CN114736298A - 用于检测自身免疫系统疾病的抗Shroom2自身抗体及其产品和应用 - Google Patents
用于检测自身免疫系统疾病的抗Shroom2自身抗体及其产品和应用 Download PDFInfo
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Abstract
本发明公开了用于检测自身免疫系统疾病的抗Shroom2自身抗体及其产品和应用。本发明制备得到的试剂盒能够实现Shroom2自身抗体的检测,并且可以用来诊断神经系统疾病,尤其是与一种或多种症状相关的神经系统疾病,所述症状包括小脑变性、精神行为异常、认知障碍、视力障碍、记忆力下降、言语障碍、运动障碍、意识水平下降、自主神经功能障碍或听觉障碍。本发明所述应用制备得到的试剂盒还可以用来区分自身免疫疾病,尤其是区分神经系统的自身免疫疾病与非自身免疫疾病。
Description
技术领域
本发明属于生物技术领域,具体涉及一种自身免疫系统疾病标志物抗Shroom2自身抗体及其产品和应用。
背景技术
神经系统自身免疫性疾病是以自身免疫细胞、免疫分子等攻击神经系统为主要致病机制的自身免疫性疾病,该类疾病可发生在中枢神经系统、周围神经系统及神经-肌肉接头处。常见的神经系统自身免疫性疾病包括:自身免疫性脑炎、中枢神经系统脱髓鞘疾病、僵人综合征、免疫介导性周围神经病、自身免疫性小脑共济失调、肌无力综合征等。在免疫反应中,作用于神经系统自身抗原的致病抗体统称为神经系统自身抗体。随着科学的发展,神经系统自身免疫性疾病谱和神经系统自身抗体谱不断完善和发展,检测患者体内自身抗体不仅能够帮助相关疾病确诊,还有助于检测疾病的进程,评价治疗效果。
Shroom是一类细胞骨架结合蛋白。1999年,Jeffrey D在小鼠中发现,Shroom是正常神经生长所需的细胞结构的关键因素[Jeffrey D.Hildebrand and PhilippeSoriano.Shroom,a PDZ Domain–Containing Actin-Binding Protein,Is Required forNeural Tube Morphogenesis in Mice.Cell,1999,99,485-497]。2005年,Jeffrey D的研究结果是Shroom在多种脊椎动物的神经管形态发生中是必不可少的,Shroom通过调节神经上皮细胞的形态促进神经生长[Jeffrey D.Hildebrand.Shroom regulates epithelialcell shape via the apical positioning of an actomyosin network.Journal ofCell Science.2005,118,5191-5203]。
shroom家族由4个成员组成,分别是Shroom1、Shroom2、Shroom3和Shroom4。Shroom2是功能上进化保守的176kDa的蛋白质,是Shroom基因家族中的一员,其表达蛋白定位于细胞周边的肌动蛋白上,为一种紧密连接蛋白,具有调节细胞内骨架和上皮细胞结构的功能。Shroom2是RhoA-ROCK通路的关键介质,该通路与肿瘤细胞的转移有关系,通过actin的重排和细胞黏着,RhoA-ROCK和Shroom2调控癌细胞转移(Jing Yuan,Lin Chen,Jingshu Xiao.SHROOM2 inhibits tumor metastasis through RhoA–ROCK pathway-dependent and independent mechanisms in nasopharyngeal carcinoma.Cell Deathand Disease.2019,10:58)。
Shroom2是视网膜黑色素上皮细胞色素沉着的中心调节因子,编码人类Shroom2的基因位于两种不同形式的眼白化病的关键区域,Shroom2突变可能是这些人类视觉系统疾病的一个关键因素(Pamela D.Fairbank,Chanjae Lee,Avegiyel Ellis.Shroom2(APXL)regulates melanosome biogenesis and localization in the retinal pigmentepithelium.Development 133.2006,4109-4118)。
Shroom2蛋白共1616个氨基酸,含有一个PDZ结构域(aa26-108),2个ASD序列(ASD1,aa684-773;ASD2,aa1317-1161)。该基因在神经细胞发育、细胞骨架形成、胃肠道发育及相关肿瘤发病中均起到一定的作用。Shroom2是高度保守的,使得即使是来自与人类关系较远的物种的Shroom2也适合于引发与人抗Shroom2抗体的特异性结合。在Shroom2同系物中,在各自物种和人类之间保守的、长度在6至12个氨基酸的区域原则上已足以引发与抗Shroom2抗体的特异性结合,构成构象表位的保守序列可能会更短。目前,尚未见Shroom2自身抗体在检测神经系统自身免疫性疾病方面的相关报道。
发明内容
本发明的目的在于提供用于检测自身免疫系统疾病标志物的抗Shroom2自身抗体及其产品和应用。
为了达到上述目的,本发明采用以下技术方案予以实现:
本发明公开了一种自身免疫性疾病标志物,所述自身免疫性疾病标志物为与Shroom2蛋白结合的自身抗体,所述Shroom2蛋白的氨基酸序列如SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3所示。
本发明还公开了用于受试者中自身免疫性疾病诊断的多肽或蛋白质,该多肽或蛋白质为包含衍生自Shroom2蛋白的一个或多个表位,所述Shroom2蛋白的氨基酸序列如SEQID NO:1、SEQ ID NO:2或SEQ ID NO:3所示。
优选地,本发明使用的多肽或蛋白质包括或具有与shroom2蛋白或根据SEQ IDNO:1、SEQ ID NO:2或SEQ ID NO:3的序列具有至少70%、至少75%、至少80%、至少90%、至少92、至少94%、至少96%、至少98%或至少99%序列同一性的氨基酸序列。在其它实施方案中,本发明涉及具有根据SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3的序列的至少20、25、30、40、50、60、70、80、90或100个连续氨基酸的shroom2蛋白或多肽的片段。在本发明进一步的实施方案中,本发明涉及与这样的片段的序列具有至少70%、至少75%、至少80%、至少90%、至少92%、至少94%、至少96%、至少98%或至少99%序列同一性的这样的片段的同系物。
更进一步优选地,所述多肽或蛋白质可以是融合其他氨基酸的融合蛋白,所述氨基酸是连接在N端或C端的,具有促进所述多肽或蛋白质的纯化、固定化、沉淀或鉴定的作用。这种氨基酸可以构成本领域已知的标签,例如,His标签、硫氧还蛋白、麦芽糖结合蛋白、谷胱甘肽-S-转移酶、flag标签、myc标签或strep标签。
根据本发明,“多肽”应理解为两个、三个、四个、五个、六个、七个、八个、十个或更多个氨基酸的聚合物。
在本发明的范围内,“表位”是能够被抗体特异性识别的多肽的一部分。所述表位可以是构象表位(由多肽的不连续氨基酸序列组成)或线性表位(由多肽的连续氨基酸序列组成)。
在本发明的范围内,对于表位,术语“衍生的”涉及由多肽或蛋白质的一级氨基酸序列的不连续或连续部分形成的表位。本领域已知的是,表位中的一个或更多个氨基酸可以被取代。因此,术语“衍生的”还涉及这样的表位:当与具有原始氨基酸序列的表位相比,尽管在氨基酸序列上具有差异,所述表位表现出不变的或基本上不变的抗体结合强度或特异性。
本发明还公开了一种核酸,所述核酸编码权利要求3所述的用于受试者中自身免疫性疾病的诊断的多肽或蛋白质,编码SEQ ID NO:1所示的多肽或蛋白质的核苷酸序列如SEQ ID NO:4所示,编码SEQ ID NO:2所示的多肽或蛋白质的核苷酸序列如SEQ ID NO:5所示,SEQ ID NO:3所示的多肽或蛋白质的核苷酸序列如SEQ ID NO:6所示。
根据本发明,“核酸”涉及DNA或RNA序列。本领域已知的是,由于遗传密码的简并性,核酸编码的某些变化不会导致其所编码的肽序列的变化。因此,术语“核酸”还包含编码相同蛋白序列、与原始核酸序列在序列上存在不同的核酸序列,即该原始序列的突变序列。
本发明公开了一种细胞,所述细胞含有能够表达上述的用于受试者中自身免疫性疾病诊断的多肽或蛋白质的载体。
根据本发明,“细胞”是能够转入载体的任何原核或真核宿主细胞。例如,细胞可以是细菌细胞(如大肠杆菌细胞)或真核细胞(如永生化人细胞,昆虫细胞,酵母)。这样的细胞包括HEK293细胞、Hela细胞、CHO、毕赤酵母、酿酒酵母、sf9、BL21、Rosetta等。
根据本发明,“载体”包括插入物(例如编码所需蛋白质的原始或突变核酸序列)和其它特征(启动子、多克隆位点、筛选标记、复制子等)的环状或线性核酸序列。这样的载体包括pTriEx载体家族、pcDNA3家族、pET系列、pBac系列等其他商品化的真核表达系统或原核表达系统的载体。
本发明还公开了一种固定化检测试剂,将上述的多肽或蛋白质固定化于固态载体上获得。
本发明范围内的“固定”指这样的分子,其与水溶液中不可溶的固态载体结合、更优选地借助共价键、静电相互作用、囊化或包埋或借助疏水相互作用,最优选地借助一个或多个共价键结合。例如载玻片、聚苯乙烯板、玻璃板、膜(尼龙膜、硝酸纤维素膜或PVDF膜)、磁珠、柱色谱介质、生物芯片、聚丙烯酰胺凝胶等。
本发明还公开了上述的自身免疫性疾病标志物、多肽或蛋白质或固定化检测试剂在制备用于自身免疫性疾病诊断的检测试剂或检测试剂盒中的应用。
优选地,检测时是检测样品与shroom2蛋白结合的自身抗体,所述样品为液体样品或组织样品,液体样品为脑脊液、血液、血浆、淋巴液或组织间液;组织样品为神经组织、肌肉组织或来自消化道的组织。
优选地,所述自身免疫性疾病包括小脑变性、精神行为异常、认知障碍、视力障碍、记忆力下降、言语障碍、运动障碍、意识水平下降、自主神经功能障碍或听觉障碍。
本发明还公开了一种自身免疫性疾病检测试剂盒,检测试剂盒中包括上述的自身免疫性疾病标志物、上述的多肽或蛋白质或上述的固定化检测试剂。
根据本发明,自身免疫性疾病,指与抗Shroom2抗体有关的小脑变性、精神行为异常、认知障碍、视力障碍、记忆力下降、言语障碍、运动障碍、意识水平下降、自主神经功能障碍,劳动能力丧失,这些症状大部分与神经疾病有关。
与现有技术相比,本发明具有以下有益效果:
本发明采用现有的检测方法进行血清抗Shroom2蛋白自身抗体的检测,可以较准确地将自身免疫系统疾病患者和正常人鉴别开来。在此背景下提供此方便、快捷、有效的检测自身免疫系统疾病的标志物及该标志物在制备用于自身免疫系统疾病检测的试剂盒用途,本发明可用于临床的自身免疫系统疾病早期诊断。
本发明提供了抗Shroom2自身抗体的试剂在制备诊断神经系统症状相关疾病的试剂盒中的应用。本发明所述应用制备得到的试剂盒能够实现Shroom2自身抗体的检测,并且可以用来诊断神经系统疾病,尤其是与一种或多种症状相关的神经系统疾病,所述症状包括小脑变性、精神行为异常、认知障碍、视力障碍、记忆力下降、言语障碍、运动障碍、意识水平下降、自主神经功能障碍或听觉障碍。本发明所述应用制备得到的试剂盒还可以用来区分自身免疫疾病,尤其是区分神经系统的自身免疫疾病与非自身免疫疾病。
附图说明
图1为血清在大鼠原代神经元上的免疫荧光法染色结果;其中,A为患者1血清染色;B为健康受试者血清染色;
图2为患者1血清免疫沉淀结果;
图3为患者1血清免疫沉淀物的WB鉴定结果;
图4为鼠脑海马组织的免疫荧光染色;其中,A为患者1血清染色;B为健康受试者血清染色;
图5为鼠脑皮层组织的免疫荧光染色;其中,A为患者1血清染色;B为健康受试者血清染色;
图6为在鼠脑海马组织上患者1血清和shroom2抗体共定位染色;其中,A为患者1血清染色;B为shroom2抗体染色、C为血清和抗体的merge图;
图7为在鼠脑皮层组织上患者1血清和shroom2抗体共定位染色;其中,A为患者1血清染色;B为shroom2抗体染色、C为血清和抗体的merge图;
图8为血清中和实验验证患者1血清在鼠脑海马组织上的信号;其中,A为加PBST中和血清;B为加入shroom2蛋白中和血清;C为加入对照蛋白中和血清;
图9为血清中和实验验证患者1血清在鼠脑皮层组织上的信号;其中,A为加PBST中和血清;B为加入shroom2蛋白中和血清;C为加入对照蛋白中和血清;
图10为CBA法检测样受试者样本中的自身抗体;其中,A为患者1血清染色;B为健康受试者血清染色;
图11为患者1血清的CBA法共染实验;其中,A为患者1血清染色;B为shroom2抗体染色;C为血清和抗体的merge图;
图12为患者2血清的CBA法共染实验;其中,A为患者2血清染色;B为shroom2抗体染色;C为血清和抗体的merge图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
需要说明的是,本发明的说明书和权利要求书中涉及的术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述:
实施例中的受试者信息
患者1和患者2的血清为医院赠予,对患者的血清进行探索性研究,已征求过患者的同意。健康受试者血清来自于医院体检中心赠送的健康体检者血清,已经体检者本人同意。
患者1:48岁,女性,一个月前出现视力模糊,行走不稳,向前或者向后倾倒以至于不能进行劳动,与人不能进行正常的沟通交流,来医院就诊,常规生化分析检测及CT正常,医生怀疑患有自身免疫性疾病,送检医院检验科检测脑炎相关项目的自身抗体(包括NMDAR、AMPAR1、AMPAR2、LGI1、CASPR2、GABABR、DPPX、IgLON5、GlyR、GABAARα1、GABAARγ2、GABAARβ3、mGluR5、D2R、Neurexin3α),结果均为阴性。因自身原因回家休息,一周后,再次入院,此时,医生怀疑其可能为其他自身抗体阳性,又将其样本送往检验科检测,经检测,该样本副肿瘤14项(包括Ri、Hu、Yo、CV2、Ma2、Amphiphysin、Titin、Ma1、SOX1、Tr、Zic4、PKCγ、Recoverin、GAD65)阴性,中枢脱髓鞘病变6项(AQP4、MBP、MOG、GFAP、AQP1、Flotillin1/2)阴性,自免性脑炎15项(包括NMDAR、AMPAR1、AMPAR2、LGI1、CASPR2、GABABR、DPPX、IgLON5、GlyR、GABAARα1、GABAARγ2、GABAARβ3、mGluR5、D2R、Neurexin3α)阴性,外周神经病12项(GM1,GD1b,GQ1b、Sulfatides、GT1b、GT1a、GD3、GD2、GD1a、GM4、GM3、GM2)阴性。
患者2:65岁,有高血压高血脂病史,3周前出现视力模糊,精神稍有异常,记忆不佳,对周围的人和事只有短暂的记忆,说话时吐字不清,行动较既往迟缓。遂入院检查,常规化验结果无明显异常,医生将其样本送往检验科,经检测,该样本副肿瘤14项(包括Ri、Hu、Yo、CV2、Ma2、Amphiphysin、Titin、Ma1、SOX1、Tr、Zic4、PKCγ、Recoverin、GAD65)阴性,中枢脱髓鞘病变6项(AQP4、MBP、MOG、GFAP、AQP1、Flotillin1/2)阴性,自免性脑炎15项(包括NMDAR、AMPAR1、AMPAR2、LGI1、CASPR2、GABABR、DPPX、IgLON5、GlyR、GABAARα1、GABAARγ2、GABAARβ3、mGluR5、D2R、Neurexin3α)阴性,外周神经病12项(GM1,GD1b,GQ1b、Sulfatides、GT1b、GT1a、GD3、GD2、GD1a、GM4、GM3、GM2)阴性。
