CN110799841B - 用于检测大肠癌的生物标志物 - Google Patents

用于检测大肠癌的生物标志物 Download PDF

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CN110799841B
CN110799841B CN201880042773.2A CN201880042773A CN110799841B CN 110799841 B CN110799841 B CN 110799841B CN 201880042773 A CN201880042773 A CN 201880042773A CN 110799841 B CN110799841 B CN 110799841B
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朝长毅
白水崇
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Denka Co Ltd
National Institutes of Biomedical Innovation Health and Nutrition
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Abstract

提供一种用于早期检测大肠癌的生物标志物。一种用于检测大肠癌的大肠癌生物标志物，其由下面的1至22的22个蛋白质中的至少一个蛋白质或者1至22的蛋白质的部分肽中的至少一个肽构成：1.膜联蛋白A11；2.膜联蛋白A3；3.膜联蛋白A4；4.腱生蛋白‑N；5.转铁蛋白受体蛋白1；6.葡萄糖转运蛋白1；7.补体成分C9；8.CD88抗原；9.78kDa葡萄糖调节蛋白；10.α‑1‑酸性糖蛋白；11.基质金属蛋白酶9；12.血管生成素‑1；13.CD67抗原；14.粘蛋白‑5B；15.衔接蛋白GRB2；16.膜联蛋白A5；17.嗅觉介导素‑4；18.中性氨基酸转运蛋白B(0)；19.三肽基肽酶1；20.热休克相关70kDa蛋白2；21.蛋白酶体亚基α型‑5；以及22.中性粒细胞明胶酶相关脂质运载蛋白。

