专利名称::用于治疗和诊断卵巢癌的组合物及方法
技术领域:
:概括地,本发明涉及卵巢癌的治疗。更具体地,本发明涉及包括至少卵巢癌蛋白质一部分的多肽,编码这些多肽的多核苷酸,及特异性识别这些多肽的抗体和免疫系统细胞。这样的多肽,多核苷酸、抗体及细胞可以用在用于卵巢癌治疗的疫苗及药物组合物中。
背景技术:
:在美国及全世界,对妇女而言,卵巢癌是严重的健康问题。尽管在这种癌症的检测和治疗方面已取得了进展,但是目前没有预防或治疗的疫苗或其它普遍成功的方法可用。目前这种疾病的治疗依靠早期诊断和侵入性治疗的联合,这可以包括各种治疗中一种或多种治疗,如手术,放射疗法,化学疗法及激素治疗。常常基于各种预后参数,包括特异肿瘤标记分析选择特定癌症的治疗疗程。然而,已建立的标记的应用常常导致难以解释的结果,而且在许多癌症患者中继续观察到高死亡率。免疫疗法有显著改进癌症治疗及存活的潜力。这种疗法可以涉及对卵巢癌抗原免疫应答的产生及增强。然而,目前为止,几乎没有卵巢癌抗原是已知的,而且也没有证明抗这种抗原的免疫应答的产生有治疗益处。因此,本领域需要鉴定卵巢肿瘤抗原及在卵巢癌治疗中使用这种抗原的改进的方法。本发明满足了这些需要而且进一步提供了其它的相关优点。发明概述简而言之,本发明提供了用于治疗癌症,如卵巢癌的组合物及方法。一方面,本发明提供了包含卵巢癌蛋白质免疫原性部分的多肽或其变异体,变异体的不同在于一个或多个置换、缺失、添加和/或插入,以致变异体与卵巢癌蛋白质特异抗血清的反应能力没有实质减弱。在一些实施方式中,卵巢癌蛋白质包括选自seqidno456-457,460-477和512-570及这些多核苷酸序列的互补序列的多核苷酸序列编码的序列。本发明进一步提供了编码上述多肽或其一部分的多核苷酸,包含这种多核苷酸的表达载体及用这种表达载体转化或转染的宿主细胞。本发明进一步提供了多肽组合物,其包含选自seqidnos394-455,458-459,478-511和571-596所述序列的氨基酸序列。另一方面,本发明提供了药物组合物及疫苗。药物组合物可以包括生理学上可接受的载体或赋形剂,联合以下物质中的一种或多种(i)包含卵巢癌蛋白质免疫原性部分的多肽,或其变异体,所述变异体的不同在于一个或多个置换、缺失、添加和/或插入,以致变异体与卵巢癌蛋白质特异抗血清的反应能力没有实质减弱,其中卵巢癌蛋白质包含如下多核苷酸编码的氨基酸序列,所述多核苷酸包含seqidnos456-457,460-477和512-570所述的任一个序列;或(ii)编码这种多肽的多核苷酸;(iii)特异结合这种多肽的抗体;(iv)表达这种多肽的抗原呈递细胞,和/或(v)与这种多肽特异反应的t细胞。疫苗可以包括非特异性免疫应答增强剂,联合以下物质中的一种或多种(i)包含卵巢癌蛋白质免疫原性部分的多肽,或其变异体,所述变异体的不同在于一个或多个置换、缺失、添加和/或插入,以致变异体与卵巢癌蛋白质特异抗血清的反应能力没有实质减弱,其中卵巢癌蛋白质包含seqidnos394-455,458-459,478-511,及571-596所述的氨基酸序列或以下多核苷酸编码的氨基酸序列,所述多核苷酸包含seqidno456-457,460-477和512-570所述的任一个序列,或(ii)编码这种多肽的多核苷酸;(iii)特异结合这种多肽的抗体特异性结合的抗独特型抗体;(iv)表达这种多肽的抗原呈递细胞,和/或(v)与这种多肽特异反应的t细胞。另一方面,本发明进一步提供了包含至少一个上述多肽的融合蛋白质,及编码这种融合蛋白质的多核苷酸。在相关方面,提供了包含融合蛋白质或编码融合蛋白质的多核苷酸及生理学上可接受载体的药物组合物。另一方面,进一步提供了疫苗,其包含融合蛋白质或编码融合蛋白质的多核苷酸及非特异性免疫增强剂。另一方面,本发明提供了抑制患者癌症发展的方法,包括给患者施用上述药物组合物或疫苗。另一方面,本发明进一步提供了刺激和/或扩增t细胞的方法,包括用(a)包含卵巢癌蛋白质免疫原性部分的多肽,或其变异体,所述变异体的不同在于一个或多个置换、缺失、添加和/或插入,以致变异体与卵巢癌蛋白质特异的抗血清的反应能力没有实质减弱,其中卵巢癌蛋白质包含seqidnos394-455,458-459,478-511,及571-596所述的氨基酸序列或如下多核苷酸编码的氨基酸序列,所述多核苷酸包含seqidno456-457,460-477和512-570所述的任一个序列;(b)编码这种多肽的多核苷酸,和/或(c)表达这种多肽的抗原呈递细胞,在足以允许t细胞刺激和/或扩增的条件和时间下接触t细胞。可以在药物组合物或疫苗中存在这样的多肽,多核苷酸和/或抗原呈递细胞以用于刺激和/或扩增哺乳动物的t细胞。另一方面,本发明提供了抑制患者卵巢癌发展的方法,包括给患者施用上述制备的t细胞。另一方面,本发明提供了抑制患者卵巢癌发展的方法,包括以下步骤(a)温育从患者分离的cd4 和/或cd8 t细胞和以下物质中的一种或多种(i)包含卵巢癌蛋白质免疫原性部分的多肽,或其变异体,变异体的不同在于一个或多个置换、缺失、添加和/或插入,以致变异体与卵巢癌蛋白质特异抗血清的反应能力没有明显减弱,其中卵巢癌蛋白质包含下述多核苷酸编码的氨基酸序列,所述多核苷酸包含seqidno456-457,460-477和512-570所述的任一个序列;(ii)编码这种多肽的多核苷酸;(iii)表达这种多肽的抗原呈递细胞;以使t细胞增殖;和(b)给患者施用有效量的增殖的t细胞,由此抑制患者卵巢癌的发育。在给患者施用前可以克隆所述增殖的细胞。另一方面,本发明也提供了鉴定分泌性肿瘤抗原的方法。这种方法包括以下步骤(a)在免疫缺陷的哺乳动物中植入肿瘤细胞;(b)在足以允许肿瘤抗原分泌进入血清的时间后,从所述免疫缺陷哺乳动物获得血清;(c)用血清免疫免疫活性的哺乳动物;(d)从免疫活性的哺乳动物获得抗血清;和(e)用抗血清筛选肿瘤表达文库,而且由此鉴定分泌性肿瘤抗原。鉴定分泌性卵巢癌抗原的优选方法包括以下步骤(a)在scid小鼠中植入卵巢癌细胞;(b)在足以允许卵巢癌抗原分泌进入血清的时间后,从该scid小鼠获得血清;(c)用该血清免疫具有免疫活性的小鼠;(d)从所述具有免疫活性的小鼠中获得抗血清;和(e)用抗血清筛选卵巢癌表达文库,和由此鉴定分泌性卵巢癌抗原。本发明也公开了o8e多克隆抗血清识别的抗体表位,所述抗体表位如本发明seqidno394-415所示。本发明进一步公开了预测与hla-0201结合的10-mer(聚体)和9-mer肽,这些肽分别是seqidno416-435和seqidno436-455。当参考下面的详细说明及附图时,本发明的这些及其它方面是明显的。本发明公开的所有参考文献都被整体引入作为参考,如每篇文献单独被引用一样。本发明另一个方面,申请人意料外地鉴定出编码o772p基因5’-末端的一系列新重复序列元件。因此,本发明提供了有xn-y所代表的结构的o772p多肽,其中x包含与seqidno596所示o772p重复序列有至少50%同一性,优选至少70%同一性,更优选至少90%同一性的序列。通常y包含与seqidno594所示o772恒定区序列有至少80%同一性,优选至少90%的同一性,更优选至少95%的同一性的序列。根据这个实施方式,通常n是从1到35的整数,优选从15到25的整数,x可以相同或不同。在一个优选实施方式中,x包含选自seqidnos574-593的序列,y包含seqidno594所示序列。在另一个优选实施方式中,示例性o772p多肽包含seqidno595所述序列,含有20个重复序列元件(即,x20),其中x元件以下列顺序排列(在o772p重复区中从n-末端到c-末端移动)seqidno574-seqidno575-seqidno576-seqidno577-seqidno578-seqidno579-seqidno580-seqidno581-seqidno582-seqidno583-seqidno584-seqidno585-seqidno586-seqidno587-seqidno588-seqidno589-seqidno590-seqidno591-seqidno592-seqidno593。根据本发明另一方面,提供了有xn-y结构的o772p多核苷酸,其中x包含选自seqidnos512-540,542-546和548-567任一个的o772p重复序列元件。通常y包含与seqidno586所示o772p恒定区序列有至少80%同一性,优选至少90%同一性,更优选至少95%同一性的序列。在这个实施方式中,通常n是从1到35,优选从15到25的整数,x可以相同或不同。在另一个实施方式中,示例性o772p多核苷酸包含seqidno569所述序列,含有20个重复序列元件(即,x20)。根据本发明另一方面,提供了o772多肽,它包含至少seqidno490-511任一个所示的抗体表位(antibodyepitope)序列。根据本发明另一方面,提供了o8e多肽,其包含至少seqidnos394-415任一个所示的抗体表位序列。序列标识符和附图的简要说明seqidno1-71是图1a-1s所示的卵巢癌抗原多核苷酸。seqidno72-74是图2a-2c所示的卵巢癌抗原多核苷酸。seqidno75是卵巢癌多核苷酸3g(图4)。seqidno76是卵巢癌多核苷酸3f(图5)。seqidno77是卵巢癌多核苷酸6b(图6)。seqidno78是卵巢癌多核苷酸8e(图7a)。seqidno79是卵巢癌多核苷酸8h(图7b)。seqidno80是卵巢癌多核苷酸12e(图8)。seqidno81是卵巢癌多核苷酸12h(图9)。seqidno82-310是图15a-15eee所示的卵巢癌抗原多核苷酸。seqidno311是卵巢癌多核苷酸o772p的全长序列。seqidno312是o772p氨基酸序列。seqidno313-384是卵巢癌抗原多核苷酸。seqidno385代表克隆o772p一种形式的cdna序列,命名为21013。seqidno386代表克隆o772p一种形式的cdna序列,命名为21003。seqidno387代表克隆o772p一种形式的cdna序列,命名为21008。seqidno388是与seqidno385对应的氨基酸序列。seqidno389是与seqidno386对应的氨基酸序列。seqidno390是与seqidno387对应的氨基酸序列。seqidno391是卵巢癌多核苷酸o8e的全长序列。seqidno392-393是o8e编码的蛋白质序列。seqidno394-415是与oe8抗体表位对应的肽序列。seqidno416-435是用oe8全长开放阅读框架预测的可能的hla-a210mer结合肽。seqidno436-455是用oe8全长开放阅读框架预测的可能的hla-a29mer结合肽。seqidno456是显示与o772p有同源性的全长genbank序列的截短核苷酸序列。seqidno457是显示与o772p有显著同源性的全长genbank序列。seqidno458是编码显示与o772p有同源性的全长genbank序列的截短形式的蛋白质。seqidno459是显示与o772p蛋白质序列有显著同源性的genbank的全长蛋白质序列。seqidno460编码包含在残基1-70中的o772p独特的n-末端部分。seqidno461包含单一序列并编码seqidno456残基1-313。seqidno462是克隆o772p的假设序列。seqidno463是克隆flj14303的cdna序列。seqidno464是克隆o772p的部分cdna序列。seqidno465是克隆o772p的部分cdna序列。seqidno466是克隆o772p的部分cdna序列。seqidno467是克隆o772p的部分cdna序列。seqidno468是克隆o772p的部分cdna序列。seqidno469是克隆o772p的部分cdna序列。seqidno470是克隆o772p的部分cdna序列。seqidno471是克隆o772p的部分cdna序列。seqidno472是克隆o772p的部分cdna序列。seqidno473是克隆o772p的部分cdna序列。seqidno474是克隆o772p的部分cdna序列。seqidno475是克隆o772p的部分cdna序列。seqidno476是克隆o772p的部分cdna序列。seqidno477代表卵巢肿瘤抗原o772p新的5’-末端。seqidno478是seqidno462编码的氨基酸序列。seqidno479是seqidno463编码的氨基酸序列。seqidno480是seqidno472编码的部分氨基酸序列。seqidno481是seqidno471可能的开放阅读框架编码的部分氨基酸序列。seqidno482是seqidno471第二个可能的开放阅读框架编码的部分氨基酸序列。seqidno483是seqidno467编码的部分氨基酸序列。seqidno484是seqidno466可能的开放阅读框架编码的部分氨基酸序列。seqidno485是seqidno466第二个可能的开放阅读框架编码的部分氨基酸序列。seqidno486是seqidno465编码的部分氨基酸序列。seqidno487是seqidno464编码的部分氨基酸序列。seqidno488代表o772p的细胞外、跨膜和细胞质区。seqidno489代表o772p的预测细胞外结构域。seqidno490代表与o772p特异抗体表位对应的肽#2的氨基酸序列。seqidno491代表与o772p特异抗体表位对应的肽#6的氨基酸序列。seqidno492代表与o772p特异抗体表位对应的肽#7的氨基酸序列。seqidno493代表与o772p特异抗体表位对应的肽#8的氨基酸序列。seqidno494代表与o772p特异抗体表位对应的肽#9的氨基酸序列。seqidno495代表与o772p特异抗体表位对应的肽#11的氨基酸序列。seqidno496代表与o772p特异抗体表位对应的肽#13的氨基酸序列。seqidno497代表与o772p特异抗体表位对应的肽#22的氨基酸序列。seqidno498代表与o772p特异抗体表位对应的肽#24的氨基酸序列。seqidno499代表与o772p特异抗体表位对应的肽#27的氨基酸序列。seqidno500代表与o772p特异抗体表位对应的肽#40的氨基酸序列。seqidno501代表与o772p特异抗体表位对应的肽#41的氨基酸序列。seqidno502代表与o772p特异抗体表位对应的肽#47的氨基酸序列。seqidno503代表与o772p特异抗体表位对应的肽#50的氨基酸序列。seqidno504代表与o772p特异抗体表位对应的肽#51的氨基酸序列。seqidno505代表与o772p特异抗体表位对应的肽#52的氨基酸序列。seqidno506代表与o772p特异抗体表位对应的肽#53的氨基酸序列。seqidno507代表与o772p特异抗体表位对应的肽#58的氨基酸序列。seqidno508代表与o772p特异抗体表位对应的肽#59的氨基酸序列。seqidno509代表与o772p特异抗体表位对应的肽#60的氨基酸序列。seqidno510代表与o772p特异抗体表位对应的肽#61的氨基酸序列。seqidno511代表与o772p特异抗体表位对应的肽#71的氨基酸序列。seqidno512(o772p重复1)代表与o772p5’可变区重复号21对应的cdna序列实例。seqidno513(o772p重复2)代表与o772p5’可变区重复号20对应的cdna序列实例。seqidno514(o772p重复3)代表与o772p5’可变区重复号19对应的cdna序列实例。seqidno515(o772p重复4)代表与o772p5’可变区重复号18对应的cdna序列实例。seqidno516(o772p重复5)代表与o772p5’可变区重复号17对应的cdna序列实例。seqidno517(hb重复1)代表与o772p5’可变区重复号21对应的cdna序列实例。seqidno518(hb重复2)代表与o772p5’可变区重复号20对应的cdna序列实例。seqidno519(hb重复3)代表与o772p5’可变区重复号19对应的cdna序列实例。seqidno520(hb重复4)代表与o772p5’可变区重复号18对应的cdna序列实例。seqidno521(hb重复5)代表与o772p5’可变区重复号17对应的cdna序列实例。seqidno522(hb重复65’-末端)代表与o772p5’可变区重复号16对应的cdna序列实例。seqidno523(1043400.1重复1)代表与o772p5’可变区重复号9对应的cdna序列实例。seqidno524(1043400.1重复2)代表与o772p5’可变区重复号10对应的cdna序列实例。seqidno525(1043400.1重复3)代表与o772p5’可变区重复号10/11对应的cdna序列实例。seqidno526(1043400.1重复4)代表与o772p5’可变区重复号11对应的cdna序列实例。seqidno527(1043400.1重复5)代表与o772p5’可变区重复号14对应的cdna序列实例。seqidno528(1043400.1重复6)代表与o772p5’可变区重复号17对应的cdna序列实例。seqidno529(1043400.3重复1)代表与o772p5’可变区重复号20对应的cdna序列实例。seqidno530(1043400.3重复2)代表与o772p5’可变区重复号21对应的cdna序列实例。seqidno531(1043400.5重复1)代表与o772p5’可变区重复号8对应的cdna序列实例。seqidno532(1043400.5重复2)代表与o772p5’可变区重复号9对应的cdna序列实例,此外还含有内含子序列。seqidno533(1043400.5重复2)代表与o772p5’可变区重复号9对应的cdna序列实例。seqidno534(1043400.8重复1)代表与o772p5’可变区重复号17对应的cdna序列实例。seqidno535(1043400.8重复2)代表与o772p5’可变区重复号18对应的cdna序列实例。seqidno536(1043400.8重复3)代表与o772p5’可变区重复号19对应的cdna序列实例。seqidno537(1043400.9重复1)代表与o772p5’可变区重复号4对应的cdna序列实例。seqidno538(1043400.9重复2)代表与o772p5’可变区重复号5对应的cdna序列实例。seqidno539(1043400.9重复3)代表与o772p5’可变区重复号7对应的cdna序列实例。seqidno540(1043400.9重复4)代表与o772p5’可变区重复号8对应的cdna序列实例。seqidno541(1043400.11重复1)代表与o772p5’可变区重复号1对应的cdna序列实例。seqidno542(1043400.11重复2)代表与o772p5’可变区重复号2对应的cdna序列实例。seqidno543(1043400.11重复3)代表与o772p5’可变区重复号3对应的cdna序列实例。seqidno544(1043400.11重复4)代表与o772p5’可变区重复号11对应的cdna序列实例。seqidno545(1043400.11重复5)代表与o772p5’可变区重复号12对应的cdna序列实例。seqidno546(1043400.12重复1)代表与o772p5’可变区重复号20对应的cdna序列实例。seqidno547(pb重复a)代表与o772p5’可变区重复号1对应的cdna序列实例。seqidno548(pb重复b)代表与o772p5’可变区重复号2对应的cdna序列实例。seqidno549(pb重复e)代表与o772p5’可变区重复号3对应的cdna序列实例。seqidno550(pb重复g)代表与o772p5’可变区重复号4对应的cdna序列实例。seqidno551(pb重复c)代表与o772p5’可变区重复号4对应的cdna序列实例。seqidno552(pb重复h)代表与o772p5’可变区重复号6对应的cdna序列实例。seqidno553(pb重复j)代表与o772p5’可变区重复号7对应的cdna序列实例。seqidno554(pb重复k)代表与o772p5’可变区重复号8对应的cdna序列实例。seqidno555(pb重复d)代表与o772p5’可变区重复号9对应的cdna序列实例。seqidno556(pb重复i)代表与o772p5’可变区重复号10对应的cdna序列实例。seqidno557(pb重复m)代表与o772p5’可变区重复号11对应的cdna序列实例。seqidno558(pb重复9)代表与o772p5’可变区重复号12对应的cdna序列实例。seqidno559(pb重复8.5)代表与o772p5’可变区重复号13对应的cdna序列实例。seqidno560(pb重复8)代表与o772p5’可变区重复号14对应的cdna序列实例。seqidno561(pb重复7)代表与o772p5’可变区重复号15对应的cdna序列实例。seqidno562(pb重复6)代表与o772p5’可变区重复号16对应的cdna序列实例。seqidno563(pb重复5)代表与o772p5’可变区重复号17对应的cdna序列实例。seqidno564(pb重复4)代表与o772p5’可变区重复号18对应的cdna序列实例。seqidno565(pb重复3)代表与o772p5’可变区重复号19对应的cdna序列实例。seqidno566(pb重复2)代表与o772p5’可变区重复号20对应的cdna序列实例。seqidno567(pb重复1)代表与o772p5’可变区重复号21对应的cdna序列实例。seqidno568代表3’恒定区cdna序列。seqidno569代表含有21个重复之共有序列,3’恒定区和3’非翻译区的cdna序列。seqidno570代表共有重复序列的cdna序列。seqidno571代表o772p5’可变区重复号1的一个可能开放阅读框架的共有氨基酸序列。seqidno572代表o772p5’可变区重复号1的第二个可能开放阅读框架的共有氨基酸序列。seqidno573代表o772p5’可变区重复号1的第三个可能开放阅读框架的共有氨基酸序列。seqidno574代表o772p5’可变区重复号2的共有氨基酸序列。seqidno575代表o772p5’可变区重复号3的共有氨基酸序列。seqidno576代表o772p5’可变区重复号4的共有氨基酸序列。seqidno577代表o772p5’可变区重复号5的共有氨基酸序列。seqidno578代表o772p5’可变区重复号6的共有氨基酸序列。seqidno579代表o772p5’可变区重复号7的共有氨基酸序列。seqidno580代表o772p5’可变区重复号8的共有氨基酸序列。seqidno581代表o772p5’可变区重复号9的共有氨基酸序列。seqidno582代表o772p5’可变区重复号10的共有氨基酸序列。seqidno583代表o772p5’可变区重复号11的共有氨基酸序列。seqidno584代表o772p5’可变区重复号12的共有氨基酸序列。seqidno585代表o772p5’可变区重复号13的共有氨基酸序列。seqidno586代表o772p5’可变区重复号14的共有氨基酸序列。seqidno587代表o772p5’可变区重复号15的共有氨基酸序列。seqidno588代表o772p5’可变区重复号16的共有氨基酸序列。seqidno589代表o772p5’可变区重复号17的共有氨基酸序列。seqidno590代表o772p5’可变区重复号18的共有氨基酸序列。seqidno591代表o772p5’可变区重复号19的共有氨基酸序列。seqidno592代表o772p5’可变区重复号20的共有氨基酸序列。seqidno593代表o772p5’可变区重复号21的共有氨基酸序列。seqidno594代表3’恒定区的氨基酸序列。seqidno595代表含有21个重复之共有序列和3’恒定区的氨基酸序列。seqidno596代表共有重复序列的氨基酸序列。图1a-1s(seqidno1-71)描述了编码代表性分泌型卵巢癌抗原的多核苷酸的部分序列。图2a-2c描述了图1的3个克隆的全部插入序列。图2表示命名为o7e(11731;seqidno72)的序列,图2b表示命名为o9e(11785;seqidno73)的序列,及图2c表示命名为o8e(13695;seqidno74)的序列。图3表示命名为o8e的卵巢癌序列微阵列表达分析结果。图4表示编码卵巢癌序列的多核苷酸的部分序列(命名为3g;seqidno75),所述的卵巢癌序列是人t-细胞白血病病毒类型i癌蛋白质tax和骨粘连蛋白的剪接融合体。图5表示命名为3f(seqidno76)的卵巢癌多核苷酸。图6表示命名为6b(seqidno77)的卵巢癌多核苷酸。图7a和7b表示命名为8e(seqidno78)和8h(seqidno79)的卵巢癌多核苷酸。图8表示命名为12c(seqidno80)的卵巢癌多核苷酸。图9表示命名为12h(seqidno81)的卵巢癌多核苷酸。图10描述了命名为3f的卵巢癌序列微阵列表达分析结果。图11描述了命名为6b的卵巢癌序列微阵列表达分析结果。图12描述了命名为8e的卵巢癌序列微阵列表达分析结果。图13描述了命名为12c的卵巢癌序列微阵列表达分析的结果。图14描述了命名为12h的卵巢癌序列微阵列表达分析的结果。图15a-15eee描述了编码代表性分泌型卵巢癌抗原的附加多核苷酸的部分序列(seqidno82-310)。图16是示例全长序列中各种部分o8e序列的位置的图解。图17是示例对o8e蛋白质的表位作图研究结果图。图18是o8e细胞表面表达的荧光激活细胞分类术(facs)分析图。图19是o8e细胞表面表达的荧光激活细胞分类术(facs)分析图。