实施例1:大鼠原代神经元细胞的免疫荧光法
步骤1大鼠原代神经元细胞分离
1、10%水合氯醛麻醉孕鼠,75%酒精浸泡消毒3min,生物安全柜取出胎鼠;
2、用预冷的HBSS(137mM NaCl,5.4mM KCl,0.6mM MgSO4·7H2O,0.5mM MgCl2·6H2O,0.3mM Na2HPO4·2H2O,0.4mM kH2PO4,5.6mM Grucose,4.2mM NaHCO3,pH7.2~7.4)冲洗胎鼠两次,在预冷的HBSS中解剖出胎脑浸泡在预冷的DMEM中;
3、弃DMEM,用眼科剪将组织块剪成1m3大小的糜状混合物。加入木瓜蛋白酶(浓度为2mg/ml)。在10cm皿中37℃消化处理30分钟,每5min轻轻摇晃一次。一般10至15个大脑组织用消化液20ml;
4、消化结束后,将细胞移到50ml离心管,加入20ml DMEM重悬,1ml枪头吹打10次,400g,4℃离心5min,
5、弃上清液,加入20ml DMEM重悬,1ml枪头吹打10次,4℃,400g离心5min,该步骤重复1次;
6、弃上清液,加入20ml Neurobasal培养基重悬细胞。
步骤2、梯度密度法分离纯化神经元细胞
本实验所采用密度介质的密度为试剂1(1.32g/ml的OptiPrep)(为Percoll的优化产品)
1、取细胞混合原液2ml,并用试剂2(Neurobasal+B27+L-glutamine)的全培养基稀释至6ml;
2、将试剂1和试剂2按照如下表1分别混合,配制介质,并按照1-2-3-4的顺序依次加入到15ml离心管底部;
表1
3、将样本加入到介质顶层;
4、800g,22℃,15min离心分离;
5、依次去除上层6ml细胞碎片部分和1ml神经元细胞和少突胶质细胞混合细胞;
6、从离心管上层取体积大约为2.5ml的神经元细胞悬液,作为后续实验材料;
7、取出神经元细胞后,用5ml的Neurobasal+B27+L-glutamine的全培养基进行稀释;
8、200g,22℃,2min离心,去除上清;
9、使用全培养基洗涤一次;
10、加入5ml Neurobasal+B27+L-glutamine的全培养基重悬;
11、血球计数板计数后按每孔60000细胞接种至48孔板中。
步骤3、对分离好的神经元进行免疫荧光染色,具体操作包括如下步骤:
1、将步骤2长有神经元细胞的48孔板从培养箱中拿出,弃上清,1×HEPES洗2次;
2、使用0.4%的多聚甲醛固定10min,弃上清,1×HEPES洗2次;
3、使用1.25M甘氨酸浸泡10min,弃上清,1×HEPES洗2次;
4、血清孵育:将患者1的血清使用PBS按照1:10的比例进行稀释,并将稀释好的样本加至含有神经元细胞的孔中,室温孵育60min,弃上清,1×HEPES洗3次;
5、二抗孵育:使用FITC标记的二抗室温孵育40min,弃上清,1×HEPES洗3次;
6、拍照:荧光显微镜下进行拍照。
实验结果:
由图1可以看出,患者1血清中存在与大鼠原代神经元结合的抗体,而健康受试者的血清未检出与神经元结合的抗体。
实施例2:shroom2的免疫沉淀
1、取6皿大鼠原代神经元细胞,弃上清,PBS洗2遍,0.4%多聚甲醛固定10min,1×HEPES洗3次;
2、血清孵育:取10ul患者1血清加至10ml DMEM中(1:1000稀释),使用0.22um滤膜过滤后加入到固定好的细胞中,室温孵育2h;
3、取15ul珠子加入到2mL管子中,平衡液洗3遍,用300ul 4%BSA封闭2h;
4、将孵育好的细胞置于冰上,弃上清,PBS洗2遍,加入500ul裂解液(150mM NaCl,1mM EDTA,100mM Tris-HCl,0.5%脱氧胆酸钠,1%TritonX-100,0.1%SDS,pH7.5),收集细胞,加入终浓度为1×的蛋白酶抑制剂,裂解30min,间隔震荡,15000rpm离心30min,取上清,测浓度;
5、将步骤4收集的上清加入到步骤3处理好的珠子中,4℃过夜旋转孵育;
6、用裂解液将所有孵育的珠子洗4次,用80ul 2*loading buffer洗脱;
7、样本处理:洗脱液中先加入5×SDS-PAGE loading buffer,再加入终浓度为0.01M的DTT,100℃加热10min,然后再加入终浓度为2%的碘乙酰胺,室温放置30min;
8、电泳:处理好的样品跑胶,使用银染试剂盒进行染色,分析结果(银染试剂盒购自于thermo)。
实验结果:
参见图2,在用患者1血清获得的来自大鼠神经元的免疫沉淀物中检出一种大约160~180KD的蛋白质(图2泳道2),该蛋白质不存在于类似制备的对照中(图2泳道1)。
实施例3:免疫印迹
1、电泳:取实施例2步骤7得到的样品进行SDS-PAGE;
2、转膜:电泳完成后,使用湿法转膜,转膜条件为200mA,90min;
3、封闭:使用5%的脱脂奶粉室温封闭1h;
4、一抗孵育:按照1:2000的比例孵育抗shroom2的多克隆抗体(抗体购自于sigma公司),室温孵育2h;
5、洗涤:TBST洗3次,每次5min;
6、二抗孵育:加入HRP标记的二抗,室温孵育1h
7、洗涤:TBST洗3次,每次5min;
8、显色:加入化学发光液,记录结果。
实验结果:
使用抗shroom2的多克隆抗体对用患者1血清所收到的,但用对照血清未收到的免疫沉淀物进行蛋白质印记分析,结果由图3可以看出,用患者1血清捕获到的免疫沉淀物中存在与shroom2抗体反应的条带,目的蛋白带处于130KD~180KD之间(目前尚不清楚WB结果上其他条带是shroom2的降解物还是杂信号),而健康受试者的免疫沉淀物中无条带出现,证明该患者体内存在与神经元蛋白反应的抗体。
实施例4:大鼠脑组织的免疫组化、血清和抗体共染以及血清中和实验验证组织上的信号
步骤1取组织,切片
1、选成年大鼠,麻醉,待老鼠四肢硬化,打开腹腔,暴露出心尖,从左心尖灌注PBS,以便全身循环;
2、取出脑组织;
3、甲醇固定10~30min;
4、脱水:将样本移入30%蔗糖溶液中,4℃放置至组织块沉底;
5、将少量包埋剂OCT滴加到标本台,置入-20℃冷冻切片机的冷冻台。待组织略微发白时用OCT在标本表面涂一薄层,继续冷冻20min,切片。
步骤2大鼠脑组织免疫组化片子的染色
1、一抗孵育:取步骤1得到的鼠脑组织片,按照1:100的比例稀释患者1血清,室温孵育1h;
2、洗涤:PBST洗3次,每次5min;
3、二抗孵育:加入FITC标记的二抗,室温孵育1h;
4、洗涤:PBST洗3次,每次5min;
5、显微镜下观察结果拍照。
步骤3血清和抗体共染验证组织上的信号
1、取步骤2经患者1血清染色的组织片,加入shroom2抗体(1:20稀释),室温孵育1h;
2、洗涤:PBST洗3次,每次5min;
3、二抗孵育:加入Alexa Fluor 594标记的二抗,室温孵育1h;
4、洗涤:PBST洗3次,每次5min;
5、显微镜下观察结果拍照。
步骤4血清中和实验验证患者1血清在鼠脑组织上染出的信号
1、中和蛋白的制备:收集过表达shroom2蛋白的HEK293细胞(过表达细胞的制备见实施例6)1皿(10cm皿),加入200ul PBS,超声破碎(破碎条件为:15%功率,破3s,停6s,共超声3次);对照细胞为转入空载pCDNA3.1的HEK293细胞,对照蛋白的制备条件与shroom2中和蛋白的制备条件相同;
2、中和实验:
(1)用PBST将患者血清按照1:100的比例稀释,稀释好的血清中分别加入20ulshroom2中和蛋白及对照蛋白,正常组加入20ul PBST,室温孵育10min;
(2)一抗孵育:将上述孵育好的样本分别加至鼠脑组织爬片上,室温孵育1h;
(3)洗涤:PBST洗3次,每次5min;
(4)二抗孵育:加入FITC标记的二抗,室温孵育1h;
(5)洗涤:PBST洗3次,每次5min;
(6)显微镜下观察结果拍照。
实验结果:
(1)鼠脑组织免疫荧光染色
结果从图4中可以看出,与健康受试者血清染色结果相比,患者1血清在鼠脑海马组织上有明显的点状信号。进一步的实验表明,该信号可与神经元marker NeuN(proteintech,货号26975)的抗体共定位。
结果从图5中可以看出,与健康受试者血清染色结果相比,患者1血清在鼠脑皮层组织上有明显的点状信号。进一步的实验表明,该信号可与神经元marker NeuN(proteintech,货号26975)的抗体共定位。
(2)患者1血清与shroom2抗体在鼠脑组织上的共染
从图6中可以看出,用shroom2抗体对经患者1血清染色的组织片进行染色(鼠脑海马组织),结果显示血清的染色和抗体的染色可共定位。
从图7中可以看出,用shroom2抗体对经患者1血清染色的组织片进行染色(鼠脑皮层组织),结果显示血清的染色和抗体的染色可共定位。
(3)血清中和实验验证鼠脑组织上的信号
用过表达shroom2的HEK239裂解物及对照裂解物(转入空载pCDNA3.1的HEK239)进行血清中和实验验证鼠脑海马组织染出的信号,结果由图8可以看出,shroom2中和蛋白明显的封闭了患者1血清在鼠脑海马组织上出现的点状信号,而对照蛋白未封闭住组织上出现的信号,表明该点状信号为鼠脑组织上表达的抗shroom2抗原的特异性信号。
用过表达shroom2的HEK239裂解物及对照裂解物(转入空载pCDNA3.1的HEK239)进行血清中和实验验证鼠脑皮层组织染出的信号,结果由图9可以看出,shroom2中和蛋白明显的封闭了患者1血清在鼠脑组织皮层上出现的点状信号,而对照蛋白未封闭住组织上出现的信号,表明该点状信号为鼠脑皮层上表达的抗shroom2抗原的特异性信号。
结论:与健康受试者血清相比,患者1血清中存在抗shroom2的自身抗体,且该自身抗体可识别鼠脑组织中的神经元上表达的shroom2蛋白。
实施例5质谱法
从实施例2SDS-PAGE胶上切下蛋白条带送去拜谱生物进行质谱法分析。
结果:靶抗原与患者1的血清首先进行免疫沉淀,然后通过SDS-PAGE电泳分离蛋白质,再进行凝胶银染(见图2)。从银染胶上切下正常血清无而患者血清有的条带,进行质谱分析。根据肽段鉴定表及肽段解读表所述,所鉴定的蛋白质序列中包括shroom2序列,质谱所鉴定的结果是可信的。
实施例6基于HEK293细胞的免疫荧光法检测患者血清中抗shroom2自身抗体及血清和抗体共染验证免疫荧光法检出的信号
步骤1、重组载体的构建
通过PCR或人工合成的方法,将shroom2基因通过分子克隆方法连至pCDNA3.1上,得到重组载体pCDNA3.1-shroom2,构建好的重组载体经测序正确后大提备用;
步骤2、细胞转染
(1)293T细胞培养:DMEM高糖培养基与FBS按9:1比例配制成10%FBS-DMEM高糖培养基,待细胞铺满时按1:5~1:6传代,置37℃,5%CO2的细胞培养箱中过夜培养;
(2)待细胞密度为30%~40%时,将pCDNA3.1-shroom2转入细胞中:
步骤3、爬片固定
(1)洗涤:将生长48h的细胞用PBS洗涤2次,
(2)固定:加入丙酮固定5min;
(3)洗涤:丙酮固定后的爬片用PBS洗涤2次,干燥后备用。
步骤4、免疫荧光染色
(1)使用PBST分别将患者1、患者2的血清稀释10倍,加至步骤3获得的细胞爬片,室温孵育1h;
(2)洗涤:PBST洗3次,每次5min;
(3)二抗孵育:加入FITC标记的二抗,室温孵育1h;
(4)洗涤:PBST洗3次,每次5min;
(5)显微镜下观察结果拍照。
步骤5、血清和抗体共染验证过表达细胞上的信号
取步骤4经患者血清染色的细胞爬片进行抗体共染实验,具体实验步骤同实施例4步骤3。
结果从图10中可以看出,与健康受试者血清染色结果相比,患者1血清在表达shroom2抗原的细胞爬片上有明显的信号。
参见如图11表达shroom2的HEK293细胞用患者1的血清(A)和抗shroom2的鼠的多克隆抗体(B)进行染色,merge后的结果(C),结果显示血清的染色和抗体的染色可共定位。
图12表达shroom2的HEK293细胞用患者2的血清(A)和抗shroom2的鼠的多克隆抗体(B)进行染色,merge后的结果(C),结果显示血清的染色和抗体的染色可共定位。
结论:与健康受试者血清相比,患者1血清中存在抗shroom2的自身抗体,且该自身抗体可识别过表达shroom2蛋白的细胞。表达shroom2蛋白的细胞可用来区分本发明所述症状的患者及健康受试者。
实施例7基于细胞的免疫荧光法验证抗shroom2自身抗体的特异性
1、取实施例6已经干燥好的过表达shroom2自身抗体的细胞爬片,备用;
2、选取具有各种神经自身抗体(抗Hu、Yo、CV2、Ma2、Amphiphysin、Ma1、SOX1、NMDAR、AMPAR1、AMPAR2、LGI1、CASPR2、GABABR、DPPX、IgLON5)的30位患者和40位健康对照的血清,进行免疫荧光染色,具体步骤如下:(1)一抗孵育:按照1:100的比例孵育患者血清,室温孵育1h;(2)洗涤:PBST洗3次,每次5min;(3)二抗孵育:加入FITC标记的二抗,室温孵育40mi;(3)洗涤:PBST洗3次,每次5min;(4);显微镜下观察结果。
3、拍照:在表达shroom2的HEK293细胞上,上述70例血清均不产生与患者血清相似的细胞形态。
实施例7结论:患有本发明描述的神经系统疾病的患者具有针对shroom2蛋白的自身抗体,而另一些患有神经系统疾病的患者和健康受试者样本没有这种抗体。本发明为实现神经系统疾病的诊断提供了待测的与自身抗体结合的新抗原。
实施例8抗shroom2自身抗体在患有本发明描述症状的患者中的检出情况
1、取实施例6步骤3已经干燥好的过表达shroom2的细胞爬片,备用;
2、选取158例具有小脑变性、精神行为异常、认知障碍、视力障碍、记忆力下降、言语障碍、运动障碍、意识水平下降、自主神经功能障碍或听觉障碍等症状中其中一种或多种症状的患者(Hu、Yo、CV2、Ma2、Amphiphysin、Ma1、SOX1、NMDAR、AMPAR1、AMPAR2、LGI1、CASPR2、GABABR、DPPX、IgLON5、D2R、Neurexin3、KCNA4、GABAARγ2、ATP1A3、Homer3、ARHGAP26、ITPR1/2、mGluR1、CARP VIII、AP3B2、septin5、GM1,GD1b,GQ1b、Sulfatides、GT1b、GT1a、GD3、GD2、GD1a、GM4、GM3、GM2自身抗体检测为阴性的患者),使用实施例6获得的细胞爬片进行免疫荧光检测;
3、结果:在158例疑似患有神经系统症状的患者中检出3例抗shroom2自身抗体阳性。
实施例8结论:抗shroom2自身抗体可在患有神经系统症状的患者中检出,说明该抗体对神经系统自身免疫疾病的诊断具有辅助作用。
以上内容仅为说明本发明的技术思想,不能以此限定本发明的保护范围,凡是按照本发明提出的技术思想,在技术方案基础上所做的任何改动,均落入本发明权利要求书的保护范围之内。
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<110> 陕西脉元生物科技有限公司
<120> 用于检测自身免疫系统疾病的抗Shroom2自身抗体及其产品和应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4851
<212> DNA
<213> 人工序列(Artificial Sequence)
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atggagggcg ccgagccccg cgcgcggccc gagcgcctgg ccgaggccga gacgcgggcg 60
gcggacggcg ggcgcctggt ggaggtgcag ctgagcggcg gcgccccgtg gggcttcacc 120
