肝脏疾病的生物标记分子及其检测方法-山东亚星游戏官网机床有限公司

专利名称：肝脏疾病的生物标记分子及其检测方法
技术领域：
本发明涉及肝脏疾病的生物标记分子及其检测方法，其是利用自身抗原筛选方法筛选出可用于检测肝脏疾病的生物标记分子，并利用这些生物标记分子发展为检测套组，藉筛选检体中自身抗体或自身抗原的存在判断是否罹患肝脏疾病。
背景技术：
免疫系统若功能不健全，便会产生免疫性疾�。颐巧硖逍矶嗉膊〉母驹蛲ǔ？勺肪坑诿庖呦低车墓δ懿唤∪�。一般可分为三种情况。第一种是抵抗力降低，身体的免疫细胞活力下降，或生产的数量不足以抵抗入侵的细菌、病毒或霉菌，因此易被感染流行�。朔�、感冒、肺炎、肠炎甚至肝炎、爱滋病等，都属于这一类。第二种的免疫不全是免疫系统反应过度，这种情况是因为入侵的物质并不是细菌而只是小花粉或是食物中大分子蛋白质，免疫系统就释出大量的抗体加以对抗，而攻势就发生在我们的细胞中，如此引起一连串的反应，也被称为过敏症，而这时候若有细菌、病毒或霉菌之类真的病原来侵犯时，免疫系统已无余力抵抗了。第三种情况是免疫细胞攻击自身的正常细胞，也就是自身免疫疾�。缋喾缡怨亟谘�、红斑性狼疮或疱疹等等，这种免疫系统疾病起因于患者本身免疫辨识系统发生问题，患者的免疫系统对本身的某些物质会产生自身抗体(或称自体抗体，autoantibody)，进而破坏身体组织造成病痛。
目前已知并非是只有自身免疫疾病才有自身抗体的存在，越来越多的相关研究指出人类对癌症所发生的免疫反应，部分存在着肿瘤自身抗原(autoantigen)与人类自身抗体的情形，因此对于发现这些可以引起人体反应的肿瘤自身抗原在癌症检测、诊断或建立预后上有一定的方向和应用性，进而应用于疾病的免疫治疗上。
在美国专利6631330、5137807、5830667、6264949、5985542等五篇中，揭露了使用生物标记分子诊断肝硬化(cirrhosis)、纤维化(fibrosis)或自身免疫性肝炎(autoimmune hepatitis，AIH)；而在美国专利4994374、5175084等两篇中则使用生物标记分子诊断肝癌(hepatcellularcarcinoma)；另外在美国专利6410724则使用和肝癌相关的DNA引物(DNAprimer)组成一检测套组，但这些生物标记分子多少都存在准确性不高或易被干扰的缺点。
在美国专利5891436及公开专利20030138860中，揭露了使用生物标记分子检测人体血清中其自身抗体的存在，以做为硬化(primary biliarycirrhosis)、癌症的判断，这说明在癌症病人中会有自身抗体(autoantibody)的产生，确立应用于筛选癌症相关的生物标记分子(biomarker)的合理性。
自1982年以来，癌症即跃居台湾地区十大死亡原因首位。其中肝癌不分男性或女性都名列前茅，这对人们的健康造成极大成胁，为有效筛检罹患肝脏疾病的患者，能够早期治疗而降低死亡率，发现高准确性及不被干扰的生物标记分子，以作为检测肝硬化及肝癌的检测套组，便成为重要的课题。

发明内容
有鉴于现有技术的缺失，本发明揭示了一种肝脏疾病的生物标记分子，由于确知其自身抗体的存在，因而可据此设计成检测套组，用于肝硬化或肝癌的诊断。
本发明的目的在于提供一种用于检测肝脏疾病的生物标记分子，其是选自SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列或其衍生物或其片段或其变异体或其组合物或其对应的自身抗体。
其中，所述肝脏疾病是肝硬化或肝癌疾病。
所述变异体是将生物标记分子的氨基酸序列中的氨基酸以一个或多个氨基酸取代、删除、插入及/或添加；变异体的氨基酸序列与生物标记分子的氨基酸序列具有大于80％的序列同源性。
本发明的另一目的是提供一种肝脏疾病的检测套组，包含一组生物标记分子，其选自SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列或其衍生物或其片段或其变异体或其组合物。
其中，所述肝脏疾病为肝硬化或肝癌。
所述检测套组还可进一步包含可辨识SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列或其衍生物或其片段或其变异体的抗体的二级抗体。
本发明的再一目的是提供一种肝脏疾病的体外筛检方法，包含下列步骤提供一检体；利用SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列或其衍生物或其片段或其变异体或其组合物的生物标记分子辨识捕捉检体中的自身抗体；及侦测自身抗体。
其中，所述肝脏疾病为肝硬化或肝癌。
所述检体为全血或血清，优选为血清。
所述生物标记分子可为各种型式，包括但不限于，检测套组、或先行固定于基材上，其中所述基材为免疫分析盘或生物芯片。
所述检体可进一步先行利用萤光标记进行标示。
所述筛检方法可增加利用二级抗体辨识吸附自身抗体的步骤。
所述二级抗体经修饰具有特殊官能团可进行显色反应、放射性侦测或萤光侦测。
所述侦测自身抗体是利用酶联免疫检测(Enzyme-linkedImmunosorbent Assay，ELISA)、放射免疫鉴定(Radioimmunoassay，RIA)或免疫萤光分析(Immunofluorescence)侦测二级抗体，或直接利用萤光扫描侦测被萤光标记标示的自身抗体。
本发明的再一目的是提供一种肝脏疾病的检测套组，包含一组可辨识具有SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列对应的抗体。
其中，所述肝脏疾病为肝硬化或肝癌。
本发明的再一目的是提供一种利用前述检测套组筛检肝脏疾病的方法，其包含下列步骤提供一检体；利用SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列对应的抗体辨识捕捉检体中的抗原；及侦测抗体与抗原的复合物。
其中，所述肝脏疾病为肝硬化或肝癌。
所述检体为全血或血清，优选为血清。
本发明的基础是利用自身抗原筛选方法，先纯化出正常人和肝硬化、肝癌病人的抗体并分别固定于不同管柱中，再将人肝疾病相关的细胞株(HepG2 C3A&SNU-387)的细胞萃取物依序通入正常人及病人的管柱，以得到和肝硬化、肝癌等肝脏疾病相关的自身抗原，再以这些自身抗原做为检测肝硬化及肝癌的生物标记套组，结合酶联免疫检测(Enzyme-linkedImmunosorbent Assay，ELISA)、放射免疫鉴定(Radioimmunoassay，RIA)或免疫萤光分析法(Immunofluorescence)等，藉检测人体血清中是否有该自身抗原(autoantigen)所相对应的自身抗体(autoantibody)的存在来判断是否罹患肝癌或肝硬化等肝脏疾病。由于这些生物标记分子都是以已存在的自身抗体筛选出来的，所以在诊断上可设计为检测套组，藉生物标记分子相对应的自身抗体是否存在，作为是否罹患该疾病的判断，这比直接筛检抗原的困难度要降低许多，并且还可有效提高准确度与灵敏度。

图1为本发明中利用自身抗原筛选方法的流程示意图。
图2为本发明中利用生物标记分子筛选自身抗体的流程示意图。
具体实施例方式
本发明提供了利用自身抗原筛选方法，筛选出可用于检测肝硬化或肝癌等肝脏疾病的生物标记分子，该自身抗原筛选方法如图1所示，包含以下步骤首先取得正常人及患者的血清样品，分别利用可捕捉抗体的亲和力管柱纯化出血清样品中所含的抗体；接着将正常人的抗体与患者的抗体分别填充入管柱中，以制得含正常人抗体的管柱及含患者抗体的管柱。抗体填充固定于管柱之中的方法，是将由血清样品纯化出来的抗体与管柱中的化学官能团形成化学键结固定于管柱中。然后取一样品，该样品可为(癌)细胞株或者病理组织的萃取物；前述患者的血清样品可为单一患者的血清样品或多数患者的血清混合样品。
将来自(癌)细胞株或病理组织萃取物的样品通入正常人抗体管柱时，可藉由正常人抗体的专一亲和力将非专一性的抗原捕捉而滞留于管柱中，通过管柱的样品将不含非专一性抗原，这个程序其实可视为利用患者抗体管柱筛选检体中自身抗原的样品前处理。经移除样品中非专一性的抗原，样品组成便剩下特有的抗原，此时再将样品通入含患者抗体的管柱中，藉由患者所具有的自身抗体来进一步筛选疾病相关的自身抗原。由于已先藉由正常人的血清抗体去除非专一性的抗原，所以利用患者的自身抗体筛选所得到的自身抗原会具有较高的专一性。
由患者抗体管柱中置换而出的自身抗原，利用质谱技术进行鉴定；所述鉴定是将质谱讯号通过数据库比较对照，以取得自身抗原的信息。
将人类肝脏疾病相关的细胞株利用上述方法进行自身抗原的纯化与鉴定，由于这些自身抗原是利用患者血清中的抗体筛选出来的，所以可利用这些自身抗原作为生物标记分子发展为检测套组，该检测套组是选自前述自身抗原筛选方法筛选出的自身抗原或其衍生物或其片段或其变异体或其组合物。藉筛选检体中自身抗体的存在，判断是否罹患肝硬化或肝癌疾病。检测套组中除生物标记分子外，还可进一步包含可辨识该生物标记分子的自身抗体的二级抗体，以利应用于相关检测方法。
运用前述检测套组筛检的方法如图2所示，包含下列步骤首先提供一检体；接着利用生物标记分子辨识捕捉检体中的自身抗体，然后侦测自身抗体。其中，所述生物标记分子选自自身抗原筛选方法筛选出的自身抗原或其衍生物或其片段或其变异体或其组合物。
其中，所述检体为全血或血清，其中优选为血清。
为有利于检测的进行，所述生物标记分子可先行固定于基材上，常见的固定基材为免疫分析盘或生物芯片。当生物标记分子捕捉检体中的自身抗体后，可利用二级抗体辨识吸附自身抗体，这些二级抗体经修饰具有特殊官能团，以进行显色反应、放射性侦测或萤光侦测。
利用二级抗体辨识吸附自身抗体后，即可加入特殊试剂进行显色反应，然后利用酶联免疫检测进行二级抗体存在的判断，进而得知自身抗体是否存在，或通过放射免疫鉴定或免疫萤光分析，同样可以得知藉由二级抗体的反应判断自身抗体是否存在，以作为是否罹患肝癌或肝硬化的依据。
若不使用二级抗体，也可将检体在与生物标记分子反应前，先利用萤光标记(例如Cy3或Cy5)标示，然后通过生物标记分子筛选自身抗体，由于自身抗体已先被萤光标记标示，所以在无须使用二级抗体情况下即可进行萤光扫瞄分析。
除了侦测自身抗体的存在作为判断，侦测抗原的存在也可作为是否罹患肝硬化或肝癌的依据，为达此目的，本发明设计了一种包含可辨识具有自身抗原筛选方法筛选出的自身抗原的抗体的检测套组，也可用于检测肝脏疾病。
利用前述检测套组筛检肝癌及肝硬化的方法包含下列步骤提供血清检体；利用所述抗体辨识捕捉血清中的抗原；及侦测抗体与抗原的复合物。
以下实施例用于进一步了解本发明的优点，但并非用于限制本发明的保护范围。
实施例1.利用肝脏疾病患者血清中的自身抗体筛选自身抗原由血清纯化自身抗体首先取肝硬化或肝癌患者的血清，将血清用键结缓冲液(bindingbuffer20mM PBS，pH7.0)以1∶10的比例稀释后，以0.45μm孔径的滤膜过滤，避免后续层析步骤造成管柱堵塞；接着以10倍管柱体积的键结缓冲液，在1ml/min的流速下清洗Protein G亲和力管柱，之后将前述经滤膜过滤的血清样本以0.2ml/min的流速通入Protein G亲和力管柱中，使抗体得以藉由与管柱的亲和力滞留于管柱中；再取5-10倍管柱体积的键结缓冲液以1ml/min的流速清洗Protein G亲和力管柱，移除血清样品中不与管柱发生亲和力键结的物质，然后取2-5倍管柱体积的洗脱缓冲液(elution buffer0.1M Glycine-HCl，pH2.7)以1ml/min的流速将抗体自管柱中洗脱出来并收集，收集抗体的试管中需预先加入60-200μl的Tris-HCl溶液(1M，pH9.0)，最后将样品置换于耦合缓冲液(coupling buffer0.2M NaHCO3，0.5M NaCl，pH8.3)中，即完成血清中自身抗体(IgG)的纯化。
由于本发明需要分别填充有正常人IgG及患者IgG的管柱各一只，所以需要取正常人及患者血清分别进行前述纯化步骤。
制备含有自身抗体的管柱取NHS-activated管柱一只，移除上端盖子后滴一滴酸化溶液(acidification solution1mM HCl(需冰浴))以避免气泡生成。接着将此管柱上端与注射器(syringe)或泵(pump)连接后移除下端扭转处，再以2倍管柱体积的酸化溶液将管柱中的异丙醇清洗出来，重复此清洗步骤3次后，将含有自身抗体的样品注入管柱中。前述的含有自身抗体的样品是由Protein-G亲和力管柱纯化血清得到的含有自身抗体的耦合缓冲液，经配制后成为总体积为1倍管柱体积，浓度为0.5-10mg/ml。将前述含有自身抗体的样品通入管柱后则将管柱密封，在25℃中反应15-30分钟，或者在4℃中反应4小时，使抗体得以与管柱产生化学键联固定于管柱中。
自身抗体与管柱完成键联反应后，先利用2倍管柱体积的封阻缓冲液(blocking buffer0.5M乙醇胺，0.5M NaCl，pH8.3)洗脱管柱，重复此步骤3次，再利用2倍管柱体积的清洗缓冲液(washing buffer0.1M醋酸盐，0.5M NaCl，pH4)清洗管柱，同样重复3次，接着再换回封阻缓冲液以2倍管柱体积洗脱管柱3次后，让管柱反应15-30分钟，使管柱中未键联自身抗体的官能团被封阻而失去活性，待封阻反应完成后以2倍管柱体积的清洗缓冲液清洗管柱，重复3次，接着以2倍管柱体积的封阻缓冲液洗脱管柱，重复3次，确保所有未键联自身抗体的官能团都能被封阻，再以2倍管柱体积的清洗缓冲液清洗管柱，重复3次后，最后以2-5倍管柱体积的一般中性pH缓冲液洗脱管柱后，完成含有自身抗体的管柱的制备。
由肝脏疾病相关的细胞株萃取物中纯化鉴定自身抗原首先将移除培养基的HepG2 C3A cells(细胞株)2.68mg以冰浴的Tris saline溶液(50mM Tris pH7.5，150mM NaCl，1.5mM PMSF，Phosphataseinhibitors)清洗两次后，加入Triton Extraction溶液1ml(15Mm TrispH7.5，120mM NaCl，25mM KCl，2mM EGTA，0.1mM DTT，0.5％TritonX-100，10μg/ml leupeptin，0.5Mm PMSF，Phosphatase inhibitors)，置于4℃、30分钟，此时细胞开始分解释出蛋白质，然后利用台式离心机于4℃以14,000rpm的转速离心15分钟，移除固态不溶的细胞结构，收集上层液进行后续的免疫亲和层析。
经收集得到的细胞萃取液与键结缓冲液(binding buffer)以1∶10的比例稀释后，利用0.45μm孔径的滤膜过滤，以避免后续通入管柱后造成管柱的阻塞，而在将样品注入具有抗体(IgG)的管柱前，需先以10倍管柱体积的键结缓冲液，在1ml/min的流速下清洗正常人及患者的抗体管柱，之后将前述经滤膜过滤的细胞萃取液以0.2ml/min的流速通入正常人抗体管柱中，再取5-10倍管柱体积的键结缓冲液以1ml/min的流速洗脱正常人抗体管柱，此时细胞萃取液中所含有的正常人抗体具有辨识捕捉的抗原会滞留其中，这一步骤的目的在于捕捉移除细胞株中非专一性的抗原，所以经管柱层析后的细胞萃取液中将不含非专一性的抗原；将此细胞萃取液收集导入患者抗体管柱，取5-10倍管柱体积的键结缓冲液以1ml/min的流速洗脱患者抗体管柱，此时细胞萃取液中所具有的自身抗原将被管柱中患者的自身抗体所捕捉而滞留于管柱之中，当细胞萃取液通过正常人抗体管柱时，一般正常人抗体可辨识捕捉的抗原将被滞留于管柱中，而将不含前述正常人抗体可辨识捕捉的抗原的细胞萃取液导入患者抗体管柱时，只有患者抗体管柱可辨识捕捉的抗原被滞留于管柱之中，这些滞留于患者抗体管柱中的抗原，藉由2-5倍管柱体积的洗脱缓冲液(elutionbuffer)以1ml/min的流速将自身抗原由患者抗体管柱中洗脱出并收集，接着使用胰蛋白酶(Trypsin)进行蛋白质水解反应，将被水解的自身抗原的多肽以质谱技术进行分析，所得的图谱可藉由数据库比较对照获知蛋白质的信息。
利用肝硬化或肝癌患者血清中的自身抗体筛选肝脏疾病相关细胞株，可得下列的自身抗原1.二磷酸核苷激酶(Nucleoside diphosphate kinase，gi|1421609，SEQ ID NO.1)2.NM23 protein(gi|35068，SEQ ID NO.2)
3.ATP合成酶beta chain(ATP synthase beta chain，mitochondrial[precursor]，gi|28940，SEQ ID NO.3)4.14-3-3蛋白zeta(14-3-3 zeta protein，tyrosine 3/tryptophan5-monooxygenase activation protein)(gi|4507953，SEQ ID NO.4)5.14-3-3蛋白epsilon(14-3-3 epsilon protein，tyrosine3/tryptophan 5-monooxygenase activation protein)(gi|4507953，SEQID NO.5)6.蛋白质二硫异构酶相关蛋白-5(Protein disulfide isomerase-related protein 5，gi|1710248，SEQ ID NO.6)7.unnamed protein product(gi|21750187，SEQ ID NO.7)8.水不溶性肌蛋白alpha 3(Tropomyosin alpha 3，gi|37403，SEQID NO.8)9.水不溶性肌蛋白alpha 4(Trypomyosin alpha 4，gi|10435300，SEQ ID NO.9)10.Calreticulin precursor(gi|4757900，SEQ ID NO.10)11.人类pre-mRNA剪接因子SF2p32(Human pre-mRNA splicing factorSF2p 32，gi|338043，SEQ ID NO.11)12.肿瘤坏死因子第一型受体的相关蛋白(Tumor necrosis factortype 1 receptor associated protein TRAP-1，gi|1082886，SEQ ID NO.12)13.肿瘤排斥抗原(gp96)(Tumor rejection antigen(gp96)1；glucose regulated protein，gi|4507677，SEQ ID NO.13)14.热休克蛋白90-beta(Heat shock protein 90-beta，gi|72222，SEQ ID NO.14)15.热休克蛋白90-alpha(Heat shock 90-alpha，gi|123678，SEQ IDNO.15)16.热休克蛋白-60(Heat shock 60kDa protein 1，gi|31542947，SEQ ID NO.16)17.HMG-1蛋白质(HMG-1，gi|968888，SEQ ID NO.17)18.KIAA0144 gene product(NICE-4 protein)(gi|13111995，SEQID NO.18)19.Valosin-containing protein(p97)；transitional endoplasmicreciculum ATPase(gi|6005942，SEQ ID NO.19)20.甘油醛三磷酸脱氢酶(Glyceraldehyde 3-phosphatedehydrogenase，liver，gi|30157565，SEQ ID NO.20)21.细胞角质蛋白(Cytokeratin，gi|1419564，SEQ ID NO.21)22.胰岛素生长因子II的mRNA结合蛋白(IGF-II mRNA-bindingprotein 1，gi|4191608，SEQ ID NO.22)23.醌还原酶(NADPHquinone reductase，gi|13236495，SEQ IDNO.23)24.热休克蛋白90的协同伴随蛋白P23(Crystal Structure Of TheHuman Co-Chaperone P23，hsp-90 co-chaperone)(gi|9257073，SEQ IDNO.24)上列由肝脏疾病相关的细胞株筛选的自身抗原如表一所示，其中左侧为蛋白质的GI编号(gi number)及名称，右侧为利用肝硬化或肝癌患者的血清在肝脏疾病相关细胞株中可被筛选出来的自身抗原，由表中显示，这些自身抗原并不局限于单一的肝脏疾病中才会被筛选出来，利用不同来源血清中的自身抗体针对不同细胞株也会重复筛选出来，显示这些抗原与肝脏疾病的相关性极为密切。表一中部分蛋白质具有两个GI编号，其是由于蛋白质及其变异体在质谱鉴定时具有近似的判别结果。
