CN111272998A - 一种同时检测中枢脱髓鞘自身抗体aqp4、mog和mbp的方法 - Google Patents
一种同时检测中枢脱髓鞘自身抗体aqp4、mog和mbp的方法 Download PDFInfo
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Abstract
本发明公开了一种同时检测中枢脱髓鞘自身抗体AQP4、MOG和MBP的方法，所述方法为：将AQP4、MOG、MBP基因分别导入HEK293T细胞中，分别与待测样品共同孵育，以转染空载体的HEK293T细胞为对照，采用免疫荧光法进行检测，若荧光信号强于对照，则待测样品为阳性；荧光信号等于或小于对照信号，则为阴性。本发明提供的在同一***同时检测中枢脱髓鞘自身抗体AQP4、MOG、MBP的方法，采用灵敏度和特异性更高的荧光免疫细胞染色技术，用于临床定性检测AQP4、MOG、MBP抗体，使检测结果更加稳定，且极大节约人力和时间成本。
Description
(一)技术领域
本发明涉及一种中枢脱髓鞘自身抗体的检测，特别涉及同时检测中枢脱髓鞘自身抗体AQP4、MOG和MBP的方法。
(二)背景技术
中枢神经***脱髓鞘疾病是以中枢神经***多灶性以及炎性脱髓鞘为主的一种自身免疫***疾病，其临床特征主要包括反复发作、多次缓解及复发。临床较为常见的中枢神经***脱髓鞘疾病包括多发性硬化症(Multiple Sclerosis,MS)、视神经脊髓炎(neuromyelitis optica,NMO)、急性散播性脑脊髓炎等。近年来中枢神经***脱髓鞘疾病发病率呈逐年升高趋势，对患者生活质量产生严重影响，对患者身心健康构成严重威胁。
多发性硬化和视神经脊髓炎是中枢神经***最为常见的脱髓鞘疾病，已成为中青年非外伤致残首要疾病之一。两种疾病均常累及视神经和脊髓，因此临床上较难区分，尤其是发病早期。目前研究认为MS是由CD4辅助T细胞(包括Th1和Th17亚群)介导的自身免疫疾病，故治疗多以免疫调节为主，如β-干扰素等。而NMO以体液免疫为主，治疗多以免疫抑制为主。因此，早期鉴别诊断对指导两种疾病的治疗和评估预后非常重要。2004年Lennon等通过间接免疫荧光染色方法检测发现视神经脊髓炎病人血清中存在着一种特异性自身抗体NMO-IgG，它在视神经炎(Optic neuritis,ON)、长节段横贯性脊髓炎(Longitudinallyextensive tranverse myelitis,LETM)患者血清中的阳性率也较高，2006年WingerchukDM修订了NMO诊断标准，将NMO-IgG抗体阳性做为诊断的重要支持条件，该诊断标准强调了水通道蛋白-4(AQP4)抗体在NMO诊断中的重要价值。在此基础上，Wingerchuk DM又于2007年提出了视神经脊髓炎疾病谱，认为ON、LETM(包括伴有自身免疫病和/或有颅内病灶)为视神经脊髓炎的限定型，连同视神经脊髓型多发性硬化(OSMS)一同被归为NMO的疾病谱。后续研究还发现，AQP4抗体滴度与患者病情的严重程度、预后、复发具有相关性，临床上用免疫球蛋白和血浆置换治疗NMO疗效较好。NMO患者尸检的病理显示病灶中血管周围有大量的免疫球蛋白和补体沉积、星型胶质细胞破坏和再生，其分布与AQP-4表达的分布相一致。通过AQP-4抗体阳性血清与转染AQP-4 cDNA的HEK293细胞孵育，并用抗人IgG示踪已结合的抗体，发现NMO中的AQP-4抗体为IgG1，而IgG1具有激活补体功能。进一步的体外实验证实AQP-4抗体与表达AQP-4分子的HEK细胞结合后能激活补体。2009年Bennett等研究发现，在已经诱导出EAE的大鼠鞘内注射人工合成的AQP-4抗体能够导致病灶内星型胶质细胞的死亡，与NMO病灶病理改变相似。这提示AQP4抗体可能不仅仅是NMO的标志物，有可能是致病性抗体。
髓鞘碱性蛋白(MBP)是构成神经髓鞘的主要成分之一，约占髓鞘蛋白总量的30％，是髓鞘中抗原性最强的蛋白质，它在中枢和周围神经***内分别由少突胶质细胞、雪旺氏细胞进行合成、分泌。抗MBP抗体诱发的自身免疫性炎症反应，被认为是包括多发性硬化(multiple sclerosis，MS)在内的多种神经***疾病的重要病因。MS患者存在多种髓鞘特异性抗体，其中重要抗体之一为抗MBP抗体。已有研究报道抗MBP抗体阳性的患者较抗体阴性的患者，缓解期短、复发率高，可作为单一脱髓鞘病灶患者诊断MS的有力佐证；抗MBP抗体阳性的临床确诊孤立综合征患者较抗体阴性患者发展为MS的时间短；因此，可将抗MBP抗体作为诊断脱髓鞘疾病的重要指标。然而，国内外关于抗MBP抗体的检测结果争议较大。有研究发现MS急性期血清中抗MBP抗体阳性率为77％；亦有研究阐明抗MBP抗体与MS的临床类型和病程有关，在首次发作阳性率仅为28％，随病情进展阳性率增高可达60％。上述研究均体现了MBP在中枢脱髓鞘疾病鉴别诊断和预后分析中的重要作用。
在过去几年中，中枢神经***(CNS)炎性脱髓鞘疾病患者血清中髓鞘少突胶质细胞糖蛋白免疫球蛋白G抗体(MOG-IgG)的作用已逐渐被重新认识。以往通过使用线性化或变性MOG肽作为抗原的酶联免疫吸附测定法的结果认为MOG抗体与多发性硬化(MS)有关，但近来新一代基于细胞的检测已经证实，全长、构象完整的人类MOG抗体与视神经炎(ON)(多为复发性)、脊髓炎、脑干脑炎以及急性播散性脑脊髓炎(ADEM)样表现等密切相关，而非经典的MS。免疫学研究表明MOG-IgG有直接致病作用，血清学检测发现几乎所有MOG-IgG阳性的患者中水通道蛋白-4(AQP4)-IgG阴性；队列研究结果显示其临床表现、辅助检查、治疗反应和预后与经典的MS和AQP4-IgG阳性的视神经脊髓炎谱系疾病(NMOSD)不同，认为其为一种新的疾病实体，被称为MOG-IgG相关性脑脊髓炎，简称为MOG脑脊髓炎(MOG-EM)。MOG-EM和MS有一定的相似性，其临床和影像学表现常有重叠：MOG-EM和MS一样，绝大多数患者病情可复发，此外，在病程中至少出现一次，分别有33％和15％的成人MOG-EM患者符合MS的McDonald和Barkhof诊断标准，因此，过去许多MOG-EM患者被误诊为MS，而结合AQP4抗体和MOG抗体的血清学检测将有助于MS的鉴别诊断。
目前国际上检测AQP-4抗体的技术主要有间接免疫荧光法(IndirectImmunofluorescence,IIF)、荧光免疫沉淀法(Fluoroimmunoprecipitation Assay,FIPA)、放射免疫沉淀法(Radioimmunoprecipitation Assay,RIPA)、细胞免疫荧光(Cell-BasedAssay,CBA)(Clin Immunol.2011Mar；138(3):239-46,Neurology.2012Feb 28；78(9):665-71)及酶联免疫吸附法(Enzyme-Linked ImmunoSorbent Assay,ELISA)。传统检测AQP-4的方法如间接免疫荧光法、酶联免疫吸附法等敏感性和特异性均不高，放射免疫沉淀法涉及放射源污染问题。传统CBA方法应用的质粒为EGFP标记的人AQP4 cDNA质粒及对照EGFPcDNA质粒，应用聚乙烯亚胺(PEI)或脂质体方法进行瞬时转染。耗时长，人力物力损耗大，且荧光效率不稳定，需要更为稳定的转染方法以获得更敏感稳定的结果。MOG、MBP自身抗体检测多使用高表达MBP抗原的神经组织切片进行染色检测，此方法存在诸多问题如由于切片组织部位难以控制，致使批次之间差异较大；组织切片多为动物来源(如欧盟公司用的猴神经组织切片)，抗原存在种属差异，降低检测的灵敏度和特异性；组织中存在除MBP的其他抗原，检测的阳性结果可能并非为MBP抗体阳性。2、Western或Elisa方法检测，虽然两种技术已较为成熟，但由于其难以维持MBP抗原的空间构象，致使其特异性不高，且费用昂贵，极大的限制了这些检测手段的推广。
(三)发明内容
本发明目的是提供一种应用免疫荧光技术同时检测中枢脱髓鞘自身抗体AQP4、MOG、MBP的方法，将稳定转染AQP-4、MOG和MBP的细胞系包被在同一载玻片，采用灵敏度和特异性较高的荧光免疫细胞染色法(CBA)检测技术，同时定性检测AQP-4、MOG及MBP抗体，使检测结果更加稳定，且极大节约人力和时间成本，有利于提高视神经脊髓炎和多发性硬化的临床诊疗水平。
本发明采用的技术方案是：
本发明提供一种同时检测中枢脱髓鞘自身抗体AQP4、MOG和MBP的方法，所述方法为：将AQP4、MOG、MBP基因分别导入HEK293T细胞中(即含AQP-4基因的HEK293T细胞系、含MBP基因的HEK293T细胞系、含MOG基因的HEK293T细胞系)，分别与待测样品共同孵育，以转染空载体的HEK293T细胞为对照，采用免疫荧光法进行检测，若待测样品荧光信号强于对照，则待测样品为阳性；荧光信号等于或小于对照信号，则为阴性。
进一步，所述检测方法为：(1)将细胞贴壁试剂与PBS以体积比1:10混合，加入含盖玻片的6孔板中，37℃，5％CO2中孵育1小时；然后加入分别转有AQP4、MOG、MBP基因的稳定HEK293T细胞系，细胞接种量均为1×106/ml，37℃，5％CO2培养至80％融合度；
(2)盖玻片(优选切割盖玻片至边长为5mm小正方形)细胞面朝上，粘贴于载玻片上(优选无影胶粘贴，紫外线照射30秒粘牢)，盖玻片即为反应区；PBST洗涤(优选洗涤细胞50ul/反应区，5分钟)；用3％BSA封闭(优选以50ul/反应区封闭30分钟)，弃去；加入经PBST以体积比1:10稀释后的待测血清(优选每孔50μl)，在37℃，5％CO2条件下孵育1小时；PBST洗涤(优选3次，每次5分钟)；加入PBST以体积比1:700稀释的生物素与链霉素结合的山羊抗人(Biotin-SP-conjugated affinipure goat anti-human IgG)(优选每孔50μl)，37℃，5％CO2条件下孵育30分钟；PBST洗涤(优选3次，每次5分钟)；加入PBST以体积比1:700稀释的荧光素488月链霉素结合物(Alex fluor 488-conjugated streptavidin)(优选每孔50μl)，PBST洗涤(优选3次，每次5分钟)；封片剂封片；同样条件下，以转染空载体的HEK293T细胞为对照；然后进行荧光显微镜检测，若荧光信号强于对照，则待测样品为阳性；荧光信号等于或小于对照信号，则为阴性。
进一步，所述AQP4、MOG、MBP基因分别以慢病毒质粒载体的形式导入HEK293T细胞，所述含AQP4基因的慢病毒质粒载体核苷酸序列为SEQ IDNO.1所示；所述含MBP基因的慢病毒质粒载体核苷酸序列为SEQ IDNO.2所示；所述含MOG基因的慢病毒质粒载体核苷酸序列为SEQ IDNO.3所示。
与现有技术相比，本发明有益效果主要体现在：本发明提供的在同一***同时检测中枢脱髓鞘自身抗体AQP4、MOG、MBP的方法，采用灵敏度和特异性更高的荧光免疫细胞染色技术，用于临床定性检测AQP4、MOG、MBP抗体，使检测结果更加稳定，且极大节约人力和时间成本。中枢脱髓鞘自身抗体检测技术的平台的建立，有利于提高视神经脊髓炎和多发性硬化的临床诊疗水平。抗体检测是自身免疫性疾病重要诊断指标，该项目的建立将极大提高视神经脊髓炎疾病谱和多发性硬化的诊疗水平，提高患者的劳动能力和生活质量，将产生很大的社会效益和经济效益。
(四)附图说明
图1为AQP4病毒载体构建示意图。
图2为MBP病毒载体构建示意图。
图3为MOG病毒载体构建示意图。。
图4为HEK293细胞感染AQP-4病毒表达载体后AQP-4蛋白表达电泳图。
图5为HEK293细胞感染MOG病毒表达载体后MOG蛋白表达电泳图。
图6为转有MBP质粒的细胞中MBP蛋白水平表达电泳图(A)和荧光图(B)。
图7为细胞免疫荧光检测待检血清AQP-4/MOG/MBP抗体表达水平的荧光图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1、转有AQP4、MOG、MBP基因的稳定HEK293T细胞的构建
1、载体构建：
(1)构建融合表达人AQP-4基因及荧光标记基因EGFP的过表达载体
以pEGFP-N1载体为模板，采用引物EGFPF和EGFPR扩增EGFP序列，所用引物如下：EGFPF:5’-CTA GGATCC ATGGTGAGCAAGGGCGAGGAG-3’和EGFPR:5’-CAGGTCGACGAATTCGCGGCCGCCTCGAGCTGCAGTTACTTGTACAGCTCGTCCATGCCG-3’，扩增片段切胶回收后连入T载体，经测序比对序列正确后将其用BamHI和SalI双酶切后与经同样酶双酶切后的pCDH-CMV-MCS-EF1-CopGFP载体片段相连接，用EGFP置换掉原载体中的EF1-copGFP序列，获得质粒载体pCDH-CMV-MCS-EGFP。
以人视神经cDNA为模板，以AQP4F和AQP4R为引物，PCR扩增AQP-4(M1+M23)序列，获得AQP-4扩增片段。
AQP4F:5’-ACTTCTAGAATGAGTGACAGACCCACAGCAAG-3’；
AQP4R：5’-CCAAGATCTTGAACCGCCTCCACCTACTGAAGACAATACCTCT CCAGATTG-3’。
将AQP-4扩增片段切胶回收后连入T载体，经测序比对序列正确后，将其用EcoRI和XhoI双酶切后与经EcoRI和XhoI双酶切后的载体pLV-Puro相连接，获得载体pLV-Puro-AQP-4；继而用XbaI和BglII双酶切后与前述获得的质粒载体pCDH-CMV-MCS-EGFP相连接，得到所述的慢病毒质粒载体pLenti-CMV-AQP-4-EGFP，核苷酸序列为SEQ IDNO.1所示，质粒见图1所示。
(2)构建融合表达人MBP基因及荧光标记基因EGFP的过表达载体
以人少突胶质细胞为模板(