ctgaagggcg gccgcgagca cggcgagccg ctggtcatca ccaagattga agagggcagt 180
aaagccgcgg cggtcgacaa gttactggct ggagatgaga tcgtcggcat caatgacatt 240
ggtctctcag ggtttagaca ggaagcgatt tgcctggtga aggggtccca taagaccctg 300
aagctggtcg tcaaaaggag gagcgagctg ggctggaggc ctcactcctg gcatgccacc 360
aagttctctg acagccaccc cgagctagcg gcctccccat tcacctccac cagcggctgt 420
ccttcctggt ccggccgaca ccacgcgagt tcttcctccc acgacctgtc cagttcctgg 480
gagcagacga acctacagcg caccttagat cacttcagct ccttggggag cgttgacagc 540
ctggaccacc cctccagtcg cctctcggtg gccaagtcca acagcagcat cgaccacctg 600
ggcagccaca gcaagcgcga ctcggcctac ggctccttct ccaccagctc tagcactcct 660
gaccacacct tgtccaaagc cgacacgtcc tccgcagaga acatcctcta cactgtgggc 720
ctctgggagg ctcccaggca gggtggccgg caggcccagg ccgcaggcga ccctcagggc 780
tcggaggaga agctcagttg tttcccgccc agggtccccg gtgacagcgg caaaggcccc 840
aggccagagt acaatgccga gcccaagctg gctgcccctg ggaggtccaa ttttgggcca 900
gtctggtatg ttcccgataa gaagaaagca ccatcatccc cacctcctcc ccctccccct 960
ctccgcagtg acagctttgc tgccaccaag agccacgaga aggcccaggg ccctgtgttc 1020
tcagaggcgg ctgcggcaca gcactttacg gccctggccc aggctcagcc tcgtggtgac 1080
cggagaccag agctcaccga tcggccttgg aggtcagcac acccggggag cctcgggaag 1140
ggatcgggag gcccgggctg cccacaggag gcccacgcag acggcagctg gccgccctcc 1200
aaggatggag cttccagtag gctgcaggcc tctctgtcca gctcagatgt gcgcttccct 1260
cagtctcctc atagcggccg acaccctccc ctatacagcg accacagccc cctctgtgct 1320
gacagccttg ggcaggagcc aggggctgcc agcttccaga acgacagccc tcctcaggtg 1380
agggggctca gcagctgtga ccagaagctg gggagcggct ggcagggtcc ccggccctgt 1440
gtgcagggag acctgcaagc agcacagctc tgggcgggat gctggccttc tgacacagcc 1500
cttggagccc tcgagagtct tcccccaccc acggtgggcc agagcccacg ccatcaccta 1560
cctcagcctg agggtcctcc ggatgcccgc gagacaggac ggtgttaccc gctggacaaa 1620
ggggccgagg gctgctccgc gggagcccag gagcctccca gggccagccg tgcagaaaaa 1680
gccagccaga ggctggcagc cagcatcacg tgggcagatg gggagagcag caggatctgc 1740
ccgcaggaga cgcccctgtt gcactccctg acccaggagg ggaagcgccg gcctgagagc 1800
agtccagagg acagcgccac cagaccgcca ccgttcgacg cccacgtggg caagcccacc 1860
cgaagaagcg accgctttgc caccaccctg cggaatgaga tccagatgca tagagccaag 1920
ctgcagaaga gccggagcac agtggctctg actgcagcag gggaggcgga ggatggcacc 1980
ggccgctgga gggccgggtt gggaggtggc acccaggaag gacccctcgc tggcacctat 2040
aaagaccacc tgaaagaggc ccaagcccgg gtcctgaggg ccacgtcctt caagcgccgc 2100
gacttggacc ccaacccagg agacctatac ccggagtcac tggaacaccg gatgggggat 2160
ccagacactg tcccccactt ctgggaggca ggcctggccc agccaccctc atctacaagt 2220
ggcgggcccc acccgccccg catcggaggc cggagacggt tcacagctga gcagaaattg 2280
aagtcctact cggaacctga gaagatgaac gaggtgggcc tcacgagggg ctacagtcct 2340
caccagcacc ccaggacatc tgaggatact gtgggcacgt ttgctgacag gtggaagttt 2400
tttgaggaaa cgagcaaacc tgttccccag aggcctgccc agaagcaagc tcttcacgga 2460
atcccgagag acaagccaga gaggccgcgg acagcgggcc gcacatgtga gggcacggag 2520
ccctggtcgc gcaccacctc ccttggggac agcctcaacg ctcacagcgc agcggagaag 2580
gcagggactt cagacctgcc gcggaggctc ggcacctttg cagagtatca ggcctcttgg 2640
aaggaacaga ggaaacctct ggaggccagg agctctgggc gctgccactc agcggatgac 2700
atcctggatg tgagcctgga cccacaggag aggccgcagc acgttcatgg gaggtcccgg 2760
tcttcaccgt ccacagacca ctacaagcag gaagcttctg tcgaactgcg aaggcaggca 2820
ggggaccccg gcgagcccag agaagagctt ccctccgcag tccgggccga ggagggacag 2880
tccacgccga gacaagcaga tgcccagtgt cgggaaggca gcccaggatc acagcagcac 2940
ccaccgagtc agaaggcacc gaacccaccc acattctctg aactatctca ctgccgggga 3000
gccccagagc tgccccggga gggccggggc cgagcgggaa ccctacctcg agattataga 3060
tactcggagg agagcacccc agcagacttg ggaccccgag cccagagccc tggctcaccc 3120
ctgcatgctc gaggacaaga ctcgtggcca gtgagctcag ccctgctctc caagaggcca 3180
gccccacaga ggccaccgcc acccaagcgc gagcccagga gatacagggc cacagacggc 3240
gcacctgctg acgcccccgt gggcgtcctc ggcaggccct tcccaacgcc atcccctgcg 3300
tccctggatg tgtatgtggc ccgcctgtcc ctctcccaca gcccctctgt gttcagcagt 3360
gcccagcccc aggacacccc gaaggccact gtctgtgagc gtggaagcca gcatgtgagc 3420
ggggacgcat cacgtcctct gccagaagca ctgctccctc ccaagcagca gcacctgcgc 3480
ctgcagacgg ccaccatgga gacctcgcgc tccccctcgc cccagttcgc cccccagaaa 3540
ctgacggaca aacctcccct gctcatccag gatgaggatt caaccagaat tgagcgggtg 3600
atggacaaca acaccacggt gaagatggtg cccatcaaga tcgtgcactc ggagagccag 3660
ccagagaagg agagccgcca gagcctggca tgccccgccg agccacctgc cctgccccac 3720
gggctggaga aagaccagat caagacgctg agcacatctg agcagttcta ctcgcgcttc 3780
tgtctgtaca cgcggcaggg tgctgagccc gaggccccac atagggccca gccggctgag 3840
ccccagcccc tgggcaccca ggtgcccccc gagaaagacc gctgcacctc ccctccaggg 3900
ctcagctaca tgaaggccaa agagaagact gtggaagacc tgaagtcgga ggagctggcc 3960
agggagatcg tggggaagga taagtccctg gccgacatcc tggatcccag tgtgaagatc 4020
aaaaccacta tggacttgat ggaaggcatc ttccccaaag acgagcacct cctggaagaa 4080
gcccagcaac ggaggaagct gctccccaaa atcccctctc ctagaagcac agaggagagg 4140
aaagaggagc ccagcgtgcc tgcggccgtg tccctggcca ccaattctac ctactacagc 4200
acgtcggccc ccaaggcgga gctgctgatc aagatgaagg acctgcagga gcagcaggag 4260
cacgaagagg attcgggaag cgacttggac cacgacctgt cggtgaagaa gcaggagctc 4320
atcgagagca tcagccgcaa gctgcaggtg ctccgggagg cccgcgagag cctgctggag 4380
gacgtgcagg ccaacaccgt gctgggggcc gaggtggagg ccatcgtgaa aggcgtctgc 4440
aagcccagcg agtttgacaa gttccggatg ttcattggag acctggacaa agtggtgaac 4500
ctcctgctgt cgctgtcagg ccgcctggcc cgggtggaga atgccctcaa taatttggac 4560
gacggcgctt ctcccggtga tcggcaatca ctgcttgaga agcagagagt cctgatccag 4620
cagcacgagg acgccaagga gctcaaggag aacctggacc gccgcgagcg catcgtcttt 4680
gacattttgg ccaactatct gagcgaggag agcctcgcgg actatgagca cttcgtgaag 4740
atgaagtcgg ccctcatcat cgagcagcgg gagctggaag ataaaatcca ccttggtgaa 4800
gagcagctga agtgcttatt ggacagcctt cagcccgaaa ggggcaaata a 4851
<210> 2
<211> 4464
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggagggcg ccgagccccg tgcacggccc gagcgcctgg ccgaagctga ggcgccggcg 60
accgacggcg tccgcctggt ggaggtgcag ctgagcggcg gcgccccttg gggcttcacc 120
ttgaagggcg gccgcgagca tggcgagcct ctggtcatca ccaagattga agaaggaagc 180
aaagctgcag cagtggacaa gctacttgct ggagatgaga tagtggccat taatgatgtg 240
agtctctcag ggttcagaca agaagcaatt tgcctggtga aaggctccca caagaccctg 300
aagcttgtgg tcaaaaggaa aagtgatcca agttggaggc cccactcctg gcacgctacc 360
aagtactttg atgtccaccc tgagccagca gcctcactgt ttctgaacac cagcggttcc 420
ccttcctgga agagccagca ccaggctagt tcttcctctc atgatttatc tggctcatgg 480
gagcatacaa gcctacagcg cacttctgac cacttcagct ccatgggcag catagacagc 540
ctcgaccaca gctcccaact ctacccatct ggacacctct catctgccaa gtccaatagc 600
agcattgacc acctgggagg ccacagcaag cgagactcag cttacggctc cttttccaca 660
tgctccagca cccctgacca caccttgccc aaggctgatg cctcttccac tgagaacatt 720
ctctataaag ttggcctttg ggaggcctcc agaccaggca gcagccgaca aagccagtct 780
acaggtgatc ctcagggact gcaggacagg ccatcatgtt tcatacctag ggttcctggt 840
aacagtagta aaagtccaag gccggaggat aatgttgaac ccaaaatagc cactcatggg 900
agatccaatt ttgggcctgt gtggtatgtt cctgacaaga aaaaagcccc ttctccccca 960