表一、由肝脏疾病相关的细胞株筛选的自身抗原

实施例2.鉴定利用自身抗原筛选方法筛选出来的自身抗原的可利用性为进一步证实实施例1.筛选出的24个自身抗原的可利用性，需进一步使用免疫分析方法(ELISA、RIA、免疫萤光法等)搭配上述24个生物标记分子，分别对正常人、肝硬化患者及肝癌患者的血清样品做进一步检测，其检测方法依循图2，首先提供一检体，然后利用SEQ ID NO1至SEQID NO24所示的任一氨基酸序列或其衍生物或其片段或其组合物的生物标记分子辨识捕捉检体中的自身抗体，最后侦测自身抗体。
以酶联免疫吸附分析(Enzyme-linked Immunosorbent Assay，ELISA)为例，其详细检测步骤如下首先将生物标记分子以披覆缓冲液(coatingbuffer，可有三种选择为a.50mM Na2HCO3，pH＝9.6或b.20mM Tris-HCl，pH＝8.5或c.10mM PBS，pH＝7.4)稀释成浓度0.5～10μg/ml，披覆缓冲液依据生物标记分子的pI值选择，优选大于pI值1-2pH值的缓冲液。在ELISA plate中加入100μl/well的生物标记分子溶液，置于4℃中反应隔夜进行固定化。
接着移除未固定化的生物标记分子，以PBST buffer清洗两次(PBSTbufferPBS buffer+0.05％Tween-20)，然后加入200μl/well的封阻缓冲液(blocking buffer可有三种选择a.Gelatin-NET0.5％Gelatin，0.15M NaCl，5mM EDTA 2Na，0.05％Tween-20，50mM Tris base或b.1％BSA-PBS，pH＝7.4或c.5％Non-fat milk-PBS，pH＝7.4)在室温下反应最少2小时进行封阻反应；反应完成后利用PBST缓冲液清洗3次，然后加入100μl/well待反应的血清溶液(血清溶液为使用封阻缓冲液稀释1000倍)，此时血清中的自身抗体将与被固定化的生物标记分子进行辨识吸附，此反应在室温下至少进行2小时后，利用PBST缓冲液清洗4次，再加入100μl/well的二级抗体(二级抗体是利用封阻缓冲液稀释5000倍)，此时二级抗体将会与自身抗体进行辨识吸附，此反应在室温下至少进行1小时后，以PBST缓冲液清洗5次，接着加入100μl/well的TMB进行30分钟显色反应，然后加入100μl/well的0.5M H2SO4后，侦测450nm的吸收度。
为确认自身抗体的表现量确实可作为肝硬化及/或肝癌的检测，可分别利用ELISA分析各自身抗原筛选正常人、肝硬化患者及肝癌患者血清中的自身抗体的吸光值数据后，以生物统计学进行分析以GADPH、NADPH、HMG-1、NM23及Cytokeratin等五个蛋白质为例，利用Wilcoxon-Mann-Whitney进行检定，在95％的信赖水准下所得结果如表二表二

正常人N＝10、肝硬化患者N＝15、肝癌患者N＝21(p＜0.05假设成立)假设各生物标记分子的自身抗体于正常人、肝硬化患者及肝癌患者中表现量具有差异性，则由表中显示这样的假设在正常人与肝硬化患者的比较，以及正常人与肝癌患者的比较的假设成立，意即各生物标记分子的自身抗体的表现量在正常人与肝硬化患者及肝癌患者中的差异，具有生物统计上的意义存在。
经由生物统计可知蛋白质GADPH的自身抗体在正常人与肝硬化患者中的表现量差异达8.375倍，在正常人与肝癌患者中的表现量差异达4.86倍；蛋白质HMG-1的自身抗体在正常人与肝硬化患者中的表现量差异达74倍；蛋白质NM23的自身抗体在正常人与肝硬化患者中的表现量达24倍，在正常人与肝癌患者中的表现量达8.545倍。这些结果证实在肝硬化及肝癌病人的血清中，本发明的24个自身抗原的相关自身抗体(autoantibodies)的表现量高于正常人，因此将所述24个自身抗原搭配免疫分析方法组合成一套检测套组，藉此筛选检体中自身抗体表现量，确实可以应用于肝硬化及肝癌的检测。
上述优选实施例，并非用以限定本发明，任何熟悉此领域的技术人员，在不脱离本发明的精神和范围内，当可作各种的更动与润饰，因此，本发明的保护范围，应以权利要求书为准。
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Cys Cys Glu Pro Arg Gly Ser Arg Ala Arg Phe Gly Cys Trp Arg Leu1 5 10 15Gln Pro Glu Phe Lys Pro Lys Gln Leu Glu Gly Thr Met Ala Asn Cys20 25 30Glu Arg Thr Phe Ile Ala Ile Lys Pro Asp Gly Val Gln Arg Gly Leu35 40 45Val Gly Glu Ile Ile Lys Arg Phe Glu Gln Lys Gly Phe Arg Leu Val50 55 60Gly Leu Lys Phe Met Gln Ala Ser Glu Asp Leu Leu Lys Glu His Tyr65 70 75 80Val Asp Leu Lys Asp Arg Pro Phe Phe Ala Gly Leu Val Lys Tyr Met85 90 95His Ser Gly Pro Val Val Ala Met Val Trp Glu Gly Leu Asn Val Val100 105 110Lys Thr Gly Arg Val Met Leu Gly Glu Thr Asn Pro Ala Asp Ser Lys115 120 125Pro Gly Thr Ile Arg Gly Asp Phe Cys Ile Gln Val Gly Arg Asn Ile130 135 140Ile His Gly Ser Asp Ser Val Glu Ser Ala Glu Lys Glu Ile Gly Leu145 150 155 160Trp Phe His Pro Glu Glu Leu Val Asp Tyr Thr Ser Cys Ala Gln Asn165 170 175Trp Ile Tyr Glu180<210>3<211>539<212>PRT<213>人属、人种<400>3Met Thr Ser Leu Trp Gly Lys Gly Thr Gly Cys Lys Leu Phe Lys Phe1 5 10 15Arg Val Ala Ala Ala Pro Ala Ser Gly Ala Leu Arg Arg Leu Thr Pro20 25 30Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu Leu Arg Ala Val Arg Arg35 40 45Arg Ser His Pro Val Arg Asp Tyr Ala Ala Gln Thr Ser Pro Ser Pro50 55 60
Lys Ala Gly Ala Ala Thr Gly Arg Ile Val Ala Val Ile Gly Ala Val65 70 75 80Val Asp Val Gln Phe Asp Glu Gly Leu Pro Pro Ile Leu Asn Ala Leu85 90 95Glu Val Gln Gly Arg Glu Thr Arg Leu Val Leu Glu Val Ala Gln His100 105 110Leu Gly Glu Ser Thr Val Arg Thr Ile Ala Met Asp Gly Thr Glu Gly115 120 125Leu Val Arg Gly Gln Lys Val Leu Asp Ser Gly Ala Pro Ile Lys Ile130 135 140Pro Val Gly Pro Glu Thr Leu Gly Arg Ile Met Asn Val Ile Gly Glu145 150 155 160Pro Ile Asp Glu Arg Gly Pro Ile Lys Thr Lys Gln Phe Ala Pro Ile165 170 175His Ala Glu Ala Pro Glu Phe Met Glu Met Ser Val Glu Gln Glu Ile180 185 190Leu Val Thr Gly Ile Lys Val Val Asp Leu Leu Ala Pro Tyr Ala Lys195 200 205Gly Gly Lys Ile Gly Leu Phe Gly Gly Ala Gly Val Gly Lys Thr Val210 215 220Leu Ile Met Glu Leu Ile Asn Asn Val Ala Lys Ala His Gly Gly Tyr225 230 235 240Ser Val Phe Ala Gly Val Gly Glu Arg Thr Arg Glu Gly Asn Asp Leu245 250 255Tyr His Glu Met Ile Glu Ser Gly Val Ile Asn Leu Lys Asp Ala Thr260 265 270Ser Lys Val Ala Leu Val Tyr Gly Gln Met Asn Gln Pro Pro Gly Ala275 280 285Arg Ala Arg Val Ala Leu Thr Gly Leu Thr Val Ala Glu Tyr Phe Arg290 295 300Asp Gln Glu Gly Gln Asp Val Leu Leu Phe Ile Asp Asn Ile Phe Arg305 310 315 320Phe Thr Gln Ala Gly Ser Glu Val Ser Ala Leu Leu Gly Arg Ile Pro325 330 335Ser Ala Val Gly Tyr Gln Pro Thr Leu Ala Thr Asp Met Gly Thr Met340 345 350Gln Glu Arg Ile Thr Thr Thr Lys Lys Gly Ser Ile Thr Ser Val Gln355 360 365
Ala Ile Tyr Val Pro Ala Asp Asp Leu Thr Asp Pro Ala Pro Ala Thr370 375 380Thr Phe Ala His Leu Asp Ala Thr Thr Val Leu Ser Arg Ala Ile Ala385 390 395 400Glu Leu Gly Ile Tyr Pro Ala Val Asp Pro Leu Asp Ser Thr Ser Arg405 410 415Ile Met Asp Pro Asn Ile Val Gly Ser Glu His Tyr Asp Val Ala Arg420 425 430Gly Val Gln Lys Ile Leu Gln Asp Tyr Lys Ser Leu Gln Asp Ile Ile435 440 445Ala Ile Leu Gly Met Asp Glu Leu Ser Glu Glu Asp Lys Leu Thr Val450 455 460Ser Arg Ala Arg Lys Ile Gln Arg Phe Leu Ser Gln Pro Phe Gln Val465 470 475 480Ala Glu Val Phe Thr Gly His Met Gly Lys Leu Val Pro Leu Lys Glu485 490 495Thr Ile Lys Gly Phe Gln Gln Ile Leu Ala Gly Glu Tyr Asp His Leu500 505 510Pro Glu Gln Ala Phe Tyr Met Val Gly Pro Ile Glu Glu Ala Val Ala515 520 525Lys Ala Asp Lys Leu Ala Glu Glu His Ser Ser530 535<210>4<211>245<212>PRT<213>人属、人种<400>4Met Asp Lys Asn Glu Leu Val Gln Lys Ala Lys Leu Ala Glu Gln Ala1 5 10 15Glu Arg Tyr Asp Asp Met Ala Ala Cys Met Lys Ser Val Thr Glu Gln20 25 30Gly Ala Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr35 40 45Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Val Ser Ser50 55 60Ile Glu Gln Lys Thr Glu Gly Ala Glu Lys Lys Gln Gln Met Ala Arg65 70 75 80
Glu Tyr Arg Glu Lys Ile Glu Thr Glu Leu Arg Asp Ile Cys Asn Asp85 90 95Val Leu Ser Leu Leu Glu Lys Phe Leu Ile Pro Asn Ala Ser Gln Ala100 105 110Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr Arg Tyr115 120 125Leu Ala Glu Val Ala Ala Gly Asp Asp Lys Lys Gly Ile Val Asp Gln130 135 140Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys Glu Met145 150 155 160Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe Ser Val165 170 175Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Glu Lys Ala Cys Ser Leu Ala180 185 190Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu195 200 205Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn210 215 220Leu Thr Leu Trp Thr Ser Asp Thr Gln Gly Asp Glu Ala Glu Ala Gly225 230 235 240Glu Gly Gly Glu Asn245<210>5<211>255<212>PRT<213>人属、人种<400>5Met Asp Asp Arg Glu Asp Leu Val Tyr Gln Ala Lys Leu Ala Glu Gln1 5 10 15Ala Glu Arg Tyr Asp Glu Met Val Glu Ser Met Lys Lys Val Ala Gly20 25 30Met Asp Val Glu Leu Thr Val Glu Glu Arg Asn Leu Leu Ser Val Ala35 40 45Tyr Lys Asn Val Ile Gly Ala Arg Arg Ala Ser Trp Arg Ile Ile Ser50 55 60Ser Ile Glu Gln Lys Glu Glu Asn Lys Gly Gly Glu Asp Lys Leu Lys65 70 75 80Met Ile Arg Glu Tyr Arg Gln Met Val Glu Thr Glu Leu Lys Leu Ile