CRL-3216TM)，以MBP F和MBP R为引物，PCR扩增MBP序列。
MBP F:5’-TTTCTAGAAATGTACAAGGAATTCGCCACCATGGCGTCACAGAAGA-3’；
MBP R:5’-TCTAGGGATCCGGGCCCGGGTTCGAACTCGAGTCAGCGTCTAGCCATGGGT-3’。
将MBP扩增片段切胶回收后连入T载体，经测序比对序列正确后，将其用EcoRI和XhoI双酶切后与经EcoRI和XhoI双酶切后的载体pLV-Puro相连接，获得载体pLV-Puro-MBP，继而用XbaI和BglII双酶切后与前述步骤(1)获得质粒载体pCDH-CMV-MCS-EGFP相连接，得到所述的慢病毒质粒载体pLV-EGFP(2)Puro-MBP-V1，核苷酸序列为SEQ IDNO.2所示，质粒见图2所示。
(3)构建融合表达人MOG基因及荧光标记基因EGFP的过表达载体
以人少突胶质细胞为模板(
CRL-3216TM)，以MOG F和MOG R为引物，PCR扩增MOG序列。
MOG F：5’-GGGTAGTTTAGAGTGATAGGATTAAGATAT-3’；
MOG R：5’-TAAAAATAAACCACCCTAAAAAAAA-3’
扩增片段经电泳后，切胶回收，回收后连入T载体，经测序比对，确认序列正确后将其用EcoRI和XhoI双酶切后与pCDH-CMV-MCS-EGFP粒载体相连接，得到过表达人MOG全长基因的慢病毒质粒载体pLenti-CMV-MOG-EGFP，核苷酸序列如SEQ ID NO.3所示，质粒见图3所示。
2、病毒包装及HEK293T稳定转染细胞系的获得
利用CaCl2法包装表达目标基因融合蛋白的慢病毒，收集病毒悬液在6μg/ml聚凝胺(Polybrene)存在的情况下感染HEK293细胞，1周后，分选阳性稳定转染细胞。继续传代培养后进行二次筛选，获得100％转染过的稳定表达融合蛋白的HEK293细胞系，具体为：
1)细胞培养：将人胚肾HEK293T细胞系接种至含有10％灭活胎牛血清的低糖DMEM培养基中，放入37℃，5％CO2孵箱中进行培养，取处于对数生长期的HEK293T细胞用含有10％灭活胎牛血清(FBS)和1％双抗(终浓度均为100μg/mL的青霉素、链霉素)的低糖DMEM培养基重悬细胞，于冻存管内，液氮保存，用于实验；
2)细胞的复苏和传代：将步骤1)冻存管取出，迅速投入37℃水浴中并不断摇动使其在最短时间内解冻，用70％酒精消毒冻存管外表后，将冻存管内细胞液转移至装有20ml低糖DMEM培养基的50ml离心管中，1000rmp离心5分钟，弃上清，沉淀置于含10％胎牛血清的低糖DMEM培养基中，37℃、5％CO2、饱和湿度下培养至汇合度达到80％；
3)细胞的消化：向步骤2)中加入1ml胰酶，消化HEK293T细胞1-2分钟，收集HEK293T细胞用含10％FBS的低糖DMEM培养基重悬，置于培养皿中，于含5％CO2的37℃温箱中孵育24小时，当细胞贴壁完全后即可开始转染。转染前2小时换液(用1.5ml完全培养基置换旧的培养基)。
4)质粒转染：取无菌1.5ml EP管，加入400μl无血清低糖DMEM培养基，分别加入6μl混合质粒(目标质粒:包装质粒VSVG:包装质粒REV＝5:3:2，体积比)，及6μl转染试剂(Lipofectamine 2000)，充分混匀，静置15分钟，获得质粒混合液412μl。将412μl的质粒混合液逐滴加入步骤3)贴壁完全的培养皿中，轻轻摇动培养皿混匀后置于含5％CO2的37℃温箱孵育。其中，目标质粒分别为慢病毒质粒载体pLenti-CMV-AQP-4-EGFP、慢病毒质粒载体pLV-EGFP(2)Puro-MBP-V1和慢病毒质粒载体pLenti-CMV-MOG-EGFP。
5)病毒收集：步骤4)孵育8小时后吸去培养基，加入2.5ml 37℃预热的低糖DMEM培养基，继续于含5％CO2的37℃温箱孵育40小时，收集培养液到1.5ml离心管中，3000rpm离心20分钟，将上清液用0.45μm滤头过滤，收集滤液，即为病毒液，分别获得AQP-4病毒液、MBP病毒液、MOG病毒液。分装后置于－80℃储存待用。
6)病毒滴度测定(稀释计数法)：
滴度单位：TU/ml，指每毫升中含有的具有生物活性的病毒颗粒数。“TU”为“transducing units”的缩写，中文为“转导单位”，表示可以感染并进入到靶细胞中的病毒基因组数。
第一天细胞准备：将步骤2)生长状态良好的HEK293T细胞消化计数后，用低糖DMEM培养基稀释至1×105/ml，加入96孔板，100ul/孔，分为3组(即AQP-4病毒液、MBP病毒液、MOG病毒液)，每组准备10个孔，放入37℃，5％CO2培养箱中培养。
第二天加病毒：在EP管中做10倍梯度稀释，连续10个稀释度，稀释方法如下：每组病毒准备10个1.5ml EP管，每管加入90ul含有10％灭活胎牛血清和1％双抗的DMEM培养液，往第一个管中加入10ul步骤5)制备的病毒液，混匀后，吸取10ul加入第二个管混匀。依此类推，做十个稀释度(10～10-8)。吸去96孔板中原有的培养基，加入等体积稀释病毒液，并做好标记。
第三天追加培养液：在每个孔再加入100ul低糖DMEM培养基，培养48小时至第五天。
第五天观察结果并计算滴度：在荧光显微镜下观察结果，并数出最后两个有荧光的荧光细胞克隆数，假设为X和Y，则滴度(TU/ml)＝(X+Y×10)×1000/2/X孔的病毒液的含量(ul)。
7)病毒感染及稳定细胞系筛选：步骤5)中的病毒液8ul与PEG8000按照体积比4：1的比例混合，每20分钟混合一次，共混合4次，4℃过夜。次日，4000g、4℃离心20分钟，弃上清，沉淀用100ul含有10％灭活胎牛血清和1％双抗的DMEM培养基重悬，获得病毒浓缩液100ul。将10ul含6μg/ml多聚胺的病毒浓缩液加入步骤1)处于对数生长期的HEK 293T细胞中，48小时后加入终浓度15ug/ml嘌呤霉素，约每2天更换一次新鲜培养基，37℃，5％CO2培养10天左右，利用单细胞克隆分选阳性稳定高表达待测蛋白的转染细胞，分别获得转染基因AQP4的HEK293T细胞、转染基因MOG的HEK 293T细胞、转染基因MBP的HEK 293T细胞，并利用定量PCR和western blot技术测定筛选的细胞与未转染293T细胞中目标抗体转录本的相对表达量及蛋白水平的表达，见图4，图5，图6所示。结果显示在稳定转染细胞系中目标抗体(AQP4、MOG、MBP)转录本比未转染的293T细胞中转录本显著增加。
实施例2、细胞免疫荧光染色法测定血清AQP4、MOG、MBP抗体
1)接种细胞：将细胞贴壁试剂(购自北京细工生物科技有限公司，货号：1027)与PBS以体积比1:10混合，加入含盖玻片的6孔板中，37℃，5％CO2中孵育1小时。然后加入分别转有AQP4、MOG、MBP基因的稳定HEK293T细胞系(即含AQP-4基因的HEK293T细胞系、含MBP基因的HEK293T细胞系、含MOG基因的HEK293T细胞系)，细胞接种量均为1×106/ml，37℃，5％CO2培养至80％融合度，用于后续检测用。同样条件下，以转染空载体的HEK293T细胞为对照。
2)细胞免疫荧光：用金刚石刻字笔切割盖玻片至边长为5mm小正方形，细胞面朝上，无影胶粘贴于载玻片上，紫外线照射30秒粘牢，盖玻片即为反应区。PBST洗涤细胞50ul/反应区，5分钟。用3％BSA以50ul/反应区封闭30分钟，弃去。相应孔分别加入经PBST以体积比1:10稀释后的待测血清50μl，在37℃，5％CO2条件下孵育1小时。PBST洗涤3次，每次5分钟。分别加入PBST稀释以体积比1:700稀释的生物素与链霉素结合的山羊抗人(Biotin-SP-conjugated affinipure goat anti-human IgG)50μl，37℃，5％CO2条件下孵育30分钟。PBST洗涤3次，每次5分钟。分别加入PBST以体积比1:700稀释的荧光素488月链霉素结合物(Alex fluor 488-conjugated streptavidin)50μl。PBST洗涤3次，每次5分钟。封片剂封片。
3)荧光显微镜分析：以转染空载体的细胞对照，在20倍物镜视野下依据红色荧光的信号强弱来标示抗体浓度的相对量。结果显示，依据荧光强度，本发明方法可以区分AQP4、MOG、MBP阳性病人中自身抗体的相对量，见图7所示。
实施例3、特异性检测
鉴于MOG抗体检测的阳性率低，为进一步促进本发明方法的临床应用，我们多中心选取了120例神经***疾病患者的血清，分别送到欧盟第三方公司检测，同时用我们的检测方法盲法检测。通过与欧盟检测方法比较，分析本发明方法的MOG阳性抗体检出率。
选取120例神经***疾病患者血清(含脱髓鞘病人30例)，用欧蒙检测方法(欧蒙医学诊断(中国)有限公司)的检出率和本发明方法(实施例2方法)的检出率进行对比。结果显示，在30例脱髓鞘患者中，欧蒙测出MOG抗体阳性血清4例，本检测方法检出6例阳性血清，且检出的MOG阳性血清的患者为中枢脱髓鞘疾病，与临床症状相符合。其余90例非脱髓鞘患者，欧蒙检测结果与本方法检测均为阴性。
序列表
<110> 天津天海新域生物科技有限公司、浙江达美生物技术有限公司
<120> 一种同时检测中枢脱髓鞘自身抗体AQP4、MOG和MBP的方法
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1710
<212> DNA
<213> 未知(Unknown)
<400> 1
atgagtgaca gacccacagc aaggcggtgg ggtaagtgtg gacctttgtg taccagagag 60
aacatcatgg tggctttcaa aggggtctgg actcaagctt tctggaaagc agtcacagcg 120
gaatttctgg ccatgcttat ttttgttctc ctcagcctgg gatccaccat caactggggt 180
ggaacagaaa agcctttacc ggtcgacatg gttctcatct ccctttgctt tggactcagc 240
attgcaacca tggtgcagtg ctttggccat atcagcggtg gccacatcaa ccctgcagtg 300
actgtggcca tggtgtgcac caggaagatc agcatcgcca agtctgtctt ctacatcgca 360
gcccagtgcc tgggggccat cattggagca ggaatcctct atctggtcac acctcccagt 420
gtggtgggag gcctgggagt caccatggtt catggaaatc ttaccgctgg tcatggtctc 480
ctggttgagt tgataatcac atttcaattg gtgtttacta tctttgccag ctgtgattcc 540
aaacggactg atgtcactgg ctcaatagct ttagcaattg gattttctgt tgcaattgga 600
catttatttg caatcaatta tactggtgcc agcatgaatc ccgcccgatc ctttggacct 660
gcagttatca tgggaaattg ggaaaaccat tggatatatt gggttgggcc catcatagga 720
gctgtcctcg ctggtggcct ttatgagtat gtcttctgtc cagatgttga attcaaacgt 780
cgttttaaag aagccttcag caaagctgcc cagcaaacaa aaggaagcta catggaggtg 840
gaggacaaca ggagtcaggt agagacggat gacctgattc taaaacctgg agtggtgcat 900
gtgattgacg ttgaccgggg agaggagaag aaggggaaag accaatctgg agaggtattg 960
tcttcagtag gtggaggcgg ttcaagatcc atggtgagca agggcgagga gctgttcacc 1020
ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg 1080
tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc 1140
accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag 1200
tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc 1260
gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc 1320
gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 1380
ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac 1440
gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac 1500
aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 1560
gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa 1620
gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc 1680
actctcggca tggacgagct gtacaagtaa 1710