cctcttggac ttcctttgcg cagtgatagc ttttctgtgg cagccagggg ccatgaaaaa 1020
gcccggggcc ctccattctc agacttggcc agtatgcagc actttatcac cctgccccat 1080
gtgcaacccc ggggggacca tagaatggaa actacagatc gtcagtggaa gctcacacac 1140
ctaagcagtg gaaaagaaat aggaaatgtg ggttaccagt cagaaggcca cctggattgc 1200
cgttggctat gctctgatga tagagcaggt aggccctcag ggcctccggg caggctccag 1260
ttctccgatg tgcactttct gaagtcttac cacgggagcc agcaccaaca gcagtgcagt 1320
gacgagagcc caagggcccc ctcatcacca agggaactgc ttcatataac tcctggtggc 1380
ggtctgcaag agcctcctga accatctcag gatgacaacc ctactcaggt gaggtggcct 1440
ggttctgctc accagaagct agatgacaga gggcggagcc actattttcc tgggtccctc 1500
aggcagcctg tacaggggag tgcccaggtt gtgatacccc gaggtgacta ttggcattca 1560
gatacaactc ctgtggacct tgaatatcct ctcttacgcc cagtaggaca gaggacctac 1620
ctacagcagc atgaggagac tccggcctct catgaaaaag aggggtatca ccagctaaat 1680
gcagggattg aaggctgctg ctctggaatc caagagcctc ctagagccag ccgcactgtg 1740
agaactggtc tgcagtgtcc tagcaatgac ttcaaattag tggatggaga gagtgggaga 1800
atttctcgtc agaggacacc catgctgcat tctctgaccc aagatggtac atggagaccc 1860
gggaacagca aggattgtgg aaatgataag ccaccactgt ttgatgccca ggtgggtaaa 1920
cccacacgga ggagtgaccg ttttgccaca acactgagga atgagatcca aatgcgtaga 1980
gcaaagctgc agaagagcaa aagtacagtg acactagctg gagacagtga agctgaagat 2040
tgtgctggag actggagagc tgatgtgggg gctgtcccag aaggttcctt ccccagcacc 2100
tataaagagc acctgaagga ggcccagaca cgtgttctga aggccacctc tttccaacgc 2160
cgagatttag atcccacccc agcagatcag tattcaggac catcagaaca caggactttt 2220
gaccacagtg cctcatcttc tttatcttct ttccctgggg agccagactc tgccccacgc 2280
ttctgtgaga caggtctggc caaggcaccc tcttctggag ttggtgtacc ccacgttctt 2340
cgaattggag gccggaaacg gttcacagca gagcagaaac taaagtccta ttctgagcca 2400
gagaaaataa atgaagtggg gctctcaggg gaccaccgtc ctcatcctac tgtcaggaca 2460
cctgaggata cagtgggtac atttgctgac aggtggaagt tttttgagga aacaagcaaa 2520
tctcttctcc agaaggcagg ccataggcaa gttcactgtg ggctccctag agagaaggct 2580
gagaggccac agacagggca ccatgaatgt gagagtacag agccctggtt ccagaagagg 2640
tcactggcca cctcctgcgg agagatcctc agtgatagaa aagtagaaaa ggcctcagag 2700
aaattgaacc cacccagaag gcttggaacc ttcgcagaat atcaagcatc ttggaaggaa 2760
cagaagaaac ctctggaagc caggagttct ggacgatacc attcagcaga tgatattctg 2820
gatgctggtc tagatcagca gcagaggcca cagtacattc atgagcggtc tcgttcatca 2880
ccgtccacag atcactactc acaggaagtg cctgttgaac caaacaggca ggcagaggac 2940
tctggtgacc acaaagaagc aattctctgt acactacaag ctgaggaggg atgctctgct 3000
ccaagctcct ctgtgctcag cagtgcccag ccgcaagaca gccagcatgt gaatgaagac 3060
acaactttcc ctcaaccaga aacccagctc tcttccaagt gtcaacacct acagacgtca 3120
gccatggaaa cttctcgctc cccttcgcct cagtttgccc cacagaagct gacagataaa 3180
cctcccctgc ttatccatga agacaactca gcaagaatcg agcgggtgat ggacaacaac 3240
accactgtga agatggtgcc cataaaaatt gtgcactcag aaagccagcc cgagaaggag 3300
agtcgtcaga gtctcgcgtg cccagctgag ctgcccccac tgcccagtgg gctggagagg 3360
gaccagatca agacactgag tacatcagag caatgctact cccgcttctg tgtgtacaca 3420
cgacaggagg tggaagctcc tcatagagcc cgccctccag agccccggcc acccagcacg 3480
cctgcacctc ctgtcagaga tagctgttcc tcccctccct cactcaacta tgggaaggcc 3540
aaggagaaaa ccatggatga cttgaagtct gaagaattag ccagggagat tgtgggaaag 3600
gacaagtctt tggctgacat cctggacccc agtgtgaaga tcaaaactac catggatctg 3660
atggagggaa tttttcccaa agatgagtac ctcctagagg aagctcagca gcggagaaag 3720
ctgctcccca aagtcccctt acccagagtc acagaggaca agaaacagga cccaggtatg 3780
ccaggggttg tgtccttggc caccaattct acctattaca gcacatcagc ccccaaagca 3840
gagcttctta tcaagatgaa ggacctgcag gagcctgaag agtattcagc aggtgacttg 3900
gatcatgacc tttctgttaa gaagcaagag ctcattgaca gtatcagccg caagctgcag 3960
gtgctccggg aagcacgtga gagcctgctg gaagacatcc aagccaacaa tgctcttggg 4020
gatgaggtgg aagccattgt gaaagatgtc tgcaagccca atgagtttga caagttccgg 4080
atgttcattg gagacctgga caaagtggtg aacctcctgc tgtcactgtc aggacgcctg 4140
gcccgtgtgg aaaatgccct taataattta gatgacaatc cttctcctgg agatcggcag 4200
tcactgttgg agaaacagag agtcctaact cagcagcacg aggatgccaa ggagcttaaa 4260
gagaacctgg accgccgtga gcgcattgtg ttcgacatcc tggctaccta cctcagcgag 4320
gagaacctgg ctgactatga gcacttcgtg aagatgaagt cagctctcat cattgagcag 4380
cgagagctgg aagataaaat ccacctgggt gaagagcagc tcaagtgttt gtttgacagc 4440
ctacagcctg agagaagcaa atga 4464
<210> 3
<211> 4818
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggagggcg ccgagccccg cgcgcggccc cagcgcctgg ccgaggccga gaccgagacg 60
cgggcggccg acggcgggcg cctggtggag gtgcagctga gcggcggcgc cccgtggggc 120
ttcaccctga agggcggccg cgagcacggc gagccgctgg tcatcaccaa gattgaagag 180
ggcagtaaag ccgcggcggt cgacaagtta ctggctggag atgagatcgt cggcatcaat 240
gacattggtc tctcagggtt tagacaggaa gcgatttgcc tggtgaaggg gtcccataag 300
accctgaaac tggtcgtcaa aaggaggagc gagctgggct ggaggcctca ctcctggcat 360
gccaccaagt tctctgacag ccaccctgag ctagcggcct ccccgttccc ctccgccagc 420
ggctgtcctt cctggtctgg ccgacaccat gcgagttctt cctcccacga cctgtccagt 480
tcctgggagc agacgaacct acagcgcacc ttagaccact tcagctccct ggggagtgtt 540
ggcagcctgg accacccctc cagtcgcctc tcggtggcca agtccaacag cagcatcgac 600
cacctgggca gccacagcaa gcgtgactca gcctacggct ccttctccac cagctctagc 660
actcctgacc acaccttgtc caaagccgac acgtcctccg cagagaacat cctctacagt 720
gtaggcctct gggaggctcc cagacagggt ggccggcagg cccaggccgc aggtgaccct 780
cagggcttgg aggagaagct aaggtgtttc ccgcccaggg tccccagtga cagcgacaaa 840
ggccccaggc cagagtacaa tgccgagccc aagctggctg tccctgggag gtccaatttt 900
gggccagtct ggtatgttcc tgataagaag aaagcaccag catccccacc ccctccccct 960
ccccctctcc gcagtgacag ctttgctgcc accaagagcc acgagaaggc ccagggccct 1020
gtgttctcag aggtggccgt ggcacagcac tttacggccc cggcccaggc tcagccccgt 1080
ggtgactgga gaccagagcc cactgatcgg ccttggaggt cagcacaccc agggagcctc 1140
gggaagggat cgggaggccc gggctgccca caggaggccc gcgcagatgg cagctggcca 1200
ccctccaagg atggagcttc cagtaggctg caggcctctc tgtccagctc agatgtgcgc 1260
cttcctcagt ctcctcgtag cggccgccac cctcccctgt acagtgacca cagcccactc 1320
tgtgctgaca gccttgggca ggagccagcg gctgccagct cccagaacga cagccttcct 1380
caggtgaggg ggctcagcag ctgtgaccag aagctgggga gcggctggca gggcccccgg 1440
ccctgtgtgc agggaggccc gcaagtggcc cagctctggg caggatgctg gccttctgac 1500
acggcccctg gagccctcaa gagtcttccc ccacccacgg tgggccagag cccacaccgt 1560
cacctacctc agcctgaggg tcctccagat gcctgcgaga caggacggtg ttacccactg 1620
gacacagggg ccgagggctg ctccgcagga gcccaggagc ctcccagggc cagccgtgta 1680
gaaaaagcca gcatcacatg ggcagatggg gagagcagca ggatctgccc gcaggagaca 1740
cccctgttgc actccctgac ccaggagggg aaacaccggc ctgagagcag cccagaggac 1800
agcgccacca gaccgccacc gttcgacgcc catgtgggca agcccacccg gagaagcgac 1860
cgctttgcga ccaccctgcg gaatgagatc cagatgcgtc gagccaagct gcagaagagc 1920
cggagcacgg tggctctgac tgcggcaggc gaggcggagg acgccaccgg ccgctggagg 1980
gtcaagttgg gaggtggcgc ccaggaagga cccttcgctg gcacctataa agaccacctg 2040
aaggaggccc aagcccgagt cctgagggcc acgtccttca agcgccgcga cttggacccc 2100
aacccagcag atctataccc ggagtcactg gaacaccgga tgggtggtcc agacactgtc 2160
cgccacttct gggaggcagg cctggcccag ccaccctcat ctacaggtgg cgggccccac 2220