85 90 95Cys Cys Asp Ile Leu Asp Val Leu Asp Lys His Leu Ile Pro Ala Ala100 105 110Asn Thr Gly Glu Ser Lys Val Phe Tyr Tyr Lys Met Lys Gly Asp Tyr115 120 125His Arg Tyr Leu Ala Glu Phe Ala Thr Gly Asn Asp Arg Lys Glu Ala130 135 140Ala Glu Asn Ser Leu Val Ala Tyr Lys Ala Ala Ser Asp Ile Ala Met145 150 155 160Thr Glu Leu Pro Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn165 170 175Phe Ser Val Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Asp Arg Ala Cys180 185 190Arg Leu Ala Lys Ala Ala Phe Asp Asp Ala Ile Ala Glu Leu Asp Thr195 200 205Leu Ser Glu Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu210 215 220Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Met Gln Gly Asp Gly Glu225 230 235 240Glu Gln Asn Lys Glu Ala Leu Gln Asp Val Glu Asp Glu Asn Gln245 250 255<210>6<211>421<212>PRT<213>人属、人种<400>6Leu Tyr Ser Ser Ser Asp Asp Val Ile Glu Leu Thr Pro Ser Asn Phe1 5 10 15Asn Arg Glu Val Ile Gln Ser Asp Ser Leu Trp Leu Val Glu Phe Tyr20 25 30Ala Pro Trp Cys Gly His Cys Gln Arg Leu Thr Pro Glu Trp Lys Lys35 40 45Ala Ala Thr Ala Leu Lys Asp Val Val Lys Val Gly Ala Val Asp Ala50 55 60Asp Lys His His Ser Leu Gly Gly Gln Tyr Gly Val Gln Gly Phe Pro65 70 75 80Thr Ile Lys Ile Phe Gly Ser Asn Lys Asn Arg Pro Glu Asp Tyr Gln85 90 95
Gly Gly Arg Thr Gly Glu Ala Ile Val Asp Ala Ala Leu Ser Ala Leu100 105 110Arg Gln Leu Val Lys Asp Arg Leu Gly Gly Arg Ser Gly Gly Tyr Ser115 120 125Ser Gly Lys Gln Gly Arg Ser Asp Ser Ser Ser Lys Lys Asp Val Ile130 135 140Glu Leu Thr Asp Asp Ser Phe Asp Lys Asn Val Leu Asp Ser Glu Asp145 150 155 160Val Trp Met Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Asn165 170 175Leu Glu Pro Glu Trp Ala Ala Ala Ala Ser Glu Val Lys Glu Gln Thr180 185 190Lys Gly Arg Val Lys Leu Ala Ala Val Asp Ala Thr Val Asn Gln Val195 200 205Leu Ala Ser Arg Tyr Gly Ile Arg Gly Phe Pro Thr Ile Lys Ile Phe210 215 220Gln Lys Gly Glu Ser Pro Val Asp Tyr Asp Gly Gly Arg Thr Arg Ser225 230 235 240Asp Ile Val Ser Arg Ala Leu Asp Leu Phe Ser Asp Asn Ala Pro Pro245 250 255Pro Glu Leu Leu Glu Ile Ile Asn Glu Asp Ile Ala Lys Arg Thr Cys260 265 270Glu Glu His Gln Leu Cys Val Val Ala Val Leu Pro His Ile Leu Asp275 280 285Thr Gly Ala Ala Gly Arg Asn Ser Tyr Leu Glu Val Leu Leu Lys Leu290 295 300Ala Asp Lys Tyr Lys Lys Lys Met Trp Gly Trp Leu Trp Thr Glu Ala305 310 315 320Gly Ala Gln Ser Glu Leu Glu Thr Ala Leu Gly Ile Gly Gly Phe Gly325 330 335Tyr Pro Ala Met Ala Ala Ile Asn Ala Arg Lys Met Lys Phe Ala Leu340 345 350Leu Lys Gly Ser Phe Ser Glu Gln Gly Ile Asn Glu Phe Leu Arg Glu355 360 365Leu Ser Phe Gly Arg Gly Ser Thr Ala Pro Val Gly Gly Gly Ala Phe370 375 380Pro Thr Ile Val Glu Arg Glu Pro Trp Asp Gly Arg Asp Gly Glu Leu
385 390 395 400Pro Val Glu Asp Asp Ile Asp Leu Ser Asp Val Glu Leu Asp Asp Leu405 410 415Gly Lys Asp Glu Leu420<210>7<211>687<212>PRT<213>人属、人种<400>7Met Val Lys Leu Ala Lys Ala Gly Lys Asn Gln Gly Asp Pro Lys Lys1 5 10 15Met Ala Pro Pro Pro Lys Glu Val Glu Glu Asp Ser Glu Asp Glu Glu20 25 30Met Ser Glu Asp Glu Glu Asp Asp Ser Ser Gly Glu Glu Val Val Ile35 40 45Pro Gln Lys Lys Gly Lys Lys Ala Ala Ala Thr Ser Ala Lys Lys Val50 55 60Val Val Ser Pro Thr Lys Lys Val Ala Val Ala Thr Pro Ala Lys Ala65 70 75 80Val Thr Thr Pro Gly Lys Lys Gly Ala Thr Pro Gly Lys Ala Leu Val85 90 95Ala Thr Pro Gly Lys Lys Gly Ala Ala Ile Pro Ala Lys Gly Ala Lys100 105 l10Asn Gly Lys Asn Ala Lys Lys Glu Asp Ser Asp Glu Glu Glu Asp Asp115 120 125Asp Ser Glu Glu Asp Glu Glu Asp Asp Glu Asp Glu Asp Glu Asp Glu130 135 140Asp Glu Ile Glu Pro Ala Ala Met Lys Ala Ala Ala Ala Ala Pro Ala145 150 155 160Ser Glu Asp Glu Asp Asp Glu Asp Asp Glu Asp Asp Glu Asp Asp Asp165 170 175Asp Asp Glu Glu Asp Asp Ser Glu Glu Glu Ala Met Glu Thr Thr Pro180 185 190Ala Lys Gly Lys Lys Ala Ala Lys Val Val Pro Val Lys Ala Lys Asn195 200 205Val Ala Glu Asp Glu Asp Glu Glu Glu Asp Asp Glu Asp Glu Asp Asp210 215 220
Asp Asp Asp Glu Asp Asp Glu Asp Asp Asp Asp Glu Asp Asp Glu Glu225 230 235 240Glu Glu Glu Glu Glu Glu Glu Glu Pro Val Lys Glu Ala Pro Gly Lys245 250 255Arg Lys Lys Glu Met Ala Lys Gln Lys Ala Ala Pro Glu Ala Lys Lys260 265 270Gln Lys Val Glu Gly Thr Glu Pro Thr Thr Ala Phe Asn Leu Phe Val275 280 285Gly Asn Leu Asn Phe Asn Lys Ser Ala Pro Glu Leu Lys Thr Gly Ile290 295 300Ser Asp Val Phe Ala Lys Asn Asp Leu Ala Val Val Asp Val Arg Ile305 310 315320Gly Met Thr Arg Lys Phe Gly Tyr Val Asp Phe Glu Ser Ala Glu Asp325 330 335Leu Glu Lys Ala Leu Glu Leu Thr Gly Leu Lys Val Phe Gly Asn Glu340 345 350Ile Lys Leu Glu Lys Pro Lys Gly Lys Asp Ser Lys Lys Glu Arg Asp355 360 365Ala Arg Thr Leu Leu Ala Lys Asn Leu Pro Tyr Lys Val Thr Gln Asp370 375 380Glu Leu Lys Glu Val Phe Glu Asp Ala Ala Glu Ile Arg Leu Val Ser385 390 395400Lys Asp Gly Lys Ser Lys Gly Ile Ala Tyr Ile Glu Phe Lys Thr Glu405 410 415Ala Asp Ala Glu Lys Thr Phe Glu Glu Lys Gln Gly Thr Glu Ile Asp420 425 430Gly Arg Ser Ile Ser Leu Tyr Tyr Thr Gly Glu Lys Gly Gln Asn Gln435 440 445Asp Tyr Arg Gly Gly Lys Asn Ser Thr Trp Ser Gly Glu Ser Lys Thr450 455 460Leu Val Leu Ser Asn Leu Ser Tyr Ser Ala Thr Glu Glu Thr Leu Gln465 470 475 480Glu Val Phe Glu Lys Ala Thr Phe Ile Lys Val Pro Gln Asn Gln Asn485 490 495Gly Lys Ser Lys Gly Tyr Ala Phe Ile Glu Phe Ala Ser Phe Glu Asp500 505 510Ala Lys Glu Ala Leu Asn Ser Cys Asn Lys Arg Glu Ile Gly Gly Arg
515 520 525Ala Ile Arg Leu Glu Leu Gln Gly Pro Arg Gly Ser Pro Asn Ala Arg530 535 540Ser Gln Pro Ser Lys Thr Leu Phe Val Lys Gly Leu Ser Glu Asp Thr545 550 555 560Thr Glu Glu Thr Leu Lys Glu Ser Phe Asp Gly Ser Val Arg Ala Arg565 570 575Ile Val Thr Asp Arg Glu Thr Gly Ser Ser Lys Gly Phe Gly Phe Val580 585 590Asp Phe Asn Ser Glu Glu Asp Ala Lys Ala Ala Lys Glu Ala Met Glu595 600 605Asp Gly Glu Ile Asp Gly Asn Lys Val Thr Leu Asp Trp Ala Lys Pro610 615 620Lys Gly Glu Gly Gly Phe Gly Gly Arg Gly Gly Gly Arg Gly Gly Phe625 630 635 640Gly Gly Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly Phe Gly Gly Arg645 650 655Gly Arg Gly Gly Phe Gly Gly Arg Gly Gly Phe Arg Gly Gly Arg Gly660 665 670Gly Gly Gly Asp His Lys Pro Gln Gly Lys Lys Thr Lys Phe Glu675 680 685<210>8<211>641<212>PRT<213>人属、人种<400>8Met Ala Gly Ile Thr Thr Ile Glu Ala Val Lys Arg Lys Ile Gln Val1 5 10 15Leu Gln Gln Gln Ala Asp Asp Ala Glu Glu Arg Ala Glu Arg Leu Gln20 25 30Arg Glu Val Glu Gly Glu Arg Arg Ala Arg Glu Gln Ala Glu Ala Glu35 40 45Val Ala Ser Leu Asn Arg Arg Ile Gln Leu Val Glu Glu Glu Leu Asp50 55 60Arg Ala Gln Glu Arg Leu Ala Thr Ala Leu Gln Lys Leu Glu Glu Ala65 70 75 80Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met Lys Val Ile Glu Asn85 90 95
Arg Ala Leu Lys Asp Glu Glu Lys Met Glu Leu Gln Glu Ile Gln Leu100 105 110Glu Glu Ala Lys His Ile Ala Glu Glu Ala Asp Arg Lys Tyr Glu Glu115 120 125Val Ala Arg Lys Leu Val Ile Ile Glu Gly Asp Leu Glu Arg Thr Glu130 135 140Glu Arg Ala Glu Leu Ala Glu Ser Arg Cys Arg Glu Met Asp Glu Gln145 150 155 160Ile Arg Leu Met Asp Gln Asn Leu Lys Cys Leu Ser Ala Ala Glu Glu165 170 175Lys Tyr Ser Gln Lys Glu Asp Lys Tyr Glu Glu Glu Ile Lys Ile Leu180 185 190Thr Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala Glu Phe Ala Glu Arg195 200 205Ser Val Ala Lys Leu Glu Lys Thr Ile Asp Asp Leu Glu Asp Thr Asn210 215 220Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe Gly225 230 235 240Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu Ser245 250 255Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe Gly260 265 270Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met Ser275 280 285Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu Gly290 295 300Lys Gly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr Phe305 310 315 320Ser Asp Ala Cys Val His His Ile Lys Arg Arg Asp Ile Val Leu Lys325 330 335Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys340 345 350His Asn Leu Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys Ala355 360 365Leu Lys Glu Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu Ala370 375 380Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg Phe Phe Gly
385 390 395 400Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met Arg405 410 415His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala Lys420 425 430Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu Gly435 440 445Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala Gly Met Val Tyr Leu450 455 460Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu465 470 475 480Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser Arg485 490 495Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met Leu500 505 510Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe Thr515 520 525Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe530 535 540Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala Ile545 550 555 560Asp Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys Pro565 570 575Pro Glu Val Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro Ser580 585 590Asn Ala Thr Ala Ser Arg Met Cys Thr Pro Gly Cys Lys Pro Trp Pro595 600 605Arg His Leu Leu Ser Thr Trp Met Ser Trp Ala Arg Gly Pro Ala Gln610 615 620Gly Leu Gly Val Val Ser Arg Asn Thr Gly Ala Cys Pro Gln His Pro625 630 635 640Pro<210>9<211>284<212>PRT<213>人属、人种<400>9