Description

用于检测大肠癌的生物标志物
技术领域
本发明涉及能够用于检测大肠癌的新型的蛋白质或者肽生物标志物。
背景技术
大肠癌(CRC)是在世界中频率最高的癌症之一，用于CRC的有效的生物标志物的开发对于提高人的生存率是不可或缺的。但是，目前使用的大肠癌的诊断法都是便潜血、CEA等精度非常低的诊断法，并未找到能够在早期高精度地诊断大肠癌的新的生物标志物，迫切需求血中生物标志物。
迄今为止，尽管通过大规模的组学研究而发现了多个癌症相关因子以及癌症的生物标志物候选，但尚未存在达到临床应用的血中生物标志物。作为证实这一点的证据，近年来的由美国食品药品监督管理局(FDA)承认的生物标志物极少，为每年两件以下。一般认为，该停滞的原因之一是在生物标志物开发的策略上存在严重的问题。
以往，在许多临床检查中，蛋白质生物标志物的检测通过基于抗体的定量化分析法(主要使用酶联抗体免疫测定法(ELISA))来进行。这些方法大大地依赖于抗体的品质，因此精度、特别是特异性存在问题，并且难以同时评价多项目的标志物候选。
由于质谱仪(MS)的最近进展，临床检查法已出现发生重大变化的可能性。作为其理由，是因为作为代表定向蛋白质组学的方法的选择反应监测/多重反应监测法(SRM/MRM)不仅能够以高精度进行生物标志物蛋白质的定量，还能够对多项目的标志物同时进行定量。在以前的报告中，Whiteaker等人使用SRM/MRM法来实施患者血浆中的多项目的乳腺癌生物标志物候选蛋白质的定量(参照非专利文献1)。除此以外，也包括本发明人等的课题组的报告在内，还报告了一些使用SRM/MRM法而验证了各种癌症的生物标志物候选蛋白质的例子(参照非专利文献2～4)。
因此，使用了SRM/MRM法的定向蛋白质组学成为用于发现生物标志物的强有力的工具。
现有技术文献
非专利文献
非专利文献1：Whiteaker,J.et al.,Nat.BIotechnol.29,625-634(2011)
非专利文献:2：Kume,H.et al.,Mol.Cell.Proteomics 13,1471-1484(2014)
非专利文献3：Muraoka,S.et al.,J.Proteome Res.11,4201-4210(2012)
非专利文献4：Narumi,R.et al.,J.Proteome Res.11,5311-5322(2012)。
发明内容
发明要解决的课题
本发明的目的在于提供一种用于早期检测大肠癌的生物标志物。
用于解决课题的方案
本发明人等首先从PubMed的文献检索中检索CRC生物标志物候选蛋白质。为了在血中检测这些生物标志物候选蛋白质，着眼于血中的细胞外囊泡(EV)。这是因为，EV不仅作为细胞间通信的中介者而起作用这点最近备受关注，而且EV被认为与各种疾病的发病和进展有关，一般认为包含较多的生物标志物候选。另外，在要使用质谱仪来检测血液中的微量的蛋白质时，由于白蛋白等血液中大量存在的夹杂蛋白质，有时会阻碍微量的标志物候选蛋白质的检测。但是，当对血液中的EV进行纯化时，大量的夹杂蛋白质被除去，因此能够检测出EV中的微量的生物标志物候选蛋白质。因此，EV含有蛋白质被认为是有希望的生物标志物候选。
本发明人等在通过文献检索而找到的生物标志物候选蛋白质中对有存在于血中EV的可能性的蛋白质缩减候选，使用SRM/MRM法来进行这些蛋白质的定量。作为其结果，确定了用于CRC的早期诊断的一些生物标志物候选，从而完成了本发明。
即，本发明的方案如下所述：
[1]一种用于检测大肠癌的大肠癌生物标志物，其由下面的1至22的22个蛋白质中的至少一个蛋白质或者1至22的蛋白质的部分肽中的至少一个肽构成：
1.膜联蛋白A11(Annexin A11)(ANXA 11)(序列号1)；
2.膜联蛋白A3(Annexin A3)(ANXA 3)(序列号2)；
3.膜联蛋白A4(Annexin A4)(ANXA 4)(序列号3)；
4.腱生蛋白-N(Tanascin-N)(TNN)(序列号4)；
5.转铁蛋白受体蛋白1(Transferrin receptor protein 1)(TFRC)(序列号5)；
6.葡萄糖转运蛋白-1(GLUT-1)(SLC2A1)(序列号6)；
7.补体成分C9(Complement component C9)(C9)(序列号7)；
8.CD88抗原(CD88 antigen)(C5AR1)(序列号8)；
9. 78kDa葡萄糖调节蛋白(78kDa glucose-regulated protein)(HSPA5)(序列号9)；
10.α-1-酸性糖蛋白(α-1-acid glycoprotein)(ORM1)(序列号10)；
11.基质金属蛋白酶9(Matrix metalloproteinase-9)(MMP9)(序列号11)；