ccgccccgca tcggaggccg gagacggttc acagctgagc agaaactgaa gtcctactcg 2280
gaacccgaga agatgaatga ggtgggcctc acgaggggct acagtcctca ccagcacccc 2340
aggacatctg aggatactgt gggcacattt gctgacaggt ggaagttttt tgaggaaacg 2400
agcaaacctg ttccccagag gcctgcccag aggcaagtgc ttcatggaat cctgagagac 2460
aagccagaga ggccgtggac agtgggccgc acgtgtgagg gcacggagcc ctggtcgcgc 2520
accacctccc ttggggacag cctcaacgct cacagcacag cggagaagac agggacgtca 2580
gacccgccgc agaggctcgg cacctttgca gagtatcagg cctcctggaa ggaacagagg 2640
aagcctctgg aggccaggag ctccgggcgc tgccactcag cggatgacat cctggatgtg 2700
ggcctggacc cgcacgagag gccgcagcac gttcatggga ggtcccggtc ttcgccatcc 2760
acagaccact acaagcagga agcttctgtc gaactgcgaa ggcaggcagg ggaccctggc 2820
gagcacagag aagagcttcc cttcgcagtc cgggccaagg agggacagtc cacgctgaga 2880
caagcagatg tccagtgttg ggaaggcagc ccaggatcac agcagcaccc accgagtcaa 2940
aacgcaccga acccacccac attctctgaa ccatctcact cccagggagc cccagagctg 3000
ccccaggagg gccggggccg agcaggaacc ctacctcgag attatagata ctcggaggag 3060
agcatcccag cagacttggg accccgagcc cacagccctg gctcacccct gcatgctcaa 3120
ggacaagact cgtggccagt gagctcagcc ctgctcgcca agaggccagc cccacagagg 3180
ccaccgccac ccaagcgcga gcccaggaga tacagggcca cagacagcgc acctgccgac 3240
gcacccctgg ccgtccttgg caggcccttc ccaacaccgt cccctgcatc cctggatgtg 3300
tgtgtggccc gtctgtccct ctcccacagc ccctctgtgt tcagcagtgc ccagccccag 3360
gacatcccaa aggccgctgc ctgtgagcgt ggaagccagc atgtgagtgg ggacacaccg 3420
ctccctccca agcagcagca cctgcgcctg cagacggcca ccatggagac ctcgcgctcc 3480
ccctcgcccc agttcgctcc ccagaagctg acggacaaac ctcccctgct catccaggat 3540
gaggattcaa ccagaattga gcgggtgatg gacaacaaca ccacggtgaa gatggtgccc 3600
atcaagatcg tgcactcgga gagccagcca gagaaggaga gccgccagag cctggcgcgc 3660
cctgctgagc cacccgccct gccccgcggg ctagagaaag accagatcaa gacgctgagc 3720
acgtcggagc agttctactc ccgcttctgc ctgtacacgc gtcagggtgc tgagcccgag 3780
gccccacata gggcccagcc agctgagccc cagcccctgg gcacccaggc gccccccgag 3840
aaggaccgct gcgcctcccc tccggggctc agctacatga aggccaaaga gaagactgtg 3900
gaagacctga agtcggagga gctggccagg gagattgtgg ggaaggataa gtccctggcc 3960
gacatcctgg atcccagtgt gaagatcaaa accaccatgg acttgatgga aggtatcttc 4020
cccaaagacg agcacctcct ggaagaagcc cagcagcgga ggaagctgct ccccaaaatc 4080
ccctctccta gaagcacaga ggagaggaaa gaggagccca gcgtgcctgc ggccgtatcc 4140
ctggccacca attctaccta ctacagcacg tcggccccca aggcggagct gctgatcaaa 4200
atgaaggaca tgcaggagca gcaggagcgt gaagaagatt cggggagcga cttggactat 4260
gacctgtcgg taaagaagca ggagctcatc gagagcatca gccgcaagct gcaggtgctt 4320
cgggaagccc gcgagagcct gctggaggac gtgcaggcca acactgtgct gggggccgag 4380
gtggaggcca tcgtgaaagg cgtctgcaag cccagcgagt ttgacaagtt ccggatgttc 4440
attggagacc tggacaaagt ggtgaacctc ctgctgtcac tgtcaggccg cttggcccgg 4500
gtggagaatg ccctcaataa tttggacgac aacgcttctc ctggtgatcg gcaatcactg 4560
cttgagaagc agagagtcct gatccagcag cacgaggacg ccaaggagct caaggagaac 4620
ctggaccgcc gcgagcgcat cgtctttgac attttggcca actatctgag cgaggagagc 4680
ctcgcggact atgagcactt cgtgaaaatg aagtcggccc tcatcatcga gcagcgggag 4740
ctggaagata aaatccacct tggtgaagag cagctgaagt gcttattcga cagccttcag 4800
cctgaaaggg gcaaataa 4818
<210> 4
<211> 1616
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Glu Gly Ala Glu Pro Arg Ala Arg Pro Glu Arg Leu Ala Glu Ala
1 5 10 15
Glu Thr Arg Ala Ala Asp Gly Gly Arg Leu Val Glu Val Gln Leu Ser
20 25 30
Gly Gly Ala Pro Trp Gly Phe Thr Leu Lys Gly Gly Arg Glu His Gly
35 40 45
Glu Pro Leu Val Ile Thr Lys Ile Glu Glu Gly Ser Lys Ala Ala Ala
50 55 60
Val Asp Lys Leu Leu Ala Gly Asp Glu Ile Val Gly Ile Asn Asp Ile
65 70 75 80
Gly Leu Ser Gly Phe Arg Gln Glu Ala Ile Cys Leu Val Lys Gly Ser
85 90 95
His Lys Thr Leu Lys Leu Val Val Lys Arg Arg Ser Glu Leu Gly Trp
100 105 110
Arg Pro His Ser Trp His Ala Thr Lys Phe Ser Asp Ser His Pro Glu
115 120 125
Leu Ala Ala Ser Pro Phe Thr Ser Thr Ser Gly Cys Pro Ser Trp Ser
130 135 140
Gly Arg His His Ala Ser Ser Ser Ser His Asp Leu Ser Ser Ser Trp
145 150 155 160
Glu Gln Thr Asn Leu Gln Arg Thr Leu Asp His Phe Ser Ser Leu Gly
165 170 175
Ser Val Asp Ser Leu Asp His Pro Ser Ser Arg Leu Ser Val Ala Lys
180 185 190
Ser Asn Ser Ser Ile Asp His Leu Gly Ser His Ser Lys Arg Asp Ser
195 200 205
Ala Tyr Gly Ser Phe Ser Thr Ser Ser Ser Thr Pro Asp His Thr Leu
210 215 220
Ser Lys Ala Asp Thr Ser Ser Ala Glu Asn Ile Leu Tyr Thr Val Gly
225 230 235 240
Leu Trp Glu Ala Pro Arg Gln Gly Gly Arg Gln Ala Gln Ala Ala Gly
245 250 255
Asp Pro Gln Gly Ser Glu Glu Lys Leu Ser Cys Phe Pro Pro Arg Val
260 265 270
Pro Gly Asp Ser Gly Lys Gly Pro Arg Pro Glu Tyr Asn Ala Glu Pro
275 280 285
Lys Leu Ala Ala Pro Gly Arg Ser Asn Phe Gly Pro Val Trp Tyr Val
290 295 300
Pro Asp Lys Lys Lys Ala Pro Ser Ser Pro Pro Pro Pro Pro Pro Pro
305 310 315 320
Leu Arg Ser Asp Ser Phe Ala Ala Thr Lys Ser His Glu Lys Ala Gln
325 330 335
Gly Pro Val Phe Ser Glu Ala Ala Ala Ala Gln His Phe Thr Ala Leu
340 345 350
Ala Gln Ala Gln Pro Arg Gly Asp Arg Arg Pro Glu Leu Thr Asp Arg
355 360 365
Pro Trp Arg Ser Ala His Pro Gly Ser Leu Gly Lys Gly Ser Gly Gly
370 375 380
Pro Gly Cys Pro Gln Glu Ala His Ala Asp Gly Ser Trp Pro Pro Ser
385 390 395 400
Lys Asp Gly Ala Ser Ser Arg Leu Gln Ala Ser Leu Ser Ser Ser Asp
405 410 415
Val Arg Phe Pro Gln Ser Pro His Ser Gly Arg His Pro Pro Leu Tyr
420 425 430
Ser Asp His Ser Pro Leu Cys Ala Asp Ser Leu Gly Gln Glu Pro Gly
435 440 445
Ala Ala Ser Phe Gln Asn Asp Ser Pro Pro Gln Val Arg Gly Leu Ser
450 455 460
Ser Cys Asp Gln Lys Leu Gly Ser Gly Trp Gln Gly Pro Arg Pro Cys
465 470 475 480
Val Gln Gly Asp Leu Gln Ala Ala Gln Leu Trp Ala Gly Cys Trp Pro
485 490 495
Ser Asp Thr Ala Leu Gly Ala Leu Glu Ser Leu Pro Pro Pro Thr Val
500 505 510
Gly Gln Ser Pro Arg His His Leu Pro Gln Pro Glu Gly Pro Pro Asp
515 520 525
Ala Arg Glu Thr Gly Arg Cys Tyr Pro Leu Asp Lys Gly Ala Glu Gly
530 535 540
Cys Ser Ala Gly Ala Gln Glu Pro Pro Arg Ala Ser Arg Ala Glu Lys
545 550 555 560
Ala Ser Gln Arg Leu Ala Ala Ser Ile Thr Trp Ala Asp Gly Glu Ser
565 570 575
Ser Arg Ile Cys Pro Gln Glu Thr Pro Leu Leu His Ser Leu Thr Gln
580 585 590
Glu Gly Lys Arg Arg Pro Glu Ser Ser Pro Glu Asp Ser Ala Thr Arg
595 600 605
Pro Pro Pro Phe Asp Ala His Val Gly Lys Pro Thr Arg Arg Ser Asp
610 615 620
Arg Phe Ala Thr Thr Leu Arg Asn Glu Ile Gln Met His Arg Ala Lys
625 630 635 640
Leu Gln Lys Ser Arg Ser Thr Val Ala Leu Thr Ala Ala Gly Glu Ala
645 650 655
Glu Asp Gly Thr Gly Arg Trp Arg Ala Gly Leu Gly Gly Gly Thr Gln
660 665 670
Glu Gly Pro Leu Ala Gly Thr Tyr Lys Asp His Leu Lys Glu Ala Gln
675 680 685
Ala Arg Val Leu Arg Ala Thr Ser Phe Lys Arg Arg Asp Leu Asp Pro
690 695 700
Asn Pro Gly Asp Leu Tyr Pro Glu Ser Leu Glu His Arg Met Gly Asp
705 710 715 720
Pro Asp Thr Val Pro His Phe Trp Glu Ala Gly Leu Ala Gln Pro Pro
725 730 735
Ser Ser Thr Ser Gly Gly Pro His Pro Pro Arg Ile Gly Gly Arg Arg
740 745 750
Arg Phe Thr Ala Glu Gln Lys Leu Lys Ser Tyr Ser Glu Pro Glu Lys