Met Glu Ala Ile Lys Lys Lys Met Gln Met Leu Lys Leu Asp Lys Glu1 5 10 15Asn Ala Ile Asp Arg Ala Glu Gln Ala Glu Ala Asp Lys Lys Ala Ala20 25 30Glu Asp Lys Cys Lys Gln Val Glu Glu Glu Leu Thr His Leu Gln Lys35 40 45Lys Leu Lys Gly Thr Glu Asp Glu Leu Asp Lys Tyr Ser Glu Asp Leu50 55 60Lys Asp Ala Gln Glu Lys Leu Glu Leu Thr Glu Lys Lys Ala Ser Asp65 70 75 80Ala Glu Gly Asp Val Ala Ala Leu Asn Arg Arg Ile Gln Leu Val Glu85 90 95Glu Glu Leu Asp Arg Ala Gln Glu Arg Leu Ala Thr Ala Leu Gln Lys100 105 110Leu Glu Glu Ala Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met Lys115 120 125Val Ile Glu Asn Arg Ala Met Lys Asp Glu Glu Lys Met Glu Ile Gln130 135 140Glu Met Gln Leu Lys Glu Ala Lys His Ile Ala Glu Glu Ala Asp Arg145 150 155 160Lys Tyr Glu Glu Val Ala Arg Lys Leu Val Ile Leu Glu Gly Glu Leu165 170 175Glu Arg Ala Glu Glu Arg Ala Glu Val Ser Glu Leu Lys Cys Gly Asp180 185 190Leu Glu Glu Glu Leu Lys Asn Val Thr Asn Asn Leu Lys Ser Leu Glu195 200 205Ala Ala Ser Glu Lys Tyr Ser Glu Lys Glu Asp Lys Tyr Glu Glu Glu210 215 220Ile Lys Leu Leu Ser Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala Glu225 230 235 240Phe Ala Glu Arg Thr Val Ala Lys Leu Glu Lys Thr Ile Asp Asp Leu245 250 255Glu Glu Lys Leu Ala Gln Ala Lys Glu Glu Asn Val Gly Leu His Gln260 265 270Thr Leu Asp Gln Thr Leu Asn Glu Leu Asn Cys Ile275 280<210>10
<211>417<212>PRT<213>人属、人种<400>10Met Leu Leu Ser Val Pro Leu Leu Leu Gly Leu Leu Gly Leu Ala Val1 5 10 15Ala Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp Gly20 25 30Trp Thr Ser Arg Trp Ile Glu Ser Lys His Lys Ser Asp Phe Gly Lys35 40 45Phe Val Leu Ser Ser Gly Lys Phe Tyr Gly Asp Glu Glu Lys Asp Lys50 55 60Gly Leu Gln Thr Ser Gln Asp Ala Arg Phe Tyr Ala Leu Ser Ala Ser65 70 75 80Phe Glu Pro Phe Ser Asn Lys Gly Gln Thr Leu Val Val Gln Phe Thr85 90 95Val Lys His Glu Gln Asn Ile Asp Cys Gly Gly Gly Tyr Val Lys Leu100 105 110Phe Pro Asn Ser Leu Asp Gln Thr Asp Met His Gly Asp Ser Glu Tyr115 120 125Asn Ile Met Phe Gly Pro Asp Ile Cys Gly Pro Gly Thr Lys Lys Val130 135 140His Val Ile Phe Asn Tyr Lys Gly Lys Asn Val Leu Ile Asn Lys Asp145 150 155 160Ile Arg Cys Lys Asp Asp Glu Phe Thr His Leu Tyr Thr Leu Ile Val165 170 175Arg Pro Asp Asn Thr Tyr Glu Val Lys Ile Asp Asn Ser Gln Val Glu180 185 190Ser Gly Ser Leu Glu Asp Asp Trp Asp Phe Leu Pro Pro Lys Lys Ile195 200 205Lys Asp Pro Asp Ala Ser Lys Pro Glu Asp Trp Asp Glu Arg Ala Lys210 215 220Ile Asp Asp Pro Thr Asp Ser Lys Pro Glu Asp Trp Asp Lys Pro Glu225 230 235 240His Ile Pro Asp Pro Asp Ala Lys Lys Pro Glu Asp Trp Asp Glu Glu245 250 255Met Asp Gly Glu Trp Glu Pro Pro Val Ile Gln Asn Pro Glu Tyr Lys260 265 270
Gly Glu Trp Lys Pro Arg Gln Ile Asp Asn Pro Asp Tyr Lys Gly Thr275 280 285Trp Ile His Pro Glu Ile Asp Asn Pro Glu Tyr Ser Pro Asp Pro Ser290 295 300Ile Tyr Ala Tyr Asp Asn Phe Gly Val Leu Gly Leu Asp Leu Trp Gln305 310 315 320Val Lys Ser Gly Thr Ile Phe Asp Asn Phe Leu Ile Thr Asn Asp Glu325 330 335Ala Tyr Ala Glu Glu Phe Gly Asn Glu Thr Trp Gly Val Thr Lys Ala340 345 350Ala Glu Lys Gln Met Lys Asp Lys Gln Asp Glu Glu Gln Arg Leu Lys355 360 365Glu Glu Glu Glu Asp Lys Lys Arg Lys Glu Glu Glu Glu Ala Glu Asp370 375 380Lys Glu Asp Asp Glu Asp Lys Asp Glu Asp Glu Glu Asp Glu Glu Asp385 390 395 400Lys Glu Glu Asp Glu Glu Glu Asp Val Pro Gly Gln Ala Lys Asp Glu405 410 415Leu<210>11<211>278<212>PRT<213>人属、人种<400>11Leu Arg Cys Val Pro Arg Val Leu Gly Ser Ser Val Ala Gly Leu Arg1 5 10 15Ala Ala Ala Pro Ala Ser Pro Phe Arg Gln Leu Leu Gln Pro Ala Pro20 25 30Arg Leu Cys Thr Arg Pro Phe Gly Leu Leu Ser Val Arg Ala Gly Ser35 40 45Glu Arg Arg Pro Gly Leu Leu Arg Pro Arg Gly Pro Cys Ala Cys Gly50 55 60Cys Gly Cys Gly Ser Leu His Thr Asp Gly Asp Lys Ala Phe Val Asp65 70 75 80Phe Leu Ser Asp Glu Ile Lys Glu Glu Arg Lys Ile Gln Lys His Lys85 90 95
Thr Leu Pro Lys Met Ser Gly Gly Trp Glu Leu Glu Leu Asn Gly Thr100 105 110Glu Ala Lys Leu Val Arg Lys Val Ala Gly Glu Lys Ile Thr Val Thr115 120 125Phe Asn Ile Asn Asn Ser Ile Pro Pro Thr Phe Asp Gly Glu Glu Glu130 135 140Pro Ser Gln Gly Gln Lys Val Glu Glu Gln Glu Pro Glu Leu Thr Ser145 150 155 160Thr Pro Asn Phe Val Val Glu Val Ile Lys Asn Asp Asp Gly Lys Lys165 170 175Ala Leu Val Leu Asp Cys His Tyr Pro Glu Asp Glu Val Gly Gln Glu180 185 190Asp Glu Ala Glu Ser Asp Ile Phe Ser Ile Arg Glu Val Ser Phe Gln195 200 205Ser Thr Gly Glu Ser Glu Trp Lys Asp Thr Asn Tyr Thr Leu Asn Thr210 215 220Asp Ser Leu Asp Trp Ala Leu Tyr Asp His Leu Met Asp Phe Leu Ala225 230 235 240Asp Arg Gly Val Asp Asn Thr Phe Ala Asp Glu Leu Val Glu Leu Ser245 250 255Thr Ala Leu Glu His Gln Glu Tyr Ile Thr Phe Leu Glu Asp Leu Lys260 265 270Ser Phe Val Lys Ser Gln275<210>12<211>661<212>PRT<213>人属、人种<400>12Arg Ala Leu Arg Arg Ala Pro Ala Leu Ala Ala Val Pro Gly Gly Lys1 5 10 15Pro Ile Leu Cys Pro Arg Arg Thr Thr Ala Gln Leu Gly Pro Arg Arg20 25 30Asn Pro Ala Trp Ser Leu Gln Ala Gly Arg Leu Phe Ser Thr Gln Thr35 40 45Ala Glu Asp Lys Glu Glu Pro Leu His Ser Ile Ile Ser Ser Thr Glu50 55 60Ser Val Gln Gly Ser Thr Ser Lys His Glu Phe Gln Ala Glu Thr Lys
65 70 75 80Lys Leu Leu Asp Ile Val Ala Arg Ser Leu Tyr Ser Glu Lys Glu Val85 90 95Phe Ile Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu Glu Lys Leu100 105 110Arg His Lys Leu Val Ser Asp Gly Gln Ala Leu Pro Glu Met Glu Ile115 120 125His Leu Gln Thr Asn Ala Glu Lys Gly Thr Ile Thr Ile Gln Asp Thr130 135 140Gly Ile Gly Met Thr Gln Glu Glu Leu Val Ser Asn Leu Gly Thr Ile145 150 155 160Ala Arg Ser Gly Ser Lys Ala Phe Leu Asp Ala Leu Gln Asn Gln Ala165 170 175Glu Ala Ser Ser Lys Ile Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser180 185 190Ala Phe Met Val Ala Asp Arg Val Glu Val Tyr Ser Arg Ser Ala Ala195 200 205Pro Gly Ser Leu Gly Tyr Gln Trp Leu Ser Asp Gly Ser Gly Val Phe210 215 220Glu Ile Ala Glu Ala Ser Gly Val Arg Thr Gly Thr Lys Ile Ile Ile225 230 235 240His Leu Lys Ser Asp Cys Lys Glu Phe Ser Ser Glu Ala Arg Val Arg245 250 255Asp Val Val Thr Lys Tyr Ser Asn Phe Val Ser Phe Pro Leu Tyr Leu260 265 270Asn Gly Arg Arg Met Asn Thr Leu Gln Ala Ile Trp Met Met Asp Pro275 280 285Lys Asp Val Gly Glu Trp Gln His Glu Glu Phe Tyr Arg Tyr Val Ala290 295 300Gln Ala His Asp Lys Pro Arg Tyr Thr Leu His Tyr Lys Thr Asp Ala305 310 315 320Pro Leu Asn Ile Arg Ser Ile Phe Tyr Val Pro Asp Met Lys Pro Ser325 330 335Met Phe Asp Val Ser Arg Glu Leu Gly Ser Ser Val Ala Leu Tyr Ser340 345 350Arg Lys Val Leu Ile Gln Thr Lys Ala Thr Asp Ile Leu Pro Lys Trp355 360 365
Leu Arg Phe Ile Arg Gly Val Val Asp Ser Glu Asp Ile Pro Leu Asn370 375 380Leu Ser Arg Glu Leu Leu Gln Glu Ser Ala Leu Ile Arg Lys Leu Arg385 390 395 400Asp Val Leu Gln Gln Arg Leu Ile Lys Phe Phe Ile Asp Gln Ser Lys405 410 415Lys Asp Ala Glu Lys Tyr Ala Lys Phe Phe Glu Asp Tyr Gly Leu Phe420 425 430Met Arg Glu Gly Ile Val Thr Ala Thr Glu Gln Glu Val Lys Glu Asp435 440 445Ile Ala Lys Leu Leu Arg Tyr Glu Ser Ser Ala Leu Pro Ser Gly Gln450 455 460Leu Thr Ser Leu Ser Glu Tyr Ala Ser Arg Met Arg Ala Gly Thr Arg465 470 475 480Asn Ile Tyr Tyr Leu Cys Ala Pro Asn Arg His Leu Ala Glu His Ser485 490 495Pro Tyr Tyr Glu Ala Met Lys Lys Lys Asp Thr Glu Val Leu Phe Cys500 505 510Phe Glu Gln Phe Asp Glu Leu Thr Leu Leu His Leu Arg Glu Phe Asp515 520 525Lys Lys Lys Leu Ile Ser Val Glu Thr Asp Ile Val Val Asp His Tyr530 535 540Lys Glu Glu Lys Phe Glu Asp Arg Ser Pro Ala Ala Glu Cys Leu Ser545 550 555 560Glu Lys Glu Thr Glu Glu Leu Met Ala Trp Met Arg Asn Val Leu Gly565 570 575Ser Arg Val Thr Asn Val Lys Val Thr Leu Arg Leu Asp Thr His Pro580 585 590Ala Met Val Thr Val Leu Glu Met Gly Ala Ala Arg His Phe Leu Arg595 600 605Met Gln Gln Leu Ala Lys Thr Gln Glu Glu Arg Ala Gln Leu Leu Gln610 615 620Pro Thr Leu Glu Ile Asn Pro Arg His Ala Leu Ile Lys Lys Leu Asn625 630 635 640His Cys Ala Gln Ala Ser Leu Ala Trp Leu Ser Cys Trp Trp Ile Arg645 650 655Tyr Thr Arg Thr Pro660