<210> 2
<211> 9496
<212> DNA
<213> 未知(Unknown)
<400> 2
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 2220
atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 2280
ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 2340
aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 2400
tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 2460
ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 2520
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 2580
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 2640
acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 2700
gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 2760
tccatagaag acaccgactc tactagagga tcgctagcgc taccggactc agatctcgaa 2820
tttctagaaa tgtacaagga attcgccacc atggcgtcac agaagagacc ctcccagagg 2880
cacggatcca agtacctggc cacagcaagt accatggacc atgccaggca tggcttcctc 2940
ccaaggcaca gagacacggg catccttgac tccatcgggc gcttctttgg cggtgacagg 3000
ggtgcgccca agcggggctc tggcaaggta ccctggctaa agccgggccg gagccctctg 3060
ccctctcatg cccgcagcca gcctgggctg tgcaacatgt acaaggactc acaccacccg 3120
gcaagaactg ctcactacgg ctccctgccc cagaagtcac acggccggac ccaagatgaa 3180
aaccccgtag tccacttctt caagaacatt gtgacgcctc gcacaccacc cccgtcgcag 3240
ggaaagggga gaggactgtc cctgagcaga tttagctggg gggccgaagg ccagagacca 3300
ggatttggct acggaggcag agcgtccgac tataaatcgg ctcacaaggg attcaaggga 3360
gtcgatgccc agggcacgct ttccaaaatt tttaagctgg gaggaagaga tagtcgctct 3420
ggatcaccca tggctagacg ctgactcgag ttcgaacccg ggcccggatc cctagataat 3480
tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc 3540
cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg 3600
gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc 3660
tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag 3720
tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag 3780
gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg 3840
gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa 3900
ggtcctccgg aggcccggca ttctgcacgc ttcgaagcgc acgtctgccg cgctgttctc 3960
ctcttcctca tctccgggcc tttcgacctg cagcccaagc ttaccgccac catggtgagc 4020
aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 4080
aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 4140
accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 4200
accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 4260
ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 4320
gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 4380
atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 4440
tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 4500
gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 4560
cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 4620
acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 4680
ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga atttggcagc 4740
ggccagtgca ccaactatgc cctgctgaaa ctggctggcg atgtggaaag caaccccgga 4800
cccatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc 4860
gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg 4920
gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc 4980
gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg 5040
gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc 5100
ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag 5160
gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg 5220
ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc 5280
ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc 5340
gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc 5400
tgaccgcgtc tggaacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat 5460
tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca 5520
tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc 5580
tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc 5640
tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt 5700
cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg 5760
gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc 5820
ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta 5880
cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg 5940
gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc 6000
cccgcctgga attaattctg cagtcgagac ctagaaaaac atggagcaat cacaagtagc 6060
aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg 6120
ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat 6180
cttagccact ttttaaaaga aaagagggga ctggaagggc taattcactc ccaacgaaga 6240
caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga ttagcagaac 6300
tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta caagctagta 6360
ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag cttgttacac 6420
cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg gaggtttgac 6480
agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt caagaactgc 6540
tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag gcgtggcctg 6600
ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc tttttgcctg 6660
tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 6720
cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 6780
gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 6840
tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat 6900
atcagagagt gagaggcctt gacattgcta gcgttttacc gtcgacctct agctagagct 6960
tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 7020
acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 7080
tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 7140
tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 7200
cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 7260
actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 7320
gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 7380
ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 7440
acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 7500
ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 7560
cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 7620
tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 7680
gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 7740
ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 7800
acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 7860
gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 7920
ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 7980
tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 8040
gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 8100
tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 8160
ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 8220
taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 8280
cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 8340
gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 8400
gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 8460
tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 8520
gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 8580
ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 8640
ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 8700
cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 8760
ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 8820
gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 8880
ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 8940
ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 9000
tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 9060
ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 9120
cacctgacgt cgacggatcg ggagatcaac ttgtttattg cagcttataa tggttacaaa 9180
taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 9240
ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaactggat aactcaagct 9300
aaccaaaatc atcccaaact tcccacccca taccctatta ccactgccaa ttacctgtgg 9360
tttcatttac tctaaacctg tgattcctct gaattatttt cattttaaag aaattgtatt 9420
tgttaaatat gtactacaaa cttagtagtt tttaaagaaa ttgtatttgt taaatatgta 9480
ctacaaactt agtagt 9496
<210> 3
<211> 22392
<212> DNA
<213> 未知(Unknown)
<400> 3
gtacagtgaa gtatacaact ttgtgcaggg caatttatca atatttattg aaattaccaa 60
aaaacatgct ctctgaacaa actattctac cagtgtagaa agcagagtaa acttcatggg 120
tgagtgacca gggcagtcac acaagggccc catgcttaga agggatactg tgtttgggtt 180
ctaaagctct gtggttcctg tcttgaaatt cttaataatt ttatctttca atttgtgtct 240
tataatgaag tccgatgaga aagcagaaca tgggctagag acttttggag cctggctcaa 300
gcgaggtcct gctccccatg cctcccagcc tccccaggac tggttttcag ctgccggctc 360
caccaccttc tgtgcaggct cgctcccagc aggggcctgg gaacagtgga aaggagggga 420
gcggtcaggc atacacacct cccttgccaa atggaaggca tggccctagg cacttgtgaa 480
gatctgcact tccccctagg tactcctgtg cctggagtgt gacattaaat taaaaaaaaa 540
aaggccgggc gcggtggctc acgcctgtaa tcccaccatt atgggaggcc aaggcaggcg 600
gatcacgagg tcaggcgatc gagaccatcc tggctaacac ggtgaaaccc cgtctccact 660
aaaaatacaa aaaaattatc tgggcatggt ggcgagcgcc tgtagtccca gctacttggg 720
aggctgagac aggagaatgg cttgaacccg ggaggcggag gttgcagtga accgagattg 780
cgccactgca ctccagcctg ggcgacagag cgagactcca tctcaaaaaa aaaaaaaaaa 840
aagaaaagaa aaaaacccca cataataggt tgacagtgga accacagaaa aaaggaaaag 900
gttgggtttt ttttctgctt tttattttct attttattat tttttaatag atttatttaa 960
ctagagatgg ggtctcacta tgttgtaaag gctggactcg agaccctggg cccgagcgat 1020
cctccaacct ggtcctccca aagtgatggg attacaggcg tgagccactg cacctggtct 1080
tttcctgcta ttaaacaagg agctccatag tttcattttg cccctcaaaa tatgtagctg 1140
gccttagtag actgatattc attgccaaat tatatgtaag agcaaaaagg ttgaaaatga 1200
tggcctgaca ttgatcaatt tgtgccttta ggtaacatat aactgtaata taactgcaat 1260
acaactagaa tataactcat aaaggcaaga atcttgtctg ccttgctgaa agttttataa 1320
tcagggccta atataaagta tgacacatag cacttgcttt taaatatgta ttgatttaaa 1380
ttaattgagt acatttttgc ttcatcctag taaaaatagg tatttaaaaa actgaaacag 1440
tctaaatgtc ttgggatgct acttaaataa ctatattata ttcatccaat aaaatattgt 1500
aagctgttta aaaataacaa ggatgttctt taggtactga taaggaaaga gcttcaagat 1560
aaattgttac catttatgta aaacaggtgg gagaagggag agggagggat gtgtgagcgc 1620
tacttgcagt actcacaggc agtgactttc gtggagcgcc ctctagtggt atatatatac 1680
aaacggaagg atttagagaa aatacagatc ggctttagct ggctgagatt tattttcaaa 1740
gcatgttact ttataagaat caatttttat ttaaaaaatt tttttgagat agggtctcac 1800
tctgtcgcac aggttggagt gcagcagcac gatcagtgct cactgcagcc tctctctctt 1860
gggctcaaca ggtgcatgtc accacgtcca gctaacaatc aattttcaaa agtacaaaaa 1920
agccatatta tgtattaatg tggaattatg aattaagtag acaacaagaa tcaaaacagg 1980
gtgtctatta tcacttctga taacataaat aatgtaaaga tacatatttt acagattatc 2040
tgtaaaagct tatacagtac tgttgctggg tatttatgta ggaaagctac catttattga 2100
atgcttacta tttcacatat ggacagcata gagcatgtta aaaaattacc acacacattt 2160
actgtattca atgtgtcact ctgaatatat tactgtgtac atggtctgtc attggacatg 2220
gtgagagatg cagattaagc tgaaattact gaggacagca acactggaag aaaattgagc 2280
tgggtgtagt ggctcagcct gtaattccaa catttcagaa ggctgaggca ggaggatcac 2340
ttgagtccag gagtttgaga ccaagggaaa gaaaagaaaa gaagctttca tttagccagg 2400
catgctggca catacctgta gtttcagcta ctcaggaggt tgaggcataa ggttcactta 2460
aacttgagag gtaaaggctg cagtgagccc tgatcacgcc actgctctcc agcctgtgac 2520
agagagagac cctgtctcaa aaacgagaaa gaaagaaaaa aagaggcaac tcaagaactc 2580
aggaatactt gcaggatctc ataacatatg ctatacaaaa tcaattaaaa taatatttaa 2640
atgctgaaag aaatgagcag ctcccagggt gatacagggt ggtttcactt cttggacaca 2700
tctacactga gctctattcc tggcaatacc tgatgttccc ataccccaga tttctttatt 2760
ttattttgag acggagtttt gctcttcttg cccaggctgg agtgcaatgg cgggatcttg 2820
gctcaccgca atctccgcct cctgggttca agggattacc ctgcctcagc ctcccgagta 2880
gctgggatta caggcgcacg ccaccatgcc cagctaattt ttgtattttt agtagaggca 2940
gggtttctcc atgttggtca ggctggtctt gaactcccaa cctcaggtaa tctgcccgct 3000
tcggcctccc aaagtgctgg gattacaggc gtgagcgggc ccagccccta taccccagat 3060
ttctgcaagt ggcaacacca ctggcttcat tttgctggtg gcccctctgg ccttcccttg 3120
tatatatcac ctttgcccaa agaccatgtc agccaaggga ctgctctcac agctccagga 3180
atcctcccct ttcaggaaat ttgaggcagt tgagggcatg aaagtaaata agctgagctc 3240
atcagaggcc ttgtattgtg gtggttaaaa gagccagttc taggactaga aagcctggct 3300
tgaaatccca gctctgccac tccctagtgg tgtgacttta gcaagttcct ttacctcttt 3360
tgtacctccc ttttctcacc tgtaagatat gggtgataat agtttaatat ttgttttgtt 3420
