410 415
Glu Asn Lys Thr Asp Glu Glu Pro Gly Tyr Ile Lys Lys Cys Cys Lys
420 425 430
Gly Phe Cys Ile Asp Ile Leu Lys Lys Ile Ser Lys Ser Val Lys Phe
435 440 445
Thr Tyr Asp Leu Tyr Leu Val Thr Asn Gly Lys His Gly Lys Lys Ile
450 455 460
Asn Gly Thr Trp Asn Gly Met Ile Gly Glu Val Val Met Lys Arg Ala
465 470 475 480
Tyr Met Ala Val Gly Ser Leu Thr Ile Asn Glu Glu Arg Ser Glu Val
485 490 495
Val Asp Phe Ser Val Pro Phe Ile Glu Thr Gly Ile Ser Val Met Val
500 505 510
Ser Arg Ser Asn Gly Thr Val Ser Pro Ser Ala Phe Leu Glu Pro Phe
515 520 525
Ser Ala Asp
530
<210> 4
<211> 535
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gly Leu Gly Pro Gly Gln Gly Glu Gln Gly Met Thr Val Ala Val Val
1 5 10 15
Phe Ser Ser Ser Gly Pro Pro Gln Ala Gln Phe Arg Ala Arg Leu Thr
20 25 30
Pro Gln Ser Phe Leu Asp Leu Pro Leu Glu Ile Gln Pro Leu Thr Val
35 40 45
Gly Val Asn Thr Thr Asn Pro Ser Ser Leu Leu Thr Gln Ile Cys Gly
50 55 60
Leu Leu Gly Ala Ala His Val His Gly Ile Val Phe Glu Asp Asn Val
65 70 75 80
Asp Thr Glu Ala Val Ala Gln Ile Leu Asp Phe Ile Ser Ser Gln Thr
85 90 95
His Val Pro Ile Leu Ser Ile Ser Gly Gly Ser Ala Val Val Leu Thr
100 105 110
Pro Lys Glu Pro Gly Ser Ala Phe Leu Gln Leu Gly Val Ser Leu Glu
115 120 125
Gln Gln Leu Gln Val Leu Phe Lys Val Leu Glu Glu Tyr Asp Trp Ser
130 135 140
Ala Phe Ala Val Ile Thr Ser Leu His Pro Gly His Ala Leu Phe Leu
145 150 155 160
Glu Gly Val Arg Ala Val Ala Asp Ala Ser His Val Ser Trp Arg Leu
165 170 175
Leu Asp Val Val Thr Leu Glu Leu Gly Pro Gly Gly Pro Arg Ala Arg
180 185 190
Thr Gln Arg Leu Leu Arg Gln Leu Asp Ala Pro Val Phe Val Ala Tyr
195 200 205
Cys Ser Arg Glu Glu Ala Glu Val Leu Phe Ala Glu Ala Ala Gln Ala
210 215 220
Gly Leu Val Gly Pro Gly His Val Trp Leu Val Pro Asn Leu Ala Leu
225 230 235 240
Gly Ser Thr Asp Ala Pro Pro Ala Thr Phe Pro Val Gly Leu Ile Ser
245 250 255
Val Val Thr Glu Ser Trp Arg Leu Ser Leu Arg Gln Lys Val Arg Asp
260 265 270
Gly Val Ala Ile Leu Ala Leu Gly Ala His Ser Tyr Trp Arg Gln His
275 280 285
Gly Thr Leu Pro Ala Pro Ala Gly Asp Cys Arg Val His Pro Gly Pro
290 295 300
Val Ser Pro Ala Arg Glu Ala Phe Tyr Arg His Leu Leu Asn Val Thr
305 310 315 320
Trp Glu Gly Arg Asp Phe Ser Phe Ser Pro Gly Gly Tyr Leu Val Gln
325 330 335
Pro Thr Met Val Val Ile Ala Leu Asn Arg His Arg Leu Trp Glu Met
340 345 350
Val Gly Arg Trp Glu His Gly Val Leu Tyr Met Lys Tyr Pro Val Trp
355 360 365
Pro Arg Tyr Ser Ala Ser Leu Gln Pro Val Val Asp Ser Arg His Leu