<210>13<211>803<212>PRT<213>人属、人种<400>13Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe1 5 10 15Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu20 25 30Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val35 40 45Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser50 55 60Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala65 70 75 80Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn85 90 95Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu100 105 110Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly115 120 125Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu130 135 140Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val145 150 155 160Lys Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn165 170 175Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile180 185 190Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys195 200 205Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu210 215 220Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr225 230 235 240Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser245 250 255
Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser260 265 270Gln Phe Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr275 280 285Val Glu Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu290 295 300Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys305 310 315 320Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met325 330 335Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu340 345 350Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp355 360 365Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys370 375 380Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu385 390 395 400Tyr Gly Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val405 410 415Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe420 425 430Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg435 440 445Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu450 455 460Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr465 470 475 480Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val485 490 495Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe500 505 510Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val515 520 525Glu Arg Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser530 535 540Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys545 550 555 560
Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys565 570 575Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala580 585 590Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg595 600 605Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp610 615 620Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu625 630 635 640Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly645 650 655Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp660 665 670Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn675 680 685Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp690 695 700Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr705 710 715 720Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly725 730 735Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp740 745 750Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu755 760 765Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val770 775 780Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys785 790 795 800Asp Glu Leu<210>14<211>724<212>PRT<213>人属、人种<400>14
Met Pro Glu Glu Val His His Gly Glu Glu Glu Val Glu Thr Phe Ala1 5 10 15Phe Gln Ala Glu Ile Ala Gln Leu Met Ser Leu Ile Ile Asn Thr Phe20 25 30Tyr Ser Asn Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser35 40 45Asp Ala Leu Asp Lys Ile Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys50 55 60Leu Asp Ser Gly Lys Glu Leu Lys Ile Asp Ile Ile Pro Asn Pro Gln65 70 75 80Glu Arg Thr Leu Thr Leu Val Asp Thr Gly Ile Gly Met Thr Lys Ala85 90 95Asp Leu Ile Asn Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Lys Ala100 105 110Phe Met Glu Ala Leu Gln Ala Gly Ala Asp Ile Ser Met Ile Gly Gln115 120 125Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val Ala Glu Lys Val Val130 135 140Val Ile Arg Lys His Asn Asp Asp Glu Gln Tyr Ala Trp Glu Ser Ser145 150 155 160Ala Gly Gly Ser Phe Thr Val Arg Ala Asp His Gly Glu Pro Ile Gly165 170 175Met Gly Thr Lys Val Ile Leu His Leu Lys Glu Asp Gln Thr Glu Tyr180 185 190Leu Glu Glu Arg Arg Val Lys Glu Val Val Lys Lys His Ser Gln Phe195 200 205Ile Gly Tyr Pro Ile Thr Leu Tyr Leu Glu Lys Glu Arg Glu Lys Glu210 215 220Ile Ser Asp Asp Glu Ala Glu Glu Glu Lys Gly Glu Lys Glu Glu Glu225 230 235 240Asp Lys Asp Asp Glu Glu Lys Pro Lys Ile Glu Asp Val Gly Ser Asp245 250 255Glu Glu Asp Asp Ser Gly Lys Asp Lys Lys Lys Lys Thr Lys Lys Ile260 265 270Lys Glu Lys Tyr Ile Asp Gln Glu Glu Leu Asn Lys Thr Lys Pro Ile275 280 285Trp Thr Arg Asn Pro Asp Asp Ile Thr Gln Glu Glu Tyr Gly Glu Phe290 295 300
Tyr Lys Ser Leu Thr Asn Asp Trp Glu Asp His Leu Ala Val Lys His305 310 315 320Phe Ser Val Glu Gly Gln Leu Glu Phe Arg Ala Leu Leu Phe Ile Pro325 330 335Arg Arg Ala Pro Phe Asp Leu Phe Glu Asn Lys Lys Lys Lys Asn Asn340 345 350Ile Lys Leu Tyr Val Arg Arg Val Phe Ile Met Asp Ser Cys Asp Glu355 360 365Leu Ile Pro Glu Tyr Leu Asn Phe Ile Arg Gly Val Val Asp Ser Glu370 375 380Asp Leu Pro Leu Asn Ile Ser Arg Glu Met Leu Gln Gln Ser Lys Ile385 390 395 400Leu Lys Val Ile Arg Lys Asn Ile Val Lys Lys Cys Leu Glu Leu Phe405 410 415Ser Glu Leu Ala Glu Asp Lys Glu Asn Tyr Lys Lys Phe Tyr Glu Ala420 425 430Phe Ser Lys Asn Leu Lys Leu Gly Ile His Glu Asp Ser Thr Asn Arg435 440 445Arg Arg Leu Ser Glu Leu Leu Arg Tyr His Thr Ser Gln Ser Gly Asp450 455 460Glu Met Thr Ser Leu Ser Glu Tyr Val Ser Arg Met Lys Glu Thr Gln465 470 475 480Lys Ser Ile Tyr Tyr Ile Thr Gly Glu Ser Lys Glu Gln Val Ala Asn485 490 495Ser Ala Phe Val Glu Arg Val Arg Lys Arg Gly Phe Glu Val Val Tyr500 505 510Met Thr Glu Pro Ile Asp Glu Tyr Cys Val Gln Gln Leu Lys Glu Phe515 520 525Asp Gly Lys Ser Leu Val Ser Val Thr Lys Glu Gly Leu Glu Leu Pro530 535 540Glu Asp Glu Glu Glu Lys Lys Lys Met Glu Glu Ser Lys Ala Lys Phe545 550 555 560Glu Asn Leu Cys Lys Leu Met Lys Glu Ile Leu Asp Lys Lys Val Glu565 570 575Lys Val Thr Ile Ser Asn Arg Leu Val Ser Ser Pro Cys Cys Ile Val580 585 590Thr Ser Thr Tyr Gly Trp Thr Ala Asn Met Glu Arg Ile Met Lys Ala
595 600 605Gln Ala Leu Arg Asp Asn Ser Thr Met Gly Tyr Met Met Ala Lys Lys610 615 620His Leu Glu Ile Asn Pro Asp His Pro Ile Val Glu Thr Leu Arg Gln625 630 635 640Lys Ala Glu Ala Asp Lys Asn Asp Lys Ala Val Lys Asp Leu Val Val645 650 655Leu Leu Phe Glu Thr Ala Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp660 665 670Pro Gln Thr His Ser Asn Arg Ile Tyr Arg Met Ile Lys Leu Gly Leu675 680 685Gly Ile Asp Glu Asp Glu Val Ala Ala Glu Glu Pro Asn Ala Ala Val690 695 700Pro Asp Glu Ile Pro Pro Leu Glu Gly Asp Glu Asp Ala Ser Arg Met705 710 715 720Glu Glu Val Asp<210>15<211>732<212>PRT<213>人属、人种<400>15Met Pro Glu Glu Thr Gln Thr Gln Asp Gln Pro Met Glu Glu Glu Glu1 5 10 15Val Glu Thr Phe Ala Phe Gln Ala Glu Ile Ala Gln Leu Met Ser Leu20 25 30Ile Ile Asn Thr Phe Tyr Ser Asn Lys Glu Ile Phe Leu Arg Glu Leu35 40 45Ile Ser Asn Ser Ser Asp Ala Leu Asp Lys Ile Arg Tyr Glu Thr Leu50 55 60Thr Asp Pro Ser Lys Leu Asp Ser Gly Lys Glu Leu His Ile Asn Leu65 70 75 80Ile Pro Asn Lys Gln Asp Arg Thr Leu Thr Ile Val Asp Thr Gly Ile85 90 95Gly Met Thr Lys Ala Asp Leu Ile Asn Asn Leu Gly Thr Ile Ala Lys100 105 110Ser Gly Thr Lys Ala Phe Met Glu Ala Leu Gln Ala Gly Ala Asp Ile115 120 125