gttgtgagga ttaaaggtgt taatgcaagt aaaccactta gaaccacagc acatagaata 3480
tctcagtaag gtggttagtt ttttttattg ttgttttcag agatgctgtg atttctccaa 3540
agtggctgtg atggctcggc aggctcctga ccctctctgc tcccacatgc ctccaccctc 3600
atcctgatct cccatccagc ttttgacagc ttgcttggtg ctggacaatt gcacacatct 3660
taccaccccc aaatcctgcc cagaagcatc ttgtgcataa ctctcctacc tgaatatgca 3720
acagggagaa agagcgtccc aggacatttt aggtttttga agaaaaaaaa acccctttgg 3780
taaaaagcca gagatccaca gcggccactt tttccatggg attgacccct gcaatcttga 3840
ctttcaacca cacagcacca gagtagccaa acattgcttg tgtccaaacg ctggctgcct 3900
tgaagggtga aagaataagc agttcccaaa ctcagctgac cttaatgtcc ttctagctcc 3960
ttacgcccat ctcggacaaa agcagaaatg tatgtctcag ttgtgtttct accccttgct 4020
gcccaatata aatttttgtg ttgcccaata taattttttg tgacgatgga aatgttctgt 4080
atttgtgttg tctgatgaga taaccactaa ctgtagtgct attgagcatt tgaaacatgg 4140
ctagtgtaat caatgaacca aatttttaat tttatttaat tgtaattaat tttaagtggc 4200
cacatgcagg gagtgactgc tgcattggac agcacggctc taaattgagc cttttttcct 4260
tatttggtga ggcatacttg ccttaagatt gggaagtcta tttttggaac ctgctaccaa 4320
tgctggtctc acacttgcaa ttctcagctg agccaagagg tgagagaaag gtcattttcc 4380
attccagatc tcactctccc ctgtgacact gaggaaactg gcaagtgatg tgaaggctgg 4440
agagcgtgtc ctgtatgctg gctctgtccc ttctgcctgt gttgactgac atagttagtt 4500
gctgcccttg ctggtctccc ttcctccaac cttgcctctc tgagcacacc tgacattcat 4560
ctcatgactt ccctaaaaac attctttggg aacaagaaac taacaaatcc caagtgacct 4620
atcacatata caaacataca gggcagagtt tggattcgcg gtagaagaaa gggaggttag 4680
acattaagaa gaatggtctg gtgatgacag ttgtgagata atagaaacag gaaaaagaaa 4740
tctaagtttt ctttcttttt ttaagaacca ataataattt ctctcttttg actagtcagt 4800
agggctgggg tggattggag gaagcttaca tattccatga acaagcctct tcctaaggtc 4860
ctgtaagtga tcctgcccca ctgattagcc cctagaagac ccttcaaagg ttggatctcc 4920
aggagggagt gggggaggaa agccctgtac caggcagcct ctgctccatt gctctggggg 4980
ggtggggaag gcaaaccctg gtcatcccct cagtctgtag cccttttgtg tgagtgcctg 5040
gcaagggtga cgtggggctg tttctgcggg cacagctgca gcaattaccg gagtggaggc 5100
agggcccagg cagcactgcc ctccaagatc ttcccttggg cttttcagca gtaaggggac 5160
atgcacccca agggcctcca cttggcctga ccttgctgcg ggggctctct gtccccagga 5220
acagtagaga tggcaagctt atcaagaccc tctctgccca gctgcctctg ctccttcctc 5280
ctcctcctcc tcctccaagt gtcttccagc tatgcaggta agacatgttt tttttcctgc 5340
cctggggaga ccctgaaaac agaaaggcta gtttcctggg gcttagctcc ttcaaacatc 5400
ctcaagttgc tatattatct ttctaaaaca tagacctact gacatgcctc ccttcctcag 5460
aaaccttccg tgggtggttc ttacagcctt caagatggag tccagactct tttttttttt 5520
tgagacagag tctccctctg ttgctcaggc tggagtgcag tggcatgatc tcggctcact 5580
gcaacctcag cctccctggt tcaagcgatt ctcctgactt ggcctcccaa gtagcggaga 5640
ctacaggcgc ctgccaccac acccagctaa attttttctt ttcttttttt tttttttttt 5700
ttttgtattt tagtacagac ggggtttcac atgttggcca ggatggtctc gatctcttga 5760
cctgctgatc cgcccgcctc agcttcccaa agtactggga ttatgggcgt gagccactgc 5820
actaggccta atttttttat ttttagtaga gatggggttt caccatgttg gccaggctgg 5880
tctggaaccc ctgacctcaa gtggtctgcc ctcctcagcc tcccaaagtg ctgagattac 5940
aggcatgagc cattgcgtct gacccagact ccttaatgtg actaactcaa ggctttcctt 6000
gaactacttc ttacttgtct ttccagcttt gtcttttcac ctctcaaatt gagataaaat 6060
aataacaacc tcttggagtt ctcatcagga ttacatgaaa tgagatatgt aacatgctta 6120
gcagtgcctg tccatagtaa atctcaataa atgtttgtgg aattataata tcttgtcatg 6180
tttgagactt tgctctgcat aatcaggcac cagtaggttt ttataaagga acccggctgt 6240
cacgtgcaga ggagaaataa acagaaagtt tcccatcctc agggagccac ctgactgaca 6300
gaggcacagt gcatccactc tccaggtcta ggggagaaag cagccttatt tcttagtagc 6360
tcagaatctg acttgagaaa cacatccaca tagaaaaaaa caaggaactt tttcgggtca 6420
gggtccggga gccacagtga ggtggaagat acaggggaag gaagagggaa atagagccat 6480
ccccagggtg gaagatctca gaagagaatt tgggaaacaa ggtatgaaca aggactgaat 6540
agtgagaagt gatggagaga cagttaaagt agatggagtg acaaaagcaa aacctctaag 6600
ggtagaatag gcagcaattt ggccaagtcc taacagggag gcccatagga ggattcaacc 6660
tcaagatgct gtgccacatt ccaagaggga acctaaaggc tgggctgaag agtcagagat 6720
ggctacagct ggcaaaaaga tgggcagatg ctgagaggag atgattgcta aaatgttctg 6780
tccaggacat tcacagtatc tctataacca gagtcttttt tgtcgttgtt gttctcaaga 6840
aggaaacttg aggccgggtg tggtggttta tgcccataat cccagcgctt tggggccaag 6900
gcaggcggat cacctgaggt caggagttcg agaccagcct ggccaacagt gtgaaacctc 6960
atctttacta aaaatacaaa aattagctgg atgcggcggt aggtgcctgt aatgccagct 7020
actcgggagg ctgaggcagg agaatcactt gaacctggga ggcggaggtt gcagggaggc 7080
ggaggttgca gtgagccaag attgcaccac tgcactccag cctgggcgac agagagtaag 7140
actgtctcaa aaaataaatg aataaataaa aaggaagaag aagaagaaga acaattgcaa 7200
tcctccctgg ctctagaatg tcatttaaaa gtcgagtgtc ttcttccttc cctgttttga 7260
agcagccctt ctcatgacag gcttgcttgc caaggttccc tctgacctta aatctcttcc 7320
ttttggtgtc ttggacaggg cagttcagag tgataggacc aagacaccct atccgggctc 7380
tggtcgggga tgaagtggaa ttgccatgtc gcatatctcc tgggaagaac gctacaggca 7440
tggaggtggg gtggtaccgc ccccccttct ctagggtggt tcatctctac agaaatggca 7500
aggaccaaga tggagaccag gcacctgaat atcggggccg gacagagctg ctgaaagatg 7560
ctattggtga gggaaaggtg actctcagga tccggaatgt aaggttctca gatgaaggag 7620
gtttcacctg cttcttccga gatcattctt accaagagga ggcagcaatg gaattgaaag 7680
tagaaggtga gtagtgccat ataatattag gtattaactg ttgggtggcc aagaacaatt 7740
attctctcaa ctgagatgag atccctcaac ccaaacatct cagtcctggg aatgatttcc 7800
ataaaaatgt acacatcaat aaacagaaac tcatgcttag ggatgtctgt tgcatcatta 7860
ttcagagtag caaggaaatt gggatcaaaa tcaatgcctt tgagtaggta agtgacagaa 7920
tgaacaatgg tagccatact gtgaatatta tgcaggcatt aaaaagatta ttttagcact 7980
aggccagatg gtttggaggc cttctataag gtattattga gtgataagag caagctgctg 8040
taggatacaa aaacaaaaac aaaaccctag ggcatggtgg tttgcctcgc agctactcag 8100
gaggctgaga cgggaggctg gcttgagccc aggggtttgc agttacagtg agctatgatt 8160
gcaccactgc actccaaccc gggtgacaga gcaaagacct tcacccccac tccctacccg 8220
tctctaaaaa aaacaaaaac aaaaacaaaa aaacccttgg gcccagcgcc gtggctcacg 8280
cctgtaatcc cagcactgtg ggaggccgag gtgggcagat cacaaggtca ggagatcgag 8340
accatcctgg ctaaaacggt gaaaccccgt ctctactaaa aatacaaaaa aaaaaaaaaa 8400
attagccagg catggtagca ggcgcctgta gtcccagcta ctcgggaggc tgaggcagga 8460