370 375 380
Thr Val Ala Thr Leu Glu Glu Arg Pro Phe Val Ile Val Glu Ser Pro
385 390 395 400
Asp Pro Gly Thr Gly Gly Cys Val Pro Asn Thr Val Pro Cys Arg Arg
405 410 415
Gln Ser Asn His Thr Phe Ser Ser Gly Asp Val Ala Pro Tyr Thr Lys
420 425 430
Leu Cys Cys Lys Gly Phe Cys Ile Asp Ile Leu Lys Lys Leu Ala Arg
435 440 445
Val Val Lys Phe Ser Tyr Asp Leu Tyr Leu Val Thr Asn Gly Lys His
450 455 460
Gly Lys Arg Val Arg Gly Val Trp Asn Gly Met Ile Gly Glu Val Tyr
465 470 475 480
Tyr Lys Arg Ala Asp Met Ala Ile Gly Ser Leu Thr Ile Asn Glu Glu
485 490 495
Arg Ser Glu Ile Val Asp Phe Ser Val Pro Phe Val Glu Thr Gly Ile
500 505 510
Ser Val Met Val Ala Arg Ser Asn Gly Thr Val Ser Pro Ser Ala Phe
515 520 525
Leu Glu Pro Tyr Ser Pro Ala
530 535
<210> 5
<211> 1623
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccgccgccgg gccctttcca agccgggcgc tcggagctgt gcccggcccc gcttcagcac 60
cgcggacagc gccggccgcg tggggctgag ccccgagccc ccgcgcacgc ttcagcgccc 120
cttccctcgg ccgacgtccc gggaccgccg ctccggggga gacgtggcgt ccgcagcccg 180
cggggccggg cgagcgcagg acggcccgga agccccgcgg gggatgcgcc gagggccccg 240
cgttcgcgcc gcgcagagcc aggcccgcgg cccgagccca tgagcaccat gcgcctgctg 300
acgctcgccc tgctgttctc ctgctccgtc gcccgtgccg cgtgcgaccc caagatcgtc 360
aacattggcg cggtgctgag cacgcggaag cacgagcaga tgttccgcga ggccgtgaac 420
caggccaaca agcggcacgg ctcctggaag attcagctca atgccacctc cgtcacgcac 480
aagcccaacg ccatccagat ggctctgtcg gtgtgcgagg acctcatctc cagccaggtg 540
ccctccccca cctccgccac ccacctcccc tctcctccat cctgcaaccc cacaccccca 600
gtttcattcc atcctttccg tgcccccttc ctccctgtaa gacaccaccc cagagtcagc 660
tggctgcttc cgggaggcct cgtctcacta ggaaccaaac accagggtct gctggctccc 720
ctatcttggc ctgagaccag tcacctgcca ccttggctgg tcctcagagg gcccctgggg 780
ctccaggccc tgactggtgt gtgtagacgt ggggctggag tgtgtcagtg tgggggtggg 840
cattccgggt aagagagtag aagcgcctgt ccagctacat gcccgccctg cagagcttta 900
aacaggacgg ggcctggggc catctttgtt tctgcttcca ggttctcctg ccctttcttt 960
cgtcccttcc ccctaccgat gggtccgcct gggaagagaa atggctcagg tgccacggca 1020
ggacgctttg tgggggtggg agtgggggtg cacacgcgag aggcatcagg gcatgggagc 1080
tgtcggcagc cagcgctgcg ggggaggacg tggctcctgg gattttgcct gtcggagctg 1140
tccgcccctg ggccgagcgc ctgctgaatt ccaatgaggc tgcaaggatc tgcaatgcag 1200
ccctttatgt aagaggcaag acagacatcc agcctagcac cgctcacacg tgcctacctg 1260
atggacacac cacatctgtg gacacacatg ctcacactca caccaaatgt tacattagca 1320
cacactcatg cacctcagca tcacacaatc aatttcatat gctcatctgc acacatgcag 1380