Ser Met Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val130 135 140Ala Glu Lys Val Thr Val Ile Thr Lys His Asn Asp Asp Glu Gln Tyr145 150 155 160Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr Val Arg Thr Asp Thr165 170 175Gly Glu Pro Met Gly Arg Gly Thr Lys Val Ile Leu His Leu Lys Glu180 185 190Asp Gln Thr Glu Tyr Leu Glu Glu Arg Arg Ile Lys Glu Ile Val Lys195 200 205Lys His Ser Gln Phe Ile Gly Tyr Pro Ile Thr Leu Phe Val Glu Lys210 215 220Glu Arg Asp Lys Glu Val Ser Asp Asp Glu Ala Glu Glu Lys Glu Asp225 230 235 240Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu Ser Glu Asp Lys Pro245 250 255Glu Ile Glu Asp Val Gly Ser Asp Glu Glu Glu Glu Lys Lys Asp Gly260 265 270Asp Lys Lys Lys Lys Lys Lys Ile Lys Glu Lys Tyr Ile Asp Gln Glu275 280 285Glu Leu Asn Lys Thr Lys Pro Ile Trp Thr Arg Asn Pro Asp Asp Ile290 295 300Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu Thr Asn Asp Trp305 310 315 320Glu Asp His Leu Ala Val Lys His Phe Ser Val Glu Gly Gln Leu Glu325 330 335Phe Arg Ala Leu Leu Phe Val Pro Arg Arg Ala Pro Phe Asp Leu Phe340 345 350Glu Asn Arg Lys Lys Lys Asn Asn Ile Lys Leu Tyr Val Arg Arg Val355 360 365Phe Ile Met Asp Asn Cys Glu Glu Leu Ile Pro Glu Tyr Leu Asn Phe370 375 380Ile Arg Gly Val Val Asp Ser Glu Asp Leu Pro Leu Asn Ile Ser Arg385 390 395 400Glu Met Leu Gln Gln Ser Lys Ile Leu Lys Val Ile Arg Lys Asn Leu405 410 415Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Leu Ala Glu Asp Lys Glu
420 425 430Asn Tyr Lys Lys Phe Tyr Glu Gln Phe Ser Lys Asn Ile Lys Leu Gly435 440 445Ile His Glu Asp Ser Gln Asn Arg Lys Lys Leu Ser Glu Leu Leu Arg450 455 460Tyr Tyr Thr Ser Ala Ser Gly Asp Glu Met Val Ser Leu Lys Asp Tyr465 470 475 480Cys Thr Arg Met Lys Glu Asn Gln Lys His Ile Tyr Tyr Ile Thr Gly485 490 495Glu Thr Lys Asp Gln Val Ala Asn Ser Ala Phe Val Glu Arg Leu Arg500 505 510Lys His Gly Leu Glu Val Ile Tyr Met Ile Glu Pro Ile Asp Glu Tyr515 520 525Cys Val Gln Gln Leu Lys Glu Phe Glu Gly Lys Thr Leu Val Ser Val530 535 540Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu Glu Lys Lys Lys545 550 555 560Gln Glu Glu Lys Lys Thr Lys Phe Glu Asn Leu Cys Lys Ile Met Lys565 570 575Asp Ile Leu Glu Lys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu580 585 590Val Thr Ser Pro Cys Cys Ile Val Thr Ser Thr Tyr Gly Trp Thr Ala595 600 605Asn Met Glu Arg Ile Met Lys Ala Gln Ala Leu Arg Asp Asn Ser Thr610 615 620Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu Ile Asn Pro Asp His625 630 635 640Ser Ile Ile Glu Thr Leu Arg Gln Lys Ala Glu Ala Asp Lys Asn Asp645 650 655Lys Ser Val Lys Asp Leu Val Ile Leu Leu Tyr Glu Thr Ala Leu Leu660 665 670Ser Ser Gly Phe Ser Leu Glu Asp Pro Gln Thr His Ala Asn Arg Ile675 680 685Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu Asp Asp Pro Thr690 695 700Ala Asp Asp Thr Ser Ala Ala Val Thr Glu Glu Met Pro Pro Leu Glu705 710 715 720
Gly Asp Asp Asp Thr Ser Arg Met Glu Glu Val Asp725 730<210>16<211>573<212>PRT<213>人属、人种<400>16Met Leu Arg Leu Pro Thr Val Phe Arg Gln Met Arg Pro Val Ser Arg1 5 10 15Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe20 25 30Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu Leu Ala35 40 45Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr Val Ile Ile50 55 60Glu Gln Ser Trp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val65 70 75 80Ala Lys Ser Ile Asp Leu Lys Asp Lys Tyr Lys Asn Ile Gly Ala Lys85 90 95Leu Val Gln Asp Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly100 105 110Thr Thr Thr Ala Thr Val Leu Ala Arg Ser Ile Ala Lys Glu Gly Phe115 120 125Glu Lys Ile Ser Lys Gly Ala Asn Pro Val Glu Ile Arg Arg Gly Val130 135 140Met Leu Ala Val Asp Ala Val Ile Ala Glu Leu Lys Lys Gln Ser Lys145 150 155 160Pro Val Thr Thr Pro Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala165 170 175Asn Gly Asp Lys Glu Ile Gly Asn Ile Ile Ser Asp Ala Met Lys Lys180 185 190Val Gly Arg Lys Gly Val Ile Thr Val Lys Asp Gly Lys Thr Leu Asn195 200 205Asp Glu Leu Glu Ile Ile Glu Gly Met Lys Phe Asp Arg Gly Tyr Ile210 215 220Ser Pro Tyr Phe Ile Asn Thr Ser Lys Gly Gln Lys Cys Glu Phe Gln225 230 235 240Asp Ala Tyr Val Leu Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Ser
245 250 255Ile Val Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro Leu Val260 265 270Ile Ile Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr Leu Val Leu275 280 285Asn Arg Leu Lys Val Gly Leu Gln Val Val Ala Val Lys Ala Pro Gly290 295 300Phe Gly Asp Asn Arg Lys Asn Gln Leu Lys Asp Met Ala Ile Ala Thr305 310 315 320Gly Gly Ala Val Phe Gly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp325 330 335Val Gln Pro His Asp Leu Gly Lys Val Gly Glu Val Ile Val Thr Lys340 345 350Asp Asp Ala Met Leu Leu Lys Gly Lys Gly Asp Lys Ala Gln Ile Glu355 360 365Lys Arg Ile Gln Glu Ile Ile Glu Gln Leu Asp Val Thr Thr Ser Glu370 375 380Tyr Glu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly385 390 395 400Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu405 410 415Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val420 425 430Glu Glu Gly Ile Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys Ile435 440 445Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp Gln Lys Ile Gly450 455 460Ile Glu Ile Ile Lys Arg Thr Leu Lys Ile Pro Ala Met Thr Ile Ala465 470 475 480Lys Asn Ala Gly Val Glu Gly Ser Leu Ile Val Glu Lys Ile Met Gln485 490 495Ser Ser Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn500 505 510Met Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val Val Arg Thr Ala515 520 525Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala Glu Val530 535 540
Val Val Thr Glu Ile Pro Lys Glu Glu Lys Asp Pro Gly Met Gly Ala545 550 555 560Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly Met Phe565 570<210>17<211>215<212>PRT<213>人属、人种<400>17Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Arg Lys Met Ser Ser Tyr1 5 10 15Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro20 25 30Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg35 40 45Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala50 55 60Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro65 70 75 80Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys85 90 95Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys100 105 110Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys115 120 125Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr130 135 140Glu Lys Lys Ala Glu Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala145 150 155 160Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val165 170 175Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Gly Glu Glu180 185 190Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu195 200 205Glu Glu Asp Asp Asp Asp Glu210 215<210>18
<211>1087<212>PRT<213>人属、人种<400>18Met Met Thr Ser Val Gly Thr Asn Arg Ala Arg Gly Asn Trp Glu Gln1 5 10 15Pro Gln Asn Gln Asn Gln Thr Gln His Lys Gln Arg Pro Gln Ala Thr20 25 30Ala Glu Gln Ile Arg Leu Ala Gln Met Ile Ser Asp His Asn Asp Ala35 40 45Asp Phe Glu Glu Lys Val Lys Gln Leu Ile Asp Ile Thr Gly Lys Asn50 55 60Gln Asp Glu Cys Val Ile Ala Leu His Asp Cys Asn Gly Asp Val Asn65 70 75 80Arg Ala Ile Asn Val Leu Leu Glu Gly Asn Pro Asp Thr His Ser Trp85 90 95Glu Met Val Gly Lys Lys Lys Gly Val Ser Gly Gln Lys Asp Gly Gly100 105 110Gln Thr Glu Ser Asn Glu Glu Gly Lys Glu Asn Arg Asp Arg Asp Arg115 120 125Asp Tyr Ser Arg Arg Arg Gly Gly Pro Pro Arg Arg Gly Arg Gly Ala130 135 140Ser Arg Gly Arg Glu Phe Arg Gly Gln Glu Asn Gly Leu Asp Gly Thr145 150 155 160Lys Ser Gly Gly Pro Ser Gly Arg Gly Thr Glu Arg Gly Arg Arg Gly165 170 175Arg Gly Arg Gly Arg Gly Gly Ser Gly Arg Arg Gly Gly Arg Phe Ser180 185 190Ala Gln Gly Met Gly Thr Phe Asn Pro Ala Asp Tyr Ala Glu Pro Ala195 200 205Asn Thr Asp Asp Asn Tyr Gly Asn Ser Ser Gly Asn Thr Trp Asn Asn210 215 220Thr Gly His Phe Glu Pro Asp Asp Gly Thr Ser Ala Trp Arg Thr Ala225 230 235 240Thr Glu Glu Trp Gly Thr Glu Asp Trp Asn Glu Asp Leu Ser Glu Thr245 250 255Lys Ile Phe Thr Ala Ser Asn Val Ser Ser Val Pro Leu Pro Ala Glu260 265 270