gaatggcgtg aacccggaag cggaggttgc agtgagccaa aatccttcca ctgcactcca 8520
gcatggggga cacagcgaga ctccgtctca aaaaaaaaaa aaaaaccctg tatttgtgag 8580
cgcacacaca cacacacaca cacacacacc tgtgcttggt cctagtgaat aagcaagtaa 8640
atcaaatgtc taaatataat tatagaaagg agatgtcact ttttggctgt acctccacta 8700
tttcattctg cagaattgca gaatttcttt ttttttttcc tttctttctt ttcttttttt 8760
ttttgacaca gagtctcgct ctgtcaccca ggctggagtg caatggcgcc ctccgcctcc 8820
tgggttcaag tgattctcct gcctcagcct cccgagtagc tgagattaca ggtgcccacc 8880
accacaccca gctaattttt gtatttttag tagagacagg gtttcaccag gttgtcaagg 8940
ttggtctcaa actcctgacc tcaggtgatc cactcgcctc agcctcccaa agtgctggga 9000
ttacaggcat gagccatggt gcccggcctc agaatttcat tttcaacatg ttttgcatga 9060
tgggtgattt tggagaatat tttttgctct atcgcaggat gattaagatg tggacaaggt 9120
gaagccgatg gagggggagc tttgaaagtt acttgctatt taattgagga actaaactgc 9180
tttgagagcc tgggggtcag atcctctgcc ttttcctcct ccccacctgc agtgcaaaca 9240
tcagacaatt gatcactatt gtatcttgga ggtgggagtg accattgcag tgctgggacc 9300
agaagatggc attgtatgtg gaacaacaaa gcactatttc tagagactgc ctgcagggat 9360
atggaaatag ctttatgtgt ctcagaatgt tcttcataca gctgttttta ttggggaaat 9420
tctacttgcc gaaaagtttg atagtgagac cctctccagt ttgcagattt ttctccttcc 9480
tgctcaacaa cttcctagct cagtaactgc ctctcccaac aaactccctc agtttcacca 9540
caccaaaaaa ggaagacaag ccggttgcgg tggctcacac ctataatccc aaaactttgg 9600
gaggccgagg cgggtggatc acctgaggtc gggagttcga gactagcctg accaacatgg 9660
agaaaccctg tctctactaa aaacacaaaa ttagcctggc gtggtggcgc attcctgtaa 9720
tcccagctgg gaggctgagg caggagaatc gcttgaaccc cggaggcgga ggttgcagtg 9780
agccaagatc gtgccattac actccagtct gggcaagaaa agtggaactc catctcaaaa 9840
aaaaaaaaaa aaaaaaaaca aggaagacaa aaagaaaagc agctaaagac tttgcctcag 9900
gggagaaagt tctcttttgg gttgctatcc acattccaac ctcctgttcc cacctcttcg 9960
tctgcatgcc taagaaactg ttttacaagt aaataaggga cgctttgtct aggctttgga 10020
gccaggaagt tgagacaaat ttaggaatga gatgaagtaa tggtattatt gcaagtctca 10080
ggtgtaacta cctctgctct ttctctgaag agtttctaat ttctcttgtt tacttatttt 10140
tttcttgtca tttttgtgat tttattacta gttgtctcta atcctttctt taaattcttc 10200
attatgaaac ataaaaacaa atgccaggcg cggcagctca cgcctgtaat cccagcactt 10260
tgggaggccg aagcgggcag atcacccgag gtcaggagtt cgagaccagc ctgatcaaca 10320
tggagaaacc ccgtctctac taaaaaatac aaaattagct aggcgtggtg gcacatgcca 10380
gtaatcccag ctacttgaga gactgaggca ggagaatcgc ttgaaccggg aggcagaggt 10440
tgcggtgagc caagatcgcg ccattgcact ccagcctggg caacaagagc aaaactctgt 10500
ctcaaaaaaa aaaaaccaca tacaaaccag agataatatt ataatgagcc tccaagtgcc 10560
taccaccttg ctgcagcact tgtcaatcca gggaccaccc acctcaccgg ctccccactc 10620
attaccaccc tcccctactc aattactgag gtaaatccta ggcagcatga tcatttcttt 10680
tttttctttt tatttatttt gagacaggat ctgtctctgt cacccaggct ggagtgtagt 10740
ggcatatctc tgctcactgc agcctctgcc tcccgggcag aagccatcct cccacctcag 10800
cctacatagt agctgggacc acaggcacac accaccacac actgctaatg ttttgtattt 10860
tttgtagaga ctgggtttta ccatgttgat caggctggtc tcaaactcct aggctcaagc 10920
aatcctccca cctcggcctc ccaaagtgct agaattacag gcgcgagcca ctgcacccag 10980
cgaagaacac tttttaaaaa ataaataggc cgggcgcggt ggctcacacc tgtaatccca 11040
gtactttggg agcccaagga gggcgaatca tgaggtcaag agattgagac catcctagct 11100
aacatggtga aaccccattt ctactacaaa tacaaaaaca aaattagcct ggcgtggtgg 11160
caggcgcctg tagtcccagc tacttgggag ctgaggcagg agaatggagt gaacccggga 11220
ggcggagctt gcagtgagct gagatcatgc cactgcactc cagcctgggg caacagagtg 11280
agactcaaaa aaaaaaaaaa aaaagccccc cctccccaca cacaataata taaataaata 11340
aataaccaca atactattat cacatcttac aaactcaaca aaaatttctt aatatcatca 11400
aatacccagt ttgtgttcaa attttcctga ttgtttcata aatatactct tacagttggt 11460
ttcttttagc gagattcaaa tgagacccac ctgttgacct ttgcccttag ggtttcccag 11520
ggtctgaatt ttgttgacga cattcccatg ttgctatgta atacggtcct ccatgccctg 11580
tgtttttctg taaactgata gatgtggagg tgcaatgaca tttgtgtttg atttactttg 11640
gcaaatatag ttcatcagtg atactctata cttcttgttg ctttacatcc ggaggctgat 11700
aatgtctgct tttctctctt ttctaattat ttgtgaaagg aaaaatgtgg ggggttggga 11760
gaaaaaaacc cttaagtaca tactcgctaa atcacattgc tacaggtaac ttccattaag 11820
aacttgaaag taaaggtagc tgcattttcc cctagggaac acaatgatag acaggagcct 11880
tagtctacag cttgaaggat tgtaattata cctaagcaac cctcctggac cagtttaatg 11940
ttattagctg tgatgtatcc ctacctttga tgtcattatc cttactcagc tcccttaaag 12000
cagagatcaa gatgaaaagg gcttcagctg cagcatggca catggagatt agagtggggc 12060
ttttggatgc tgaggagcag acctagaatg ggaaatagat gggagccaca gaagtgaagg 12120
tccccctccc tcattgctca acctactcca catctccagg tctgcacatc tgttcagtta 12180
ctgaatcctg tgtaagctac cttctttttc ttttttcttt tatttattta tttatttttt 12240
ttttgagatg gagttttgct cttgttaccc aggctggagt gcaatggtgc aatctcggct 12300
cactgcaccc tccaactccc aggttcatgc aattctcctc cctcagcctt ccaagtagct 12360
gggattacag gctgcaccac catgtctggc taatttttgt attatcagta gagagagggt 12420
ttcaccatgt tggccaagcc ggtctcgaac tcctgacctc aagtgatcca cccaccttgg 12480
cctcccaaaa tgctgggatt acaggtgtga gccaccatgc ccgctgtaaa ctaccttctt 12540
aaaagctcta gaagagggct cttaaccttt tgttgtgtgt catgcacctt ccgcaagctg 12600
atgaagttga tagacccatc tcagaatttt tttttttttt ttgagacagt gtctcactct 12660
gtcacccagg attggttgca gtggcacgat catggctcat tgcagcctcc acctcccagg 12720
ctcaagtgat cctcctgact cagcctcttg aatagctgag accacaggct tgtgtcacca 12780
tgcccaggta atttttaatt ttttttcgta gaggcagggt ctcacattat gttgcccagt 12840
ctggcctcga gaactcctgg gctcaagcaa tcttcctgcc ttggcctccc aaagtggtgg 12900
gattacaggg gagagccacc acacctagcc agaagaatgt tttaaataca ccaaataaaa 12960
catttatacc aaaatacagt tatcaaaata ttaaattaac aagagttagg gtgaccctat 13020
taattagtgt aatttcaaaa tagtaatgaa cataagtgat agtttgagat ttctgtgact 13080
tttctaatgt gacgtgaaaa tatttgtgat ttttcttttt cttttttttt tttgagatgg 13140
agtttcgctc ttgttgccca ggctggagtg caatggcaag atctcggctc acctcaacct 13200
ccgcctcctg ggttcaagcg attctcctgc ctcagcctct tgagtagctg ggattacagg 13260
actgtgccac cacgtccagc taattttgta tttttagtag aaacagggtt tctccatgtt 13320
ggtcaggctg gtcttgaact cccaacctca ggcgatccgc ccgcctcggc ctcccaaagt 13380
gctgggatta caggtgtgag ccaccgcacc tggccaatat ttgtgatttt tattgacgac 13440