图20表示o8e转染的hek293细胞的荧光激活细胞分类术(facs)分析,证明了o8e的细胞表面表达。图21表示skbr3乳腺肿瘤细胞的荧光激活细胞分类术(facs)分析,证明了o8e的细胞表面表达。图22表示hek293细胞中的o8e表达。用抗o8e兔多克隆抗血清#233l探测细胞。图23表示抗o8e兔血清的elisa分析。图24表示亲和纯化的兔抗o8e多克隆抗体的elisa分析。图25是测定抗o8emab(单克隆抗体)的抗体内化的图,表示抗氨基酸61-80的mabs诱导了配体内化。发明详述如上所述,概括地,本发明涉及用于癌症(如卵巢癌)治疗的组合物和方法。本发明所述组合物可以包括免疫原性多肽,编码这种多肽的多核苷酸,结合多肽的结合剂如抗体,抗原呈递细胞(apcs)和/或免疫系统细胞(例如,t细胞)。本发明多肽通常包含至少卵巢癌蛋白质或其变异体的免疫原性部分。用免疫测定技术已鉴定出一些卵巢癌蛋白质,而且在本发明中被称为卵巢癌抗原。“卵巢癌抗原”是卵巢肿瘤细胞(优选人细胞)以至少高于正常卵巢细胞水平至少2倍的水平表达的蛋白质。某些卵巢癌抗原可检测地(在免疫测定中,如elisa或westernblot)与抗植入了人卵巢肿瘤的免疫缺陷动物血清产生的抗血清反应。这样的卵巢癌抗原从卵巢肿瘤流出或分泌进入免疫缺陷动物的血清。因此,本发明提供的一些卵巢癌抗原是分泌型抗原。本发明的一些核酸序列通常包含编码这种多肽全部或一部分的dna或rna序列,或是这种序列的互补序列。本发明进一步提供了用评估卵巢肿瘤中表达改变的技术鉴定的卵巢癌序列。这样的序列可以是多核苷酸或蛋白质序列。卵巢癌序列在卵巢肿瘤中通常以高于正常卵巢组织中表达水平至少2倍,优选至少5倍的水平表达,这是用这里提供的代表性测定法测定的。本发明提供了一些部分卵巢癌多核苷酸序列。包含这样的多核苷酸序列(或其互补序列)的基因编码的蛋白质也认为是卵巢癌蛋白质。抗体通常是免疫系统蛋白质,或其抗原结合片段,它们能结合本文所述卵巢癌多肽的至少一部分。本发明提供的组合物中可以应用的t细胞通常是对这种多肽特异的t细胞(例如,cd4 和/或cd8 )。本文所述的一些方法进一步利用了表达这里提供的卵巢癌多肽的抗原呈递细胞(如树突细胞或巨噬细胞)。卵巢癌多核苷酸编码本文所述卵巢癌蛋白质或其一部分或其它变异体的任何多核苷酸都包括在本发明中。优选的多核苷酸包含编码卵巢癌蛋白质一部分的至少15个连续核苷酸,优选至少30个连续核苷酸,更优选至少45个连续核苷酸。更优选,多核苷酸编码卵巢癌蛋白质,如卵巢癌抗原的免疫原性部分。与任何这样的序列互补的多核苷酸也包括在本发明中。多核苷酸可以是单链(编码链或反义链)或双链,而且可以是dna(基因组、cdna或合成的)或rna分子。附加的编码或非编码序列可以,但不是必需,存在于本发明多核苷酸中,而且多核苷酸可以,但不是必需,与其它分子和/或支持材料连接。多核苷酸可以包含天然序列(即,编码卵巢癌蛋白质或其部分的内源序列)或可以包含这样的序列的变异体。多核苷酸变异体可以包含一个或多个置换、添加、缺失和/或插入,使得与天然卵巢癌蛋白质比较,所述编码多肽的免疫原性不减少。通常可以如本文所述评估对编码多肽的免疫原性的影响。变异体优选显示与编码天然卵巢癌蛋白质或其部分的多核苷酸有至少约70%同一性,更优选有至少约80%同一性,最优选有至少约90%同一性。通过用本领域技术人员熟知的计算机算法,如megalign,用默认参数,比较序列可以容易地检测两个多核苷酸或多肽序列的同一性百分率。通常通过“比较窗口”比较序列以鉴定和比较序列相似性的局部区域进行两个序列间比较。本文所用“比较窗口”指至少约20个连续位置,通常30到约75个,或40到约50个,其中两个序列最佳对齐(aligned)后,序列可以与同样数目连续位置的参照序列比较。可以用,例如,lasergene这套生物信息学软件(dnastar,inc.,madison,wi)的megalign程序,用默认参数进行比较序列的最佳对齐。优选地,通过在至少20个位置的比较窗口比较两个最佳对齐序列检测序列同一性的百分数,其中窗口中多核苷酸或多肽序列部分与参照序列(不包含添加或缺失)比较可以包含20%或以下,通常5-15%,或10-12%的添加或缺少(即缺口)。可以通过测定在两个序列中出现相同核酸碱基或氨基酸残基的位置数得出匹配位置数,匹配位置数除以参照序列总位置数(即,窗口大小),用100乘以所得结果得出序列同一性的百分数,来计算同一性百分数。变异体也可以,或作为替代方案,与天然基因,或其部分或互补序列显著同源。在中度严格条件下,这样的多核苷酸变异体能与编码天然卵巢癌蛋白质(或互补序列)的天然存在的dna序列杂交。适合的中度严格条件包括在5×ssc,0.5%sds,1.0mmedta(ph8.0)溶液中预洗;在50℃-65℃,5×ssc中杂交过夜;随后65℃,用含有0.1%sds的2×,0.5×和0.2×ssc各洗2次20分钟。本领域技术人员将理解,做为遗传密码简并的结果,存在许多编码本发明所述多肽的核苷酸序列。这些多核苷酸中一些与任何天然基因的核苷酸序列具有最小限度的同源性。因此,本发明特别考虑了由于密码子使用不同而改变的多核苷酸。而且,包含本文提供的多核苷酸序列的基因的等位基因在本发明范围内。等位基因是由于一个或多个突变,如核苷酸缺失、添加和/或置换而改变的内源基因。由此产生的mrna和蛋白质可以,但不是必需,有改变的结构或功能。可以用标准技术(例如杂交、扩增和/或数据库序列比较)鉴定等位基因。可以用各种技术中任一种制备多核苷酸。例如,如下文将详述的,可以通过用如下抗血清筛选晚期传代卵巢肿瘤表达文库鉴定卵巢癌多核苷酸,所述抗血清是在用植入晚期传代卵巢肿瘤的scid小鼠血清注射免疫活性的小鼠后用这种小鼠的血清产生的。卵巢癌多核苷酸也可以用目的是评估差异基因表达的各种技术中任一种鉴定。或者,可以从卵巢肿瘤细胞制备的cdna扩增多核苷酸。可以通过聚合酶链式反应(pcr)扩增这样的多核苷酸。对于这个方法而言,可以基于本发明提供的序列设计、可以购买或合成序列特异的引物。可以用扩增后的部分,利用公知技术,从合适文库(例如,卵巢癌cdna文库)分离全长基因。在这样的技术中,用一个或多个多核苷酸探针或适合扩增的引物筛选文库(cdna或基因组)。优选地,按大小筛选文库以包括较大分子。也可以优选随机引物文库以鉴定基因5’和上游区域。优选基因组文库以获得内含子和延伸5’序列。对于杂交技术,可以用公知技术标记(例如,通过用32p切口平移或末端标记)部分序列。随后通过用标记探针(参见sambrook等,molecularcloningalaboratorymanual,coldspringharborlaboratories,coldspringharbor,ny,1989)杂交含有变性细菌菌落(或含有噬菌斑的菌苔)的滤膜筛选细菌或细菌噬菌体文库。筛选和扩大杂交菌落或噬菌斑,分离dna进行进一步分析。可以通过,例如,用部分序列上的引物和载体引物的pcr分析cdna克隆以检测附加序列的量。可以产生限制性图谱和部分序列上的以鉴定一个和多个重叠克隆。然后可以用标准技术测定完整序列,这包括产生一系列缺失克隆。随后,装配由此产生的重叠序列为单一连续序列。用公知技术,通过连接适合片段可以产生全长cdna分子。作为替代方案,存在从部分cdna序列获得全长编码序列的大量扩增技术。在这些技术中,通常用pcr进行扩增。可以用各种商用试剂盒的任一个进行扩增步骤。可以用,例如,本领域公知的软件设计引物。优选22-30核苷酸长度,有至少50%gc含量和在约68-72℃温度下与靶序列退火的引物。如上所述可以测序扩增区域,而且可以装配重叠序列为连续序列。这种扩增技术之一是反向pcr(参见triglia等,nucl.acidsres.168186,1988),其用限制性内切酶在基因已知区产生片段。然后通过分子内连接环化这个片段,用这个片段作为模板,用来源于已知区的不同方向的引物进行pcr。在可替换的方法中,可以通过用接头序列的引物和对已知区域特异的引物扩增获得邻接部分序列的序列。扩增后的序列通常用同样的接头引物和对已知区域特异的第二个引物进行第二轮扩增。这个方法的改变是利用在已知序列相反方向开始延伸的两个引物,这在wo96/38591中描述了。另外的技术包括捕获pcr(capturepcr)(lagrstrom等,pcrmethodsapplic.1111-19,1991)和步行pcr(walkingpcr)(parker等,nucl.acids.res.193055-60,1991)。也可以利用其它采用扩增的方法以获得全长cdna序列。在一些情况下,通过已表达序列标志(est)数据库中提供的序列的分析可能获得全长cdna序列,可从genbank中得到已表达序列标志(est)数据库。用公知程序(例如,ncbiblast检索)通常可以进行重叠ests的检索,而且可以用这样的ests产生连续的全长序列。图1a-1s(seqidno1-71)和图15a-15eee(seqidno82-310)提供了编码部分卵巢癌抗原的cdna分子的一些核酸序列。图1a-1s提供的序列看来是新的。对于图15a-15eee的序列,数据库检索表明了有显著同一性的配对。这些多核苷酸是用目的是鉴定分泌的肿瘤抗原的技术,通过卵巢肿瘤cdna表达文库的血清学筛选分离的。简而言之,在载体λ-screen(novagen)中从衍生于scid的人卵巢肿瘤(ov9334)制备晚期传代卵巢肿瘤表达文库。通过用植入了晚期传代卵巢肿瘤的scid小鼠的血清注射免疫活性小鼠获得用于筛选的血清。这个技术允许编码分泌性肿瘤抗原免疫原性部分的cdna分子的鉴定。具体地,本发明包括这里所述的多核苷酸,及包含这种序列的全长多核苷酸,这种全长多核苷酸的其它部分,和与这样全长分子的全部或部分互补的序列。这个技术也可以用于其它类型肿瘤分泌的抗原的鉴定,这对本领域技术人员显而易见。图4-9(seqidno75-81)及seqidno313-384提供了编码部分卵巢癌蛋白质的cdna分子的其它核酸序列。这些序列是通过筛选肿瘤相关表达的微阵列cdna(即,用本文的代表性测定法测定,卵巢肿瘤中表达至少高于正常卵巢组织5倍)鉴定的。根据制造商说明(而且基本上如schena等,proc.natl.acad.sci.usa9310614-10619,1996和heller等,proc.natl.acad.sci.usa942150-2155所述)用synteni微阵列(paloaltoca)进行这样的筛选。seqidno311和319提供了包含这些核酸序列中一些的全长序列。各种公知技术中任何技术都可以用于评估cdna的肿瘤相关表达。例如,可以应用利用标记多核苷酸探针的杂交技术。可供选择地,或此外,可以用扩增技术,如实时(real-time)pcr(参见gibson等,genomeresearch6995-1001,1996;heid等,genomeresearch6986-994,1996)。实时pcr是评估扩增期间pcr产物积累水平的技术。这个技术允许多个样品中mrna水平的定量评估。简而言之,从肿瘤和正常组织提取mrna,而且用标准技术制备cdna。例如,用perkinelmer/appliedbiosystem(fostercity,ca)7700prism仪器可以进行实时pcr。用例如perkinelmer/appliedbiosystem(fostercity,ca)提供的primerexpress程序,可以为目的基因设计配对引物及荧光探针。本领域技术人员可以最初确定引物和探针的最佳浓度,而且可以,例如从perkinelmer/appliedbiosystem(fostercity,ca)购得对照(例如β-肌动蛋白)引物和探针。为了测定样品中特定rna的量,同时用含有目的基因的质粒产生标准曲线。可以用实时pcr测定的ct值产生标准曲线,所述值与用在测定法中的起始cdna浓度有关。通常范围为10-106目的基因拷贝的标准稀释是足够的。此外,产生对照序列的标准曲线。这允许组织样品起始rna含量相对于对照量的标准化,以便于比较。通常可以通过本领域公知的任何方法制备多核苷酸变异体,包括通过例如固相亚磷酰胺化学合成的化学合成。用标准诱变技术,如寡核苷酸定向位点特异诱变(参见adelman等,dna2183,1983)也可以在核苷酸序列中引入修饰。或者,假如dna与适合的rna聚合酶启动子(如t7或sp6)一起被引入载体,那么通过编码卵巢癌抗原或其部份的dna序列体外或体内转录可以产生rna分子。如本发明所述,一些部分可以用于制备编码多肽。此外,或作为替代方案,可以给患者施用一部分以便体内产生编码多肽。与编码序列互补的序列的一部分(即反义多核苷酸)也可以用做探针或调节基因表达。可以转录成反义rna的cdna构建体可以被引入细胞或组织以有利于反义rna产生。也可以用本发明所述反义多核苷酸抑制卵巢癌蛋白质的表达。可以用反义技术通过三股螺旋的形成控制基因表达,三股螺旋的形成损坏双螺旋充分打开以结合聚合酶、转录因子或调节分子的能力(参见gee等,在huber和carr,molecularandimmunologicapproaches,futurapublishingco.(mt.kisco,ny;1994)中的描述)。可备选地,可以设计反义分子与基因控制区(例如,启动子、增强子或转录起始位点)杂交,而且阻止基因转录;或通过抑制转录物与核糖体结合阻止翻译。任何多核苷酸都可以进一步被修饰以增强体内稳定性。可能的修饰包括,但不限于,在5’和/或3’末端增加侧翼序列;在主链中用硫代磷酸酯或2’o-甲基,而不是磷酸二酯酶键接;和/或包括非常规的碱基如次黄嘌呤核苷,queosine,wybutosine,及腺嘌呤,胞嘧啶核苷,鸟嘌呤,胸腺嘧啶和脲嘧啶核苷的乙酰-,甲基-,硫代和其它修饰形式。用已建立的重组dna技术可以将本发明所述的核苷酸序列和各种其它核苷酸序列连接。例如,可以克隆多核苷酸进入各种克隆载体,包括质粒、噬菌粒、λ-噬菌体衍生物和粘粒。特别有用的载体包括表达载体,复制载体,探针产生载体和测序载体。一般地,载体将包含在至少一种生物中起作用的复制起点,方便的限制性内切酶位点和一个和多个筛选标记。其它元件取决于期望的用途,而且对本领域技术人员显而易见。在一些具体实施例方式中,可以配制多核苷酸以允许进入哺乳动物细胞,并在其中表达。这样的制剂对治疗目的特别有用,如下文所述。本领域技术人员将理解存在许多使多核苷酸在靶细胞中表达的方法,而且可以利用任何合适的方法。例如,可以将多核苷酸引入病毒载体,如,但不限于腺病毒,腺相关病毒,反转录病毒,或痘苗或其它痘病毒(例如,禽痘病毒)。引入dna进入这类载体的技术是本领域技术人员公知的。反转录病毒可以附加地转移或引入筛选标记基因(以帮助转导细胞的鉴定或筛选)和/或靶组成成分,如编码特定靶细胞之受体的配体的基因,以使载体有靶特异性。也可以用抗体,通过本领域技术人员公知的方法完成靶向。其它用于治疗目的的制剂包括胶体分散系统,如大分子复合物、纳米胶囊、微球体、珠粒和基于脂质的系统,包括水包油乳剂、微胶粒、混合微胶粒和脂质体。用做体外和体内递送载体的优选胶体系统是脂质体(即人工膜泡囊)。这类系统的制备和用途是本领域公知的。卵巢癌多肽在本
发明内容中,多肽可以包含至少卵巢癌蛋白质或其变异体的免疫原性部分,如本文所述。如上所述,一些卵巢癌蛋白质是卵巢肿瘤细胞表达的卵巢癌抗原,而且在免疫测定(如elisa)中可检测地与如下抗血清反应,所述抗血清是针对植入了卵巢肿瘤的免疫缺陷动物的血清产生的。本文所述的卵巢癌多核苷酸编码其它卵巢癌蛋白质。本发明所述多肽可以是任何长度。可以存在衍生于天然蛋白质和/或异源序列的附加序列,而且这样的序列可以(但不是必须)有进一步的免疫原性或抗原性。本文所用“免疫原性部分”是b细胞和/或t细胞表面抗原受体识别(即特异结合)的抗原部分。这样的免疫原性部分通常包含卵巢癌蛋白质或其变异体的至少5个氨基酸残基,更优选地至少10,而且更优选地至少20个氨基酸残基。优选的免疫原性部分是按本文所述分离的cdna分子编码的。更多的免疫原性部分通常可以用公知技术鉴定,如在paul,fundamentalimmunology,第三版,243-247(ravenpress,1993)中及其所引参考文献所概括的。这样的技术包括筛选能与卵巢癌蛋白质特异的抗体,抗血清和/或t细胞系或克隆反应的多肽。如果抗血清和抗体特异结合卵巢癌蛋白质(即在elisa或其它免疫测定中它们与卵巢癌蛋白质反应,而不与其它不相关蛋白质可检测地反应),那么如本文所用的抗血清和抗体是“卵巢癌蛋白质特异的”。用公知技术,可以如本文所述制备这样的抗血清、抗体和t细胞。天然卵巢癌蛋白质的免疫原性部分是与这样的抗血清、抗体和/或t细胞以不显著少于全长多肽反应性的水平(即在elisa和/或t细胞反应性测定法中)反应的部分。在这样的测定法中,这样的免疫原性部分可以以相似或高于全长蛋白质反应性的水平反应。通常可以用本领域技术人员公知的方法进行这样筛选,如在harlow和lane,antibodiesalaboratorymanual,coldspringharborlaboratory,1988所述。例如,可以在固相载体上固定多肽,用患者血清接触以允许在血清内的抗体与固定的多肽结合。然后除去未结合的血清,而且用,例如125i标记的蛋白质a检测结合的抗体。如上所述,组合物可以包括天然卵巢癌蛋白质的变异体。多肽“变异体”,如本文所述,是通过一个或多个置换、缺失、和/或插入而不同于天然卵巢癌蛋白质的多肽,这使得该多肽免疫原性没有显著减弱。换而言之,与天然卵巢癌蛋白质比较,变异体与卵巢癌蛋白质特异的抗血清反应的能力可能加强或无变化,或与天然卵巢癌蛋白质比较可能减弱少于50%,优选少于20%。通过修饰上述多肽序列之一和评估修饰多肽与如本发明所述卵巢癌蛋白质特异的抗体或抗血清的反应性,通常可以鉴定这样的变异体。优选的变异体包括其中除去了一个或多个部分,如n-末端前导序列或跨膜结构域的变异体。其它优选的变异体包括从成熟蛋白质n-末端和/或c-末端除去一小部分(例如,1-30个氨基酸,优选5-15个氨基酸)的变异体。优选多肽变异体显示与天然多肽有至少约70%,更优选至少约90%,而且最优选至少约95%的同一性。优选地,变异体包含保守置换。“保守置换”是与一个氨基酸与另一个有相似特性的氨基酸置换,因此肽化学领域的技术人员将预期多肽的二级结构和亲水性没有显著变化。通常可以在残基极性、电荷、溶解度、疏水性、亲水性和/或两亲性相似的基础上进行氨基酸置换。例如,带负电荷氨基酸包括天冬氨酸和谷氨酸;带正电荷氨基酸包括赖氨酸和精氨酸;有相似亲水性值,不带电荷极性头基团的氨基酸包括亮氨酸、异亮氨酸和缬氨酸;甘氨酸和丙氨酸;天冬酰胺和谷氨酰胺;和丝氨酸、苏氨酸、苯丙氨酸和酪氨酸。其它可以代表保守改变的氨基酸组包括(1)丙氨酸、脯氨酸、甘氨酸、谷氨酸、天冬氨酸、谷氨酰胺、天冬酰胺、丝氨酸、苏氨酸;(2)半胱氨酸、丝氨酸、酪氨酸、苏氨酸;(3)缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、丙氨酸、苯丙氨酸;(4)赖氨酸、精氨酸、组氨酸;和(5)苯丙氨酸、酪氨酸、色氨酸、组氨酸。变异体也可以,或可备选地包含非保守改变。例如通过对多肽免疫原性、二级结构和亲水性有轻微影响的氨基酸的缺失或添加也可以(或可备选地)修饰变异体。如上所述,多肽可以在蛋白质端n-末端包含共翻译或翻译后引导蛋白质转移的信号(或前导)序列。多肽也可以与接头或其它易于多肽合成、纯化或鉴定,或增强多肽与固相载体结合的序列缀合(conjugated)。例如多肽可以与免疫球蛋白fc区域缀合。可以用各种公知方法中任一种制备多肽。用本领域技术人员公知的各种表达载体中任一种,从dna序列可以容易地制备本发明所述dna序列编码的重组多肽。可以在包含重组多肽编码dna分子的表达载体转化或转染的适合宿主细胞中完成表达。适合宿主细胞包括原核生物、酵母、高等真核细胞。优选地,所用宿主细胞是大肠杆菌(e.coli)、酵母或哺乳动物细胞系,如cos或cho。可以用商业可得到的过滤器首先浓缩从分泌重组蛋白质或多肽进入培养基的适合宿主/载体系统得到的上清液。浓缩后,可以应用浓缩物于适合的纯化基质上,如亲和性基质或离子交换树脂。最后,利用一个或多个反相hplc步骤进一步纯化重组多肽。用本领域技术人员公知技术,通过合成方法也可以产生有少于约100个氨基酸,通常少于约50个氨基酸的部分和其它变异体。例如,可以用任何商业可获得的固相技术,如merrifield固相合成方法合成这样的多肽,其中氨基酸顺序加入至生长中的氨基酸链。参见merrifield,j.am.chem.soc.852149-2146,1963。从供应商,如appliedbiosystem,inc.(fostercity,ca)可购得多肽自动合成设备,而且可以根据制造商的说明书操作。在一些特定实施方式中,多肽可以是包含多个本发明所述多肽,或包含一个本发明所述多肽和公知肿瘤抗原,如卵巢癌蛋白质或这种蛋白质的变异体的融合蛋白质。融合伴侣可以,例如有助于提供t辅助细胞表位(thelperepitopes)(免疫融合伴侣),优选人识别的t辅助细胞表位,或可以有助于以高于天然重组蛋白质的产量表达蛋白质(表达增强子)。一些优选的融合伴侣既是免疫学的又是表达增强的融合伴侣。可以选择其它融合伴侣以增加蛋白质溶解度或使蛋白质靶向期望的细胞内区室。进一步的融合伴侣包括有利于蛋白质纯化的亲和标记。通常可以用标准技术,包括化学缀合(chemicalconjugation),制备融合蛋白质。优选地,在表达系统中,融合蛋白质作为重组蛋白质表达,与非融合蛋白质比较允许增高水平的产生。简而言之,可以单独装配编码多肽组分的dna序列,并连入适合表达载体。在有或无肽接头时,编码一个多肽组分的dna序列3’-末端与编码第二多肽组分的dna序列5’-末端连接,使得可读框架是同相的。这样可以翻译成保留两种组成多肽生物活性的单一融合蛋白。可以利用肽接头序列以足够间距分开第一和第二多肽组分,确保每个多肽折叠成它的二级和三级结构。用本领域公知标准技术在融合蛋白中引入这样的肽接头序列。合适的肽接头序列可以基于下列因素选择(1)它们能形成可弯曲的延伸构象;(2)它们不能形成能与第一和第二多肽功能表位相互作用的二级结构;和(3)缺少可能与多肽功能表位反应的疏水或带电残基。优选的多肽接头序列包含甘氨酸、天冬酰胺和丝氨酸残基。其它接近中性的氨基酸,如苏氨酸和丙氨酸也可以用于接头序列。可以有用地用作接头的氨基酸序列包括在maratea等,gene4039-46,1985;murphy等,proc.natl.acid.sci.usa838258-8262,1986;美国专利号4,935,233和美国专利号4,751,180中所述的那些。接头序列长度通常可以从1到约50个氨基酸。如果第一和第二多肽有非必需n-末端氨基酸区,这些非必需n-末端氨基酸区能用于分离功能结构域和阻止空间干扰,则不需要接头序列。连接的dna序列可操作地与合适的转录或翻译调节元件连接。负责dna表达的调节元件仅位于编码第一多肽的dna序列5’。相似地,终止翻译所需要的终止密码子和转录终止信号仅出现在编码第二多肽的dna序列3’。也提供了包含本发明多肽和不相关免疫原性蛋白质的融合蛋白质。优选地,所述免疫原性蛋白质能激发记忆应答。这种蛋白质的例子包括破伤风、肺结核和肝炎蛋白质(参见,例如,stoute等,newengl.j.med.,33686-91,1997)。在优选实施方式中,免疫融合伴侣来源于蛋白质d,革兰氏阴性细菌流感嗜血杆菌(haemophilusinfluenza)b(wo91/18926)的表面蛋白质。优选地,蛋白质d衍生物大约包含蛋白质的头1/3(例如,头n-末端100-110个氨基酸),而且可以脂质化蛋白质d衍生物。在一些优选实施方式中,脂蛋白d融合伴侣的最初109个残基包括在n-末端以给多肽提供附加外源t细胞表位和增加在大肠杆菌(e.coli)中的表达水平(这样起到表达增强子作用)。脂质尾部确保抗原对抗原呈递细胞的最佳呈递。其它融合伴侣包括流感病毒的非结构蛋白质,ns1(血凝素)。尽管可以用包括t辅助细胞表位的不同部分,但通常用n-末端81个氨基酸。在另一个实施方式中,免疫融合伴侣是称为lyta的蛋白质,或它的部分(优选c-末端部分)。lyta来源于肺炎链球菌,其合成n-乙酰-l-丙氨酸酰胺酶,称为酰胺酶lyta(被lyta基因编码;gene43265-292,1986)。lyta是特异分解肽聚糖主链某些键的自溶素。lyta蛋白质的c-末端结构域与胆碱或一些胆碱类似物,如deae的亲和有关。已利用这个特性研发对融合蛋白质表达有用的大肠杆菌(e.coli)c-lyta表达质粒。已描述了(参见biotechnology10795-798,1992)在氨基末端含有c-lyta片段的杂合蛋白质的纯化。在优选实施方式中,可以向融合蛋白质引入lyta重复部分。在c-末端区域,从178个残基开始发现了重复部分。特别优选的重复部分包含残基188-305。一般而言,如本发明所述的多肽(包括融合蛋白质)和多核苷酸是分离的。“分离的”多肽或多核苷酸是从其原始环境移出的多肽或多核苷酸。例如,如果天然存在的蛋白质从一些或所有自然系统中共存物质中被分离出来,那么该天然存在的蛋白质是分离的。优选地,这样的多肽有至少约90%的纯度,更优选至少约95%纯度,最优选至少约99%纯度。如果,例如多核苷酸被克隆进不是天然环境一部分的载体中,就认为多核苷酸被分离了。结合剂本发明进一步提供了试剂,如抗体和其抗原结合片段,其能特异地与卵巢癌蛋白质结合。用在这里的抗体,或其抗原结合片段,如果它以可检测水平与卵巢癌蛋白质反应(例如,在elisa中),而在相似条件下,不与不相关蛋白质可检测地反应,就是“特异结合”卵巢癌蛋白质。用在这里的“结合”指两个分离分子间非共价连接,以致形成“复合物”。例如,通过测定复合物形成的结合常数可以评估结合能力。结合常数是当复合物浓度除以组分产物浓度时获得的值。一般地,当复合物形成的结合常数超过约103l/mol时,在本发明上下文中两个化合物称为“结合”。