atccattgac acctgctcat gtgccacaca cggcttggca tgcattccca gaggcacgtg 1440
caaacatgca catttacaca catggttcca gtcattcaca cgcatgtaca cgaacagaca 1500
tgccagggca tgtgatgcac ataaccatac cctagcacac gcgtgaacac ctgcatggtc 1560
acacacggac ctacgggtct ttgccaagca cctctgggtg caggctggaa gcaagagctg 1620
ggg 1623
<210> 6
<211> 1599
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gcgctcagag gaccgggcag ttgccgtccg gagtggggca ggaagcggag ctagggatct 60
tggaggggag ctcctagggc ttggccggat catgggaccg ggtgagcgct gagaatcgcg 120
gccgcagcca tcagccctgg agatgaccag gagcggccac tgctgagaac tatgtggaga 180
gaggctgcgg tgagtgctag cgcgggacgc cgggcctggg aggccccctt gggaaggggg 240
gaggtgggag gcctgaagag ggaggaagcg gcaggatcac aagagggtcg ctgcgtggga 300
aagatccacc tccctcgggg aaccgggacc gcgggagccc gcgtccttcc ctccccctct 360
cctactgctg gctcgtcaca aactcagcca gcgccccccc accctcgtta cctcctcttt 420
ctctccctac ctccctcgct cagcagctcc cggtcgcaca actcccagca gccggcgctg 480
gggaggtggt agggggtgcg ggtggaggtg cccagttagc ttctcgtggg tggccaacct 540
ggggctccca ttctcctgtg caccctggag tggagctccc gctatcagcc acaggggctg 600
aggatccgca ctgggggcgt cgtgggcatc gggcttcagc ttcccccctc ttgtctagcc 660
cccctgggcg catagggggg cttgtgagca ctgcagactg cagccttccc ctcaaccctc 720
cctcccctcg ttggtgggct gaccctgcct ctccttctct cctccgcccc gttagagccc 780
tgctgcagag cctccggctg ggatagccgc cccccgtggg ggcgatgcgg acagcgcggg 840
acagccaggg gagcgcgctg gggccgcagc atgcgggaac ccgctaaacc cggtggctgc 900
tgaggcggcc gagatgctcg tgcgcgcagc gcgccccact gcatcctcga ccttctcggg 960
ctacaggtac gtcccagagg gagatcaggg ctccttgggc cggggaggag ggagactcga 1020
ccggagggag agcgggagag ggatcaggct agagagggag agggaaagcg ggagggagcc 1080
cagcggcgaa gtgcaggagg ggcaccaggg ggcgctgcgg ctccgcgctc cgcggcgctt 1140
acccaggaac gtcgaggcct cttggagttc gcctcaactt cccccggctc cagcagcgtc 1200
cgtcccgaga ggcgcgcgtc agggcacgcg tgcacgaccg tgctggtatt tgcaggtacg 1260
cagggctgtg cacctgccgc agttcatgcg tgtgcaccac ctgaggacat gtgaggactc 1320
cggctggccg tgggcaccgc tacagcagtc tgaggaagtg cgcagaaaag cgcgcgtgcc 1380
tatgagcgtg tgcggcccac tgcccacgtg cacctaggac ttggcaacag cgcccgcagt 1440
agaaaggcta gggaggggct gtacagtcca cgtccgtctg ggagcttctt gggagcctac 1500
ctttccccct acgcacgttc tttgccagct cctacgcgct ggcccgcggg gggacgcgcc 1560
tactctccag aattcagcat tgcacacggc gcgcgtgag 1599
<210> 7
<211> 1593
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cactaacgct ccaaaaacca aaaaccgtaa ttgccagaag aagcgttaaa aatctattcc 60
agccactaac ctcacatgca cacggaataa ttactctgga ttctgcattg tgagctgctc 120