755 760 765
Met Asn Glu Val Gly Leu Thr Arg Gly Tyr Ser Pro His Gln His Pro
770 775 780
Arg Thr Ser Glu Asp Thr Val Gly Thr Phe Ala Asp Arg Trp Lys Phe
785 790 795 800
Phe Glu Glu Thr Ser Lys Pro Val Pro Gln Arg Pro Ala Gln Lys Gln
805 810 815
Ala Leu His Gly Ile Pro Arg Asp Lys Pro Glu Arg Pro Arg Thr Ala
820 825 830
Gly Arg Thr Cys Glu Gly Thr Glu Pro Trp Ser Arg Thr Thr Ser Leu
835 840 845
Gly Asp Ser Leu Asn Ala His Ser Ala Ala Glu Lys Ala Gly Thr Ser
850 855 860
Asp Leu Pro Arg Arg Leu Gly Thr Phe Ala Glu Tyr Gln Ala Ser Trp
865 870 875 880
Lys Glu Gln Arg Lys Pro Leu Glu Ala Arg Ser Ser Gly Arg Cys His
885 890 895
Ser Ala Asp Asp Ile Leu Asp Val Ser Leu Asp Pro Gln Glu Arg Pro
900 905 910
Gln His Val His Gly Arg Ser Arg Ser Ser Pro Ser Thr Asp His Tyr
915 920 925
Lys Gln Glu Ala Ser Val Glu Leu Arg Arg Gln Ala Gly Asp Pro Gly
930 935 940
Glu Pro Arg Glu Glu Leu Pro Ser Ala Val Arg Ala Glu Glu Gly Gln
945 950 955 960
Ser Thr Pro Arg Gln Ala Asp Ala Gln Cys Arg Glu Gly Ser Pro Gly
965 970 975
Ser Gln Gln His Pro Pro Ser Gln Lys Ala Pro Asn Pro Pro Thr Phe
980 985 990
Ser Glu Leu Ser His Cys Arg Gly Ala Pro Glu Leu Pro Arg Glu Gly
995 1000 1005
Arg Gly Arg Ala Gly Thr Leu Pro Arg Asp Tyr Arg Tyr Ser Glu Glu
1010 1015 1020
Ser Thr Pro Ala Asp Leu Gly Pro Arg Ala Gln Ser Pro Gly Ser Pro
1025 1030 1035 1040
Leu His Ala Arg Gly Gln Asp Ser Trp Pro Val Ser Ser Ala Leu Leu
1045 1050 1055
Ser Lys Arg Pro Ala Pro Gln Arg Pro Pro Pro Pro Lys Arg Glu Pro
1060 1065 1070
Arg Arg Tyr Arg Ala Thr Asp Gly Ala Pro Ala Asp Ala Pro Val Gly
1075 1080 1085
Val Leu Gly Arg Pro Phe Pro Thr Pro Ser Pro Ala Ser Leu Asp Val
1090 1095 1100
Tyr Val Ala Arg Leu Ser Leu Ser His Ser Pro Ser Val Phe Ser Ser
1105 1110 1115 1120
Ala Gln Pro Gln Asp Thr Pro Lys Ala Thr Val Cys Glu Arg Gly Ser
1125 1130 1135
Gln His Val Ser Gly Asp Ala Ser Arg Pro Leu Pro Glu Ala Leu Leu
1140 1145 1150
Pro Pro Lys Gln Gln His Leu Arg Leu Gln Thr Ala Thr Met Glu Thr
1155 1160 1165
Ser Arg Ser Pro Ser Pro Gln Phe Ala Pro Gln Lys Leu Thr Asp Lys
1170 1175 1180
Pro Pro Leu Leu Ile Gln Asp Glu Asp Ser Thr Arg Ile Glu Arg Val
1185 1190 1195 1200
Met Asp Asn Asn Thr Thr Val Lys Met Val Pro Ile Lys Ile Val His
1205 1210 1215
Ser Glu Ser Gln Pro Glu Lys Glu Ser Arg Gln Ser Leu Ala Cys Pro
1220 1225 1230
Ala Glu Pro Pro Ala Leu Pro His Gly Leu Glu Lys Asp Gln Ile Lys
1235 1240 1245
Thr Leu Ser Thr Ser Glu Gln Phe Tyr Ser Arg Phe Cys Leu Tyr Thr
1250 1255 1260
Arg Gln Gly Ala Glu Pro Glu Ala Pro His Arg Ala Gln Pro Ala Glu
1265 1270 1275 1280
Pro Gln Pro Leu Gly Thr Gln Val Pro Pro Glu Lys Asp Arg Cys Thr
1285 1290 1295
Ser Pro Pro Gly Leu Ser Tyr Met Lys Ala Lys Glu Lys Thr Val Glu
1300 1305 1310
Asp Leu Lys Ser Glu Glu Leu Ala Arg Glu Ile Val Gly Lys Asp Lys
1315 1320 1325
Ser Leu Ala Asp Ile Leu Asp Pro Ser Val Lys Ile Lys Thr Thr Met
1330 1335 1340
Asp Leu Met Glu Gly Ile Phe Pro Lys Asp Glu His Leu Leu Glu Glu
1345 1350 1355 1360
Ala Gln Gln Arg Arg Lys Leu Leu Pro Lys Ile Pro Ser Pro Arg Ser
1365 1370 1375
Thr Glu Glu Arg Lys Glu Glu Pro Ser Val Pro Ala Ala Val Ser Leu
1380 1385 1390
Ala Thr Asn Ser Thr Tyr Tyr Ser Thr Ser Ala Pro Lys Ala Glu Leu
1395 1400 1405
Leu Ile Lys Met Lys Asp Leu Gln Glu Gln Gln Glu His Glu Glu Asp
1410 1415 1420
Ser Gly Ser Asp Leu Asp His Asp Leu Ser Val Lys Lys Gln Glu Leu
1425 1430 1435 1440
Ile Glu Ser Ile Ser Arg Lys Leu Gln Val Leu Arg Glu Ala Arg Glu
1445 1450 1455
Ser Leu Leu Glu Asp Val Gln Ala Asn Thr Val Leu Gly Ala Glu Val
1460 1465 1470
Glu Ala Ile Val Lys Gly Val Cys Lys Pro Ser Glu Phe Asp Lys Phe
1475 1480 1485
Arg Met Phe Ile Gly Asp Leu Asp Lys Val Val Asn Leu Leu Leu Ser
1490 1495 1500
Leu Ser Gly Arg Leu Ala Arg Val Glu Asn Ala Leu Asn Asn Leu Asp
1505 1510 1515 1520
Asp Gly Ala Ser Pro Gly Asp Arg Gln Ser Leu Leu Glu Lys Gln Arg
1525 1530 1535
Val Leu Ile Gln Gln His Glu Asp Ala Lys Glu Leu Lys Glu Asn Leu
1540 1545 1550
Asp Arg Arg Glu Arg Ile Val Phe Asp Ile Leu Ala Asn Tyr Leu Ser
1555 1560 1565
Glu Glu Ser Leu Ala Asp Tyr Glu His Phe Val Lys Met Lys Ser Ala
1570 1575 1580
Leu Ile Ile Glu Gln Arg Glu Leu Glu Asp Lys Ile His Leu Gly Glu
1585 1590 1595 1600
Glu Gln Leu Lys Cys Leu Leu Asp Ser Leu Gln Pro Glu Arg Gly Lys
1605 1610 1615
<210> 5
<211> 1487
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Glu Gly Ala Glu Pro Arg Ala Arg Pro Glu Arg Leu Ala Glu Ala
1 5 10 15
Glu Ala Pro Ala Thr Asp Gly Val Arg Leu Val Glu Val Gln Leu Ser
20 25 30
Gly Gly Ala Pro Trp Gly Phe Thr Leu Lys Gly Gly Arg Glu His Gly
35 40 45
Glu Pro Leu Val Ile Thr Lys Ile Glu Glu Gly Ser Lys Ala Ala Ala
50 55 60
Val Asp Lys Leu Leu Ala Gly Asp Glu Ile Val Ala Ile Asn Asp Val
65 70 75 80
Ser Leu Ser Gly Phe Arg Gln Glu Ala Ile Cys Leu Val Lys Gly Ser
85 90 95
His Lys Thr Leu Lys Leu Val Val Lys Arg Lys Ser Asp Pro Ser Trp
100 105 110
Arg Pro His Ser Trp His Ala Thr Lys Tyr Phe Asp Val His Pro Glu
115 120 125
Pro Ala Ala Ser Leu Phe Leu Asn Thr Ser Gly Ser Pro Ser Trp Lys
130 135 140
Ser Gln His Gln Ala Ser Ser Ser Ser His Asp Leu Ser Gly Ser Trp
145 150 155 160
Glu His Thr Ser Leu Gln Arg Thr Ser Asp His Phe Ser Ser Met Gly
165 170 175
Ser Ile Asp Ser Leu Asp His Ser Ser Gln Leu Tyr Pro Ser Gly His
180 185 190
Leu Ser Ser Ala Lys Ser Asn Ser Ser Ile Asp His Leu Gly Gly His
195 200 205
Ser Lys Arg Asp Ser Ala Tyr Gly Ser Phe Ser Thr Cys Ser Ser Thr
210 215 220
Pro Asp His Thr Leu Pro Lys Ala Asp Ala Ser Ser Thr Glu Asn Ile
225 230 235 240
Leu Tyr Lys Val Gly Leu Trp Glu Ala Ser Arg Pro Gly Ser Ser Arg
245 250 255
Gln Ser Gln Ser Thr Gly Asp Pro Gln Gly Leu Gln Asp Arg Pro Ser
260 265 270
Cys Phe Ile Pro Arg Val Pro Gly Asn Ser Ser Lys Ser Pro Arg Pro
275 280 285
Glu Asp Asn Val Glu Pro Lys Ile Ala Thr His Gly Arg Ser Asn Phe
290 295 300
Gly Pro Val Trp Tyr Val Pro Asp Lys Lys Lys Ala Pro Ser Pro Pro
305 310 315 320
Pro Leu Gly Leu Pro Leu Arg Ser Asp Ser Phe Ser Val Ala Ala Arg
325 330 335
Gly His Glu Lys Ala Arg Gly Pro Pro Phe Ser Asp Leu Ala Ser Met
340 345 350
Gln His Phe Ile Thr Leu Pro His Val Gln Pro Arg Gly Asp His Arg
355 360 365
Met Glu Thr Thr Asp Arg Gln Trp Lys Leu Thr His Leu Ser Ser Gly
370 375 380
Lys Glu Ile Gly Asn Val Gly Tyr Gln Ser Glu Gly His Leu Asp Cys
385 390 395 400
Arg Trp Leu Cys Ser Asp Asp Arg Ala Gly Arg Pro Ser Gly Pro Pro
405 410 415
Gly Arg Leu Gln Phe Ser Asp Val His Phe Leu Lys Ser Tyr His Gly
420 425 430
Ser Gln His Gln Gln Gln Cys Ser Asp Glu Ser Pro Arg Ala Pro Ser
435 440 445
Ser Pro Arg Glu Leu Leu His Ile Thr Pro Gly Gly Gly Leu Gln Glu
450 455 460
Pro Pro Glu Pro Ser Gln Asp Asp Asn Pro Thr Gln Val Arg Trp Pro
465 470 475 480
Gly Ser Ala His Gln Lys Leu Asp Asp Arg Gly Arg Ser His Tyr Phe
485 490 495
Pro Gly Ser Leu Arg Gln Pro Val Gln Gly Ser Ala Gln Val Val Ile
500 505 510
Pro Arg Gly Asp Tyr Trp His Ser Asp Thr Thr Pro Val Asp Leu Glu
515 520 525