可以用本领域公知方法测定结合常数。用本文提供的代表性测定法,结合剂进一步能区分患癌症(如卵巢癌)的患者和没患癌症的患者。换而言之,在患这种疾病的至少约20%的患者中,结合卵巢癌抗原的抗体或其它结合剂将产生表明癌症存在的信号,而且在没患癌症的至少约90%的个体中将产生表明这种疾病不存在的阴性信号。为了检测结合剂是否满足这种要求,可以如本文所述测定患癌症(用标准诊断方法测定的)和没患癌症患者生物样品(例如血液、血清、leukophoresis、尿和/或肿瘤活检)中与结合剂结合的多肽的存在。显而易见,应该测定统计学上显著的患病和没患病样品数。每个结合剂应该满足上述标准;然而,本领域技术人员将认识到可以联合使用结合剂以改进敏感性。满足上述要求的任何试剂都可以是结合剂。例如,结合剂可以是有或无肽组分的核糖体,rna分子或多肽。在优选实施方式中,结合剂是抗体或其抗原结合片段。可以通过本领域技术人员公知各种技术任一种制备抗体。参见,例如harlow和lane,antibodiesalaboratorymanual,coldspringharborlaboratory,1988。一般地,可以通过细胞培养技术产生抗体,包括本文所述单克隆抗体的产生,或通过抗体基因转染进适合细菌或哺乳动物细胞宿主,以允许产生重组抗体。在一个技术中,包含多肽的免疫原最初被注射进广泛种类的哺乳动物中任一种(例如,小鼠、大鼠、兔子、绵羊和山羊)。在这步,本发明多肽可以用作免疫原,而不用修饰。可备选地,特别对于相对短的肽,如果肽与载体蛋白质连接,如牛血清白蛋白或匙孔_血蓝蛋白,可能激发强免疫应答。给动物宿主注射免疫原,优选地,根据预先确定的包含一个或多个加强免疫的方案。定期取动物血液。随后,例如,用与合适固相载体连接的多肽,通过亲和层析,从这样的抗血清中纯化多肽特异的多克隆抗体。例如,用kohler和milstein,eur.j.immunol.6511-519,1976和其改进的技术可以制备对目的抗原多肽特异的单克隆抗体。简而言之,这些方法包括能产生有期望特异性(即与目的多肽的反应性)的抗体的永生细胞系的制备。例如,可以从上述免疫动物得到的脾脏细胞产生这样细胞系。随后,脾脏细胞通过,例如与骨髓瘤细胞融合伴侣,优选与免疫动物同系的骨髓瘤细胞融合伴侣融合,这样脾脏细胞可以永生化。可以利用各种融合技术。例如脾脏细胞和骨髓瘤细胞可以与非离子去污剂混合几分钟,然后低密度放置于支持杂交细胞生长,而不支持骨髓瘤细胞生长的选择培养基上。优选筛选技术用hat(次黄嘌呤,氨基喋呤,胸腺嘧啶脱氧核苷)筛选。在足够时间后,通常约1到2周,观察到杂种集落。筛选单集落,测定它们培养上清液的对多肽的结合活性。优选有高反应性和特异性的杂交瘤。从生长杂交瘤集落的上清液中分离单克隆抗体。另外,可以利用各种技术增加产量,如注射杂交瘤细胞系进入适合脊椎动物宿主,如小鼠的腹腔内。然后从腹水液体或血液中收集单克隆抗体。通过传统技术,如层析、凝胶过滤、沉淀和提取除去抗体中污染物。本发明多肽也可以在纯化过程,例如亲和层析步骤中使用。在一些实施方式中,优选应用抗体的抗原结合片段。这样片段包括fab片段,其可用标准技术制备。简而言之,可以从兔血清中通过蛋白质a珠粒柱(harlow和lane,antibodiesalaboratorymanual,coldspringharborlaboratory,1988)亲和层析纯化免疫球蛋白,而且用木瓜蛋白酶消化产生fab和fc片段。通过蛋白质a玻珠柱亲和层析可以分离fab和fc片段。本发明单克隆抗体可以与一个或多个治疗剂偶连。在这方面适合的治疗剂包括放射性核素、分化诱导剂、药物、毒素和其衍生物。优选的放射性核素包括90y,123i,125i,131i,186re,188re,211at和212bi。优选药物包括氨甲喋呤,和嘧啶和嘌呤类似物。优选的分化诱导剂包括佛波酯和丁酸。优选的毒素包括蓖麻毒素,相思豆毒蛋白,白喉毒素,霍乱毒素,gelonin,假单胞菌外毒素,志贺菌毒素,和商陆抗病毒蛋白质。治疗剂可以直接或间接(例如,通过接头基团)与适合单克隆抗体偶连(例如,共价键合)。当治疗剂和抗体每个都有能互相反应的取代基时,两者间的直接反应是可能的。例如,在一个上的亲核基团,如氨基或巯基,能与在另一个上含有羰基的基团如酸酐或酸卤化物反应,或与含有容易离去基团(如卤化物)的烷基反应。可备选地,可期望通过接头基团偶连治疗剂和抗体。接头基团起间隔区作用以间隔抗体和治疗剂以避免干扰结合能力。接头基团也起到增加治疗剂或抗体上取代基化学反应性的作用,因此增加了偶连效率。化学反应性的增加也可以有助于治疗剂,或治疗剂上功能基团的应用,反之则是不可能的。对本领域技术人员而言显而易见,各种双功能或多功能试剂,包括同种或异种功能的(如在piercechemicalco.,rockford,il目录中所述),都可以用做接头基团。例如通过氨基,羧基,巯基或氧化碳水化合物残基可以实现耦连。有大量描述这种方法学的参考文献,例如美国专利号4,671,958,rodwell等。当没有本发明免疫缀合物抗体部分,治疗剂更有效时,可期望用在内在化进入细胞时或期间可切割的接头基团。已描述了大量不同的可切割接头基团。从这些接头基团中细胞内释放治疗剂的机理包括通过二硫键还原(例如,美国专利号4,489,710,spitler),通过光不稳定键的辐射(例如,美国专利号4,625,014,senter等),通过衍生氨基酸侧链的水解(例如,美国专利号4,638,045,kohn等),通过血清补体介导的水解(例如,美国专利号4,671,958,rodwell等),和通过酸催化的水解(例如,美国专利号4,569,789,blattler等)的裂解。可能期望偶连到抗体上一个以上的治疗剂。在一个具体实施方式中,多个治疗剂分子与一个抗体分子偶连。在另一具体实施方式中,一种以上类型的治疗剂可以与一个抗体偶连。不管特定实施方式,可以通过各种方法制备有一个以上治疗剂的免疫缀合物。例如一个以上治疗剂可以直接与抗体分子偶连,或可以使用提供用于结合的多位点的接头。载体可以通过各种方法带有治疗剂,包括直接或通过接头基团的共价结合。适合载体包括蛋白质,如白蛋白(例如,美国专利号4,507,234,kato等),肽和多糖,如氨基葡聚糖(例如,美国专利号4,699,784,shih等)。载体也可以通过非共价结合或通过如在脂质体泡中胶囊化(例如,美国专利号4,429,008和4,873,088)带有治疗剂。对放射性核素剂特异的载体包括放射性卤化小分子和螯合化合物。例如,美国专利号4,735,792公开了代表性的放射性卤化小分子及它们的合成。可以从螯合化合物形成放射性核素螯合物,所述螯合化合物包括含有氮原子和硫原子做为结合金属,或氧化金属,放射性核素的供体原子的那些。例如,美国专利号4,673,562,davison等公开了代表性螯合化合物及其合成。可以用各种给药抗体和免疫缀合物的途径。典型地,给药将是静脉内,肌内,皮下或切除肿瘤的基床上。显而易见,抗体/免疫缀合物的精确剂量将随所用抗体、肿瘤上的抗原密度和抗体清除比率而变化。本发明也提供了模仿卵巢癌蛋白质免疫原性部分的抗独特型抗体。用公知技术,可以针对特异结合卵巢癌蛋白质免疫原性部分的抗体或其抗原结合片段产生这种抗体。如本发明所述,模仿卵巢癌蛋白质免疫原性部分的抗独特型抗体是与抗体或其抗原结合片段结合的那些抗体,所述抗体或其抗原结合片段特异结合卵巢癌蛋白质免疫原性部分。t细胞免疫治疗组合物也可以,或可备选地,包括卵巢癌蛋白质特异的t细胞。通常可以在体外或用离体标准技术制备这样的细胞。例如,用商业上可得到的细胞分离系统,如可从cellproinc.,bothellwa得到的cepratetm系统(也参见美国专利号5,240,856;美国专利号5,215,926;wo89/06280;wo91/16116;wo92/07243),t细胞可以存在于哺乳动物,如患者的骨髓,外周血或骨髓或外周血的一部分中(或从其中分离)。可备选地,可以从相关或不相关的人、非人的动物,细胞系或培养物中得到t细胞。可以用卵巢癌多肽,编码卵巢癌多肽的多核苷酸和/或表达这样多肽的抗原呈递细胞(apc)刺激t细胞。在足以允许多肽特异的t细胞产生的条件下和时间内进行这种刺激。优选地,卵巢癌多肽或多核苷酸存在于递送载体,如微球体中以有利于特异t细胞的产生。如果t细胞杀死卵巢癌多肽包被的或表达编码这样多肽的基因的靶细胞,那么认为t细胞对卵巢癌多肽是特异的。可以用各种标准技术任一种评估t细胞特异性。例如,在铬释放测定法或增殖测定法中,与阴性对照比较,裂解和/或增殖高于2倍增加的刺激指数表明t细胞特异性。例如,可以如chen等,cancerres.541065-1070,1994所述进行这样的测定方法。可备选地,通过各种公知技术可以完成t细胞增殖检测。例如,通过测定增加的dna合成速率能检测t细胞增殖(例如,通过氚胸腺嘧啶脱氧核苷脉冲标记t细胞培养,而且测定掺入dna中的氚胸腺嘧啶脱氧核苷量)。与卵巢癌多肽(200ng/ml-100μg/ml,优选100ng/ml-25μg/ml)接触3-7天应该引起t细胞增殖至少2倍增高和/或如上述接触2-3小时应该引起t细胞活化,这用标准细胞因子测定法测定,其中细胞因子释放(例如tnf或ifn-γ)水平2倍的增加表明了t细胞活化(参见coligan等,currentprotocolsinimmunology,卷1,wileyinterscience(greene1998))。响应卵巢癌多肽,多核苷酸或表达卵巢癌多肽的apc活化的t细胞可以是cd4 和/或cd8 。可以用标准技术扩增卵巢癌多肽特异的t细胞。在优选实施方式中,从患者或相关或不相关供体得到t细胞,而且在刺激和扩增(expansion)后,给患者施用t细胞。为了治疗目的,能在体外或体内大量扩增响应卵巢癌多肽,多核苷酸或apc而增殖的cd4 或cd8 t细胞。通过各种方法可以完成这种t细胞的体外增殖。例如,在有或无添加t细胞生长因子,如白细胞介素-2,和/或合成卵巢癌多肽的刺激细胞时,t细胞能再次暴露于卵巢癌多肽。可备选地,通过克隆,能大量扩增在卵巢癌多肽存在情况下增殖的一个或多个t细胞。克隆细胞的方法是本领域公知的,包括有限稀释。扩增后,可以给患者返回细胞,例如,如chang等,crit.rev.oncol.hematol.22213,1996所述。药物组合物和疫苗在一些方面,药物组合物或疫苗可以包括本发明所述多肽,多核苷酸,结合剂和/或免疫系统细胞。药物组合物包含一个或多个这样的化合物或细胞和生理学上可接受的载体。疫苗可以包含一个和多个这样化合物或细胞和非特异免疫应答增强剂。非特异免疫应答增强剂可以是增强对外源抗原免疫应答的任何物质。非特异免疫应答增强剂例子包括佐剂,可生物降解微球体(如polylacticgalactide)和脂质体(其中包括化合物;参见,例如,fullerton,美国专利号4,235,877)。通常,疫苗制备如,例如m.f.powell和m.j.newman主编,“vaccinedesign(thesubunitandadjuvantapproach),”plenumpress(ny1995)所述。本发明范围内的药物组合物和疫苗也可以包含生物活性或生物惰性的其它化合物。例如,组合物或疫苗中可以存在其它肿瘤抗原的一个或多个免疫原性部分,或掺入融合蛋白中或做为单独化合物。药物组合物或疫苗可以包含编码上述一个或多个多肽的dna,由此原位产生多肽。如上述,dna可以存在于本领域技术人员公知的各种递送系统任一个中,包括核酸表达系统,细菌和病毒表达系统。合适的核酸表达系统包含在患者中表达必需的dna序列(如适合的启动子和终止信号)。细菌递送系统包括细菌的给予(如卡介苗),所述细菌在其细胞表面表达多肽的免疫原性部分。在优选实施方式中,可以用病毒表达载体系统(例如,痘苗或其它痘病毒,反转录病毒或腺病毒)引入dna,病毒表达载体系统可以包括非致病性(缺陷的),有复制能力病毒的应用。许多文献公开了适合的系统,例如fisher-hoch等,pnas86317-321,1989;flexner等,ann.n.y.acad.sci.56986-103,1989;flexner等,vaccines817-21,1990;美国专利号4,603,112,4,769,330,和5,017,487;wo89/01973;美国专利号4,777,127;gb2,200,651;ep0,345,242;wo91/02805;berkner,biotechniques6616-627,1988;rosenfeld等,science252431-434,1991;kolls等,pans91215-219,1994;kass-eisler等,pans9011498-11502,1993;guzman等,circulation882838-2848,1993;和guzman等,cir.res.731202-1207,1993。引入dna进入这样表达系统的技术是本领域技术人员公知的。dna也可以是“裸的”,例如,如ulmer等,science2591745-1749,1993和cohen,science2591691-1692,1993所述。通过在生物降解珠粒上包被dna,可以增加裸dna的摄取,有效地转运裸dna进入细胞。尽管本发明药物组合物可以利用本领域技术人员公知的任何适合载体,但是载体类型取决于给药方式而变化。本发明组合物可以配制用于任何适合的给药方式,包括例如,局部,口,鼻,静脉内,颅内,腹膜内,皮下和肌内给药。对于肠胃外给药,例如皮下注射,优选的载体包括水,盐水,乙醇,脂肪,蜡或缓冲液。对于口服给药,可以利用任何一种上述载体或固体载体,如甘露醇,乳糖,淀粉,硬脂酸镁,糖精钠,滑石粉,纤维素,葡萄糖,蔗糖和碳酸镁。也可以用生物降解微球体(例如,polylactatepolyglycolate)做为本发明药物组合物的载体。适合的生物降解微球体公开了,例如,美国专利号4,897,268和5,075,109。这样的组合物也可以包括缓冲液(例如中性缓冲盐水或磷酸缓冲盐水),碳水化合物(例如葡萄糖,甘露糖,蔗糖或葡聚糖),甘露醇,蛋白质,多肽或氨基酸如甘氨酸,抗氧化剂,螯合剂如edta或谷胱甘肽,佐剂(例如,氢氧化铝)和/或防腐剂。可备选地,本发明的组合物可以形成冻干粉。也可以用公知技术在脂质体中包囊化化合物。各种非特异免疫应答增强剂中任一种都可以用在本发明疫苗中。例如可以包括佐剂。大多数佐剂含有目的是保护抗原免受快速分解代谢的物质,如氢氧化铝或矿物油,和免疫应答刺激剂,如脂质a,bortadellapertussis或结核分支杆菌中得到的蛋白质。适合的佐剂商业可得到,例如弗氏(freund’s)不完全佐剂和完全佐剂(difcolaboratories,detroit,mi),merckadjuvant65(merckandcompany,inc.,rahway,nj),明矾,生物降解微球体,单磷酰脂质a和quila。细胞因子,如gm-csf或白细胞介素-2,-7,或-12,也可以用做佐剂。在本发明提供的疫苗中,优选设计为主要诱导th1型免疫应答的佐剂组合物。高水平的th1型细胞因子(例如,ifn-γ,il-2和il-12)趋向促进细胞介导的对给药抗原免疫应答的诱导。相反,高水平的th2型细胞因子(例如,il-4,il-5,il-6,il-10和tnf-β)趋向促进体液免疫应答的诱导。应用本发明提供的疫苗后,患者将支持诱导th1-和th2-型应答的免疫应答。在优选实施方式中,应答主要是th1型,th1型细胞因子水平比th2型细胞因子水平将增加更大程度。用标准测定法可以容易估定这些细胞因子水平。对于细胞因子家族的综述参见mosmann和coffman,ann.rev.immunol.7145-173,1989。用于激发主要是th1型应答的优选佐剂包括,例如,单磷酰脂质a,优选3-脱-o-酰化单磷酰脂质a(3d-mpl)和铝盐的结合。可以从ribiimmunochemresearchinc.(hamilton,mt;参见美国专利号4,436,727;4,877,611;4,866,034和4,912,094)得到mpl佐剂。也优选的是as-2(smithklinebeecham)。含有cpg的寡核苷酸(其中cpg二核苷酸未被甲基化)也诱导主要是th1应答。这种寡核苷酸是公知的,而且,例如,wo96/02555中所述。另一个优选佐剂是皂甙,优选qs21,其可以单独使用或与其它佐剂联合使用。例如,加强系统包括单磷酰脂质a和皂甙衍生物的联合,如qs21和3d-mpl联合,如wo94/00153所述,或弱反应原性组合物,其中用胆固醇猝灭qs21,如wo96/33739所述。其它优选制剂包括水包油乳剂和生育酚。wo95/17210描述了水包油乳剂中包含qs21,3d-mpl和生育酚的特别有效的佐剂制剂。可以用使抗原,免疫应答增强剂和适合载体或赋形剂联合的公知方法制备本发明所述的任何疫苗。本发明描述的组合物可以做为持续释放制剂的一部分(即,给药后,引起化合物缓慢释放的制剂如胶囊或海绵)给药。通常可以用公知技术制备这种制剂,通过例如,口,直肠或皮下植入,或通过在期望靶位点植入给药这种制剂。持续释放的制剂可以包含分散在载体基质和/或包含在速率控制膜包裹的贮槽内的多肽,多核苷酸或抗体。用于这种制剂的载体是生物兼容的,而且也可以是生物降解的;优选提供相对恒定的活性成分释放水平的制剂。包含在持续释放制剂中的活性化合物的量取决于植入位点,释放速率及期望的释放期间和将要处理及预防疾病的性质。在药物组合物及疫苗中可以利用各种递送载体中任一种,以有利用靶向肿瘤细胞的抗原特异免疫应答的产生。递送载体包括抗原呈递细胞(apcs),如树突细胞,巨噬细胞,b细胞,单核细胞及基因工程改造为有效apcs的其它细胞。这些细胞可以,但不是必须,被遗传修饰以增加提呈抗原能力,以改进t细胞应答的活化和/或维持,以使本身有抗肿瘤效果和/或与接受者免疫相容(即匹配的hla单倍型)。apcs通常可以从各种生物液体或器官分离,包括肿瘤和肿瘤周边组织,apc也可以是自体,同种异体的,同源的或异种的细胞。本发明一些优选实施方式用树突细胞或其祖先细胞做为抗原呈递细胞。树突细胞是高度有效的呈递细胞(banchereau和steinman,nature392245-251,1998),而且已证明其做为激发预防或治疗性抗肿瘤免疫的生理学佐剂是有效的(参见timmerman和levy,ann.rev.med.50507-529,1999)。一般,基于树突细胞的通常形状(在原位是星状,体外有明显胞质突起(树突)可见)和树突细胞缺少b细胞(cd19和cd20),t细胞(cd3),单核细胞(cd14)和天然杀伤细胞(cd56)用标准测定法测定的分化标记,可以鉴定树突细胞。当然也可以基因工程改造树突细胞以表达通常在体内或体外树突细胞没有发现的特异表面细胞受体或配体,而且本发明考虑到了这样修饰的树突细胞。做为树突细胞的替代物,也可以在疫苗中用分泌性小泡载抗原的树突细胞(称为exosome)(参见zitvogel等,naturemed.4594-600,1998)。可以从外周血,骨髓,肿瘤浸润细胞,肿瘤周边组织浸润细胞,淋巴结,脾脏,皮肤,脐带血或任何其它适合的组织或液体获得树突细胞和祖先细胞。例如,通过加细胞因子联合物,如gm-csf,il-4,il-13和/或tnfα到从外周血收集的单核细胞培养物上,可以离体分化树突细胞。可备选地,通过向培养基中加入gm-csf,il-3,tnfα,cd40配体,lps,flt3配体和/或诱导树突细胞成熟和增殖的其它化合物联合,从外周血,脐带血或骨髓收集的cd34阳性细胞可以分化成树突细胞。为了方便,树突细胞分类为“不成熟”和“成熟”细胞,这允许用简单方法区别两种表征良好的表型。然而,这种命名法不应该解释为排除所有可能的分化中间阶段。不成熟树突细胞被表征为apc,具有高能力的抗原摄取和加工,这与fcγ受体,甘露糖受体和dec-205标记相关。成熟表型通常以这些标记的低表达为特征,而与t细胞活化有关的细胞表面分子,如类型imhc和类型iimhc,粘附分子(例如,cd54和cd11)和共同刺激性分子(例如,cd40,cd80和cd86)高表达。通常可以用编码卵巢癌抗原(或它的部分或它的其它变异体)的多核苷酸转染apcs,由此在细胞表面表达抗原,或其免疫原性部分。可以离体进行这样的转染,而且如本发明所述,包括这样转染细胞的组合物或疫苗随后可以用于治疗目的。可备选地,可以给患者施用靶向树突或其它抗原呈递细胞的基因递送载体,引起发生在体内的转染。例如,通常可以用本领域公知的任何方法进行树突细胞的体内和离体转染,如wo92/24447所述方法,或mahvi等,immunologyandcellbiology75456-460,1997所述的基因枪方法。通过树突细胞或先祖细胞与多肽,dna(裸的或质粒载体中)或rna温育;或与抗原表达重组细菌或病毒(例如,痘苗,禽痘,腺病毒或慢病毒载体)温育可以得到载有抗原的树突细胞。在加载前,多肽可以共价缀合提供t细胞辅助的免疫伴侣。可备选地,可以用非缀合的免疫伴侣单独地或在多肽存在的情况下脉冲树突细胞。癌症治疗本发明的进一步方面,本发明描述的组合物可以用于癌症,如卵巢癌的免疫治疗。在这样的方法中,通常给患者施用药物组合物和疫苗。本文所用“患者”指任何温血动物,优选人。患者可以患有癌症,也可以没有患癌症。因此,上述药物组合物和疫苗可以用于预防癌症发展或治疗患有癌症的患者。在一些优选实施方式中,患者患有卵巢癌。用这个领域通常接受的标准可以诊断这样的癌症,包括恶性肿瘤的出现。可以在外科除去原发肿瘤和/或治疗,如给予放射性治疗或传统化学治疗药物前或后给药药物组合物和疫苗。在一些实施方式中,免疫治疗可以是主动免疫治疗,其中用免疫应答修饰剂(如肿瘤疫苗,细菌佐剂和/或细胞因子)给药,治疗取决于内生宿主免疫系统的体内刺激以发生抗肿瘤反应。在其它实施方式中,免疫治疗可以是被动免疫治疗,其中治疗涉及有明确肿瘤免疫反应性的药剂(如效应细胞或抗体)的递送,所述有明确肿瘤免疫反应性的药剂能直接或间接介导抗肿瘤效应,而且不必依靠完整宿主免疫系统。效应细胞的例子包括t淋巴细胞(如cd8 细胞毒性t淋巴细胞和cd4 t辅助肿瘤浸润淋巴细胞),杀伤细胞(如天然杀伤细胞和淋巴因子激活的杀伤细胞),b细胞和表达本发明提供的多肽的抗原呈递细胞(如树突细胞和巨噬细胞)。为过继免疫治疗,可以克隆,表达对本发明所述多肽特异的t细胞受体和抗体受体并转移进其它载体或效应细胞。也可以用本发明多肽产生抗体或抗独特型的抗体(如上述和美国专利号4,918,164所述)用于被动免疫治疗。如本文所述,通过体外生长,通常可以获得足够数量用于过继免疫治疗的效应细胞。将单个抗原特异效应细胞扩增至数十亿个同时又保留体内抗原识别的培养条件是本领域公知的。这种体外培养条件通常是用抗原间歇刺激,常常是在细胞因子(如il-2)和非分裂的饲养细胞存在的情况下。如上述,本发明提供的免疫反应性多肽可以用于快速扩增抗原特异的t细胞培养物以产生足够免疫治疗量的细胞。特别地,用本领域公知的标准技术,可以用一个或多个多核苷酸转染或用免疫反应多肽脉冲抗原呈递细胞,如树突细胞,巨噬细胞或b细胞。例如,可以用多核苷酸转染抗原呈递细胞,所述多核苷酸有适合增加在重组病毒或其它表达系统中表达的启动子。培养的用于治疗中的效应细胞必须能在体内生长和广泛分布,而且能长期存活。研究表明通过用补加il-2的抗原重复刺激(参见,例如cheever等,immunologicalreviews157177,1997),能诱导培养的效应细胞在体内生长,而且以相当大的数量长期存活。可备选地,可以向干细胞引入表达本发明所述多肽的载体,干细胞来源于患者,而且体外克隆繁殖以自体移植回相同的患者。给药的途径和频率及剂量随不同个体而不同,而且用标准技术可以容易地确定。一般地,药物组合物和疫苗可以通过注射(例如皮内,肌内,静脉内和皮下),鼻内(吸入),口服或切除肿瘤的基床给药。优选地,可以在52周的期间内给药1到10个剂量。优选地,给药6次剂量,间隔为1个月,而且其后可以定期给予加强接种。对于各个患者可以适用替换方案。合适剂量是当如上述给药时,能提高抗肿瘤免疫应答,而且至少高于基线水平(即,未治疗)10-50%的化合物的量。通过测定患者抗肿瘤抗体或通过能体外杀死患者肿瘤细胞的依赖疫苗的溶细胞效应细胞的产生,能检测这样的应答。与没有接种的患者比较,这样疫苗也应该能引起在接种患者中产生改进临床结果的免疫应答(例如,更经常的缓解,完全或部分或较长的无疾病存活)。一般地,对于包括一个或多个多肽的药物组合物和疫苗,出现在剂量中每个多肽量的范围为每kg主体从约100μg到5mg。适合的剂量体积随着患者体积而变化,但通常范围从约0.1ml到约5ml。一般地,合适的剂量和治疗方案提供了足以提供治疗和/或预防益处的量的活性化合物。与没有治疗的患者比较,通过确定治疗患者中改进的临床结果(例如,更经常的缓解,完全或部分或较长的无疾病存活),能测定这样的应答。预先存在的卵巢癌抗原免疫应答的增加通常与改进的临床结果相关。用标准增殖,细胞毒性或细胞因子测定法通常可以评估这样的免疫应答,用从治疗前后患者获得的样品可以进行这样的测定。筛选以鉴定分泌性卵巢癌抗原本发明提供了鉴定分泌性肿瘤抗原的方法。在该方法中,植入免疫缺陷动物,如scid小鼠以肿瘤,同时维持肿瘤一段时间,足以允许肿瘤抗原分泌进入血清。一般地,通常可以皮下或在免疫缺陷动物性腺脂垫中植入肿瘤,而且维持1-9个月,优选1-4个月。通常如wo97/18300所述进行植入。然后,用标准和本文所述技术,在免疫活性的小鼠中用含有分泌的抗原的血清制备抗血清。简而言之,50-100μl血清(从三组免疫缺陷动物中混合的,每组有不同scid来源的人卵巢癌肿瘤)可以与适合佐剂,如ribi-mpl或mpt tdm(sigachemicalco.,st.louis,mo)1∶1(体积∶体积)混合,腹膜内注射进同系免疫活性动物,以月为间隔,总计5个月。第三,四和五次免疫后,通过抽血从以这种方法免疫的动物获得抗血清。得到的抗血清通常预先清除大肠杆菌(e.coli)和噬菌体抗原,而且用于(通常在稀释后,如1∶200)血清学表达筛选。