tccataccct gaattacctt tgaattaaat cttttttttt ttgaatttgc atctcttcaa 180
gacacaagat taaaacaaaa tttacgctaa attggatttt aaattatctt ccgttcattt 240
atccttcgtc tttcttatgt ggatatgcaa gcgagaagaa gggactggac attcccaaca 300
tgctcactcc cttaatctgt ccgtctagag gtttggcttc tacaaaccaa ggtagggcaa 360
attctatttt attttttcct cacctggggt agggattcct cattttatct atctcttcga 420
gtccctcaag tggatctttt tggaagaaaa ggtcacaaga tatgaaaata atgctgtttt 480
gggaaaaaag gcttattaca aagaaggaaa tctcccatgt tttttctttt tcttctctca 540
tctaacaggt tttgggcttt tggggggcag tgggggtgga tgtatgcagg cacaaaagat 600
gttggatggc atcaggggaa tgtatctata tctatagaca tagccagcca ggagacctgt 660
gtgattttcc agttaaatag cattttattc acccagaact aaagctccga tttttatggg 720
aatctagaaa tccttttttt tttttttttt caggttggct ttattcccct tgtcaccttc 780
atgcctctct ccctgtctcc tgtctttacc tttctaggca tcctttctat gccctttaaa 840
acagataaaa ataatgattg agattactgt gattgttgcc ctgaaatcca cttgctgtct 900
aaagggttga caaggggccc agagatcccg ggaggcctat tgcagagggg taggtctagg 960
tggggatcgg gctggtctca gaacacctgg aattgaagga tgctgcaggt taaacgactt 1020
aaatcttaag ctcggaaagg aggaattctg tcaatattct gcttttaatt tgtatttttt 1080
cctgtgctat tatttttgag ctgtaaattt ctccttctgt ctacatatct atttataccc 1140
acacatcctc caagcaaact gttacttcta cactcttgca aaatggctat cttgcagagt 1200
tctaaaatgc tgtgcatgtg gaaggggtgc agaattcgac acgtgagtct ttgtgttaaa 1260
caaagagatg tgctgtttgc atgtattagt cttgtaccct caaaaatgga attaatactt 1320
taacattttc cctttcatca atttttacaa tatgtgaaat gtgattaggc agtattttaa 1380
tgtacttgag aagagaaata gcaggagaaa tggaaaagac ttagctaatg actcttcagc 1440
tagtcactca tgcaatccaa gacaactctt gagagaatca gatcattcat gggttggagg 1500
gccatgatgg tgagtgggct gctcctttcc ttgctccact gaccatatgt ccctgaactc 1560
tgaaggcgtc taaatgcagg gatcaggttg gga 1593
<210> 8
<211> 1605
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggcggccggc gcgggctctc gtcgcgggcg cagcgcccct tcccctcggt aagtggcgag 60
cggcgggcga ggggcaccaa cttcgccgcc aacttggggc tgggttcccg cggcgcgcga 120
gcaaggacgg cggagacaga ggcgggacac ccggggggtt ggacccacgc aggactctcg 180
gggctcaagg agggggcggc tggaggaccc ggaccctcaa gaacctggcc ttgggttttg 240
acccgggcgg ccggagagaa ggcgaagaag aggccgagca gacaggcgcg cggcggacgc 300
gagagaagga cgcgggcgaa gcgtccaaga cgttcgtccc cagggtcgcc gagccctcct 360
cctccttcgc ttggcgcggc agcaccgcgg gaccagggga gtctgggctg ccgcggaccc 420
aaactgttgc tgctggaagc cgccggggcg gagagcgggg aacagggccg ggagtggggg 480