Tyr Pro Leu Leu Arg Pro Val Gly Gln Arg Thr Tyr Leu Gln Gln His
530 535 540
Glu Glu Thr Pro Ala Ser His Glu Lys Glu Gly Tyr His Gln Leu Asn
545 550 555 560
Ala Gly Ile Glu Gly Cys Cys Ser Gly Ile Gln Glu Pro Pro Arg Ala
565 570 575
Ser Arg Thr Val Arg Thr Gly Leu Gln Cys Pro Ser Asn Asp Phe Lys
580 585 590
Leu Val Asp Gly Glu Ser Gly Arg Ile Ser Arg Gln Arg Thr Pro Met
595 600 605
Leu His Ser Leu Thr Gln Asp Gly Thr Trp Arg Pro Gly Asn Ser Lys
610 615 620
Asp Cys Gly Asn Asp Lys Pro Pro Leu Phe Asp Ala Gln Val Gly Lys
625 630 635 640
Pro Thr Arg Arg Ser Asp Arg Phe Ala Thr Thr Leu Arg Asn Glu Ile
645 650 655
Gln Met Arg Arg Ala Lys Leu Gln Lys Ser Lys Ser Thr Val Thr Leu
660 665 670
Ala Gly Asp Ser Glu Ala Glu Asp Cys Ala Gly Asp Trp Arg Ala Asp
675 680 685
Val Gly Ala Val Pro Glu Gly Ser Phe Pro Ser Thr Tyr Lys Glu His
690 695 700
Leu Lys Glu Ala Gln Thr Arg Val Leu Lys Ala Thr Ser Phe Gln Arg
705 710 715 720
Arg Asp Leu Asp Pro Thr Pro Ala Asp Gln Tyr Ser Gly Pro Ser Glu
725 730 735
His Arg Thr Phe Asp His Ser Ala Ser Ser Ser Leu Ser Ser Phe Pro
740 745 750
Gly Glu Pro Asp Ser Ala Pro Arg Phe Cys Glu Thr Gly Leu Ala Lys
755 760 765
Ala Pro Ser Ser Gly Val Gly Val Pro His Val Leu Arg Ile Gly Gly
770 775 780
Arg Lys Arg Phe Thr Ala Glu Gln Lys Leu Lys Ser Tyr Ser Glu Pro
785 790 795 800
Glu Lys Ile Asn Glu Val Gly Leu Ser Gly Asp His Arg Pro His Pro
805 810 815
Thr Val Arg Thr Pro Glu Asp Thr Val Gly Thr Phe Ala Asp Arg Trp
820 825 830
Lys Phe Phe Glu Glu Thr Ser Lys Ser Leu Leu Gln Lys Ala Gly His
835 840 845
Arg Gln Val His Cys Gly Leu Pro Arg Glu Lys Ala Glu Arg Pro Gln
850 855 860
Thr Gly His His Glu Cys Glu Ser Thr Glu Pro Trp Phe Gln Lys Arg
865 870 875 880
Ser Leu Ala Thr Ser Cys Gly Glu Ile Leu Ser Asp Arg Lys Val Glu
885 890 895
Lys Ala Ser Glu Lys Leu Asn Pro Pro Arg Arg Leu Gly Thr Phe Ala
900 905 910
Glu Tyr Gln Ala Ser Trp Lys Glu Gln Lys Lys Pro Leu Glu Ala Arg
915 920 925
Ser Ser Gly Arg Tyr His Ser Ala Asp Asp Ile Leu Asp Ala Gly Leu
930 935 940
Asp Gln Gln Gln Arg Pro Gln Tyr Ile His Glu Arg Ser Arg Ser Ser
945 950 955 960
Pro Ser Thr Asp His Tyr Ser Gln Glu Val Pro Val Glu Pro Asn Arg
965 970 975
Gln Ala Glu Asp Ser Gly Asp His Lys Glu Ala Ile Leu Cys Thr Leu
980 985 990
Gln Ala Glu Glu Gly Cys Ser Ala Pro Ser Ser Ser Val Leu Ser Ser
995 1000 1005
Ala Gln Pro Gln Asp Ser Gln His Val Asn Glu Asp Thr Thr Phe Pro
1010 1015 1020
Gln Pro Glu Thr Gln Leu Ser Ser Lys Cys Gln His Leu Gln Thr Ser
1025 1030 1035 1040
Ala Met Glu Thr Ser Arg Ser Pro Ser Pro Gln Phe Ala Pro Gln Lys
1045 1050 1055
Leu Thr Asp Lys Pro Pro Leu Leu Ile His Glu Asp Asn Ser Ala Arg
1060 1065 1070
Ile Glu Arg Val Met Asp Asn Asn Thr Thr Val Lys Met Val Pro Ile
1075 1080 1085
Lys Ile Val His Ser Glu Ser Gln Pro Glu Lys Glu Ser Arg Gln Ser
1090 1095 1100
Leu Ala Cys Pro Ala Glu Leu Pro Pro Leu Pro Ser Gly Leu Glu Arg
1105 1110 1115 1120
Asp Gln Ile Lys Thr Leu Ser Thr Ser Glu Gln Cys Tyr Ser Arg Phe
1125 1130 1135
Cys Val Tyr Thr Arg Gln Glu Val Glu Ala Pro His Arg Ala Arg Pro
1140 1145 1150
Pro Glu Pro Arg Pro Pro Ser Thr Pro Ala Pro Pro Val Arg Asp Ser
1155 1160 1165
Cys Ser Ser Pro Pro Ser Leu Asn Tyr Gly Lys Ala Lys Glu Lys Thr
1170 1175 1180
Met Asp Asp Leu Lys Ser Glu Glu Leu Ala Arg Glu Ile Val Gly Lys
1185 1190 1195 1200
Asp Lys Ser Leu Ala Asp Ile Leu Asp Pro Ser Val Lys Ile Lys Thr
1205 1210 1215
Thr Met Asp Leu Met Glu Gly Ile Phe Pro Lys Asp Glu Tyr Leu Leu
1220 1225 1230
Glu Glu Ala Gln Gln Arg Arg Lys Leu Leu Pro Lys Val Pro Leu Pro
1235 1240 1245
Arg Val Thr Glu Asp Lys Lys Gln Asp Pro Gly Met Pro Gly Val Val
1250 1255 1260
Ser Leu Ala Thr Asn Ser Thr Tyr Tyr Ser Thr Ser Ala Pro Lys Ala
1265 1270 1275 1280
Glu Leu Leu Ile Lys Met Lys Asp Leu Gln Glu Pro Glu Glu Tyr Ser
1285 1290 1295
Ala Gly Asp Leu Asp His Asp Leu Ser Val Lys Lys Gln Glu Leu Ile
1300 1305 1310
Asp Ser Ile Ser Arg Lys Leu Gln Val Leu Arg Glu Ala Arg Glu Ser
1315 1320 1325
Leu Leu Glu Asp Ile Gln Ala Asn Asn Ala Leu Gly Asp Glu Val Glu
1330 1335 1340
Ala Ile Val Lys Asp Val Cys Lys Pro Asn Glu Phe Asp Lys Phe Arg
1345 1350 1355 1360
Met Phe Ile Gly Asp Leu Asp Lys Val Val Asn Leu Leu Leu Ser Leu
1365 1370 1375
Ser Gly Arg Leu Ala Arg Val Glu Asn Ala Leu Asn Asn Leu Asp Asp
1380 1385 1390
Asn Pro Ser Pro Gly Asp Arg Gln Ser Leu Leu Glu Lys Gln Arg Val
1395 1400 1405
Leu Thr Gln Gln His Glu Asp Ala Lys Glu Leu Lys Glu Asn Leu Asp
1410 1415 1420
Arg Arg Glu Arg Ile Val Phe Asp Ile Leu Ala Thr Tyr Leu Ser Glu
1425 1430 1435 1440
Glu Asn Leu Ala Asp Tyr Glu His Phe Val Lys Met Lys Ser Ala Leu
1445 1450 1455
Ile Ile Glu Gln Arg Glu Leu Glu Asp Lys Ile His Leu Gly Glu Glu
1460 1465 1470
Gln Leu Lys Cys Leu Phe Asp Ser Leu Gln Pro Glu Arg Ser Lys
1475 1480 1485
<210> 6
<211> 1605
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Glu Gly Ala Glu Pro Arg Ala Arg Pro Gln Arg Leu Ala Glu Ala
1 5 10 15
Glu Thr Glu Thr Arg Ala Ala Asp Gly Gly Arg Leu Val Glu Val Gln
20 25 30
Leu Ser Gly Gly Ala Pro Trp Gly Phe Thr Leu Lys Gly Gly Arg Glu
35 40 45
His Gly Glu Pro Leu Val Ile Thr Lys Ile Glu Glu Gly Ser Lys Ala
50 55 60
Ala Ala Val Asp Lys Leu Leu Ala Gly Asp Glu Ile Val Gly Ile Asn
65 70 75 80
Asp Ile Gly Leu Ser Gly Phe Arg Gln Glu Ala Ile Cys Leu Val Lys
85 90 95
Gly Ser His Lys Thr Leu Lys Leu Val Val Lys Arg Arg Ser Glu Leu
100 105 110
Gly Trp Arg Pro His Ser Trp His Ala Thr Lys Phe Ser Asp Ser His
115 120 125
Pro Glu Leu Ala Ala Ser Pro Phe Pro Ser Ala Ser Gly Cys Pro Ser
130 135 140
Trp Ser Gly Arg His His Ala Ser Ser Ser Ser His Asp Leu Ser Ser
145 150 155 160
Ser Trp Glu Gln Thr Asn Leu Gln Arg Thr Leu Asp His Phe Ser Ser
165 170 175
Leu Gly Ser Val Gly Ser Leu Asp His Pro Ser Ser Arg Leu Ser Val
180 185 190
Ala Lys Ser Asn Ser Ser Ile Asp His Leu Gly Ser His Ser Lys Arg
195 200 205
Asp Ser Ala Tyr Gly Ser Phe Ser Thr Ser Ser Ser Thr Pro Asp His
210 215 220
Thr Leu Ser Lys Ala Asp Thr Ser Ser Ala Glu Asn Ile Leu Tyr Ser
225 230 235 240
Val Gly Leu Trp Glu Ala Pro Arg Gln Gly Gly Arg Gln Ala Gln Ala
245 250 255
Ala Gly Asp Pro Gln Gly Leu Glu Glu Lys Leu Arg Cys Phe Pro Pro
260 265 270
Arg Val Pro Ser Asp Ser Asp Lys Gly Pro Arg Pro Glu Tyr Asn Ala
275 280 285
Glu Pro Lys Leu Ala Val Pro Gly Arg Ser Asn Phe Gly Pro Val Trp
290 295 300
Tyr Val Pro Asp Lys Lys Lys Ala Pro Ala Ser Pro Pro Pro Pro Pro
305 310 315 320
Pro Pro Leu Arg Ser Asp Ser Phe Ala Ala Thr Lys Ser His Glu Lys
325 330 335
Ala Gln Gly Pro Val Phe Ser Glu Val Ala Val Ala Gln His Phe Thr
340 345 350
Ala Pro Ala Gln Ala Gln Pro Arg Gly Asp Trp Arg Pro Glu Pro Thr
355 360 365
Asp Arg Pro Trp Arg Ser Ala His Pro Gly Ser Leu Gly Lys Gly Ser
370 375 380
Gly Gly Pro Gly Cys Pro Gln Glu Ala Arg Ala Asp Gly Ser Trp Pro
385 390 395 400
Pro Ser Lys Asp Gly Ala Ser Ser Arg Leu Gln Ala Ser Leu Ser Ser
405 410 415
Ser Asp Val Arg Leu Pro Gln Ser Pro Arg Ser Gly Arg His Pro Pro
420 425 430