Asn Val Thr Ile Thr Ala Gly Gln Arg Ile Asp Leu Ala Val Leu Leu275 280 285Gly Lys Thr Pro Ser Thr Met Glu Asn Asp Ser Ser Asn Leu Asp Pro290 295 300Ser Gln Ala Pro Ser Leu Ala Gln Pro Leu Val Phe Ser Asn Ser Lys305 310 315 320Gln Thr Ala Ile Ser Gln Pro Ala Ser Gly Asn Thr Phe Ser His His325 330 335Ser Met Val Ser Met Leu Gly Lys Gly Phe Gly Asp Val Gly Glu Ala340 345 350Lys Gly Gly Ser Thr Thr Gly Ser Gln Phe Leu Glu Gln Phe Lys Thr355 360 365Ala Gln Ala Leu Ala Gln Leu Ala Ala Gln His Ser Gln Ser Gly Ser370 375 380Thr Thr Thr Ser Ser Trp Asp Met Gly Ser Thr Thr Gln Ser Pro Ser385 390 395 400Leu Val Gln Tyr Asp Leu Lys Asn Pro Ser Asp Ser Ala Val His Ser405 410 415Pro Phe Thr Lys Arg Gln Ala Phe Thr Pro Ser Ser Thr Met Met Glu420 425 430Val Phe Leu Gln Glu Lys Ser Pro Ala Val Ala Thr Ser Thr Ala Ala435 440 445Pro Pro Pro Pro Ser Ser Pro Leu Pro Ser Lys Ser Thr Ser Ala Pro450 455 460Gln Met Ser Pro Gly Ser Ser Asp Asn Gln Ser Ser Ser Pro Gln Pro465 470 475 480Ala His Gln Lys Leu Lys Gln Gln Lys Lys Lys Ala Ser Leu Thr Ser485 490 495Lys Ile Pro Ala Leu Ala Val Glu Met Pro Gly Ser Ala Asp Ile Ser500 505 510Gly Leu Asn Leu Gln Phe Gly Ala Leu Gln Phe Gly Ser Glu Pro Val515 520 525Leu Ser Asp Tyr Glu Ser Thr Pro Thr Thr Ser Ala Ser Ser Ser Gln530 535 540Ala Pro Ser Ser Leu Tyr Thr Ser Thr Ala Ser Glu Ser Ser Ser Thr545 550 555 560Ile Ser Ser Asn Gln Ser Gln Glu Ser Gly Tyr Gln Ser Gly Pro Ile
565 570 575Gln Ser Thr Thr Tyr Thr Ser Gln Asn Asn Ala Gln Gly Pro Leu Tyr580 585 590Glu Gln Arg Ser Thr Gln Thr Arg Arg Tyr Pro Ser Ser Ile Ser Ser595 600 605Ser Pro Gln Lys Asp Leu Thr Gln Ala Lys Asn Gly Phe Ser Ser Val610 615 620Gln Ala Thr Gln Leu Gln Thr Thr Gln Ser Val Glu Gly Ala Thr Gly625 630 635 640Ser Ala Val Lys Ser Asp Ser Pro Ser Thr Ser Ser Ile Pro Pro Leu645 650 655Asn Glu Thr Val Ser Ala Ala Ser Leu Leu Thr Thr Thr Asn Gln His660 665 670Ser Ser Ser Leu Gly Gly Leu Ser His Ser Glu Glu Ile Pro Asn Thr675 680 685Thr Thr Thr Gln His Ser Ser Thr Leu Ser Thr Gln Gln Asn Thr Leu690 695 700Ser Ser Ser Thr Ser Ser Gly Arg Thr Ser Thr Ser Thr Leu Leu His705 710 715 720Thr Ser Val Glu Ser Glu Ala Asn Leu His Ser Ser Ser Ser Thr Phe725 730 735Ser Thr Thr Ser Ser Thr Val Ser Ala Pro Pro Pro Val Val Ser Val740 745 750Ser Ser Ser Leu Asn Ser Gly Ser Ser Leu Gly Leu Ser Leu Gly Ser755 760 765Asn Ser Thr Val Thr Ala Ser Thr Arg Ser Ser Val Ala Thr Thr Ser770 775 780Gly Lys Ala Pro Pro Asn Leu Pro Pro Gly Val Pro Pro Leu Leu Pro785 790 795 800Asn Pro Tyr Ile Met Ala Pro Gly Leu Leu His Ala Tyr Pro Pro Gln805 810 815Val Tyr Gly Tyr Asp Asp Leu Gln Met Leu Gln Thr Arg Phe Pro Leu820 825 830Asp Tyr Tyr Ser Ile Pro Phe Pro Thr Pro Thr Thr Pro Leu Thr Gly835 840 845Arg Asp Gly Ser Leu Ala Ser Asn Pro Tyr Ser Gly Asp Leu Thr Lys850 855 860
Phe Gly Arg Gly Asp Ala Ser Ser Pro Ala Pro Ala Thr Thr Leu Ala865 870 875 880Gln Pro Gln Gln Asn Gln Thr Gln Thr His His Thr Thr Gln Gln Thr885 890 895Phe Leu Asn Pro Ala Leu Pro Pro Gly Tyr Ser Tyr Thr Ser Leu Pro900 905 910Tyr Tyr Thr Gly Val Pro Gly Leu Pro Ser Thr Phe Gln Tyr Gly Pro915 920 925Ala Val Phe Pro Val Ala Pro Thr Ser Ser Lys Gln His Gly Val Asn930 935 940Val Ser Val Asn Ala Ser Ala Thr Pro Phe Gln Gln Pro Ser Gly Tyr945 950 955 960Gly Ser His Gly Tyr Asn Thr Gly Val Ser Val Thr Ser Ser Asn Thr965 970 975Gly Val Pro Asp Ile Ser Gly Ser Val Tyr Ser Lys Thr Gln Gln Ser980 985 990Phe Glu Lys Gln Gly Phe His Ser Gly Thr Pro Ala Ala Ser Phe Asn995 10001005Leu Pro Ser Ala Leu Gly Ser Gly Gly Pro Ile Asn Pro Ala Thr101010151020Ala Ala Ala Tyr Pro Pro Ala Pro Phe Met His Ile Leu Thr Pro102510301035His Gln Gln Pro His Ser Gln Ile Leu His His His Leu Gln Gln104010451050Asp Gly Gln Thr Gly Ser Gly Gln Arg Ser Gln Thr Ser Ser Ile105510601065Pro Gln Lys Pro Gln Thr Asn Lys Ser Ala Tyr Asn Ser Tyr Ser107010751080Trp Gly Ala Asn1085<210>19<211>806<212>PRT<213>人属、人种<400>19Met Ala Ser Gly Ala Asp Ser Lys Gly Asp Asp Leu Ser Thr Ala Ile1 5 10 15Leu Lys Gln Lys Asn Arg Pro Asn Arg Leu Ile Val Asp Glu Ala Ile
20 25 30Asn Glu Asp Asn Ser Val Val Ser Leu Ser Gln Pro Lys Met Asp Glu35 40 45Leu Gln Leu Phe Arg Gly Asp Thr Val Leu Leu Lys Gly Lys Lys Arg50 55 60Arg Glu Ala Val Cys Ile Val Leu Ser Asp Asp Thr Cys Ser Asp Glu65 70 75 80Lys Ile Arg Met Asn Arg Val Val Arg Asn Asn Leu Arg Val Arg Leu85 90 95Gly Asp Val Ile Ser Ile Gln Pro Cys Pro Asp Val Lys Tyr Gly Lys100 105 110Arg Ile His Val Leu Pro Ile Asp Asp Thr Val Glu Gly Ile Thr Gly115 120 125Asn Leu Phe Glu Val Tyr Leu Lys Pro Tyr Phe Leu Glu Ala Tyr Arg130 135 140Pro Ile Arg Lys Gly Asp Ile Phe Leu Val Arg Gly Gly Met Arg Ala145 150 155 160Val Glu Phe Lys Val Val Glu Thr Asp Pro Ser Pro Tyr Cys Ile Val165 170 175Ala Pro Asp Thr Val Ile His Cys Glu Gly Glu Pro Ile Lys Arg Glu180 185 190Asp Glu Glu Glu Ser Leu Asn Glu Val Gly Tyr Asp Asp Ile Gly Gly195 200 205Cys Arg Lys Gln Leu Ala Gln Ile Lys Glu Met Val Glu Leu Pro Leu210 215 220Arg His Pro Ala Leu Phe Lys Ala Ile Gly Val Lys Pro Pro Arg Gly225 230 235 240Ile Leu Leu Tyr Gly Pro Pro Gly Thr Gly Lys Thr Leu Ile Ala Arg245 250 255Ala Val Ala Asn Glu Thr Gly Ala Phe Phe Phe Leu Ile Asn Gly Pro260 265 270Glu Ile Met Ser Lys Leu Ala Gly Glu Ser Glu Ser Asn Leu Arg Lys275 280 285Ala Phe Glu Glu Ala Glu Lys Asn Ala Pro Ala Ile Ile Phe Ile Asp290295 300Glu Leu Asp Ala Ile Ala Pro Lys Arg Glu Lys Thr His Gly Glu Val305 310 315 320
Glu Arg Arg Ile Val Ser Gln Leu Leu Thr Leu Met Asp Gly Leu Lys325 330 335Gln Arg Ala His Val Ile Val Met Ala Ala Thr Asn Arg Pro Asn Ser340 345 350Ile Asp Pro Ala Leu Arg Arg Phe Gly Arg Phe Asp Arg Glu Val Asp355 360 365Ile Gly Ile Pro Asp Ala Thr Gly Arg Leu Glu Ile Leu Gln Ile His370 375 380Thr Lys Asn Met Lys Leu Ala Asp Asp Val Asp Leu Glu Gln Val Ala385 390 395 400Asn Glu Thr His Gly His Val Gly Ala Asp Leu Ala Ala Leu Cys Ser405 410 415Glu Ala Ala Leu Gln Ala Ile Arg Lys Lys Met Asp Leu Ile Asp Leu420 425 430Glu Asp Glu Thr Ile Asp Ala Glu Val Met Asn Ser Leu Ala Val Thr435 440 445Met Asp Asp Phe Arg Trp Ala Leu Ser Gln Ser Asn Pro Ser Ala Leu450 455 460Arg Glu Thr Val Val Glu Val Pro Gln Val Thr Trp Glu Asp Ile Gly465 470 475 480Gly Leu Glu Asp Val Lys Arg Glu Leu Gln Glu Leu Val Gln Tyr Pro485 490 495Val Glu His Pro Asp Lys Phe Leu Lys Phe Gly Met Thr Pro Ser Lys500 505 510Gly Val Leu Phe Tyr Gly Pro Pro Gly Cys Gly Lys Thr Leu Leu Ala515 520 525Lys Ala Ile Ala Asn Glu Cys Gln Ala Asn Phe Ile Ser Ile Lys Gly530 535 540Pro Glu Leu Leu Thr Met Trp Phe Gly Glu Ser Glu Ala Asn Val Arg545 550 555 560Glu Ile Phe Asp Lys Ala Arg Gln Ala Ala Pro Cys Val Leu Phe Phe565 570 575Asp Glu Leu Asp Ser Ile Ala Lys Ala Arg Gly Gly Asn Ile Gly Asp580 585 590Gly Gly Gly Ala Ala Asp Arg Val Ile Asn Gln Ile Leu Thr Glu Met595 600 605Asp Gly Met Ser Thr Lys Lys Asn Val Phe Ile Ile Gly Ala Thr Asn610 615 620
Arg Pro Asp Ile Ile Asp Pro Ala Ile Leu Arg Pro Gly Arg Leu Asp625 630 635 640Gln Leu Ile Tyr Ile Pro Leu Pro Asp Glu Lys Ser Arg Val Ala Ile645 650 655Leu Lys Ala Asn Leu Arg Lys Ser Pro Val Ala Lys Asp Val Asp Leu660 665 670Glu Phe Leu Ala Lys Met Thr Asn Gly Phe Ser Gly Ala Asp Leu Thr675 680 685Glu Ile Cys Gln Arg Ala Cys Lys Leu Ala Ile Arg Glu Ser Ile Glu690 695 700Ser Glu Ile Arg Arg Glu Arg Glu Arg Gln Thr Asn Pro Ser Ala Met705 710 715 720Glu Val Glu Glu Asp Asp Pro Val Pro Glu Ile Arg Arg Asp His Phe725 730 735Glu Glu Ala Met Arg Phe Ala Arg Arg Ser Val Ser Asp Asn Asp Ile740 745 750Arg Lys Tyr Glu Met Phe Ala Gln Thr Leu Gln Gln Ser Arg Gly Phe755 760 765Gly Ser Phe Arg Phe Pro Ser Gly Asn Gln Gly Gly Ala Gly Pro Ser770 775 780Gln Gly Ser Gly Gly Gly Thr Gly Gly Ser Val Tyr Thr Glu Asp Asn785 790 795 800Asp Asp Asp Leu Tyr Gly805<210>20<211>260<212>PRT<213>人属、人种<400>20Met Val Lys Val Lys Val Glu Val Asn Gly Phe Gly His Thr Gly Arg1 5 10 15Leu Val Thr Arg Ala Ala Phe Asn Ser Gly Lys Val Asp Ile Val Thr20 25 30Ile Asn Asp Pro Phe Ile Asp Leu Asn Tyr Pro Phe Ile Asp Leu Asn35 40 45Tyr Met Ile Tyr Met Phe Gln Tyr Asp Ser Met Ala Asn Ser Met Ala50 55 60
Pro Ser Arg Leu Arg Met Gly Ser Leu Ser Ser Arg Glu Ile Pro Ser65 70 75 80Pro Ser Ser Arg Ser Glu Ile Pro Pro Lys Ser Asn Gly Gly Glu Ala85 90 95Lys Arg Ile Ile Ile Ser Ala Pro Ser Ala Asp Ala Pro Met Phe Met100 105 110Met Gly Ile Asn Arg Glu Lys Tyr Asp Asn Ser Leu Glu Ile Ile Ser115 120 125Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys Val Ile130 135 140His Asp Asn Phe Gly Ile Met Glu Gly Leu Met Thr Thr Val His Ala145 150 155 160Ile Ala Ala Thr Gln Lys Thr Val Asp Ser Pro Ser Gly Lys Leu Trp165 170 175Cys Asp Gly Cys Arg Ala Leu Gln Asn Ile Ile Pro Ala Ser Thr Gly180 185 190Ser Ser Leu Ala Lys Pro Ser Val Val Pro Thr Thr Asn Val Ser Val195 200 205Val Asp Leu Thr Cys His Leu Glu Lys Pro Ala Lys Tyr Asp Asp Ile210 215 220Lys Lys Trp Cys Ser Arg His Gln Lys Ala Pro Ser Arg Ala Ser Trp225 230 235 240Ala Thr Leu Ser Thr Cys Asn Arg Val Val Asp Leu Met Ala His Met245 250 255Ala Ser Lys Glu260<210>21<211>279<212>PRT<213>人属、人种<400>21Met Asn Lys Val Glu Leu Glu Ser Arg Leu Glu Gly Leu Thr Asp Glu1 5 10 15Ile Asn Phe Leu Arg Gln Leu Tyr Glu Glu Glu Leu Arg Glu Leu Gln20 25 30Ser Gln Ile Ser Asp Thr Ser Val Val Leu Ser Met Asp Asn Ser Arg35 40 45Ser Leu Asp Met Asp Ser Ile Ile Ala Glu Val Lys Ala Gln Tyr Glu