ccggggctgg gatccaccag agcgaggggt tggaaaggag tcgagaaggt gaaagggggc 540
tgtgcggctc cccagcccag tgccccagtt tccctaggtc tgctgggcac ctgctgcgca 600
ctcgcctcgg cgcccagacc ccttcccccg ccgcgagtcc cgcttcttcc ccttccagct 660
ctgccccatc tcctttgccc cagcctagct ctctccttcc tgcgacctgg gagaatcgga 720
gtgggcttcc agagtagtgg catcttgcag atgctggccc gtgcccggct cccgggcttc 780
cagagcgcaa agggaagggt aggcagagag ggcactgggc gagcaagacc tggcgctggg 840
ccctggctgc gcaccagccg cctgcgccgg tctctggggg cttcgcccag gatccccgac 900
ggcggggagg ggaagccccg agtctcctgt ttcctgccta cgcgggcagg gcggggacac 960
gcggagcaaa ggcacccggc aacctgaacc cccaggtgcg tcccgcgacc ctgcaaggag 1020
ctgctgggtg agcaagacgg gaattcccgc caggccctga tgcccacccc attcgtccac 1080
tcggtttccc agccctggtc gtccaagggg acttctggct cggtccccca gccctcggat 1140
ggtggcgtta tcttagttca gtccctgacc ccgggccaca ggaaaatcga ggaaagacac 1200
ttatttgggg agggtaccat tccgctggct gtatttcaga gcccgtccag gtcttggcgg 1260
tgctagtgac tttgagcctg tttctaaact cccgaagaat aagagcgtgc caggggcagg 1320
taggcaggtc cgggacacct gggttcgccc agggcccccg ggcgggcctg ggatggtact 1380
ggggccgcct gccaggctgg cttggtcccc ccactgcggg actcggggtc acgacccagc 1440
ttgggggagt gggggtgggg gccccggagc cgcggacgcc ggcggggggc agcgtggggc 1500
ggggggcgcc gggtgcgtgg tgctcagctt ggccgccagg aggcgtggcg cgggtccccg 1560
cccggagccg ccgccgccgc cgccgccgcc gcatcgcgag tgtcc 1605
Claims (6)
1.一种抗NMDAR脑炎重组抗原组合,其特征在于,包括NR1蛋白的N端结构域、NR2A蛋白的N端结构域、NR2B蛋白的N端结构域和NR2C蛋白的N端结构域,所述N端结构域不包含相应蛋白的跨膜片段、跨膜区域和C端结构域;
所述NR1蛋白的N端结构域的氨基酸序列如SEQ ID NO:1所示;
NR2A蛋白的N端结构域的氨基酸序列如SEQ ID NO:2所示;
NR2B蛋白的N端结构域的氨基酸序列如SEQ ID NO:3所示;
NR2C蛋白的N端结构域的氨基酸序列如SEQ ID NO:4所示。
2.编码如权利要求1所述的一种抗NMDAR脑炎重组抗原组合的基因片段组合。
3.一种含权利要求2所述的基因片段组合的重组蛋白表达载体。
4.一种试剂盒,其特征在于,包含如权利要求1所述的抗NMDAR脑炎重组抗原组合。
5.根据权利要求4所述的一种试剂盒,其特征在于,所述试剂盒为包含如权利要求1所述的抗NMDAR脑炎重组抗原组合的胶体金免疫层析试剂盒、免疫印迹检测试剂盒、酶联免疫检测试剂盒、纳米磁微粒管式化学发光检测试剂盒。
6.如权利要求1所述的抗NMDAR脑炎重组抗原组合在制备用于检测抗NMDAR脑炎抗体试剂盒的应用。
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CN114144427A (zh) * | 2019-07-16 | 2022-03-04 | 德国神经退行性疾病中心 | 用于检测和分离nmdar自身抗体的nmda受体构建体 |
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CN114144427A (zh) * | 2019-07-16 | 2022-03-04 | 德国神经退行性疾病中心 | 用于检测和分离nmdar自身抗体的nmda受体构建体 |
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Denomination of invention: A recombinant antigen against NMDAR encephalitis and its application Granted publication date: 20240426 Pledgee: Chengdu Branch of China CITIC Bank Co.,Ltd. Pledgor: Sichuan light carrying Biotechnology Co.,Ltd. Registration number: Y2024980030579 |