Leu Tyr Ser Asp His Ser Pro Leu Cys Ala Asp Ser Leu Gly Gln Glu
435 440 445
Pro Ala Ala Ala Ser Ser Gln Asn Asp Ser Leu Pro Gln Val Arg Gly
450 455 460
Leu Ser Ser Cys Asp Gln Lys Leu Gly Ser Gly Trp Gln Gly Pro Arg
465 470 475 480
Pro Cys Val Gln Gly Gly Pro Gln Val Ala Gln Leu Trp Ala Gly Cys
485 490 495
Trp Pro Ser Asp Thr Ala Pro Gly Ala Leu Lys Ser Leu Pro Pro Pro
500 505 510
Thr Val Gly Gln Ser Pro His Arg His Leu Pro Gln Pro Glu Gly Pro
515 520 525
Pro Asp Ala Cys Glu Thr Gly Arg Cys Tyr Pro Leu Asp Thr Gly Ala
530 535 540
Glu Gly Cys Ser Ala Gly Ala Gln Glu Pro Pro Arg Ala Ser Arg Val
545 550 555 560
Glu Lys Ala Ser Ile Thr Trp Ala Asp Gly Glu Ser Ser Arg Ile Cys
565 570 575
Pro Gln Glu Thr Pro Leu Leu His Ser Leu Thr Gln Glu Gly Lys His
580 585 590
Arg Pro Glu Ser Ser Pro Glu Asp Ser Ala Thr Arg Pro Pro Pro Phe
595 600 605
Asp Ala His Val Gly Lys Pro Thr Arg Arg Ser Asp Arg Phe Ala Thr
610 615 620
Thr Leu Arg Asn Glu Ile Gln Met Arg Arg Ala Lys Leu Gln Lys Ser
625 630 635 640
Arg Ser Thr Val Ala Leu Thr Ala Ala Gly Glu Ala Glu Asp Ala Thr
645 650 655
Gly Arg Trp Arg Val Lys Leu Gly Gly Gly Ala Gln Glu Gly Pro Phe
660 665 670
Ala Gly Thr Tyr Lys Asp His Leu Lys Glu Ala Gln Ala Arg Val Leu
675 680 685
Arg Ala Thr Ser Phe Lys Arg Arg Asp Leu Asp Pro Asn Pro Ala Asp
690 695 700
Leu Tyr Pro Glu Ser Leu Glu His Arg Met Gly Gly Pro Asp Thr Val
705 710 715 720
Arg His Phe Trp Glu Ala Gly Leu Ala Gln Pro Pro Ser Ser Thr Gly
725 730 735
Gly Gly Pro His Pro Pro Arg Ile Gly Gly Arg Arg Arg Phe Thr Ala
740 745 750
Glu Gln Lys Leu Lys Ser Tyr Ser Glu Pro Glu Lys Met Asn Glu Val
755 760 765
Gly Leu Thr Arg Gly Tyr Ser Pro His Gln His Pro Arg Thr Ser Glu
770 775 780
Asp Thr Val Gly Thr Phe Ala Asp Arg Trp Lys Phe Phe Glu Glu Thr
785 790 795 800
Ser Lys Pro Val Pro Gln Arg Pro Ala Gln Arg Gln Val Leu His Gly
805 810 815
Ile Leu Arg Asp Lys Pro Glu Arg Pro Trp Thr Val Gly Arg Thr Cys
820 825 830
Glu Gly Thr Glu Pro Trp Ser Arg Thr Thr Ser Leu Gly Asp Ser Leu
835 840 845
Asn Ala His Ser Thr Ala Glu Lys Thr Gly Thr Ser Asp Pro Pro Gln
850 855 860
Arg Leu Gly Thr Phe Ala Glu Tyr Gln Ala Ser Trp Lys Glu Gln Arg
865 870 875 880
Lys Pro Leu Glu Ala Arg Ser Ser Gly Arg Cys His Ser Ala Asp Asp
885 890 895
Ile Leu Asp Val Gly Leu Asp Pro His Glu Arg Pro Gln His Val His
900 905 910
Gly Arg Ser Arg Ser Ser Pro Ser Thr Asp His Tyr Lys Gln Glu Ala
915 920 925
Ser Val Glu Leu Arg Arg Gln Ala Gly Asp Pro Gly Glu His Arg Glu
930 935 940
Glu Leu Pro Phe Ala Val Arg Ala Lys Glu Gly Gln Ser Thr Leu Arg
945 950 955 960
Gln Ala Asp Val Gln Cys Trp Glu Gly Ser Pro Gly Ser Gln Gln His
965 970 975
Pro Pro Ser Gln Asn Ala Pro Asn Pro Pro Thr Phe Ser Glu Pro Ser
980 985 990
His Ser Gln Gly Ala Pro Glu Leu Pro Gln Glu Gly Arg Gly Arg Ala
995 1000 1005
Gly Thr Leu Pro Arg Asp Tyr Arg Tyr Ser Glu Glu Ser Ile Pro Ala
1010 1015 1020
Asp Leu Gly Pro Arg Ala His Ser Pro Gly Ser Pro Leu His Ala Gln
1025 1030 1035 1040
Gly Gln Asp Ser Trp Pro Val Ser Ser Ala Leu Leu Ala Lys Arg Pro
1045 1050 1055
Ala Pro Gln Arg Pro Pro Pro Pro Lys Arg Glu Pro Arg Arg Tyr Arg
1060 1065 1070
Ala Thr Asp Ser Ala Pro Ala Asp Ala Pro Leu Ala Val Leu Gly Arg
1075 1080 1085
Pro Phe Pro Thr Pro Ser Pro Ala Ser Leu Asp Val Cys Val Ala Arg
1090 1095 1100
Leu Ser Leu Ser His Ser Pro Ser Val Phe Ser Ser Ala Gln Pro Gln
1105 1110 1115 1120
Asp Ile Pro Lys Ala Ala Ala Cys Glu Arg Gly Ser Gln His Val Ser
1125 1130 1135
Gly Asp Thr Pro Leu Pro Pro Lys Gln Gln His Leu Arg Leu Gln Thr
1140 1145 1150
Ala Thr Met Glu Thr Ser Arg Ser Pro Ser Pro Gln Phe Ala Pro Gln
1155 1160 1165
Lys Leu Thr Asp Lys Pro Pro Leu Leu Ile Gln Asp Glu Asp Ser Thr
1170 1175 1180
Arg Ile Glu Arg Val Met Asp Asn Asn Thr Thr Val Lys Met Val Pro
1185 1190 1195 1200
Ile Lys Ile Val His Ser Glu Ser Gln Pro Glu Lys Glu Ser Arg Gln
1205 1210 1215
Ser Leu Ala Arg Pro Ala Glu Pro Pro Ala Leu Pro Arg Gly Leu Glu
1220 1225 1230
Lys Asp Gln Ile Lys Thr Leu Ser Thr Ser Glu Gln Phe Tyr Ser Arg
1235 1240 1245
Phe Cys Leu Tyr Thr Arg Gln Gly Ala Glu Pro Glu Ala Pro His Arg
1250 1255 1260
Ala Gln Pro Ala Glu Pro Gln Pro Leu Gly Thr Gln Ala Pro Pro Glu
1265 1270 1275 1280
Lys Asp Arg Cys Ala Ser Pro Pro Gly Leu Ser Tyr Met Lys Ala Lys
1285 1290 1295
Glu Lys Thr Val Glu Asp Leu Lys Ser Glu Glu Leu Ala Arg Glu Ile
1300 1305 1310
Val Gly Lys Asp Lys Ser Leu Ala Asp Ile Leu Asp Pro Ser Val Lys
1315 1320 1325
Ile Lys Thr Thr Met Asp Leu Met Glu Gly Ile Phe Pro Lys Asp Glu
1330 1335 1340
His Leu Leu Glu Glu Ala Gln Gln Arg Arg Lys Leu Leu Pro Lys Ile
1345 1350 1355 1360
Pro Ser Pro Arg Ser Thr Glu Glu Arg Lys Glu Glu Pro Ser Val Pro
1365 1370 1375
Ala Ala Val Ser Leu Ala Thr Asn Ser Thr Tyr Tyr Ser Thr Ser Ala
1380 1385 1390
Pro Lys Ala Glu Leu Leu Ile Lys Met Lys Asp Met Gln Glu Gln Gln
1395 1400 1405
Glu Arg Glu Glu Asp Ser Gly Ser Asp Leu Asp Tyr Asp Leu Ser Val
1410 1415 1420
Lys Lys Gln Glu Leu Ile Glu Ser Ile Ser Arg Lys Leu Gln Val Leu
1425 1430 1435 1440
Arg Glu Ala Arg Glu Ser Leu Leu Glu Asp Val Gln Ala Asn Thr Val
1445 1450 1455
Leu Gly Ala Glu Val Glu Ala Ile Val Lys Gly Val Cys Lys Pro Ser
1460 1465 1470
Glu Phe Asp Lys Phe Arg Met Phe Ile Gly Asp Leu Asp Lys Val Val
1475 1480 1485
Asn Leu Leu Leu Ser Leu Ser Gly Arg Leu Ala Arg Val Glu Asn Ala
1490 1495 1500
Leu Asn Asn Leu Asp Asp Asn Ala Ser Pro Gly Asp Arg Gln Ser Leu
1505 1510 1515 1520
Leu Glu Lys Gln Arg Val Leu Ile Gln Gln His Glu Asp Ala Lys Glu
1525 1530 1535
Leu Lys Glu Asn Leu Asp Arg Arg Glu Arg Ile Val Phe Asp Ile Leu
1540 1545 1550
Ala Asn Tyr Leu Ser Glu Glu Ser Leu Ala Asp Tyr Glu His Phe Val
1555 1560 1565
Lys Met Lys Ser Ala Leu Ile Ile Glu Gln Arg Glu Leu Glu Asp Lys
1570 1575 1580
Ile His Leu Gly Glu Glu Gln Leu Lys Cys Leu Phe Asp Ser Leu Gln
1585 1590 1595 1600
Pro Glu Arg Gly Lys
1605
Claims (10)
1.一种自身免疫性疾病标志物,其特征在于,所述自身免疫性疾病标志物为与Shroom2蛋白结合的自身抗体,所述Shroom2蛋白的氨基酸序列如SEQ ID NO:1、SEQ ID NO:2或SEQID NO:3所示。
2.用于受试者中自身免疫性疾病诊断的多肽或蛋白质,其特征在于,该多肽或蛋白质为包含衍生自Shroom2蛋白的一个或多个表位,所述Shroom2蛋白的氨基酸序列如SEQ IDNO:1、SEQ ID NO:2或SEQ ID NO:3所示。
3.根据权利要求2所述的用于受试者中自身免疫性疾病的诊断的多肽或蛋白质,其特征在于,该多肽或蛋白质还融合其他氨基酸构成融合蛋白,所述氨基酸是连接在N端或C端的,具有促进所述多肽或蛋白质的纯化、固定化、沉淀或鉴定的作用。
4.一种核酸,其特征在于,所述核酸编码权利要求3所述的用于受试者中自身免疫性疾病的诊断的多肽或蛋白质,编码SEQ ID NO:1所示的多肽或蛋白质的核苷酸序列如SEQ IDNO:4所示,编码SEQ ID NO:2所示的多肽或蛋白质的核苷酸序列如SEQ ID NO:5所示,SEQID NO:3所示的多肽或蛋白质的核苷酸序列如SEQ ID NO:6所示。
5.一种细胞,其特征在于,所述细胞含有能够表达权利要求2所述的用于受试者中自身免疫性疾病诊断的多肽或蛋白质的载体。
6.一种固定化检测试剂,其特征在于,将权利要求2~4中任意一项所述的多肽或蛋白质固定化于固态载体上获得。
7.权利要求1所述的自身免疫性疾病标志物、权利要求2所述的多肽或蛋白质或权利要求6所述的固定化检测试剂在制备用于自身免疫性疾病诊断的检测试剂或检测试剂盒中的应用。
8.如权利要求7所述的应用,其特征在于,检测时是检测样品与shroom2蛋白结合的自身抗体,所述样品为液体样品或组织样品,液体样品为脑脊液、血液、血浆、淋巴液或组织间液;组织样品为神经组织、肌肉组织或来自消化道的组织。
9.如权利要求7所述的应用,其特征在于,所述自身免疫性疾病包括小脑变性、精神行为异常、认知障碍、视力障碍、记忆力下降、言语障碍、运动障碍、意识水平下降、自主神经功能障碍或听觉障碍。
10.一种自身免疫性疾病检测试剂盒,其特征在于,检测试剂盒中包括权利要求1所述的自身免疫性疾病标志物、权利要求2所述的多肽或蛋白质或权利要求6所述的固定化检测试剂。
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