文库通常是含有植入scid小鼠中之类型的一个或多个肿瘤的cdnas的表达文库。可以在任何适合的载体中制备该表达文库,如在λ-screen(novagen)中。用标准技术可以鉴定编码与抗血清反应的多肽的cdnas,并对其测序。这种cdna分子可以被进一步表征以评估在肿瘤和正常组织中的表达,及评估患者的抗原分泌。本发明提供的方法有优于其它肿瘤抗原发现方法的优点。特别地,通过这个方法鉴定的所有抗原应该通过肿瘤细胞坏死分泌和释放。这种抗原可能在肿瘤细胞表面存在足够长的一段时间,以允许接种后被免疫系统靶向和杀死。检测癌症的方法一般地,基于从患者得到的生物样品(如血液,血清,尿和/或肿瘤活检)中存在一个或多个卵巢癌蛋白质和/或编码这种蛋白质的多核苷酸,可以检测患者的癌症。换而言之,可以用这样的蛋白质做为标记以表明存在或不存在癌症,如卵巢癌。此外,这样的蛋白质可以用于其它癌症的检测。本发明提供的结合剂通常允许检测生物样品中与该结合剂结合的蛋白质水平。可以用多核苷酸引物和探针检测编码肿瘤蛋白质的mrna水平,mrna水平也表明了癌症存在或不存在。一般地,肿瘤组织中卵巢癌相关序列应该以高于正常组织至少3倍的水平出现。有各种本领域技术人员公知的用结合剂检测样品中多肽标记的测定形式。参见,例如harlow和lane,antibodiesalaboratorymanual,coldspringharborlaboratory,1988。一般地,患者存在或不存在癌症,可以通过以下方法确定(a)用结合剂接触从患者得到的生物样品;(b)检测样品中与结合剂结合的多肽的水平;和(c)比较该多肽水平和预先确定的截断值。在优选实施方式中,测定法包括用固定在固相载体上的结合剂结合和分离样品残余部分中的多肽。然后,可以用含有报告基团的检测试剂检测结合多肽,该检测试剂可特异地与结合剂/多肽复合物结合。这样的检测试剂可包括,例如,特异结合所述多肽的结合剂,或特异性与该结合剂结合的或抗体其它试剂,如抗免疫球蛋白,蛋白质g,蛋白质a或凝集素。可备选地,可以利用竞争测定法,其中用报告基团标记多肽,而且在结合剂和样品温育后允许多肽与固定的结合剂结合。样品组分抑制标记多肽与结合剂结合的程度表明了样品与固定化结合剂的反应性。用于这种测定法中合适的多肽包括结合剂结合的全长卵巢癌蛋白质和其部分,如上所述。固相载体可以是本领域技术人员公知的可以结合肿瘤蛋白质的任何物质。例如,固相载体可以是微量滴定板中的检测孔或硝化纤维素或其它适合的膜。可备选地,载体可以是珠粒或盘,如玻璃,玻璃纤维,乳胶或塑料材料,如聚苯乙烯或聚氯乙烯。载体也可以是磁性颗粒或纤维光学传感器,如,例如美国专利号5,359,681所公开的。用本领域技术人员公知的各种技术,结合剂可以固定在固相载体上,这在专利和科学文献中充分描述了。在本发明上下文中,术语“固定”指非共价连接,如吸附,和共价结合(其中可以是试剂和在载体上的功能性基团直接连接,或可以以交联剂方式连接)。优选吸附到滴定板的孔或膜上的固定。在这种情况下,通过在合适缓冲液中接触结合剂与固相载体适合的时间完成吸附。接触时间随温度变化,但通常在约1小时到约1天。一般地,用范围从约10ng到约10μg,优选从约100ng到约1μg量的结合剂,接触塑料微量滴定板的孔(如聚苯乙烯或聚氯乙烯)足以固定足够量的结合剂。首先通过载体和双功能试剂反应,双功能试剂将与载体和在结合剂上的功能基团,如羟基或氨基反应,通常可以取得结合剂与固相载体的共价结合。例如用苯醌,或通过载体上醛基与结合伴侣上胺和活性氢的缩合,结合剂可以与有适合聚合物包被的载体共价结合(参见,例如pierceimmunotechnologycatalogandhandbook,1991,在a12-13)。在一些实施方式中,测定法是双抗体夹层测定法。通过首先将固定在固相载体上的抗体与样品接触,所述固相载体通常是微量滴定板的孔,使得样品中多肽允许与固定的抗体结合而进行这样测定。然后从固定的多肽-抗体复合物中除去未结合样品,而且加入含有报告基团的检测试剂(优选能结合多肽不同位点的第二抗体)。然后,用适于特异报告基团的方法测定与固相载体保持结合的检测试剂的量。更特别地,如上述,一旦抗体固定于载体上,载体上残存的蛋白质结合位点通常被封闭。可以使用本领域技术人员公知的任何适合封闭试剂,如牛血清白蛋白或tween20tm(sigmachemicalco.,st.louis,mo)。然后温育固定的抗体与样品,而且允许多肽与抗体结合。温育前可以用适合的稀释剂,如磷酸缓冲盐溶液(pbs)稀释样品。一般地,适合的接触时间(即温育时间)是足以检测从卵巢癌个体获得的样品中多肽的时间。优选地,接触时间足以达到这样的结合水平,所述结合水平至少是结合和未结合多肽间平衡时达到的结合水平的约95%。本领域技术人员将意识到通过测定出现在一段时间内的结合水平可以容易地测定达到平衡需要的时间。室温下,通常约30分钟的温育时间是足够的。然后,通过用适合的缓冲液,如含有0.1%tween20tm的pbs洗固相载体来除去未结合的样品。随后,向固相载体加入含有报告基团的第二抗体。优选报告基团包括上述的报告基团。然后,检测试剂与固定的抗体-多肽复合物温育一段时间,这段时间足以检测结合多肽。通过测定出现在一段时间内的结合水平,通常可以确定合适的时间。随后,除去未结合的检测试剂,用报告基团检测结合的检测试剂。所用检测报告基团的方法取决于报告基团的性质。对于放射性基团,通常闪烁计数或放射自显影方法是适合的。可以用分光光度方法检测染料,发光基团和荧光基团。可以用与不同报告基团(通常是放射活性或荧光基团或酶)偶连的抗生物素蛋白检测生物素。通过添加底物(通常是特定的时间),之后进行反应产物的分光光度或其它分析通常可以检测酶报告基团。为了检测存不存在癌症,如卵巢癌,通常比较与固相载体保持结合的报告基团的检测信号和与预先确定截断值对应的信号。在优选实施方式中,癌症检测截断值是当固定的抗体与来源于未患癌症患者的样品温育时获得的平均中间信号。一般地,产生高于预先确定截断值3个标准偏差之信号的样品认为是癌症阳性。在替换优选实施方式中,用receiveroperator曲线,根据sackett等,clinicalepidemiologyabasicscienceforclinicalmedicne,littlebrownandco.,1985,106-7页的方法测定截断值。简而言之,在这个实施方式中,从与用于诊断检测结果的每个可能截断值对应的真阳性率(即敏感性)和假阳性率(100%特异性)对的图表,可以测定截断值。在图表中最接近左上角(即,包围最大面积的值)的截断值是最精确的截断值,而且可以认为产生比这种方法测定的截断值高的信号的样品是阳性。可备选地,可以沿图移动截断值到左边以最小化假阳性率,或到右边以最小化假阴性率。一般地,认为产生比这种方法测定的截断值高的信号的样品是癌症阳性。在相关实施方式中,以流通或带试验形式进行测定,其中在膜,如硝化纤维素上固定结合剂。在流通试验中,当样品通过膜时,样品中多肽结合固定的结合剂。然后,当含有第二结合剂的溶液流经膜时,第二个标记结合剂与结合剂-多肽复合物结合。如上所述,随后可以进行结合的第二结合剂的检测。在带试验形式中,浸入结合剂结合的膜的一个末端到含有样品的溶液中。样品通过含有第二结合剂的区域沿膜移动,而且到达固定的结合剂区域。第二结合剂在固定的抗体区浓缩表明癌症的存在。通常,第二结合剂在此位点浓缩产生肉眼可见的型式,如线。缺少这样的型式表明阴性结果。一般地,选择固定在膜上结合剂的量,以便当生物样品含有足以以上述方式在双抗体夹层测定法中产生阳性信号的多肽水平时,产生视觉可辨别的型式。用在这样测定法中优选的结合剂是抗体和其抗原结合片段。优选地,固定在膜上抗体的量范围从约25ng到约1μg,更优选从约50ng到约500ng。通常用非常少量的生物样品可以进行这样的试验。当然,存在适用于本发明肿瘤蛋白质或结合剂的大量其它测定方案。上面说明仅仅是示例性的。例如可以容易修改上述方案以用卵巢癌多肽检测生物样品中结合这样多肽的抗体,这对本领域技术人员是显而易见的。这种卵巢癌蛋白质特异抗体的检测可能与癌症存在相关。也可以,或可备选地,基于生物样品中特异与卵巢癌蛋白质反应的t细胞的存在检测癌症。在一些方法中,从患者分离的包括cd4 和/或cd8 t细胞的生物样品与卵巢癌蛋白质,编码这样多肽的多核苷酸和/或表达这样多肽至少免疫原性部分的抗原呈递细胞(apc)温育,并检测是否存在t细胞的特异活化。适合的生物样品包括,但不限于分离的t细胞。例如,通过常规技术(如通过外周血淋巴细胞的ficoll/hypaque密度梯度离心)可以从患者分离t细胞。t细胞可以体外,37℃与卵巢癌蛋白质(例如,5-25μg/ml)温育2-9天(通常4天)。可能期望在不存在卵巢癌蛋白质的情况下,温育另一等份t细胞样品做为对照。对于cd4 t细胞,优选通过估计t细胞增殖检测激活。对于cd8 t细胞,优选通过估计溶细胞活性检测激活。增殖水平高于无疾病患者至少两倍和/或溶细胞活性水平高于无疾病患者至少20%表明患者患有癌症。如上所述,也可以,或可备选地,基于生物样品中编码卵巢癌蛋白质的mrna水平检测癌症。例如,为扩增来源于生物样品的卵巢癌蛋白质cdna,可以在基于聚合酶链式反应(pcr)的试验中,利用至少两个寡核苷酸引物,其中至少一个寡核苷酸引物对编码卵巢癌蛋白质的多核苷酸特异(即与其杂交)。然后分离扩增的cdna,用本领域公知技术,如凝胶电泳检测。相似地,与编码卵巢癌蛋白质的多核苷酸特异杂交的寡核苷酸探针也可以用于杂交测定中以检测生物样品中编码肿瘤蛋白质的多核苷酸存在。为了允许测定条件下的杂交,寡核苷酸引物和探针应该包括与编码卵巢癌蛋白质多核苷酸部分至少有约60%,优选至少约75%,更优选至少约90%同一性的寡核苷酸序列,所述多核苷酸部分的长度至少是10个核苷酸,优选至少20个核苷酸。优选地,寡核苷酸引物和/或探针与编码本发明所述多肽的多核苷酸在中度严格条件下杂交,如上述定义。可以在本发明所述诊断方法中有用地利用的寡核苷酸引物和/或探针,优选长度至少是10-40个核苷酸。在优选实施方式中,寡核苷酸引物包含具有本发明提供序列的dna分子的至少10个连续核苷酸,更优选至少15个连续核苷酸。基于pcr的测定和杂交测定技术是本领域公知的(参见,例如,mullis等,coldspringharborsymp.quant.biol.,51263,1987;erlich主编,pcrtechnology,stocktonpress,ny,1989)。一个优选测定法利用rt-pcr,其中pcr与反转录结合应用。通常,从生物样品,如活检组织提取rna,而且反转录产生cdna分子。用至少一个特异引物pcr扩增产生cdna分子,可以分离cdna分子并用例如,凝胶电泳显现cdna分子。可以对从试验患者和未患癌症个体提取的生物样品进行扩增。可以对跨越2个数量级的cdna的几个稀释进行扩增反应。通常认为试验患者样品几个稀释的表达比非患癌样品相同稀释的表达有2倍或更高的增加是阳性。在另一个实施方式中,可以用卵巢癌蛋白质和编码这样蛋白质的多核苷酸做为检测癌症进展趋势的标记。在这个实施方式中,可以随时间推移进行上述癌症诊断测定,而且评估反应性多肽水平的变化。例如,在6个月到1年的期间,每24-72小时进行测定,而且此后根据需要进行测定。一般地,患者中通过结合剂检测的多肽水平随时间推移而增加,癌症正在这些患者中进展。相反,当反应性多肽水平随时间推移或保持恒定或减少时,癌症没有进展。可以直接对癌症进行一些体内诊断测定。一个这样的测定包括用结合剂接触肿瘤细胞。然后可以通过报告基团直接或间接检测结合的结合剂。也可以在组织学应用中利用这样结合剂。可备选地,多核苷酸探针可以用于这样的应用。如上所述,为了改进敏感性,可以在已知样品中测定多个卵巢癌蛋白质标记。显而易见,在单个测定中可以联合对本发明提供的不同蛋白质特异的结合剂。而且,同时可以用多个引物和探针。可以基于常规试验进行肿瘤蛋白质标记筛选以检测引起最佳敏感性的联合。此外,或可备选地,本发明所提供肿瘤蛋白质的测定法可以与其它公知肿瘤抗原测定法联合。诊断试剂盒本发明进一步提供了用于任一上述诊断方法的试剂盒。通常这样的试剂盒包括两种或多种进行诊断测定必需的组分。组分可以是化合物,试剂,容器和/或设备。例如,试剂盒的一个容器可以含有特异结合卵巢癌蛋白质的单克隆抗体或其片段。如上述,可以提供与载体材料附着的这样抗体或片段。一个或多个其它容器可以包含用于检测的成分,如试剂或缓冲液。这样试剂盒也可以,或可备选地包含上述检测试剂,检测试剂含有适于直接或间接检测抗体结合的报告基团。可备选地,可以设计检测生物样品中编码卵巢癌蛋白质的mrna水平的试剂盒。这样的试剂盒通常包括至少一个与编码卵巢癌蛋白质的多核苷酸杂交的寡核苷酸探针或引物,如上述。例如,在pcr或杂交测定中,可以利用这样的寡核苷酸引物。可存在于这样试剂盒中的其它组分包括第二寡核苷酸和/或诊断试剂或容器以方便编码卵巢癌蛋白质多核苷酸的检测。通过示例方式,而不是限制方式提供了下面的实施例。实施例实施例1代表性卵巢癌蛋白质cdna的鉴定这个实施例示例说明了编码卵巢癌蛋白质的cdna分子的鉴定。预先清除抗scid小鼠血清(针对带有晚期传代卵巢癌(latepassageovariancarcinoma)的scid小鼠的血清产生的)的大肠杆菌(e.coli)和噬菌体抗原,而且在血清表达筛选中以1∶200的稀释度使用。用定向rh寡聚(dt)引发cdna文库构建试剂盒和λscreen载体(novagen)从源于scid的人卵巢肿瘤(ov9334)制备筛选文库。利用细菌噬菌体λ筛选。筛选了约400,000pfu(噬斑形成单位)的扩增的ov9334文库。分离了196个阳性克隆。在图1a-1s和seqidno1到71中提供了一些看似新的序列。图2a-2c(seqidno72到74)所示是三个完整插入序列。其它具有已知序列的克隆如图15a-15eee(seqidno82到310)所示。数据库检索鉴定了与图15a-15eee所示序列显著同一的下面序列。用微阵列技术进一步表征这些克隆以检测各种肿瘤和正常组织中mrna的表达水平。用synteni(paloalto,ca)微阵列,根据制造商的说明进行这样分析。在载波片上排列pcr扩增产物,在阵列中每个产物占有唯一的位置。从组织样品中提取待试验mrna,反转录和产生荧光标记的cdna探针。用标记cdna探针探测微阵列和扫描载波片以检测荧光强度。用synteni’s提供的gemtools软件分析数据。一个克隆(13695,也称为o8e)结果如图3所示。实施例2用微阵列技术鉴定卵巢癌cdna这个实施例示例了通过pcr扣除和微阵列分析进行的卵巢癌多核苷酸的鉴定。用synteni(paloalto,ca)微阵列,根据制造商的说明(而且基本上如schena等,proc.natl.acad.sci.usa9310614-10619,1996和heller等,proc.natl.acad.sci.usa942150-2155,1997所述)分析cdnas微阵列的卵巢肿瘤特异表达。用包括四个卵巢肿瘤(其中3个是转移性肿瘤)cdna的被测者(tester)和5个正常组织(肾上腺,肺,胰腺,脾脏和脑)cdna的驱动者(driver)进行pcr扣除。用从这个扣除回收的cdna片段进行dna阵列分析,其中pcr扩增该片段,与芯片粘附,而且与源于人卵巢肿瘤和各种正常人组织mrnas的荧光标记探针杂交。在这个分析中,扫描载波片,而且检测荧光强度,用synteni的gemtools软件分析数据。一般地,认为在肿瘤细胞中(相对于正常细胞)显示至少5倍表达增加的序列是卵巢肿瘤抗原。分析荧光结果,而且通过dna测序和数据库检索进一步表征那些在卵巢肿瘤中显示增加表达的克隆,以确定序列的新颖性。用这样的测定,鉴定了一个卵巢癌肿瘤抗原,它是人t-细胞白血病病毒类型i癌蛋白质tax(参见jin等,cell9381-91,1998)和称为骨粘连蛋白的细胞外基质蛋白质的剪接融合物。在融合点存在剪接接头序列。这个克隆序列如图4和seqidno75所示。从这样测定中也单独鉴定到了没有剪接和没有改变的骨粘连蛋白。通过这种方法鉴定的进一步克隆在本发明中称为3f,6b,8e,8h,12c和12h。这些克隆的序列如图5到图9和seqidno76到81所示。如上所述进行微阵列分析,而且如图10到14所示。通过筛选卵巢肿瘤(scid来源的)cdna文库获得包含克隆3f,6b,8e和12h的全长序列。这个2996碱基对序列(命名为o772p)如seqidno311所示,而且编码的914氨基酸蛋白质序列如seqidno312所示。psort分析表明了定位到质膜的类型1a跨膜蛋白质。除了上述的一些序列外,该筛选鉴定了表1详述的下列序列表1这个筛选进一步鉴定了克隆o772p的多种形式,本发明称作21013,21003和21008。psort分析表明21003(seqidno386;翻译为seqidno389)和21008(seqidno387;翻译为seqidno390)代表了o772p的类型1a跨膜蛋白质形式。21013(seqidno385;翻译为seqidno388)看似该蛋白质的截短形式,而且通过prort分析预测是分泌的蛋白质。其它序列分析产生了o8e全长克隆(2627bp,符合northern分析观察到的信息大小;seqidno391)。如下所述获得这个核苷酸序列发现原始o8e序列(origo8econs)与est克隆(image#1987589)序列有33个核苷酸的重叠。这个克隆提供了原始o8e序列上游的1042个附加核苷酸。用单一est和o8e序列引物(estxo8epcr),通过测序从卵巢原始肿瘤文库产生的多个pcr片段,证实了est和o8e间的连接。当卵巢肿瘤文库锚定pcr给出了几个都终止在推定的起始甲硫氨酸上游,而不能产生任何附加序列信息的克隆(锚定pcrcons)时,进一步表明了全长状态。图16表示示例全长o8e序列中每个部分序列位置的图。从全长o8e序列可以翻译两个蛋白质序列。对于“a”(seqidno393)起始于推定的起始甲硫氨酸。第二个形式“b”seqidno392)在核苷酸序列的5’末端包括27个附加上游残基。实施例3这个实施例公开了o8e多克隆抗血清识别的抗体表位(antibodyepitopes)的鉴定和表征。产生抗源于大肠杆菌(e.coli)的o8e重组蛋白质的兔抗血清,而且检测对与o8e氨基酸序列对应的20或21mer肽的抗体表位识别。来源于亲和纯化抗o8e血清的酸洗脱峰和/或盐洗脱峰识别全长o8e蛋白质的跨越氨基酸区31到65,76到110,136到200和226到245的肽。因此上述肽对应的氨基酸序列构成了亲和纯化抗o8e抗体识别的抗体表位。抗o8e兔血清的elisa分析如图23所示,而且亲和纯化兔抗o8e多克隆抗体的elisa分析如图24所示。为了表位作图,合成了对应于o8e蛋白质的20或21mer肽。为了抗体亲和纯化,将兔抗o8e血清过o8e琼脂糖凝胶柱,然后用含有0.5mnacl和20mmpo4的盐缓冲液洗脱抗体,随后用0.2m甘氨酸,ph2.3进行酸洗脱步骤。通过1mtris,ph8的添加中和纯化的抗体,并将缓冲液转换为磷酸缓冲盐水(pbs)。为了酶连免疫吸附测定法(elisa)分析,室温下(rt),在96孔平底板上以2μg/ml包被o8e肽和o8e重组蛋白质2小时。然后用pbs 0.1%tween20洗板5次,用pbs 1%牛血清白蛋白(bsa)封闭1小时。然后以1μg/ml加酸或盐洗脱部分的亲和纯化抗o8e抗体到孔中,室温(rt)温育1小时。再一次洗板,接着加驴抗兔ig辣根过氧化物酶(hrp)抗体,室温1小时。洗板,然后通过添加生色底物3’,3’,5’,5’-四甲基联苯胺(tmb)(bos等,j.ofimmunoassay2187-204(1981);可从sigma(st.louis,mo)得到)显色。室温温育反应15分钟,通过加1nh2so4停止反应。在自动板读数器上,在可见密度450(od450)下读取板。与oe8抗体表位对应的肽序列这里公开在seqidno394-415。图17公开了o8e多克隆抗血清识别的抗体表位。实施例4这个实施例公开了卵巢癌组织样品中o8e表达的ihc分析。对于免疫组织化学研究,切石蜡包埋甲醛固定的卵巢癌组织为8微米切片。用在0.1m柠檬酸钠缓冲液(ph6.0)中蒸汽加热诱导表位恢复(steamheatinducedepitoperetrieval,shier)作为最佳染色条件。切片与10%血清/pbs温育5分钟。加入每个切片第一抗体(抗-o8e兔亲和纯化多克隆抗体),25分钟,随后与抗兔生物素标记的抗体温育25分钟。通过用过氧化氢进行三个1.5分钟的温育封闭内源过氧化物酶活性。抗生物素蛋白-生物素复合物/辣根过氧化物系统与dab色原体一起使用以显现抗原表达。用苏木精复染载波片。六个卵巢癌组织切片中一个(乳头状浆液癌(papillaryserouscarcinoma))显示了o8e免疫反应性。当优化染色条件时,用o8e多克隆抗体,4/5卵巢癌样品染色阳性。o8e表达定位到质膜。用抗o8e兔多克隆抗体分析六个卵巢癌组织。六个卵巢癌组织样品中一个(乳头状浆液癌)染色为o8e表达阳性。o8e表达定位到表面膜。实施例5这个实施例公开了预测与hla-a2结合,同时对人cd8t细胞应答有免疫原性的o8e肽。通过使用o8e全长开放阅读框架(orf)和通过用“episeek”(用于预测mhc结合肽的程序)运行它,预测o8e可能的hla-a2结合肽。所用程序是以parker,k.c.等,j.immunol.152(1)163-175(1994)(已整体引入本发明作为参考)公开的算法为基础。预测与hla-0201结合的10mer和9mer肽这里分别公开为seqidno416-435和seqidno436-455。实施例6这个实施例公开了通过荧光激活细胞分选(facs)检测o8e表面表达。对于facs分析,用冰冷的染色缓冲液(pbs/1%bsa/叠氮化物)洗细胞。下一步,在冰上细胞与10μg/ml亲和纯化兔抗b305d多克隆抗体温育30分钟。用染色缓冲液洗细胞3次,然后冰上与1∶100稀释的山羊抗兔ig(h l)-fitc试剂(southernbiotechnolgy)温育30分钟。洗3次后,在含有碘化丙锭(prodiumiodide)的染色缓冲液中重悬浮细胞,该活体染料允许可渗透细胞的鉴定,之后通过facs分析细胞。在稳定表达o8ecdna的skbr3乳腺癌细胞和hek293细胞上证实了o8e表面表达。用对照不相关质粒dna稳定转染的mb415细胞或hek293细胞均没有显示o8e表面表达(图18和19)。实施例7这个实施例进一步评估了o8e的表达和表面定位。为了表达和纯化用于免疫的抗原,37℃,在振荡培养箱中,在大肠杆菌(e.coli)重组表达系统中表达的o8e在有适合抗生素的lb肉汤中过夜生长。第二天早上,在2l带挡板三角瓶中,加10ml过夜培养物到500ml加有适合抗生素的2×yt中。当培养物光密度(在560nm)达到0.4-0.6时,用iptg(1mm)诱导细胞。与iptg温育后4小时,离心收集细胞。然后用磷酸缓冲盐洗细胞,再一次离心。弃去上清液,或冷冻细胞备用或立刻处理。向细胞沉淀中20ml加入溶解缓冲液,涡旋。为了粉碎大肠杆菌细胞,然后在16,000psi压力下,将这个混合物通过弗氏压碎器。然后,再一次离心细胞,通过sds-page检查上清液和沉淀中重组蛋白质的分配。对于定位于细胞沉淀的蛋白质,在10mmtrisph8.0,1%chaps中重悬浮沉淀,而且洗包含体沉淀,再一次离心。再重复这个过程2次。用含有添加10mm咪唑的ph8.010mmtris的8m尿素或6m盐酸胍溶解洗过的包含体沉淀。加溶解蛋白质到5ml镍螯合物树脂(qiagen)中,室温下温育45分钟到1小时,同时持续搅拌。温育后,倾注树脂和蛋白质混合物通过一次性的柱子,收集流过物。然后用10-20倍柱体积的溶解缓冲液洗柱子。随后,用8m尿素,10mmtrisph8.0和300mm咪唑从柱子上洗脱抗原,收集在3ml级分中。进行sds-page凝胶以确定合并哪些级分以进一步纯化。做为最后纯化步骤,用适合缓冲液平衡强阴离子交换树脂,如hi-prepq(biorad),而且加样上面合并的级分到柱上。用逐步增加的盐梯度洗脱柱上抗原。当过柱时,收集级分,而且进行另一个sds-page凝胶以检测合并柱上的哪些级分。用10mmtrisph8.0透析合并的级分。然后评估这个物质的sds-page或hplc测定的可接受纯度,劳里(lowry)测定法或氨基酸分析测定的浓度,氨基酸末端蛋白质序列测定的同一性,及鲎(lal)测定法测定的内毒素水平。然后,通过0.22μm过滤器过滤后,装瓶蛋白质,并在需要免疫前冷冻抗原。为了产生多克隆抗血清,每种前列腺抗原400μg与100μg胞壁酰二肽(mdp)混合。加入等体积不完全弗氏(freund’s)佐剂(ifa),然后混合。每4周,用100μg与等体积ifa混合的抗原加强免疫。每次加强免疫后7天,取动物血液。在4℃温育血液12-24小时,离心,产生血清。为了表征多克隆抗血清,通过与50μl(典型地是1μg)抗原在4℃温育20小时包被96孔板。孔中加入250μlbsa封闭缓冲液,室温温育2小时。用pbs/0.01%tween洗板6次。在pbs中稀释抗o8e兔血清或亲和纯化抗o8e抗体。每个孔加入50μl稀释抗体,室温下温育30分钟。如上述洗板,之后以1∶10000稀释度加入50μl山羊抗兔辣根过氧化物酶(hrp),室温下温育30分钟。如上述洗板,每个孔加入100μltmb微孔过氧化物酶底物。在暗处室温温育15分钟后,用100μl1nh2so4终止比色反应,在450nm下立即读数。所有多克隆抗体均显示了对o8e抗原的免疫反应性。为了在哺乳动物hek293细胞中重组表达,亚克隆全长o8ecdna进入哺乳动物表达载体pcdna3.1 和pcep4(invitrogen),所述载体被修饰分别含有组氨酸和flag表位标签。用fugene6试剂(roche)转染这些构建体进入hek293细胞(atcc,美国典型培养物保藏中心)。简而言之,hek293细胞以100,000细胞/ml密度在含有10%fbs(hyclone)的dmem(gibco)中接种并过夜培养。