aaagtcaaag gttctcttca tattattgtg gtgtatcgcc tacaagcata attaaaataa 13500
acactaaatt tcagtttaaa gtttactgaa aataaatatg tattttttat tccctattta 13560
agctttgaat cccctgactt cctataccat taccactgtc ctagttcagg ttcatgttgt 13620
tttttacttt aattgttatc acagtctctt aacatttctc cctatgttct ccagtcctgt 13680
aggtgctaaa tctgacgtgg tcacttctca gcttggaatc cttcagtgca ccaccacagc 13740
cttgaactac atatttgaaa tacatattta ttttcagtaa actttaaact gaaatttagt 13800
gtttatttta attatgcttg taggcgatac accacaataa tatgaagaga acctttgact 13860
ttgtcgtcaa taaaaagtcc cttgagggac ttcagatgta agtcccttag ctgctcgtta 13920
aaactccccc agcctgaccc aatacacaat cttgacttta aaccacttgt cattctaaat 13980
cactagcatt tcctggaaaa aaaagccatt tttccttcag ggctaagctc agggaccaat 14040
tctgtgtcac cttctttgaa tcctgatgat attcacttct ttatttgacc tgatttattg 14100
ggccccagac accatgctga gtgttgggga ttcagctctg gacaatgtca aatgtcagtc 14160
ctgcctttca gatcctttct actgggtgag ccctggagtg ctggttctcc tcgcggtgct 14220
gcctgtgctc ctcctgcaga tcactgttgg cctcatcttc ctctgcctgc agtacagact 14280
gagaggtaca gggcagaggg tgggtggatc aggatccttt ctttaaatga gctggcttct 14340
tggagctaca ccacttaaca tgtatttgtg agtgacttct gggttcagaa gttcttctca 14400
ctattgagtg ataaagaaaa aaaataactc catgatgaaa gagttttaca tcttacggaa 14460
tgctttcata tgaataatcg gacctagcat ttccctatga gctaactatg ccatatagta 14520
accccatttt acagaggata caactgaggc caggagtagt tcagtgactt actcaaaccg 14580
atataactta taagtggtag agctgaggcc tctgtatcat acctagcagc tccatgcaac 14640
ttgggagagt gtgagcttcg aagtcagaca ggtctaggct attaggagtt ttgaataaag 14700
atactgaagt gaaagtctct accacacagt aggcgttcga aaattgtttc ctctttctcc 14760
attcaacact gaggactcag gttcagctgc tgatgaagct cctctttttt gcctagagct 14820
ttcattctga gccttctcct cctaccaagt gtctccccaa tgccagagca ggaagagtct 14880
tcactcctcc ccatgcccca cctcccattt gttactaaga ggagaggaga aagtagcaag 14940
gagggtatgg ggaatgttct gggggaatgg gtgttggtgc gatcaacaac aaagtccttt 15000
ctctcacctt gaattcatcc cagatgcctg cttgtttact tcttccacac aaaaaaaggc 15060
cttcagccct catggctgag cagaaagaat ctgaatgtta gagtcaggca gcctgggttt 15120
gaattccatc tcaggtactg aactctatag caaaattctt agattctcca agcttcagtt 15180
gccttgtctg tcaaatagag aaaacatcct tcgtcctaaa ttgtagggag gattaaagtc 15240
atgcaaagtg cctactacaa atccagtcac aaagtagcta gctactcact aaatgttcag 15300
ctcctccctc ctcattcaga tgggaagtgg ctttagataa acaaagtggc aacgcagtgg 15360
gctggagcag ctctgtgaac tgagaatcca agaaaagggg cgaagagcag ctgggatgta 15420
ttggatgctt gtgctggctt ggagcattgc tcacattctt tattcgctat tgtatctaga 15480
ctatagctag agaaagagcc gcaaccattg gctttaaatc cagtgctctt cctactctcc 15540
tgaggttgtt tccaggctgc agagaaatag cctgcacaag gggcccaggc gctgggtgtg 15600
ggagggtccc caccgagagc cagaacatgc aggaactaaa atgttgcctt tttctatttt 15660
aggaaaactt cgagcagaga taggtgagtt ccagtcatcg tttctcccaa ttcttgcctt 15720
ttggtttttt ggcataacgg aaatggtccc gttcttggac cgtctctccc tctcaatacc 15780
ctgttttccc ctcagtttcc ctttctctac agtgggtgtg tcgtgcctag aacaagtttt 15840
aagtaattaa ataacaaaga ctcaggataa aagatccttt ttgagtgccc tactaaatcc 15900
atttccattt gtttctcttt cagagaatct ccaccggact tttggtaagt tccggcatgt 15960
ctaggccctc ccaggtcaac ttggtatttc actctagttc cagtcacctg ggggaacaag 16020
gacccctggc tcctggttga gtcccttcct ctcttctctt ttctttcttt aaataagaag 16080
tcatttgcat ttaggattgg taaaatcata ataaaaatac tcatgtactg tttttatgtg 16140
ccaggcacta ttctaactac tttacaaaaa tgttatctta ttctgtttaa ctccttatgc 16200
acatgatctc tcttttcagg aatggcaaaa cagaggtaaa tagatcgttt acacgtaaac 16260
ctgatgtctg gttggggagg tgaaacaaac agaaacaaga cacaactgta tcacctgtac 16320
ttatatttct gctttacaaa ctcaggatgt ttccatgagt acagaacatg actaatcaga 16380
gaagacctca tagaggaata gaaaagccac caagccccac taggaattga cccctcaagg 16440
acatggtttc tagccttttt gttcactgca gattgcccaa tgcctaaaga taatggcaac 16500
agaagagcac ccaaatattt gttagataaa tgttgcagac actagaaggt gtcattaggg 16560
cacagatggt accttctctg agcaaacttc cttcacagct cctcctcccg aggctgtagg 16620
tgactctact cttgtcacct ggcacacaga gttctatcgt acgatttagg aaattagacc 16680
agtgtgtgga ccacacacac acacatcttt acacacccaa agaggaggaa tagtatcttt 16740
gttttggagg acttgactat gaaaggtctt aactcctttt tgtaccatga atctctctgg 16800
cactccagtg aagtctaaag gacccctttg cagaatgttt ttaaatatac acataaaata 16860
gaacacatag gattgcaaaa acaatcattg tactaaaata cagttatcaa ccgataatca 16920
catttgtgat atagtaacat aaatgtttct tttttttttt ttttgaggca gagttttgct 16980
cttgtcaccc aggctggagt gcaatggcgc gatctaggct cactgaaacc tctgcctccc 17040
gggttcaagc gattctcagc ctcctgagta gctgggatta caggtgcccg ccaccacacc 17100
cagctaattt ttgtattttt agtagagact aggtttcacc aggttggcca ggctggcctc 17160
gaactcctga cctcaggtga tccacctgcc ttggcctccc aaagtgctgg gattacgggc 17220
atgagccacc gtgcccggcc ataaatattt ctttagccaa agtaatacat taagtaatgt 17280
agcagcaagt ctaataacct gtaatttctt tctttctttc tttctttctt tttttttgag 17340
atgaagtttt tttgagatgg agtgcaatgg cacaatctcg gctcactgca acctccacct 17400
cctgggttca agcgattctc ctgcctcagc ctcccaagtt gctggaacta caggcgcatg 17460
ccaccatgcc cagctaattt ttgtattttt agtagagacg gggtttcacc atgttggcca 17520
ggctggtctt gaacccctga cctcaggtga tctgcctgcc ttggccttcc aaagtgctgg 17580
gattacaggc atgagccacc aggcccagcc caataacctt taatttcaac atactaataa 17640
acataaacag tatttcaaga tttctgcaat aactctaatg ggaatgaaaa catctgtggc 17700
ttccattggt aattaagtca caggtactgc tcatattgtg gttagttgta aaatgttttg 17760
gtttgttttg ttttttccaa gacttggggg aatgggtgtt ggtgggatca acaagagtct 17820
tgctctgtgg cccaggctgg agtgcagggg caggatcttg gctcactgca acctccgcct 17880
cccaggttca agcgattctc ctgcctcagc ctcctgagta gctggcatta caggcatgtg 17940
ccaccacgcc cacctaattt ttacattttt agtagagatg gggtttcacc atgttggcct 18000
ggctggtctt gaactcttgg cctcatgatc cacccgtctc ggactcccag agtgttggga 18060
ttacaggcat gagccaccac acctggcagt tgttacattt ttaatgaaag aaaatgttaa 18120
atccagttat tgaaaataag gaggcagtac ttttctcatc caagttcatg gactttctga 18180
attttgtccc cagagtcctt tggtgttcta ggaccccagg ttaaggaacc aaaaaagaca 18240
ggtgggtggg gcatgagggg gaacacatgt taaccctgtt tgttctggtg aacaattcag 18300
atccccactt tctgagggtg ccctgctgga agataaccct gtttgtaatt gtgccggttc 18360
ttggaccctt ggttgccttg atcatctgct acaactggct acatcgaaga ctagcaggtg 18420