50 55 60Asp Ile Ala Asn Arg Ser Arg Ala Glu Ala Glu Ser Met Tyr Gln Ile65 70 75 80Lys Tyr Glu Glu Leu Gln Ser Leu Ala Gly Lys His Gly Asp Asp Leu85 90 95Arg Arg Thr Lys Thr Glu Ile Ser Glu Met Asn Arg Asn Ile Ser Arg100 105 110Leu Gln Ala Glu Ile Glu Gly Leu Lys Gly Gln Arg Ala Ser Leu Glu115 120 125Ala Ala Ile Ala Asp Ala Glu Gln Arg Gly Glu Leu Ala Ile Lys Asp130 135 140Ala Asn Ala Lys Leu Ser Glu Leu Glu Ala Ala Leu Gln Arg Ala Lys145 150 155 160Gln Asp Met Ala Arg Gln Leu Arg Glu Tyr Gln Glu Leu Met Asn Val165 170 175Lys Leu Ala Met Asp Ile Glu Ile Ala Thr Tyr Arg Lys Leu Leu Glu180 185 190Gly Glu Glu Ser Arg Leu Glu Ser Gly Met Gln Asn Met Ser Ile His195 200 205Thr Lys Thr Thr Gly Gly Tyr Ala Gly Gly Leu Ser Ser Ala Tyr Gly210 215 220Gly Leu Ala Ser Pro Gly Leu Ser Tyr Ser Leu Gly Ser Ser Phe Gly225 230 235 240Ser Gly Ala Gly Ser Ser Ser Phe Ser Arg Thr Ser Ser Ser Arg Ala245 250 255Val Val Val Lys Lys Ile Glu Thr Arg Asp Gly Lys Leu Val Ser Glu260 265 270Ser Ser Asp Val Leu Pro Lys275<210>22<211>577<212>PRT<213>人属、人种<400>22Met Asn Lys Leu Tyr Ile Gly Asn Leu Asn Glu Ser Val Thr Pro Ala1 5 10 15Asp Leu Glu Lys Val Phe Ala Glu His Lys Ile Ser Tyr Ser Gly Gln20 25 30
Phe Leu Val Lys Ser Gly Tyr Ala Phe Val Asp Cys Pro Asp Glu His35 40 45Trp Ala Met Lys Ala Ile Glu Thr Phe Ser Gly Lys Val Glu Leu Gln50 55 60Gly Lys Arg Leu Glu Ile Glu His Ser Val Pro Lys Lys Gln Arg Ser65 70 75 80Arg Lys Ile Gln Ile Arg Asn Ile Pro Pro Gln Leu Arg Trp Glu Val85 90 95Leu Asp Ser Leu Leu Ala Gln Tyr Gly Thr Val Glu Asn Cys Glu Gln100 105 110Val Asn Thr Glu Ser Glu Thr Ala Val Val Asn Val Thr Tyr Ser Asn115 120 125Arg Glu Gln Thr Arg Gln Ala Ile Met Lys Leu Asn Gly His Gln Leu130 135 140Glu Asn His Ala Leu Lys Val Ser Tyr Ile Pro Asp Glu Gln Ile Ala145 150 155 160Gln Gly Pro Glu Asn Gly Arg Arg Gly Gly Phe Gly Ser Arg Gly Gln165 170 175Pro Arg Gln Gly Ser Pro Val Ala Ala Gly Ala Pro Ala Lys Gln Gln180 185 190Gln Val Asp Ile Pro Leu Arg Leu Leu Val Pro Thr Gln Tyr Val Gly195 200 205Ala Ile Ile Gly Lys Glu Gly Ala Thr Ile Arg Asn Ile Thr Lys Gln210 215 220Thr Gln Ser Lys Ile Asp Val His Arg Lys Glu Asn Ala Gly Ala Ala225 230 235 240Glu Lys Ala Ile Ser Val His Ser Thr Pro Glu Gly Cys Ser Ser Ala245 250 255Cys Lys Met Ile Leu Glu Ile Met His Lys Glu Ala Lys Asp Thr Lys260 265 270Thr Ala Asp Glu Val Pro Leu Lys Ile Leu Ala His Asn Asn Phe Val275 280 285Gly Arg Leu Ile Gly Lys Glu Gly Arg Asn Leu Lys Lys Val Glu Gln290 295 300Asp Thr Glu Thr Lys Ile Thr Ile Ser Ser Leu Gln Asp Leu Thr Leu305 310 315 320Tyr Asn Pro Glu Arg Thr Ile Thr Val Lys Gly Ala Ile Glu Asn Cys
325 330 335Cys Arg Ala Glu Gln Glu Ile Met Lys Lys Val Arg Glu Ala Tyr Glu340 345 350Asn Asp Val Ala Ala Met Ser Leu Gln Ser His Leu Ile Pro Gly Leu355 360 365Asn Leu Ala Ala Val Gly Leu Phe Pro Ala Ser Ser Ser Ala Val Pro370 375 380Pro Pro Pro Ser Ser Val Thr Gly Ala Ala Pro Tyr Ser Ser Phe Met385 390 395 400Gln Ala Pro Glu Gln Glu Met Val Gln Val Phe Ile Pro Ala Gln Ala405 410 415Val Gly Ala Ile Ile Gly Lys Lys Gly Gln His Ile Lys Gln Leu Ser420 425 430Arg Phe Ala Ser Ala Ser Ile Lys Ile Ala Pro Pro Glu Thr Pro Asp435 440 445Ser Lys Val Arg Met Val Ile Ile Thr Gly Pro Pro Glu Ala Gln Phe450 455 460Lys Ala Gln Gly Arg Ile Tyr Gly Lys Leu Lys Glu Glu Asn Phe Phe465 470 475 480Gly Pro Lys Glu Glu Val Lys Leu Glu Thr His Ile Arg Val Pro Ala485 490 495Ser Ala Ala Gly Arg Val Ile Gly Lys Gly Gly Lys Thr Val Asn Glu500 505 510Leu Gln Asn Leu Thr Ala Ala Glu Val Val Val Pro Arg Asp Gln Thr515 520 525Pro Asp Glu Asn Asp Gln Val Ile Val Lys Ile Ile Gly His Phe Tyr530 535 540Ala Ser Gln Met Ala Gln Arg Lys Ile Arg Asp Ile Leu Ala Gln Val545 550 555 560Lys Gln Gln His Gln Lys Gly Gln Ser Asn Gln Ala Gln Ala Arg Arg565 570 575Lys<210>23<211>329<212>PRT<213>人属、人种<400>23
Met Ala Thr Gly Gln Lys Leu Met Arg Ala Val Arg Val Phe Glu Phe1 5 10 15Gly Gly Pro Glu Val Leu Lys Leu Arg Ser Asp Ile Ala Val Pro Ile20 25 30Pro Lys Asp His Gln Val Leu Ile Lys Val His Ala Cys Gly Val Asn35 40 45Pro Val Glu Thr Tyr Ile Arg Ser Gly Thr Tyr Ser Arg Lys Pro Leu50 55 60Leu Pro Tyr Thr Pro Gly Ser Asp Val Ala Gly Val Ile Glu Ala Val65 70 75 80Gly Asp Asn Ala Ser Ala Phe Lys Lys Gly Asp Arg Val Phe Thr Ser85 90 95Ser Thr Ile Ser Gly Gly Tyr Ala Glu Tyr Ala Leu Ala Ala Asp His100 105 110Thr Val Tyr Lys Leu Pro Glu Lys Leu Asp Phe Lys Gln Gly Ala Ala115 120 125Ile Gly Ile Pro Tyr Phe Thr Ala Tyr Arg Ala Leu Ile His Ser Ala130 135 140Cys Val Lys Ala Gly Glu Ser Val Leu Val His Gly Ala Ser Gly Gly145 150 155 160Val Gly Leu Ala Ala Cys Gln Ile Ala Arg Ala Tyr Gly Leu Lys Ile165 170 175Leu Gly Thr Ala Gly Thr Glu Glu Gly Gln Lys Ile Val Leu Gln Asn180 185 190Gly Ala His Glu Val Phe Asn His Arg Glu Val Asn Tyr Ile Asp Lys195 200 205Ile Lys Lys Tyr Val Gly Glu Lys Gly Ile Asp Ile Ile Ile Glu Met210 215 220Leu Ala Asn Val Asn Leu Ser Lys Asp Leu Ser Leu Leu Ser His Gly225 230 235 240Gly Arg Val Ile Val Val Gly Ser Arg Gly Thr Ile Glu Ile Asn Pro245 250 255Arg Asp Thr Met Ala Lys Glu Ser Ser Ile Ile Gly Val Thr Leu Phe260 265 270Ser Ser Thr Lys Glu Glu Phe Gln Gln Tyr Ala Ala Ala Leu Gln Ala275 280 285Gly Met Glu Ile Gly Trp Leu Lys Pro Val Ile Gly Ser Gln Tyr Pro
290 295 300Leu Glu Lys Val Ala Glu Ala His Glu Asn Ile Ile His Gly Ser Gly305 310 315 320Ala Thr Gly Lys Met Ile Leu Leu Leu325<210>24<211>125<212>PRT<213>人属、人种<400>24Met Gln Pro Ala Ser Ala Lys Trp Tyr Asp Arg Arg Asp Tyr Val Phe1 5 10 15Ile Glu Phe Cys Val Glu Asp Ser Lys Asp Val Asn Val Asn Phe Glu20 25 30Lys Ser Lys Leu Thr Phe Ser Cys Leu Gly Gly Ser Asp Asn Phe Lys35 40 45His Leu Asn Glu Ile Asp Leu Phe His Cys Ile Asp Pro Asn Asp Ser50 55 60Lys His Lys Arg Thr Asp Arg Ser Ile Leu Cys Cys Leu Arg Lys Gly65 70 75 80Glu Ser Gly Gln Ser Trp Pro Arg Leu Thr Lys Glu Arg Ala Lys Leu85 90 95Asn Trp Leu Ser Val Asp Phe Asn Asn Trp Lys Asp Trp Glu Asp Asp100 105 110Ser Asp Glu Asp Met Ser Asn Phe Asp Arg Phe Ser Glu115 120 12权利要求
1.一种用于检测肝脏疾病的生物标记分子，其选自SEQ ID NO1至SEQ IDNO24所示的任一氨基酸序列或其衍生物或其片段或其变异体或其组合物及其对应的自身抗体。
2.如权利要求1所述的检测肝脏疾病的生物标记分子，其中所述肝脏疾病为肝硬化或肝癌。
3.如权利要求1所述的检测肝脏疾病的生物标记分子，其中所述变异体与所述SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列具有大于80％的序列同源性。
4.一种肝脏疾病的检测套组，包含生物标记分子，其选自SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列或其衍生物或其片段或其变异体或其组合物。
5.如权利要求4所述的肝脏疾病的检测套组，其中所述肝脏疾病为肝硬化或肝癌。
6.如权利要求4所述的肝脏疾病的检测套组，其中所述检测套组进一步包含可辨识SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列或其衍生物或其片段或其组合物的自身抗体的二级抗体。
7.一种肝脏疾病的体外筛检方法，包含下列步骤提供一检体；利用SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列或其衍生物或其片段或其变异体或其组合物的生物标记分子辨识捕捉检体中的自身抗体；及侦测自身抗体。
8.如权利要求7所述的体外筛检方法，其中所述检体包含全血或血清。
9.如权利要求8所述的体外筛检方法，其中所述检体为血清。
10.如权利要求7所述的体外筛检方法，其中所述生物标记分子可制成检测套组。
11.如权利要求7所述的体外筛检方法，其中所述生物标记分子可进一步先行固定于基材上。
12.如权利要求11所述的体外筛检方法，其中所述基材为免疫分析盘或生物芯片。
13.如权利要求7所述的体外筛检方法，其中所述检体进一步先行以萤光标记进行标示。
14.如权利要求7所述的体外筛检方法，其中可增加一利用二级抗体辨识吸附自身抗体的步骤。
15.如权利要求14所述的体外筛检方法，其中所述二级抗体经修饰具有特殊官能团可进行显色反应、放射性侦测或萤光侦测。
16.如权利要求7所述的体外筛检方法，其中所述侦测自身抗体是利用萤光扫瞄分析侦测具有萤光标记的自身抗体。
17.如权利要求7所述的体外筛检方法，其中所述侦测自身抗体是利用酶联免疫检测、放射免疫分析或免疫萤光分析侦测二级抗体。
18.一种肝脏疾病的检测套组，包含一组可辨识具有SEQ ID NO1至SEQ IDNO24所示的任一氨基酸序列的抗体。
19.如权利要求18所述的肝脏疾病的检测套组，其中所述肝脏疾病为肝硬化或肝癌。
20.一种肝脏疾病的体外筛检方法，包含下列步骤提供一检体；利用SEQ ID NO1至SEQ ID NO24所示的任一氨基酸序列对应的抗体辨识捕捉检体中的抗原；及侦测抗体与抗原的复合物。
21.如权利要求20所述的体外筛检方法，其中所述检体为全血或血清。
22.如权利要求21所述的体外筛检方法，其中所述检体为血清。
全文摘要
本发明涉及肝脏疾病的生物标记分子及其检测方法，所述生物标记分子，为选自SEQ ID NO1至SEQ NO24所示的任一氨基酸序列或其衍生物或其片段或其变异体或其组合物或其对应的自身抗体；利用这些生物标记分子发展为检测套组，藉筛选检体中自身抗体或自身抗原的存在判断是否罹患肝脏疾�。捎行岣咦既范扔肓槊舳�。
文档编号G01N33/68GK1632589SQ20031012179
公开日2005年6月29日申请日期2003年12月24日优先权日2003年12月24日
发明者曾锱翎, 郑平福申请人:财团法人工业技术研究院

亚星游戏官网-www.yaxin868.com

肝脏疾病的生物标记分子及其检测方法