下一天,向100μl不含fbs的dmem中加入2μlfugene6,室温温育15分钟。向1μgo8e/pcep4或o8e/pcdna3.1质粒dna中加入fugene6/dmem混合物。然后加fugene/dna混合物到hek293细胞,37℃,7%co2中温育48-72小时。用pbs润洗细胞,然后通过离心收集和沉淀。为了western印迹分析,通过冰上在含有triton-x100的裂解缓冲液中温育细胞,产生全细胞裂解物。通过10,000rpm,4℃离心5分钟澄清裂解物。用含有β-巯基乙醇的sds-page上样缓冲液稀释样品,然后在加载至sds-page凝胶前煮沸10分钟。转移蛋白质到硝酸纤维素上,而且用以1∶750稀释的抗o8e兔多克隆血清#2333l探测。用与hrp偶联的山羊抗兔ig并随后在ecl底物中温育显示印迹。对于facs分析,进一步用冰冷却染色缓冲液(pbs 1%bsa 叠氮化物)洗细胞。下一步,在冰上,细胞与10μg/ml蛋白质a纯化的抗o8e多克隆血清温育30分钟。用染色缓冲液洗细胞3次,然后冰上与1∶100稀释的山羊抗兔ig(h l)-fitc试剂(southernbiotehnology)温育30分钟。洗3次后,在含有碘化丙锭(pi)的染色缓冲液中重悬浮细胞,该活体染料允许可渗透细胞的鉴定,之后通过facs分析细胞。从这些试验中(图20-21示例了结果),用兔抗o8e血清,通过facs分析检测到转染的hek293细胞和skbr3细胞表面上o8e表达。通过westernblot分析,也在转染的hek293细胞裂解物中检测到表达(图22)。实施例8抗o8emabs(单克隆抗体)的产生和表征如下所述,产生抗源于大肠杆菌(e.coli)的o8e蛋白质的小鼠单克隆抗体。用含有50μg重组o8e的完全弗氏佐剂(cfa)腹膜内(ip)免疫a/j小鼠,随后用含有10μg重组o8e蛋白质的不完全弗氏佐剂(ifa)腹膜内(ip)加强免疫。切除脾脏前3天,用约50μg溶解的o8e重组蛋白质静脉内免疫小鼠。除去对o8e有阳性效价的小鼠的脾脏,制备单细胞悬浮液,用于与sp2/0骨髓瘤细胞融合以产生b细胞杂交瘤。通过elias检测杂种克隆上清液对重组o8e的特异性,而且用跨整个o8e序列的肽进行表位作图。还通过流式细胞术测试mabs检测稳定转染o8e的细胞表面上和乳腺肿瘤细胞系表面上o8e的能力。对于elisa分析,分别用重组o8e蛋白质或跨整个o8e分子的重叠20mer肽,在1-2μg/ml或10μg/ml浓度下包被96孔板。包被后,用冲洗缓冲液(pbs 0.1%tween20)洗板5次,用含有0.5%bsa,0.4%tween20的pbs封闭。然后加杂种上清液或纯化mabs,室温温育板60分钟。用冲洗缓冲液洗板5次,加与辣根过氧化物酶(hrp)连接的第二抗体,驴抗鼠ig(jacksonimmunoresearch),60分钟。在冲洗缓冲液中再一次洗板5次,随后加辣根过氧化物酶底物。产生的杂交瘤克隆中,有15个识别整个o8e蛋白质的分泌的mabs。表位作图显示在这15个克隆中,14个分泌的mabs识别o8e氨基酸残基61-80,一个克隆分泌识别氨基酸残基151-170的mab。对于流式细胞计量术分析,收集稳定转染o8e的hek293细胞和表达o8emrna的skbr3细胞,在流动染色缓冲液(pbs 1%bsa 叠氮化物)中洗涤。在冰上,细胞与mab杂合体上清液温育30分钟,随后,用染色缓冲液洗3次。在冰上,细胞与山羊抗鼠ig-fitc温育30分钟,随后在含有碘化丙锭的冲洗缓冲液中重悬浮前用染色缓冲液洗3次。流式细胞计量术分析表明15/15mabs能检测表达在o8e转染的hek293细胞表面上的o8e蛋白质。在skbr3细胞上试验的6/6mabs能识别表面表达的o8e。实施例9序列o772p的延长dna和蛋白质序列分析通过筛选卵巢肿瘤(scid来源的)cdna文库获得包含克隆3f,6b,8e和12的全长序列,详细如实施例2所述。这个2996碱基对序列,命名为o722p,如序列seqidno311所示,而且编码的914氨基酸蛋白质序列如seqidno312所示。利用公共数据库,包括genbank,检索dna序列o772p,它显示了与genbank登记号ak024365(seqidno457)的显著命中。发现这个genbank序列长度是3557碱基对,编码长度为1156氨基酸的蛋白质(seqidno459)。这个序列的截短形式,残基25-3471(seqidno456),编码长度是1148个氨基酸的蛋白质(seqidno458),其中残基25对应于genbank序列中第一个atg起始密码子。公开的dna序列(seqidno457)与o772p不同在于它有与seqidno457的碱基958-962对应的5个碱基对插入。这个插入引起读框移位,以致与o772p(seqidno312)相比,seqidno457编码附加的n-末端蛋白质序列。此外,o772p编码包含在残基1-79(seqidno460)中的单一n-末端部分。seqidno456的n-末端部分残基1-313也含有单一序列(uniquesequence),如seqidno461所列。实施例10产生多克隆抗体用于o772p分子细胞相关表达型式的免疫化学和流式细胞计量术分析在本申请实施例2和9中鉴定了o772p分子。为了评估在各种组织中抗原表达的亚细胞定位和特异性,产生抗o772p的多克隆抗体。为了产生这些抗体,在大肠杆菌(e.coli)重组表达系统中表达o772p-1(序列seqidno312的氨基酸44-772)和o772p-2(序列seqidno312的氨基酸477-914),37℃在lb肉汤中过夜生长。下一天,加入10ml过夜培养物到500ml含有适合抗生素的2×yt中。当培养物光密度(在560nm)达到0.4-0.6时,用iptg(1mm)诱导细胞。温育后,收集,洗涤,裂解细胞,在16,000psi压力下,通过弗氏压碎器。然后,离心细胞,通过sds-page检查重组蛋白质的分配。对于定位于细胞沉淀的蛋白质,在10mmtrisph8.0,1%chaps中重悬浮沉淀,而且洗涤包含体沉淀,离心。用含有添加10mm咪唑的ph8.010mmtris的8m尿素或6m胍hcl溶解洗过的包含体沉淀。加溶解蛋白质到5ml镍螯合树脂(qiagen)中,室温下温育45分钟。温育后,倾注树脂和蛋白质混合物通过柱子,收集通过的液流。然后用10-20倍柱体积的缓冲液洗柱子。随后,用8m尿素,10mmtrisph8.0和300mm咪唑从柱子上洗脱抗原,收集在3ml的级分中。进行sds-page凝胶以确定合并哪些级分以进一步纯化。做为最后纯化步骤,用适合缓冲液平衡强阴离子交换树脂,而且加样上面合并的级分到柱上。用逐步增加的盐梯度洗脱柱上抗原。收集级分并用sds-page凝胶分析以确定合并柱上的哪些级分。用10mmtrisph8.0透析合并的级份,呈上由此产生的蛋白质用于最后释放的质量控制。释放的标准是(a)sds-page或hplc测定的纯度,(b)劳里(lowry)测定法或氨基酸分析测定的浓度,(c)氨基酸末端蛋白质序列测定的同一性,及(d)鲎(lal)测定法测定的内毒素水平。然后,通过0.22μm过滤器过滤蛋白质后,在需要免疫前,冷冻抗原。为了产生多克隆抗血清,将400μgo772p-1或o772p-2与100μg胞壁酰二肽(mdp)混合。每4周,用100μg与等体积不完全弗氏(freund’s)佐剂(ifa)混合的抗原免疫兔。每次加强免疫后7天,取动物血液。在4℃温育血液12-24小时,离心,产生血清。为了鉴定多克隆抗血清的特性,用抗原包被96孔板,随后用bsa封闭。在pbs中稀释抗兔血清,加入每个孔。然后洗板和加入山羊抗兔辣根过氧化物酶(hrp)。再一次洗板,每个孔加入tmb微孔过氧化物酶底物。温育后,终止比色反应,在450nm下立即对板读数。所有多克隆抗体均显示了对合适抗原的免疫反应性。在石蜡包埋甲醛固定的组织上进行o772p表达的免疫组织化学分析。发现o772p在正常卵巢和卵巢癌中表达,而不在心脏,肾,结肠,肺或肝中表达。此外,免疫组织化学和流式细胞计量术分析表明o772p是质膜相关分子。o772p1含有预测由氨基酸859-880编码的跨质膜结构域1。o772p的n-末端是细胞外的,而且是通过氨基酸1-859编码,而c-末端是细胞内的。序列分析表明有17个可能的n-连接的糖基化作用位点。实施例11o772p在原发卵巢肿瘤细胞表面上表达为了在哺乳动物细胞中重组表达,分别亚克隆o772p-21008(seqidno387)和o772p全长cdna(seqidno311,编码seqidno312蛋白质)进入哺乳动物表达载体pbib或pcep4。用fugene6(roche)转染这些构建体进入hek293细胞。然后,hek293细胞以100,000细胞/ml密度在含有胎牛血清(fbs)的dmem中接种,过夜生长。第2天,加入2μlfugene6到100μldmem中,dmem中不含有fbs,室温下温育15分钟。向1μgo772p/pbib或o772p/pcep4质粒dna中加入fugene6/dmem混合物,室温温育另外15分钟。然后,将fugene6/dna混合物加到hek293细胞中,37℃,7%co2中培养48-72小时。洗涤细胞,然后通过离心沉淀。为了western印迹分析,通过在裂解缓冲液中温育细胞,产生全细胞裂解液,随后离心澄清。稀释样品,进行sds-page。然后转移凝胶到硝化纤维素上,而且用纯化抗o772p-2兔多克隆抗体探测。用与hrp结合的山羊抗兔ig在ecl底物中温育后显示印迹。westernblot分析表明能在o772p转染的hek293细胞中检测到o772p-21008。为了检测细胞中o772p的细胞表达分布图,在scid小鼠中生长原发卵巢癌肿瘤细胞。从小鼠中取回细胞,通过流式细胞计量术分析。简而言之,在含有pbs,1%bsa,和叠氮化钠的冷却染色缓冲液中洗细胞。细胞与10μg/ml纯化的抗o772p-1和o772p-2多克隆血清温育30分钟。温育后,在染色缓冲液中洗细胞3次,用与fitc缀合的山羊抗兔ig(h l)(southernbiotechnology)温育。在含有碘化丙锭(pi)的染色缓冲液中洗涤和重悬浮细胞,该pi是鉴定非存活细胞的活体染料。然后用荧光激活细胞分类术(facs)分析细胞。facs分析表明,o772p在细胞表面存在。肿瘤细胞上的o772p表面表达允许通过治疗抗体进行免疫靶向。实施例12抗o8e单克隆抗体的功能表征如实施例8所述,产生小鼠抗源于大肠杆菌(e.coli)的o8e的单克隆抗体(mab),并检测该单克隆抗体促进o8e抗原内在化的能力。体外用细胞毒性测定法测定抗体内在化。简而言之,在50ng/孔纯化抗o8e或对照抗体存在的情况下,hek293和o8e/hek转染细胞在含有添加10%热失活fbs的dme的96孔板中接种。抗o8emab的同种型如下11a6-igg1/κ,15c6-igg2b/κ,18a8-igg2b/κ,及14f1-igg2a/κ。w6/32是用作阳性对照的黑猩猩(pan)抗人mhc类型i小鼠单克隆抗体,还包括做为阴性对照的两个不相关mab,ir-pharm和ir-crxa。与o8e特异抗体或相关对照抗体温育后,以100ng/ml的浓度加mab-zap(一种山羊抗小鼠ig-皂草素缀合的第二抗体)(advancedtargetingsystems)到一半孔中,37℃,在7%co2培养箱中温育板48到72小时。这个测定法利用了皂草素(核酶失活蛋白质)的有毒特性,当其内在化时皂草素有细胞毒性作用。与mab-zap温育后,通过添加mts试剂,随后在微板elisa读数器上对板od490读数来定量内在化。图25描述了从这些测定得到的结果。上图代表没有被o8e转染的hek细胞,因此o8e抗体不应该结合和被内在化。无论样品是否与mab-zap温育,除了阳性对照ab,w6/32外,所有样品的增殖水平是同样的。下图代表被o8e转染的hek细胞,因此应该结合o8e特异抗体。来源于杂交瘤11h6,14f1,和15c6的,识别o8e氨基酸61-80的抗体能促进o8e表面蛋白质内在化,这是通过由于mab-zap的毒性引起增殖水平降低检测到的。实施例13卵巢肿瘤抗原o772p的表征上文(例如实施例2和9)已描述了卵巢肿瘤抗原o772p多种形式的cdna和蛋白质序列。genbank检索表明o772p同flj14303(登记号ako24365;seqidno457和463)有高度相似性。在序列seqidno478和479中分别公开了与o772p和flj14303对应的蛋白质序列。flj14303与o772p大部分相同,3’-末端大量序列,显示了100%同源性。然而,flj143035’-末端比o772p进一步延伸5’。此外,flj14303含有5bp插入序列(seqidno457),这个插入引起了氨基末端蛋白质序列的读框移位,以致与o772p比较,flj14303利用了不同起始甲硫氨基酸,因此编码不同蛋白质。在基因组序列中存在这个插入,而且在显示与这个区域有同一性的所有est克隆中可见这个插入,表明flj14303(seqidno457)代表了o772p的剪接变体,同时有含有延伸和不同氨基末端的orf。附加的5’核苷酸序列包括在o772p基因组作图期间鉴定的重复序列。o772p的5’末端和flj14303的相应区域显示90-100%的同源性。总的看来,这表明o772p和flj14303是相同基因不同剪接的变异体,不同单一重复序列被剪接进入基因的5’-末端。在染色体19的相同区域内另外10个或更多个重复序列的鉴定,表明由于不同重复序列的不同剪接,可以有许多种形式的o772p,每个都有不同的5’-末端。o772p的northern印迹分析证明了不同大小的多个与o772p杂交的转录物,一些超过了10kb。进一步分析鉴定了13个另外o772p相关序列,它们的cdna和氨基酸序列如表2所述。表2nd=未检测最初,认为这些序列代表了o772p剪接形式的重叠和/或不连续序列,其能编码o772p特定剪接形式所独特的多肽。然而,这些序列的核苷酸对齐(alignment)未能在重复元件内鉴定出相同区域。这表明这些序列可能代表单o772p基因的不同特定区域,该基因含有16或更多重复结构域,它们的全部形成了单一线性转录物。序列ls#104300.10(表2;seqidno465)的5’-末端对o772p和flj14303都是唯一的,而且不含重复元件,表明这个序列可能代表o772p的5’-末端。以前,跨膜预测分析表明o772p含有1至3个跨膜的横跨结构域。通过用免疫组织化学和流式细胞计量术证实了这点,也证明了代表o772p的质膜相关分子的存在。然而免疫组织化学也表明了分泌形式的o772p的存在,它可能从o772p的可变剪接形式或从翻译后切割事件产生。表2所示几个序列的分析表明了序列1043400b.12,1043400.8b和1043400.11b都含有跨膜区域,而1043400.8a,1043400.10,1043400.1,1043400.11a和1043400.9都缺少跨膜区域,表明这些蛋白质可能是分泌的。分析表明,部分o772p被表达和/或保留在质膜上,使o772p成为定向抗这种蛋白质的特异免疫治疗,例如治疗性抗体的有吸引力的目标靶。在序列seqidno489中公开了预测的o772p细胞外结构域,而且由于在以下序列中的切割事件,o772p的分泌可能会出现slveqvfldktlnasfhwlgstyqlvdihvtemessvyqp.在seqidno489位置10的赖氨酸(k)处最可能出现蛋白酶切割。o772p的细胞外,跨膜和细胞质区域都在序列seqidno488中公开了细胞外的slveqvfldktlnasfhwlgstyqlvdihvtemessvyqptsssstqhfylnftitnlpysqdkaqpgttnyqrnkrniedalnqlfrnssiksyfsdcqvstfrsvpnrhhtgvdslcnfsplarrvdrvaiyeeflrmtrngtqlqnftldrssvlvdgyfpnrnepltgnsdlpf跨膜的waviliglagllglitclicgvlvtt细胞质的rrrkkegeynvqqqcpgyyqshldledlq实施例14在卵巢癌和正常组织中o8e表达的免疫组织化学(ihc)分析为了检测哪些组织表达卵巢癌抗原o8e,用对o8e特异的多克隆和单克隆抗体,在各种组织切片进行ihc分析。o8e特异性单克隆抗体的产生如实施例8中详细所述。用于染色的单克隆抗体是11a6和14f1,它们是o8e的氨基酸61-80特异的,所用单克隆抗体还有18a8,它识别oe8的氨基酸151-170(其产生详见实施例12)。为了染色,在甲醛溶液中固定组织样品12-24小时,在切成8μm切片前在石蜡中包埋。用在0.1m柠檬酸钠缓冲液中蒸汽加热诱导表位恢复(sheir)做为最佳染色条件。切片与10%血清/pbs温育5分钟。然后每个切片加入第一抗体,25分钟,随后与抗兔或抗小鼠生物素标记的抗体温育25分钟。通过用过氧化氢进行三个1.5分钟的温育封闭内源过氧化物酶活性。抗生物素蛋白-生物素复合物/辣根过氧化物酶(abc/hrp)系统与dab色原体一起使用以观察抗原表达。用苏木精复染载波片以观察细胞核。用o8e兔亲和纯化多克隆抗体(氨基酸29-283;该抗体的产生细节,参见实施例3)得到的结果如表3所示。用3个单克隆抗体得到的结果如表4所示。表3用多克隆抗体进行的o8e免疫组织化学分析表4用单克隆抗体进行的o8e免疫组织化学分析0=没有染色,1=轻度染色,2=中度染色,n/a=没有得到实施例15o772p多克隆抗体的表位作图为了进行o772p的表位作图,产生了肽,这些肽的序列来自o772p序列。这些肽是15mers,有5个氨基酸重叠,通过在膜载体上化学合成而产生。所述肽通过其c-末端与whatman50纤维素载体共价结合,而n-末端不结合。为了测定表位特异性,用100%乙醇湿润膜1分钟,随后在tbs/tween/triton缓冲液(50mmtris,137mmnacl,2.7mmkcl,0.5%bsa,0.05%tween20,0.05%tritonx-100,ph7.5)中封闭16小时。然后用两个o772p特异抗体,o772p-1(seqidno312的氨基酸44-772)和o772p-2(seqidno312的477-914;参见实施例10中抗体产生的详述)及不相关兔抗体做为对照探测肽。稀释抗体为1μg/ml,室温下与膜温育2小时。然后在tbs/tween/triton缓冲液中洗膜30分钟,之后与1∶10,000稀释的hrp-连接的抗兔第二抗体温育2小时。再一次在tbs/tween/triton中洗膜30分钟。用ecl观察抗肽反应性。在表5中描述了特定表位对每个o772p多克隆抗体的结合特异性。表5*=相对的反应水平,-未结合,****最大结合实施例16o772p相关的新n-末端重复结构的鉴定如上所述,已鉴定和表征了各种o772pcdna和蛋白质形式(例如,实施例1,2,9和14)。重要的是,相对于正常组织,证明了o772prna和蛋白质在卵巢癌组织中过量表达,因此是卵巢癌诊断和治疗应用的有吸引力的靶标。利用以前鉴定为与o772p有同源性的基因组核苷酸序列的开放阅读框架(orfs)的生物信息学分析,在编码o772p蛋白质的基因的5’区域鉴定出了多个核苷酸重复序列。用对各重复特异的引物,通过rt-pcr证实了这些重复序列中的许多。从cdna扩增了含有多个重复的片段,因而证实了特定重复的存在及允许确定这些重复的顺序。意外地,当分析来源于不同数据库和实验室来源的各组o772p序列时,在o772p基因的5’-区域和o772p蛋白质的相应n-末端区域鉴定到了至少20个不同重复结构,每个重复结构相互之间都有实质水平的同一性(参见表6)。每个重复包含编码多肽单元的连续开放阅读框架,所述多肽单元能与一个或多个其它重复剪接,这样形成了不同数量和顺序的重复串连体。有趣地是,在科学文献中描述了有类似于本发明所述o772p的重复结构域的其它分子。例如蛋白质粘蛋白家族,它们是下述粘液的主要糖蛋白组分,所述粘液包被呼吸道,消化道和泌尿生殖道被覆细胞表面。已证明不同粘蛋白是由不同数量,长度和氨基酸序列的串连重复序列组成(perez-vilar和hill,j.biol.chem.274(45)31751-31754,1999)。预期本文所述各种鉴定的重复结构很可能通过可变剪接产生多种形式的o772p。已鉴定的重复的cdna序列如seqidno513-540,542-546和548-567所述。所述重复的编码氨基酸序列如seqidnos574-593所述。在许多情况下,这些氨基酸序列代表共有序列,所述共有序列是从一个以上通过试验得到的序列的对齐得到的。这些剪接形式中每一个都能编码有多个重复结构域的唯一o772p蛋白质,所述多个重复结构域与含有跨膜区的o772p恒定羧基末端蛋白质部分连接。o772p恒定区的cdna序列如seqidno568所述,而且编码的氨基酸序列如seqidno594所述。分解所有得到的可用o772p序列为其可鉴定的重复。用加权残基重量表(weightedresideweighttable)及cluster方法(dnastar序列分析包中的megalign软件)比较这些序列以鉴定重复序列间关系。利用这个信息,通过rt-pcr提供的排序数据,和用seqman(dnastar)进行的序列对齐(自动和人工),鉴定出了一个包含20个明显不同重复单元的示例性共有序列全长o772p重叠群。这个o772pcdna重叠群的cdna如seqidno569所述,而且编码氨基酸序列如seqidno595所述。这种形式的o772p蛋白质包括按以下顺序的下述共有重复结构seqidno572-seqidno574-seqidno575-seqidno576-seqidno577-seqidno578-seqidno579-seqidno580-seqidno581-seqidno582-seqidno583-seqidno584-seqidno585-seqidno586-seqidno587-seqidno588-seqidno589-seqidno590-seqidno591-seqidno592-seqidno593。因此,seqidno595代表了o772p蛋白质一个示例性全长共有序列。然而,如上所述,基于对这个蛋白质目前的知识和基于描述含有类似重复结构的蛋白质的科学文献,预期存在许多有或多或少重复的其它形式o772p。另外,预期许多形式的o772p有不同排列,例如不同顺序的这些n-末端重复结构。通过o772p的northern分析支持了有不同数量重复的多种形式o772p的存在。在这个研究中,o772p特异探针的northern杂交产生了多个o772p杂交性转录物的成片条带,一些超过了10kb。因此,o772p蛋白质的可变重复区可以用xn-y结构示例性地表示,其中x包含与seqidno596所示共有重复序列有至少50%同一性的重复结构;n是蛋白质中存在的重复数,预期典型地是从1到约35的整数;y包含seqidno594所述的恒定区序列,或与seqidno594有至少80%的同一性的序列。o772p分子xn重复区中存在的每个x是不同的。为了确定20个重复区中每个的共有序列,对齐经试验测定的分立重复区的序列并确定共有序列。除了确定每个重复区的共有序列外,也确定了共有重复序列。通过对齐20个各共有序列获得这个序列。通过对齐来源于各重复区每一个的共有氨基酸序列和总的重复共有序列,检测重复的变异性。同一性数据如表6所示。表6与共有重复序列比较,重复序列的同一性百分率从上述描述可以理解,尽管为了举例说明的目的这里描述本发明特定实施方式,但是可以进行各种修改,而不背离本发明的精神和范围。因此,本发明仅受所附权利要求书的限制。