cagtggctgg gcagcaggca agaccaccaa atagtggggg accaagtcag ctctgaatgg 18480
gaagccaaaa gagaatagaa ccaggactca agattagggg agctgggatt tccttattcc 18540
tctgtcccca tgcccaaccc caggctcttc tgagaaactg tgaagagaac cacttactgg 18600
atctgtggga tcccccagtg gaaagggcag tgtgggtcac tccaaatgtc catagggagg 18660
atgtggggaa ggtgctattc atcttccact aatcacatat ttgtttcttt ttgttttcag 18720
ggcaattcct tgaagagcta cgtaagttct cttctctctg ttataagcag agaataaaaa 18780
gccaggaaag ggagacagaa gcaacaagag gaagaggcgg gctattgagg gatcacattc 18840
ccagaggaaa ggaggagctg gagagcctgg gtggagggaa gactcctcct gggaggtaga 18900
gggcaaagaa gccagctgtt agagacacat ttacaggtgg cagagaagct ggaggcactc 18960
ctatctgcca cctgatccat tcctccttca ctgcccctaa gcaggaatcc aaccctagct 19020
ggtctcattg cccattccac agcaactgcc cagtgcctca cctctcagat caaccattga 19080
ggcaggaatg gagacaagat gaccccaagg gcttttcttc tccctagttc aatggtttta 19140
tgatacaaac tactgacata cgtttttcaa gttattttct ccttcttcta ggaaatccct 19200
tctgagtgat gtcacatctt ggcaggggtg gaggagagcc tggttgccca gggatttgtc 19260
cttggggaca tctcatccat caagttgcac actcactggc atctttgcta tggggacatt 19320
ccaatttgca ctttcaggaa cactctgaat tccaagtaga attgatttcc cttcttctgt 19380
catctacctt ttctcttcat tttcccattt ttattaccct tctttccatt tctctctcca 19440
gtcttccacc tggaagccct ctctggctaa ggacaggcag gtgcccctct ctccatcaga 19500
ggacacctgt actggagagc aacacaggat ggtctctgcc atgaactgga ggccaggaat 19560
ctcctcactg aaaattacag tatggtaact ttgcaaatgg tggttgtttc ttccaagact 19620
ccagccctga ttgcgcaaaa ctgaaaggca tgtgaaggga aggaagagga agagtgcaaa 19680
acattgaaga gagagctgag tgagctgaag agtgaggata tgagtagccc caacccaaac 19740
ctggagatgg ggagaaacct acagaatact agccagagct cctccttgtc ttggcagcct 19800
actagggacc tggggaagca aaaacgaaag ctgggcaaca tgcctgcttt agaatgtttt 19860
ccttctactt acacatcttc cacaggtctc agaatctttc cttcctctca tccttttctc 19920
ctatcttcat atctatcaga gtatccactg tttattcaac aactactact tgatggtcag 19980
acacaaacaa acaagctagg tgctaattaa taaagatacg agttttggcc gggtgcggtg 20040
gctcacgcct gtaatcccag cactttggga ggccgaggcg ggcgaatcac gaggtcagga 20100
gttcaagacc agcctggcca acatggtgaa accccatctc tactaaaaat acaaacaatt 20160
aactgagcat agtggtgggc acctataata ccagctactc cggaggctga ggcaggagaa 20220
tcgcttgaac ccaggaggca gaggttgcag tgagctgaga tcgtgccact gcactctagc 20280
cggagtgaca gagtaagact ctgtctcaaa aataaataaa taaataaata aataaataaa 20340
taaataaata aataaaaaat aataatacaa gttttcataa gcacacttct aaccccttgt 20400
cttttatgta tttccttcct tatccacgca cctgtctccc tctactccag cctcattacc 20460
ccagaggtca gtcctcagga aaactaaaca caaagaaaga gctcagtcag aaaggccatt 20520
tatttatgtt tcaagatgct cactgcctcc tttgttttgt ctcctttgca ggccttctct 20580
cttaggcctc ttctcctggg ggtatggatc ctggggggag attgatcacc tccatgcttc 20640
cattcctccc cagccatagt ggggacatca tgagagaagc caagccactg gcccaggatc 20700
acccggcatt tatggtggct gctctggcac aggtccttgc ctttatagcc cctccagtga 20760
tccataaggc cctctttctc cccaaaggag aggtcacaga tagggcaaag gtagctcttc 20820
tgcttccagt gggtctgctg gtgtctgacc agcctggaaa atgagctgaa agacttgctg 20880
caatggaagc agtagttggg cggctctgtg aggtgggcct tctggtgtct ggagagatag 20940
gatttcttgc taaaagtcaa agaacaatgg gggcaacaga agacattgag tcttgagggc 21000
ttcactggat gagagttgga tctggcatcc tgacagaggg ttccagtgat gggtgcctgg 21060
gtcctggtca caggtgcttg gttcttaagt acagatgcct ggttctgggc cataggaccc 21120
tcagttctaa atatgggttc ctgggacctg gccactggtg catggttcac atccaaaagc 21180
ccctggatgg acctctggct tctggcgatg ggtgtctgga attcagcctg ggtgcctgga 21240
atcctcaaag tacactcctg gtttccatcc actggctcct ggttttggtg tatcttctgg 21300
tggcgtttga gctcagactg gtcccggaag ctcttcccac acacagagca tgaatggggc 21360
cggtaaccca gatggacgcg gcggtgacga cttagtccag aagcatcaca gtaggtcttg 21420
tcacagagcg tgcaacagaa gggcctctcc ccaagatgca tgcgtctgtg atagctgagg 21480
gacttggggc tccgaaacaa cttcccacac tgactgcagc tgttagtcag cttgggattg 21540
tgaacaaact ggtggctata gaggtaggag cgcctgctga aacatttgcc acaggtgtag 21600
caaaaaaagg gtggcccagc ctgggatgct tgaagcaccc gggtcctgtc catagtccca 21660
gctggggcag atagggggca ctggccggcc cctctgcatg caaggaagac cttgtcatca 21720
ctagtcccct catctctcag actgggatgt tgttctcgaa gctctttctt cttgccttct 21780
acagtgaatg aggaagaata acacaaaatt cactgtaaga actccaacag aggcttggca 21840
tggtggctca cacctgtaat cccagcactt tgggaggccg aggccagcgg atcacctgag 21900
gttaggagtt cgaaaccagc ctgaccaaca tggtgaaacc ctgtctctac tacaaataca 21960
aaaattagct gggcgtcatg gcatctgcct gtaatctcag ctactaggga gactgaggca 22020
ggacaatcac tcgaacccgg gaggcggagg ttgcagtgag ccaagatggt gccactgcac 22080
tcctgcctgg gcaactagag tgaaactctg tctcaaaaaa aaaaaaagaa agaaagaaaa 22140
agaagaagaa gaaggagaag gagaagaagg agaaggagaa gagaaggaga agaagaagaa 22200
gaaggaagaa gaagaagaag aaaagaaaag aagaagaaga agaagacgaa gacgaagaag 22260
aagaagaaga ggaagaagaa gaactccaac acagcactcc attcagccta acacacttct 22320
tgtctctgcc cttgctctcc cacccaacac attcatcctt acccttgggc ctcataggct 22380
agaaataaga ag 22392
Claims (9)
1.一种同时检测中枢脱髓鞘自身抗体AQP4、MOG和MBP的方法，其特征在于所述方法为：将AQP4、MOG、MBP基因分别导入HEK293T细胞中，分别与待测样品共同孵育，以转染空载体的HEK293T细胞为对照，采用免疫荧光法进行检测，若荧光信号强于对照，则待测样品为阳性；荧光信号等于或小于对照信号，则为阴性。
2.如权利要求1所述的方法，其特征在于所述检测的方法为：(1)将细胞贴壁试剂与PBS以体积比1:10混合，加入含盖玻片的6孔板中，37℃，5％CO2中孵育1小时；然后加入分别转有AQP4、MOG、MBP基因的稳定HEK293T细胞系，37℃，5％CO2培养至80％融合度；
(2)盖玻片细胞面朝上，粘贴于载玻片上，PBST洗涤，3％BSA封闭，弃去；加入经PBST以体积比1:10稀释后的待测血清，在37℃，5％CO2条件下孵育1小时；PBST洗涤；加入PBST以体积比1:700稀释的生物素与链霉素结合的山羊抗人IgG 50μl，37℃，5％CO2条件下孵育30分钟；PBST洗涤；加入PBST以体积比1:700稀释的荧光素488月链霉素结合物，PBST洗涤；封片；同样条件下，以转染空载体的HEK293T细胞为对照；然后进行荧光显微镜检测，若荧光信号强于对照，则待测样品为阳性；荧光信号等于或小于对照信号，则为阴性。
3.如权利要求2所述的方法，其特征在于步骤(1)细胞接种量1×106/ml。
4.如权利要求2所述的方法，其特征在于步骤(2)所述盖玻片用无影胶粘贴与载玻片上，紫外照射固定。
5.如权利要求2所述的方法，其特征在于步骤(2)所述稀释后的待测血清，每孔加入50μl；所述山羊抗人IgG和荧光素488月链霉素结合物加入量均为50μl/孔。
6.如权利要求1所述的方法，其特征在于所述AQP4、MOG、MBP基因分别以慢病毒质粒载体的形式导入HEK293T细胞，所述含AQP4基因的慢病毒质粒载体核苷酸序列为SEQ ID NO.1所示；所述含MBP基因的慢病毒质粒载体核苷酸序列为SEQ ID NO.2所示；所述含MOG基因的慢病毒质粒载体核苷酸序列为SEQ ID NO.3所示。
7.如权利要求6所述的方法，其特征在于所述含AQP4基因的慢病毒质粒载体是将AQP4基因转入质粒载体pCDH-CMV-MCS-EGFP构建而成。
8.如权利要求6所述的方法，其特征在于所述含MBP基因的慢病毒质粒载体是将MBP基因转入质粒载体pCDH-CMV-MCS-EGFP构建而成。
9.如权利要求6所述的方法，其特征在于所述含MOG基因的慢病毒质粒载体是将MOG基因转入质粒载体pCDH-CMV-MCS-EGFP构建而成。
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