序列表<110>corixacorporationmitcham,jenniferl.king,gordone.algate,paula.fling,stevenp.retter,marcw.fanger,garyrichardreed,steveng.vedvick,thomass.carter,darrickhill,paulalbone,earl<120>用于治疗和诊断卵巢癌的组合物和方法<130>210121.46201pc<140>pct<141>2001-07-17<160>596<170>fastseqforwindowsversion4.0<210>1<211>461<212>dna<213>人(homosapiens)<400>1ttagagaggcacagaaggaagaagagttaaaagcagcaaagccgggtttttttgttttgt60tttgttttgttttgttttgagatggagtctcactctgttgcccaagctggagtacaacgg120catgatctcagctcgctgcaacctccgcctcccacgttcaagtgattctcctgcctcagc180ctcccaagtagctgggattacaggcgcccgccaccacgctcagctaattttttttgtatt240tttagtagagacagggtttcaccaggttggccaggctgctcttgaactcctgacctcagg300tgatccacccgcctcggcctcccaaagtgctgggattacaggcgtgagccaccacgcccg360gcccccaaagctgtttcttttgtctttagcgtaaagctctcctgccatgcagtatctaca420taactgacgtgactgccagcaagctcagtcactccgtggtc461<210>2<211>540<212>dna<213>人<400>2taggatgtgttggaccctctgtgtcaaaaaaaacctcacaaagaatcccctgctcattac60agaagaagatgcatttaaaatatgggttattttcaactttttatctgaggacaagtatcc120attaattattgtgtcagaagagattgaatacctgcttaagaagcttacagaagctatggg180aggaggttggcagcaagaacaatttgaacattataaaatcaactttgatgacagtaaaaa240tggcctttctgcatgggaacttattgagcttattggaaatggacagtttagcaaaggcat300ggaccggcagactgtgtctatggcaattaatgaagtctttaatgaacttatattagatgt360gttaaagcagggttacatgatgaaaaagggccacagacggaaaaactggactgaaagatg420gtttgtactaaaacccaacataatttcttactatgtgagtgaggatctgaaggataagaa480aggagacattctcttggatgaaaattgctgtgtagagtccttgcctgacaaagatggaaa540<210>3<211>461<212>dna<213>人<400>3ttagagaggcacagaaggaagaagagttaaaagcagcaaagccgggtttttttgttttgt60tttgttttgttttgttttgagatggagtctcactctgttgcccaagctggagtacaacgg120catgatctcagctcgctgcaacctccgcctcccacgttcaagtgattctcctgcctcagc180ctcccaagtagctgggattacaggcgcccgccaccacgctcagctaattttttttgtatt240tttagtagagacagggtttcaccaggttggccaggctgctcttgaactcctgacctcagg300tgatccacccgcctcggcctcccaaagtgctgggattacaggcgtgagccaccacgcccg360gcccccaaagctgtttcttttgtctttagcgtaaagctctcctgccatgcagtatctaca420taactgacgtgactgccagcaagctcagtcactccgtggtc461<210>4<211>531<212>dna<213>人<220><221>misc_feature<222>454,492,526<223>n=a,t,c或g<400>4tctttttctttcgatttccttcaatttgtcacgtttgattttatgaagttgttcaagggc60taactgctgtgtattatagctttctctgagttccttcagctgattgttaaatgaatccat120ttctgagagcttagatgcagtttctttttcaagagcatctaattgttctttaagtctttg180gcataattcttccttttctgatgactttttatgaagtaaactgatccctgaatcaggtgt240gttactgagctgcatgtttttaattctttcgtttaatagctgcttctcagggaccagata300gataagcttattttgatattccttaagctcttgttgaagttgtttgatttccataatttc360caggtcacactgtttatccaaaacttctagctcagtcttttgtgtttgctttctgatttg420gacatcttgtagtctgcctgagatctgctgatgntttccattcactgcttccagttccag480gtggagactttnctttctggagctcagcctgacaatgccttcttgntccct531<210>5<211>531<212>dna<213>人<400>5agccagatggctgagagctgcaagaagaagtcaggatcatgatggctcagtttcccacag60cgatgaatggagggccaaatatgtgggctattacatctgaagaacgtactaagcatgata120aacagtttgataacctcaaaccttcaggaggttacataacaggtgatcaagcccgtactt180ttttcctacagtcaggtctgccggccccggttttagctgaaatatgggccttatcagatc240tgaacaaggatgggaagatggaccagcaagagttctctatagctatgaaactcatcaagt300taaagttgcagggccaacagctgcctgtagtcctccctcctatcatgaaacaacccccta360tgttctctccactaatctctgctcgttttgggatgggaagcatgcccaatctgtccattc420atcagccattgcctccagttgcacctatagcaacacccttgtcttctgctacttcaggga480ccagtattcctcccctaatgatgcctgctcccctagtgccttctgttagta531<210>6<211>531<212>dna<213>人<400>6aatagatttaatgcagagtgtcaacttcaattgattgatagtggctgcctagagtgctgt60gttgagtaggtttctgaggatgcaccctggcttgaagagaaagactggcaggattaacaa120tatctaaaatctcacttgtaggagaaaccacaggcaccagagctgccactggtgctggca180ccagctccaccaaggccagcgaagagcccaaatgtgagagtggcggtcaggctggcacca240gcactgaagccaccactggtgctggcactggcactggcactgttattggtactggtactg300gcaccagtgctggcactgccactctcttgggctttggctttagcttctgctcccgcctgg360atccgggctttggcccagggtccgatatcagcttcgtcccagttgcagggcccggcagca420ttctccgagccgagcccaatgcccattcgagctctaatctcggccctagccttggcttca480gctgcagcctcagctgcagccttcaaatccgcttccatcgcctctcggtac531<210>7<211>531<212>dna<213>人<400>7gccaagaaagcccgaaaggtgaagcatctggatggggaagaggatggcagcagtgatcag60agtcaggcttctggaaccacaggtggccgaagggtctcaaaggccctaatggcctcaatg120gcccgcagggcttcaaggggtcccatagccttttgggcccgcagggcatcaaggactcgg180ttggctgcttgggcccggagagccttgctctccctgagatcacctaaagcccgtaggggc240aaggctcgccgtagagctgccaagctccagtcatcccaagagcctgaagcaccaccacct300cgggatgtggcccttttgcaagggagggcaaatgatttggtgaagtacctttt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gccccgccctggtgtcacagaggctactattactggcctggaaccggg180aaccgaatatacaatttatgtcattgccctgaagaataatcagaagagcgagcccctgat240tggaaggaaaaagacagacgagcttccccaactggtaacccttccacaccccaatcttca300tggaccananancttggatngtcctttcacnggttnaaaaaacccttttcgcccccccac360cttggggattaaccttgggaaanggggatttnaccnttcc400<210>250<211>400<212>dna<213>人<220><221>misc_feature<222>338,357,361,369,388,394<223>n=a,t,c或g<400>250tcgagcggccgcccgggcaggtcctgtcagagtggcactggtagaagttccaggaaccct60gaactgtaagggttcttcatcagtgccaacaggatgacatgaaatgatgtactcagaagt120gtcctggaatggggcccatgagatggttgtctgagagagagcttcttgtcctacattcgg180cgggtatggtcttggcctatgccttatgggggtggccgttgtgggcggtgtggtccgcct240aaaaccatgttcctcaaagatcatttgttgcccaacactgggttgctgaccagaagtgcc300aggaagctgaataccatttccagtgtcatacccagggngggtgaccaaagggggtcnttt360ngacctggngaaaggaaccatccaaaanctctgncccatg400<210>251<211>514<212>dna<213>人<220><221>misc_feature<222>8,107,312,338,351,352,357,363,366,373,380,405,421,444,508<223>n=a,t,c或g<400>251agcgtggncgcggccgaggtctgaggatgtaaactcttcccaggggaaggctgaagtgct60gaccatggtgctactgggtccttctgagtcagatatgtgactgatgngaactgaagtagg120tactgtagatggtgaagtctgggtgtccctaaatgctgcatctccagagccttccatcat180taccgtttcttcttttgctatgggatgagacactgttgagtattctctaaagtcaccact240gaaatcttcctccaaaggaaaacctgtggaaaagccccttatttctgccccataatttgg300ttctcctaatcnctctgaaatcactatttccctggaangtttgggaaaaanngggcnacc360tgncantggaaantggatanaaagatcccaccattttacccaacnagcagaaagtgggaa420nggtaccgaaaagctccaagtaanaaaaaggagggaagtaaaggtcaagtgggcaccagt480ttcaaacaaaactttccccaaactatanaaccca514<210>252<211>501<212>dna<213>人<220><221>misc_feature<222>20,21,25,44,343,347,356,362,387,391,398,409,428,430,453,494<223>n=a,t,c或g<400>252aagcggccgcccgggcaggnncagnagtgccttcgggactgggntcacccccaggtctgc60ggcagttgtcacagcgccagccccgctggcctccaaagcatgtgcaggagcaaatggcac120cgagatattccttctgccactgttctcctacgtggtatgtcttcccatcatcgtaacacg180ttgcctcatgagggtcacacttgaattctccttttccgttcccaagacatgtgcagctca240tttggctggctctatagtttggggaaagtttgttgaaactgtgccactgacctttacttc300ctccttctctactggagctttccgtaccttccacttctgctgntggnaaaaagggnggaa360cntcttatcaatttcattggacagtancccnctttctncccaaaacatncaagggaaaat420attgattncnagagcggattaaggaacaacccnaattatgggggccagaaataaaggggg480cttttccacaggtnttttcct501<210>253<211>226<212>dna<213>人<400>253tcgagcggccgcccgggcaggtctgcaggctattgtaagtgttctgagcacatatgagat60aacctgggccaagctatgatgttcgatacgttaggtgtattaaatgcacttttgactgcc120atctcagtggatgacagccttctcactgacagcagagatcttcctcactgtgccagtggg180caggagaaagagcatgctgcgactggacctcggccgcgaccacgct226<210>254<211>226<212>dna<213>人<400>254agcgtggtcgcggccgaggtccagtcgcagcatgctctttctcctgcccactggcacagt60gaggaagatctctgctgtcagtgagaaggctgtcatccactgagatggcagtcaaaagtg120catttaatacacctaacgtatcgaacatcatagcttggcccaggttatctcatatgtgct180cagaacacttacaatagcctgcagacctgcccgggcggccgctcga226<210>255<211>427<212>dna<213>人<220><221>misc_feature<222>327,403<223>n=a,t,c或g<400>255cgagcggccgcccgggcaggtccagactccaatccagagaaccaccaagccagatgtcag60aagctacaccatcacaggtttacaaccaggcactgactacaagatctacctgtacacctt120gaatgacaatgctcggagctcccctgtggtcatcgacgcctccactgccattgatgcacc180atccaacctgcgtttcctggccaccacacccaattccttgctggtatcatggcagccgcc240acgtgccaggattaccggctacatcatcaagtatgagaagcctgggtctcctcccagaga300agtggtccctcggccccgccctggtgncacagaagctactattactggcctggaaccggg360aaccgaatatacaatttatgtcattgccctgaagaataatcanaagagcgagcccctgat420tggaagg427<210>256<211>535<212>dna<213>人<220><221>misc_feature<222>347,456,475<223>n=a,t,c或g<400>256agcgtggtcgcggccgaggtcctgtcagagtggcactggtagaagttccaggaaccctga60actgtaagggttcttcatcagtgccaacaggatgacatgaaatgatgtactcagaagtgt120cctggaatggggcccatgagatggttgtctgagagagagcttcttgtcctgtctttttcc180ttccaatcaggggctcgctcttctgattattcttcagggcaatgacataaattgtatatt240cggttcccggttccaggccagtaatagtagcctctgtgacaccagggcggggccgaggga300ccacttctctgggaggagacccaggcttctcatacttgatgatgtanccggtaatcctgg360caccgtggcggctgccatgataccagcaaggaattgggtgtggtggccaagaaacgcagg420ttggatggtgcatcaatggcagtggaggcgtcgatnaccacaggggagctccgancattg480tcattcaaggtggacaggtagaatcttgtaatcaggtgcctggtttgtaaacctg535<210>257<211>544<212>dna<213>人<220><221>misc_feature<222>495,511<223>n=a,t,c或g<400>257tcgagcggccgcccgggcaggtttcgtgaccgtgacctcgaggtggacaccaccctcaag60agcctgagccagcagatcgagaacatccggagcccagagggcagccgcaagaaccccgcc120cgcacctgccgtgacctcaagatgtgccactctgactggaagagtggagagtactggatt180gaccccaaccaaggctgcaacctggatgccatcaaagtcttctgcaacatggagactggt240gagacctgcgtgtaccccactcagcccagtgtggcccagaagaactggtacatcagcaag300aaccccaaggacaagaagcatgtctggttcggcgaaagcatgaccgatggattccagttc360gagtatggcggccagggctccgaccctgccgatgtggacctcggccgcgaccacgctaag420cccgaattccagcacactggcggccgttactagtgggatccgagcttcggtaccaagctt480ggcgtaatcatgggncatagctgtttcctgngtgaaaatggtattccgcttcacaatttc540ccac544<210>258<211>418<212>dna<213>人<400>258agcgtggtcgcggccgaggtccacatcggcagggtcggagccctggccgccatactcgaa60ctggaatccatcggtcatgctctcgccgaaccagacatgcctcttgtccttggggttctt120gctgatgtaccagttcttctgggccacactgggctgagtggggtacacgcaggtctcacc180agtctccatgttgcagaagactttgatggcatccaggttgcagccttggttggggtcaat240ccagtactctccactcttccagtcagagtggcacatcttgaggtcacggcaggtgcgggc300ggggttcttgcggctgccctctgggctccggatgttctcgatctgctggctcaagctctt360gaagggtggtgtccacctcgaggtcacggtcacgaaacctgcccgggcggccgctcga418<210>259<211>377<212>dna<213>人<220><221>misc_feature<222>320,326,342,352<223>n=a,t,c或g<400>259agcgtggtcgcggccgaggtcaagaaccccgcccgcacctgccgtgacctcaagatgtgc60cactctgactggaagagtggagagtactggattgaccccaaccaaggctgcaacctggat120gccatcaaagtcttctgcaacatggagactggtgagacctgcgtgtaccccactcagccc180agtgtggcccagaagaactggtacatcagcaagaaccccaaggacaagaggcatgtctgg240ttcggcgagagcatgaccgatggattccagttcgagtatggcggccagggctccgaccct300gccgatgtggacctgcccgngccggnccgctcgaaaagcccnaatttccagncacacttg360gccggccgttactactg377<210>260<211>332<212>dna<213>人<400>260tcgagcggccgcccgggcaggtccacatcggcagggtcggagccctggccgccatactcg60aactggaatccatcggtcatgctctcgccgaaccagacatgcctcttgtccttggggttc120ttgctgatgtaccagttcttctgggccacactgggctgagtggggtacacgcaggtctca180ccagtctccatgttgcagaagactttgatggcatccaggttgcagccttggttggggtca240atccagtactctccactcttccagtcagagtggcacatcttgaggtcacggcaggtgcgg300gcggggttcttgacctcggccgcgaccacgct332<210>261<211>94<212>dna<213>人<400>261cgagcggccgcccgggcaggtccccccccttttttttttttttttttttttttttttttt60tttttttttttttttttttttttttttttttttt94<210>262<211>650<212>dna<213>人<220><221>misc_feature<222>412,582,612,641,646<223>n=a,t,c或g<400>262agcgtggtcgcggccgaggtctggcattccttcgacttctctccagccgagcttcccaga60acatcacatatcactgcaaaaatagcattgcatacatggatcaggccagtggaaatgtaa120agaaggccctgaagctgatggggtcaaatgaaggtgaattcaaggctgaaggaaatagca180aattcacctacacagttctggaggatggttgcacgaaacacactggggaatggagcaaaa240cagtctttgaatatcgaacacgcaaggctgtgagactacctattgtagatattgcaccct300atgacattggtggtcctgatcaagaatttggtgtggacgttggccctgtttgctttttat360aaaccaaactctatctgaaatcccaacaaaaaaaatttaactccatatgtgntcctcttg420ttctaatcttggcaaccagtgcaagtgaccgacaaaattccagttatttatttccaaaat480gtttggaaacagtataatttgacaaagaaaaaaggatacttctctttttttggctggtcc540accaaatacaattcaaaaggctttttggttttatttttttanccaattccaatttcaaaa600tgtctcaatggngcttataataaaataaactttcacccttnttttntgat650<210>263<211>573<212>dna<213>人<220><221>misc_feature<222>453,458,544<223>n=a,t,c或g<400>263agcgtggtcgcggccgaggtctgggatgctcctgctgtcacagtgagatattacaggatc60acttacggagaaacaggaggaaatagccctgtccaggagttcactgtgcctgggagcaag120tctacagctaccatcagcggccttaaacctggagttgattataccatcactgtgtatgct180gtcactggccgtggagacagccccgcaagcagcaagccaatttccattaattaccgaaca240gaaattgacaaaccatcccagatgcaagtgaccgatgttcaggacaacagcattagtgtc300aagtggctgccttcaagttcccctgttactggttacagaagtaaccaccactcccaaaaa360tggaccaggaccaacaaaaactaaaactgcaggtccagatcaaacagaaaatggactatt420gaaggcttgcagcccacagtggaagtatgtggntaggngtctatgctcagaatcccaagc480cggagaaagtcagccttctggtttagactgcagtaaccaacattgatcgccctaaaggac540tggncattcacttggatggtggatgtccaattc573<210>264<211>550<212>dna<213>人<220><221>misc_feature<222>39,174,352,526<223>n=a,t,c或g<400>264tcgagcggccgcccgggcaggtccttgcagctctgcagngtcttcttcaccatcaggtgc60agggaatagctcatggattccatcctcagggctcgagtaggtcaccctgtacctggaaac120ttgcccctgtgggctttcccaagcaattttgatggaatcgacatccacatcagngaatgc180cagtcctttagggcgatcaatgttggttactgcagtctgaaccagaggctgactctctcc240gcttggattctgagcatagacactaaccacatactccactgtgggctgcaagccttcaat300agtcatttctgtttgatctggacctgcagttttaagtttttggtggtcctgncccatttt360tgggaagtggggggttactctgtaaccagtaacaggggaacttgaaggcagccacttgac420actaatgctgttgtcctgaacatcggtcacttgcatctggggatggttttgacaatttct480ggttcggcaaattaatggaaattggcttgctgcttggcggggctgnctccacgggccagt540gacagcatac550<210>265<211>596<212>dna<213>人<220><221>misc_feature<222>347,352,353,534,555,587<223>n=a,t,c或g<400>265tcgagcggccgcccgggcaggtccttgcagctctgcagtgtcttcttcaccatcaggtgc60agggaatagctcatggattccatcctcagggctcgagtaggtcaccctgtacctggaaac120ttgcccctgtgggctttcccaagcaattttgatggaatcgacatccacatcagtgaatgc180cagtcctttagggcgatcaatgttggttactgcagtctgaaccagaggctgactctctcc240gcttggattctgagcatagacactaaccacatactccactgtgggctgcaagccttcaat300agtcatttctgtttgatctggacctgcagttttaagtttttgttggncctgnnccatttt360tggggaaggggtggttactcttgtaaccagtaacaggggaacttgaagcagccacttgac420actaatgctggtggcctgaacatcggtcacttgcatctgggatggtttggtcaatttctg480ttcggtaattaatgggaaattggcttactggcttgcgggggctgtctccacggncagtga540caagcatacacaggngatgggtataatcaactccaggtttaaggccnctgatggta596<210>266<211>506<212>dna<213>人<220><221>misc_feature<222>393,473<223>n=a,t,c或g<400>266agcgtggtcgcggccgaggtctgggatgctcctgctgtcacagtgagatattacaggatc60acttacggagaaacaggaggaaatagccctgtccaggagttcactgtgcctgggagcaag120tctacagctaccatcagcggccttaaacctggagttgattataccatcactgtgtatgct180gtcactggccgtggagacagccccgcaagcagtaagccaatttccattaattaccgaaca240gaaattgacaaaccatcccagatgcaagtgaccgatgttcaggacaacagcattagtgtc300aagtggctgccttcaagttcccctgttactggttacagagtaaccaccactcccaaaaat360gggaccaggaccaacaaaaaactaaaactgcanggtccagatcaaacagaaatgactatt420gaaggcttgcagcccacagtggagtatgtgggttagtgtctatgctcagaatnccaagcg480gagagagtcagcctctggttcagact506<210>267<211>548<212>dna<213>人<220><221>misc_feature<222>346,358,432,510,512<223>n=a,t,c或g<400>267tcgagcggccgcccgggcaggtcagcgctctcaggacgtcaccaccatggcctgggctct60gctcctcctcaccctcctcactcagggcacagggtcctgggcccagtctgccctgactca120gcctccctccgcgtccgggtctcctggacagtcagtcaccatctcctgcactggaaccag180cagtgacgttggtgcttatgaatttgtctcctggtaccaacaacacccaggcaaggcccc240caaactcatgatttctgaggtcactaagcggccctcaggggtccctgatcgcttctctgg300ctccaagtctggcaacacggcctccctgaccgtctctgggctccangctgaggatgangc360tgattattactggaagctcatatgcaggcaacaacaattgggtgttcggcggaagggacc420aagctgaccgtnctaaggtcaagcccaaggcttgcccccctcggtcactctgttcccacc480ctcctctgaagaagctttcaagccaacaangncacactgggtgtgtctcataagtggact540ttctaccc548<210>268<211>584<212>dna<213>人<220><221>misc_feature<222>98,380,421,454,495,506,512,561,565,579<223>n=a,t,c或g<400>268agcgtggtcgcggccgaggtctgtagcttctgtgggacttccactgctcaggcgtcaggc60tcaggtagctgctggccgcgtacttgttgttgctttgnttggagggtgtggtggtctcca120ctcccgccttgacggggctgctatctgccttccaggccactgtcacggctcccgggtaga180agtcacttatgagacacaccagtgtggccttgttggcttgaagctcctcagaggagggtg240ggaacagagtgaccgagggggcagccttgggctgacctaggacggtcagcttggtccctc300cgccgaacacccaattgttgttgcctgcatatgagctgcagtaataatcagcctcatcct360cagcctggagcccagagacngtcaagggaggcccgtgtttgccaagacttggaagccaga420naagcgatcagggacccctgagggccgctttacngacctcaaaaaatcatgaatttgggg480ggcctttgcctgggngttggttggtnaccagnaaaacaaaatttcataaagcaccaacgt540cactgctggtttccagtgcangaanatggtgaactgaantgtcc584<210>269<211>368<212>dna<213>人<220><221>misc_feature<222>265,329<223>n=a,t,c或g<400>269agcgtggtcgcggccgaggtccagcatcaggagccccgccttgccggctctggtcatcgc60ctttctttttgtggcctgaaacgatgtcatcaattcgcagtagcagaactgccgtctcca120ctgctgtcttataagtctgcagcttcacagccaatggctcccatatgcccagttccttca180tgtccaccaaagtacccgtctcaccatttacaccccaggtctcacagttctcctgggtgt240gcttggcccgaagggaggtaagtanacggatggtgctggtcccacagttctggatcaggg300tacgaggaatgacctctagggcctgggcnacaagccctgtatggacctgcccgggcgggc360ccgctcga368<210>270<211>368<212>dna<213>人<220><221>misc_feature<222>54,163,219,229,316<223>n=a,t,c或g<400>270tcgagcggccgcccgggcaggtccatacagggctgttgcccaggccctagaggncattcc60ttgtaccctgatccagaactgtgggaccagcaccatccgtctacttacctcccttcgggc120caagcacacccaggagaactgtgagacctggggtgtaaatggngagacgggtactttggt180ggacatgaaggaactgggcatatgggagccattggctgngaagctgcanacttataagac240agcagtggagacggcagttctgctactgcgaattgatgacatcgtttcaggccacaaaaa300gaaaggcgatgaccanagccggcaaggcggggcttcctgatgctggacctcggccgccga360ccacgctt368<210>271<211>424<212>dna<213>人<220><221>misc_feature<222>279,329,362,384,400<223>n=a,t,c或g<400>271agcgtggtcgcggccgaggtccactagaggtctgtgtgccattgcccaggcagagtctct60gcgttacaaactcctaggagggcttgctgtgcggagggcctgctatggtgtgctgcggtt120catcatggagagtggggccaaaggctgcgaggttgtggtgtctgggaaactccgaggaca180gagggctaaatccatgaagtttgtggatggcctgatgatccacagcggagaccctgttaa240ctactacgttgacactgctgtgcgccacgtgttgctcanacagggtgtgctgggcatcaa300ggtgaagatcatgctgccctgggacccanctggcaaaaatggcccttaaaaaccccttgc360cntgaccacgtgaaccatttgtgngaaccccaagatgaanatacttgcccaccacccccc420attc424<210>272<211>541<212>dna<213>人<220><221>misc_feature<222>422,442,510,513,515,525<223>n=a,t,c或g<400>272tcgagcggccgcccgggcaggtctgccaaggagaccctgttatgctgtggggactggctg60gggcatggcaggcggctctggcttcccacccttctgttctgagatgggggtggtgggcag120tatctcatctttgggttccacaatgctcacgtggtcaggcaggggcttcttagggccaat180cttaccagttgggtcccagggcagcatgatcttcaccttgatgcccagcacaccctgtct240gagcaacacgtggcgcacagcagtgtcaacgtagtagttaacagggtctccgctgtggat300catcaggccatccacaaacttcatggatttagccctctgtcctcggagtttcccaaaaca360ccacaacctcgccagcctttgggccccacttcttcatgaatgaaaccgcagcacaccatt420ancaaggcccttccgcacaggnaagcccttcctaaggagttttgtaaacgcaaaaaactc480ttgcctggggcaaatgggcacacagacctntantnggaccttggnccgcgaaccaccgct540t541<210>273<211>579<212>dna<213>人<220><221>misc_feature<222>223,265,277,308,329,346,360,366,429,448,517,524,531,578<223>n=a,t,c或g<400>273agcgtggtcgcggccgaggtctggccctcctggcaaggctggtgaagatggtcaccctgg60aaaacccggacgacctggtgagagaggagttgttggaccacagggtgctcgtggtttccc120tggaactcctggacttcctggcttcaaaggcattaggggacacaatggtctggatggatt180gaagggacagcccggtgctcctggtgtgaagggtgaacctggngcccctggtgaaaatgg240aactccaggtcaaacaggagcccgngggcttcctggngagagaggacgtgttggtgcccc300tggcccanacctgcccgggcggccgctcnaaaagccgaaatccagnacactggcggccgn360tactantggaatccgaacttcggtaccaaagcttggccgtaatcatggccatagcttgtt420ccctggggnggaaattggtattccgctnccaattccacacaacataccgaacccggaaag480cattaaagtgtaaaagccctgggggggcctaaatgangtgagcntaactcncatttaatt540ggcgttgcgcttcactgccccgcttttccagtccgggna579<210>274<211>330<212>dna<213>人<220><221>misc_feature<222>171<223>n=a,t,c或g<400>274tcgagcggccgcccgggcaggtctgggccaggggcaccaacacgtcctctctcaccagga60agcccacgggctcctgtttgacctggagttccattttcaccaggggcaccaggttcaccc120ttcacaccaggagcaccgggctgtcccttcaatccatccagaccattgtgncccctaatg180cctttgaagccaggaagtccaggagttccagggaaaccacgagcaccctgtggtccaaca240actcctctctcaccaggtcgtccgggttttccagggtgaccatcttcaccagccttgcca300ggagggccagacctcggccgcgaccacgct330<210>275<211>97<212>dna<213>人<220><221>misc_feature<222>2,35,72<223>n=a,t,c或g<400>275ancgtggtcgcggccgaggtcctcaccagaggtgncacctacaacatcatagtggaggca60ctgaaagaccancagaggcataaggttcgggaagagg97<210>276<211>610<212>dna<213>人<220><221>misc_feature<222>358,360,363,382,424,433,464,468,477,491,499,511,558,584,588,590<223>n=a,t,c或g<400>276tcgagcggccgcccgggcaggtccattttctccctgacggtcccacttctctccaatctt60gtagttcacaccattgtcatggcaccatctagatgaatcacatctgaaatgaccacttcc120aaagcctaagcactggcacaacagtttaaagcctgattcagacattcgttcccactcatc180tccaacggcataatgggaaactgtgtaggggtcaaagcacgagtcatccgtaggttggtt240caagccttcgttgacagagttgtccacggtaacaacctcttcccgaaccttatgcctctg300ctggtctttcagtgcctccactatgatgttgtaggtggcacctctggtgaggacctcngn360ccngaacaacgcttaagcccgnattctgcagaataatcccatcacacttggcggccgctt420cgancatgcatcntaaaaggggccccaatttcccccttataagngaanccgtatttncca480atttcactggncccgccgnttttacaaacgncggtgaactggggaaaaaccctggcggtt540acccaactttaatcgccnttggcagcacaatccccccttttcgnccancntgggcgtaaa600taaccgaaaa610<210>277<211>38<212>dna<213>人<220><221>misc_feature<222>2,5,18,21,31<223>n=a,t,c或g<400>277ancgnggtcgcggccgangtnttttttcttnttttttt38<210>278<211>443<212>dna<213>人<220><221>misc_feature<222>156,212,233,245,327,331,336,361,364,381,391,397,419,437<223>n=a,t,c或g<400>278agcgtggtcgcggccgaggtctgaggttacatgcgtggtggtggacgtgagccacgaaga60ccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaa120gccgcgggaggagcagtacaacagcacgtaccgggnggtcagcgtcctcaccgtcctgca180ccagaattggttgaatggcaaggagtacaagngcaaggtttccaacaaagccntcccagc240ccccntcgaaaaaaccatttccaaagccaaagggcagccccgagaaccacaggtgtacac300cctgcccccatcccgggaggaaaagancaanaaccnggttcagccttaacttgcttggtc360naangctttttatcccaacgnacttcccccntggaantgggaaaaaccaatgggccaanc420cgaaaaacaattacaanaacccc443<210>279<211>348<212>dna<213>人<220><221>misc_feature<222>219,256,291,297,307,314,317<223>n=a,t,c或g<400>279tcgagcggccgcccgggcaggtgtcggagtccagcacgggaggcgtggtcttgtagttgt60tctccggctgcccattgctctcccactccacggcgatgtcgctgggatagaagcctttga120ccaggcaggtcaggctgacctggttcttggtcatctcctcccgggatgggggcagggtga180acacctggggttctcggggcttgccctttggttttgaanatggttttctcgatgggggct240ggaagggctttgttgnaaaccttgcacttgactccttgccattcacccagncctggngca300ggacggngaggacnctnaccacacggaaccgggctggtggactgctcc348<210>280<211>149<212>dna<213>人<220><221>misc_feature<222>18,34,51,118,120,140<223>n=a,t,c或g<400>280agcgtggtcgcggacgangtcctgtcagagtggnactggtagaagttccangaaccctga60actgtaagggttcttcatcagtgccaacaggatgacatgaaatgatgtactcagaagngn120cctggaatggggcccatganatggttgcc149<210>281<211>404<212>dna<213>人<220><221>misc_feature<222>383,386,388,393<223>n=a,t,c或g<400>281tcgagcggccgcccgggcaggtccaccacacccaattccttgctggtatcatggcagccg60ccacgtgccaggattaccggctacatcatcaagtatgagaagcctgggtctcctcccaga120gaagtggtccctcggccccgccctggtgtcacagaggctactattactggcctggaaccg180ggaaccgaatatacaatttatgtcattgccctgaagaataatcagaagagcgagcccctg240attggaaggaaaaagacagacgagcttccccaactggtaacccttccacaccccaatctt300catggaccagagatcttggatgttccttccacagttcaaaagacccctttcggcaccccc360cctgggtatgaacctgggaaaanggnanttaanctttcctggca404<210>282<211>507<212>dna<213>人<220><221>misc_feature<222>320,341,424,450,459,487,498<223>n=a,t,c或g<400>282agcgtggtcgcggccgaggtctgggatgctcctgctgtcacagtgagatattacaggatc60acttacggagaaacaggaggaaatagccctgtccaggagttcactgtgcctgggagcaag120tctacagctaccatcagcggccttaaacctggagttgattataccatcactgtgtatgct180gtcactggccgtggagacagccccgcaagcagcaagccaatttccattaattaccgaaca240gaaattgacaaaccatcccagatgcaagtgaccgatgttcaggacaacagcattagtgtc300aagtggctgccttcaaggtnccctggtactgggttacagantaaccaccactcccaaaaa360tggaccaggaaccacaaaaacttaaactgcagggtccagatcaaaacagaaatgactatt420gaangcttgcagcccacagtgggagtatgngggtagtgnctatgcttcagaatccaagcg480gaaaaangtcaagccttntgggttcaa507<210>283<211>325<212>dna<213>人<220><221>misc_feature<222>216,292,303,304<223>n=a,t,c或g<400>283tcgagcggccgcccgggcaggtccttgcagctctgcagtgtcttcttcaccatcaggtgc60agggaatagctcatggattccatcctcagggctcgagtaggtcaccctgtacctggaaac120ttgcccctgtgggctttcccaagcaattttgatggaatcgacatccacatcagtgaatgc180cagtcctttagggcgatcaatgttggttactgcagnctgaaccagaggctgactctctcc240gcttggattctgagcatagacactaaccacatactccactgtgggctgcaanccttcaat300aanncatttctgtttgatctggacc325<210>284<211>331<212>dna<213>人<220><221>misc_feature<222>54,59,63,121,312,327<223>n=a,t,c或g<400>284tcgagcggccgcccgggcaggtctggtggggtcctggcacacgcacatgggggngttgnt60ctnatccagctgcccagcccccattggcgagtttgagaaggtgtgcagcaatgacaacaa120naccttcgactcttcctgccacttctttgccacaaagtgcaccctggagggcaccaagaa180gggccacaagctccacctggactacatcgggccttgcaaatacatccccccttgcctgga240ctctgagctgaccgaattcccccttgcgcatgcgggactggctcaagaaccgtcctggca300cccttgtatganagggatgaagacacnaccc331<210>285<211>509<212>dna<213>人<220><221>misc_feature<222>316,319,327,329,339,344,357,384,398,427,443,450,478<223>n=a,t,c或g<400>285agcgtggtcgcggccgaggtctgtcctacagtcctcaggactctactccctcagcagcgt60ggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaa120gcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacac180atgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttcccccg240catcccccttccaaacctgcccgggcggccgctcgaaagccgaattccagcacactggcg300gccggtactagtgganccnaacttggnanccaacctggnggaantaatgggcataanctg360tttctggggggaaattggtatccngtttacaattcccncacaacatacgagccggaagca420taaaagngtaaaagcctgggggnggcctantgaagtgaagctaaactcacattaattngc480gttgccgctcactggcccgcttttccagc509<210>286<211>336<212>dna<213>人<220><221>misc_feature<222>188,251,267<223>n=a,t,c或g<400>286tcgagcggccgcccgggcaggtttggaagggggatgcgggggaagaggaagactgacggt60ccccccaggagttcaggtgctgggcacggtgggcatgtgtgagttttgtcacaagatttg120ggctcaactctcttgtccaccttggtgttgctgggcttgtgatctacgttgcaggtgtag180gtctgggngccgaagttgctggagggcacggtcaccacgctgctgagggagtagagtcct240gaggactgtangacagacctcggccgngaccacgctaagccgaattctgcagatatccat300cacactggcggccgctccgagcatgcattttagagg336<210>287<211>30<212>dna<213>人<220><221>misc_feature<222>8,18<223>n=a,t,c或g<400>287agcgtggncgcggacgangacaacaacccc30<210>288<211>316<212>dna<213>人<220><221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tccaccaccagc8280actcctgggacccccacagtgtatctgggagcatctaagactccagcctcgatatttggc8340ccttcagctgccagccatctcctgatactattcaccctcaacttcaccatcactaacctg8400cggtatgaggagaacatgtggcctggctccaggaagttcaacactacagagagggtcctt8460cagggcctgctaaggcccttgttcaagaacaccagtgttggccctctgtactctggctgc8520aggctgaccttgctcaggccagagaaagatggggaagccaccggagtggatgccatctgc8580acccaccgccctgaccccacaggccctgggctggacagagagcagctgtatttggagctg8640agccagctgacccacagcatcactgagctgggcccctacacactggacagggacagtctc8700tatgtcaatggtttcacccatcggagctctgtacccaccaccagcaccggggtggtcagc8760gaggagccattcacactgaacttcaccatcaacaacctgcgctacatggcggacatgggc8820caacccggctccctcaagttcaacatcacagacaacgtcatgaagcacctgctcagtcct8880ttgttccagaggagcagcctgggtgcacggtacacaggctgcagggtcatcgcactaagg8940tctgtgaagaacggtgctgagacacgggtggacctcctctgcacctacctgcagcccctc9000agcggcccaggtctgcctatcaagcaggtgttccatgagctgagccagcagacccatggc9060atcacccggctgggcccctactctctggacaaagacagcctctaccttaacggttacaat9120gaacctggtcyagatgagcctcctacaactcccaagccagccaccacattcctgcctcct9180ctgtcagaagccacaacagccatggggtaccacctgaagaccctcacactcaacttcacc9240atctccaatctccagtattcaccagatatgggcaagggctcagctacattcaactccacc9300gagggggtccttcagcacctgctcagacccttgttccagaagagcagcatgggccccttc9360tacttgggttgccaactgatctccctcaggcctgagaaggatggggcagccactggtgtg9420gacaccacctgcacctaccaccctgaccctgtgggccccgggctggacatacagcagctt9480tactgggagctgagtcagctgacccatggtgtcacccaactgggcttctatgtcctggac9540agggatagcctcttcatcaatggctatgcaccccagaatttatcaatccggggcgagtac9600cagataaatttccacattgtcaactggaacctcagtaatccagaccccacatcctcagag9660tacatcaccctgctgagggacatccaggacaaggtcaccacactctacaaaggcagtcaa9720ctacatgacacattccgcttctgcctggtcaccaacttgacgatggactccgtgttggtc9780actgtcaaggcattgttctcctccaatttggaccccagcctggtggagcaagtctttcta9840gataagaccctgaatgcctcattccattggctgggctccacctaccagttggtggacatc9900catgtgacagaaatggagtcatcagtttatcaaccaacaagcagctccagcacccagcac9960ttctaccygaatttcaccatcaccaacctaccatattcccaggacaaagcccagccaggc10020accaccaattaccagaggaacaaaaggaatattgaggatgcgctcaaccaactcttccga10080aacagcagcatcaagagttatttttctgactgtcaagtttcaacattcaggtctgtcccc10140aacaggcaccacaccggggtggactccctgtgtaacttctcgccactggctcggagagta10200gacagagttgccatctatgaggaatttctgcggatgacccggaatggtacccagctgcag10260aacttcaccctggacaggagcagtgtccttgtggatgggtattytcccaacagaaatgag10320cccttaactgggaattctgaccttcccttctgggctgtcatcytcatcggcttggcagga10380ctcctgggactcatcacatgcctgatctgcggtgtcctggtgaccacccgccggcggaag10440aaggaaggagaatacaacgtccagcaacagtgcccaggctactaccagtcacacctagac10500ctggaggatctgcaatgactggaacttgccggtgcctggggtgcctttcccccagccagg10560gtccaaagaagcttggctggggcagaaataaaccatattggtcggacacaaaaaaaaaaa10620aa10622<210>570<211>469<212>dna<213>人<220><221>misc_feature<222>71,92,93,120,124,168,178,218,230,300,321,350,387,412,414,415,422,423,451<223>n=a,t,c或g<400>570gtttcacccatcggagctctgtgcccaccaccagcactcctgggacctccacagtggacc60tgggaacctcwgggactccatcctccctcccyrgccccacagctgctggccctctcctgr120tgcyattcaccctcaacttcaccatcaccaacctgcagtatgaggagracatgcatcrcc180ctggctccaggaagttcaacaccacggagagggtcctkcagggtctgctyaggtcccttg240ttcaagaacaccagtgttggccctctgtactctggctgcagactgaccttgctcaggccy300gagaaggatggggcagccacyggagtggatgccatctgcacccaccgccytgaccccaaa360agccctggactggacagagagcagctrtactgggagctgagccagctgaccmayrgcatc420amwgagctgggcccctacaccctggacaggracagtctctatgtcaatg469<210>571<211>130<212>prt<213>人<220><221>variant<222>69,107,110<223>xaa=任何氨基酸<400>571hisproglnleugluglnglnproglnserhissertrpcyshisser51015proserthrserthrhishisglnproalavalargglyglyhisala202530alaproglyserarglyspheasnalahisarggluargthralagly354045sercysserasnproargserglyilealavaltrpasnthrserile505560glnalaalaaspxaaprohisserglyglnargargilealaglnpro65707580argglntrpmetproseralahisilealaleuthrleulysthrser859095asptrpthrgluseraspcysthrglyserxaaalailexaaglnmet100105110alaserargsertrpalaprothrprotrpthrglythrvalsermet115120125sermet130<210>572<211>130<212>prt<213>人<220><221>variant<222>1,58,78,92,94<223>xaa=任何氨基酸<400>572xaaileproserserasnserserhisserproilehisglyalaile51015hisproglnleuglnleuilethrasnleuglntyrglugluaspmet202530arghisleuvalproglyserserthrargthrgluarggluleugln354045glyargalaglnthrleuaspglngluxaaglnserglyileproleu505560pheargleuglnthrserleuthrglnalaarggluglyxaaleuser65707580hisglyserglycyshisleuhisthrserproxaaproxaaargpro859095argthrglyglnargalathrvalleuglyalagluglnserasplys100105110trphisproglyalaglyproleuhisproglyprogluglnserleu115120125cysgln130<210>573<211>130<212>prt<213>人<220><221>variant<222>1,54<223>xaa=任何氨基酸<400>573xaaserproalaargthralaalathrvalprophemetvalprophe51015thrleuasnpheasnserserprothrcysserthrargargthrcys202530glythrtrppheglngluvalglnargalaglnarggluasncysarg354045valvalleulysproxaaileargasnserserleuglutyrleutyr505560serglycysargleualaserleuargproglulysaspserserala65707580thralavalaspalailecysthrhisargproaspprogluaspleu859095glyleuasparggluargleutyrtrpgluleuserasnleuthrasn100105110glyileglngluleuglyprotyrthrleuaspargasnserleutyr115120125valasn130<210>574<211>156<212>prt<213>人<220><221>variant<222>101<223>xaa=任何氨基酸<400>574glyphethrhisargsersermetprothrthrserthrproglythr51015serthrvalaspvalglythrserglythrproserserserproser202530prothrthralaglyproleuleumetprophethrleuasnphethr354045ilethrasnleuglntyrglugluaspmetargargthrglyserarg505560lyspheasnthrmetgluservalleuglnglyleuleulysproleu65707580phelysasnthrservalglyproleutyrserglycysargleuthr859095leuleuargproxaalysaspglyalaalathrglyvalaspalaile100105110cysthrhisargleuaspprolysserproglyleuasnargglugln115120125leutyrtrpgluleuserlysleuthrasnaspileglugluleugly130135140protyrthrleuaspargasnserleutyrvalasn145150155<210>575<211>158<212>prt<213>人<220><221>variant<222>103<223>xaa=任何氨基酸<400>575glyphethrhisglnserservalserthrthrserthrproglythr51015serthrvalaspleuargthrservalthrproserserleuserser202530prothrilemetalaalaglyproleuleuvalprophethrleuasn354045phethrilethrasnleuglntyrglygluaspmetglyhisprogly505560serarglyspheasnthrthrgluargvalleuglnglyleuleugly65707580proilephelysasnthrservalglyproleutyrserglycysarg859095leuthrserleuargserxaalysaspglyalaalathrglyvalasp100105110alailecysilehishisleuaspprolysserproglyleuasnarg115120125gluargleutyrtrpgluleuserglnleuthrasnglyilelysglu130135140leuglyprotyrthrleuaspargasnserleutyrvalasn145150155<210>576<211>122<212>prt<213>人<400>576alaalaglyproleuleuvalleuphethrleuasnphethrilethr51015asnleulystyrglugluaspmethisargproglyserarglysphe202530asnthrthrgluargvalleuglnthrleuargglyprometphelys354045asnthrserglyglyleuleutyrserglycysargleuthrleuleu505560argserglulysaspglyalaalathrglyvalaspalailecysthr65707580hisargleuaspprolysserproglyvalasparggluglnleutyr859095trpgluleuserglnleuthrasnglyilelysgluleuglyprotyr100105110thrleuaspargasnserleutyrvalasn115120<210>577<211>156<212>prt<213>人<220><221>variant<222>11,106,151<223>xaa=任何氨基酸<400>577glyphethrhisargthrservalprothrxaaserthrproglythr51015serthrvalaspleuglythrserglythrpropheserleuproser202530proalathralaglyproleuleuvalleuphethrleuasnphethr354045ilethrasnleulystyrglugluaspmethisargproglyserarg505560lyspheasnthrthrgluargvalleuglnthrleuleuglypromet65707580phelysasnthrservalglyleuleutyrserglycysargleuthr859095leuleuargserglulysaspglyalaxaathrglyvalaspalaile100105110cysthrhisargleuaspprolysserproglyvalaspargglugln115120125leutyrtrpgluleuserglnleuthrasnglyilelysgluleugly130135140protyrthrleuaspargxaaserleutyrvalasn145150155<210>578<211>155<212>prt<213>人<400>578glyphethrhistrpileprovalprothrserserthrproglythr51015serthrvalaspleuglyserglythrproserserleuproserpro202530thrthralaglyproleuleuvalprophethrleuasnphethrile354045thrasnleuglntyrglugluaspmethishisproglyserarglys505560pheasnthrthrgluargvalleuglnglyleuleuglyprometphe65707580lysasnthrservalglyleuleutyrserglycysargleuthrleu859095leuargproglulysasnglyalaalathrglymetaspalailecys100105110serhisargleuaspprolysserproglyleuasnarggluglnleu115120125tyrtrpgluleuserglnleuthrhisglyilelysgluleuglypro130135140tyrthrleuasparghisserleutyrvalasn145150155<210>579<211>155<212>prt<213>人<220><221>variant<222>52,138<223>xaa=任何氨基酸<400>579glyphethrhistrpileprovalprothrserserthrproglythr51015serthrvalaspleuglyserglythrproserserleuproserpro202530thrthralaglyproleuleuvalprophethrleuasnphethrile354045thrasnleuxaatyrglugluaspmethiscysproglyserarglys505560pheasnthrthrgluargvalleuglnserleuleuglyprometphe65707580lysasnthrservalglyproleutyrserglycysargleuthrleu859095leuargserglulysaspglyalaalathrglyvalaspalailecys100105110thrhisargleuaspprolysserproglyvalasparggluglnleu115120125tyrtrpgluleuserglnleuthrasnxaailelysgluleuglypro130135140tyrthrleuaspserasnserleutyrvalasn145150155<210>580<211>156<212>prt<213>人<220><221>variant<222>23<223>xaa=任何氨基酸<400>580glyphethrhisglnthrseralaproasnthrserthrproglythr51015serthrvalaspleuglyxaaserglythrproserserleuproser202530prothrseralaglyproleuleuvalprophethrleuasnphethr354045ilethrasnleuglntyrglugluaspmethishisproglyserarg505560lyspheasnthrthrgluargvalleuglnglyleuleuglypromet65707580phelysasnthrservalglyleuleutyrserglycysargleuthr859095leuleuargproglulysasnglyalaalathrglymetaspalaile100105110cysserhisargleuaspprolysserproglyleuasnargglugln115120125leutyrtrpgluleuserglnleuthrhisglyilelysgluleugly130135140protyrthrleuaspargasnserleutyrvalasn145150155<210>581<211>156<212>prt<213>人<400>581glyphethrhisargserservalalaprothrserthrproglythr51015serthrvalaspleuglythrserglythrproserserleuproser202530prothrthralavalproleuleuvalprophethrleuasnphethr354045ilethrasnleuglntyrglygluaspmetarghisproglyserarg505560lyspheasnthrthrgluargvalleuglnglyleuleuglyproleu65707580phelysasnserservalglyproleutyrserglycysargleuile859095serleuargserglulysaspglyalaalathrglyvalaspalaile100105110cysthrhishisleuasnproglnserproglyleuaspargglugln115120125leutyrtrpglnleuserglnmetthrasnglyilelysgluleugly130135140protyrthrleuaspargasnserleutyrvalasn145150155<210>582<211>156<212>prt<213>人<220><221>variant<222>151<223>xaa=任何氨基酸<400>582glyphethrhisargserserglyleuthrthrserthrprotrpthr51015serthrvalaspleuglythrserglythrproserprovalproser202530prothrthralaglyproleuleuvalprophethrleuasnphethr354045ilethrasnleuglntyrglugluaspmethisargproglyserarg505560lyspheasnalathrgluargvalleuglnglyleuleuserproile65707580phelysasnserservalglyproleutyrserglycysargleuthr859095serleuargproglulysaspglyalaalathrglymetaspalaval100105110cysleutyrhisproasnprolysargproglyleuaspargglugln115120125leuty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