12.血管生成素-1(Angiopoietin-1)(ANGPT1)(序列号12)；
13.CD67抗原(CD67 antigen)(CEACAM8)(序列号13)；
14.粘蛋白-5B(Mucin-5B)(MUC5B)(序列号14)；
15.衔接蛋白GRB2(Adapter protein GRB2)(GRB2)(序列号15)；
16.膜联蛋白A5(Annexin A5)(ANXA 5)(序列号16)；
17.嗅觉介导素-4(Olfactomedin-4)(OLFM4)(序列号17)；
18.中性氨基酸转运蛋白B(0)(Neutral amino acid transporter B(0))(SLC1A5)(序列号18)；
19.三肽基肽酶-1(Tripeptidyl-peptidase 1)(TPP1)(序列号19)；
20.热休克相关70kDa蛋白2(Heat shock-related 70kDa protein 2)(HSPA2)(序列号20)；
21.蛋白酶体亚基α型-5(Proteasome subunit alpha type-5)(PSMA5)(序列号21)；或者
22.中性粒细胞明胶酶相关脂质运载蛋白(Neutrophil gelatinase-associatedlipocalin)(LCN2)(序列号22)。
[2]根据[1]的用于检测大肠癌的大肠癌生物标志物，其中，是将[1]的1至22的22个蛋白质中的两个以上的蛋白质或者1至22的蛋白质的部分肽中的两个以上的肽组合而成的。
[3]根据[1]或[2]的用于检测大肠癌的大肠癌生物标志物，其中，[1]的1至22的蛋白质的部分肽是由通过序列号23至59表示的氨基酸序列构成的肽。
[4]根据[1]的用于检测大肠癌的大肠癌生物标志物，其中，由下面的12个蛋白质中的至少一个蛋白质或者该12个蛋白质的部分肽中的至少一个肽构成：
1.膜联蛋白A11(序列号1)；
2.膜联蛋白A3(序列号2)；
3.膜联蛋白A4(序列号3)；
4.腱生蛋白-N(序列号4)；
5.转铁蛋白受体蛋白1(序列号5)；
6.葡萄糖转运蛋白-1(序列号6)；
8.CD88抗原(序列号8)；
11.基质金属蛋白酶9(序列号11)；
16.膜联蛋白A5(序列号16)；
17.嗅觉介导素-4(序列号17)；
19.三肽基肽酶1(序列号19)；或者
22.中性粒细胞明胶酶相关脂质运载蛋白(序列号22)
[5]根据[4]的用于检测大肠癌的大肠癌生物标志物，其中，是将[4]的12个蛋白质中的2个以上的蛋白质或者该12个蛋白质的部分肽中的2个以上的肽组合而成的。
[6]根据[4]或[5]的用于检测大肠癌的大肠癌生物标志物，其中，[4]的12个蛋白质的部分肽是由通过序列号23～34、37、38、43、44、53、54、56及59表示的氨基酸序列构成的肽。
[7]根据[1]的用于检测大肠癌的大肠癌生物标志物，其中，所述大肠癌生物标志物由膜联蛋白A4或膜联蛋白A11或者它们的部分肽构成。
[8]根据[7]的用于检测大肠癌的大肠癌生物标志物，其中，是将膜联蛋白A4和膜联蛋白A11或者将膜联蛋白A4的部分肽和膜联蛋白A11的部分肽组合而成的。
[9]根据[7]或[8]记载的用于检测大肠癌的大肠癌生物标志物，其中膜联蛋白A4及膜联蛋白A11的部分肽是由通过序列号23、24、27及28表示的氨基酸序列构成的肽。
[10]一种用于检测大肠癌的大肠癌生物标志物，其是将[1]至[9]中任一项的大肠癌生物标志物和CEA组合而成的。
[11]一种大肠癌的检测方法，其包括对生物体试样中的[1]～[10]任一项的大肠癌生物标志物进行测定。
[12]一种大肠癌检测方法，其对生物体试样中的[1]～[10]任一项的大肠癌生物标志物进行测定，当该生物标志物与健康正常人相比较以高浓度存在时，判断为患有大肠癌。
[13]根据[11]或[12]的大肠癌检测方法，其中，生物体试样为血液中的细胞外囊泡(EV)。
[14]根据[11]～[13]中任一项的大肠癌检测方法，其中，检测通过免疫学测定法或者质谱法来进行。
[15]一种大肠癌检测试剂盒，其包含针对生物体试样中的[1]～[10]中任一项的大肠癌生物标志物的抗体。
本说明书包含作为本申请的优先权的基础的日本专利申请第2017-129941号的公开内容。
发明的效果
能够通过本发明的生物标志物来高灵敏度且高特异性地检测大肠癌，特别是能够检测早期的大肠癌。并且，通过将生物标志物进行组合，能够进一步高灵敏度且高特异性地进行检测。
附图说明
图1是示出用于探索细胞外囊泡(EV)中的大肠癌标志物的策略的图。在图中示出在各个工序中挑选出的候选蛋白质的数量。
图2是示出在EV中的、用于CRC生物标志物候选蛋白质的鸟枪法蛋白质组学分析的方法的图。图2a示出EV的制备及MS分析的工序，图2b示出通过鸟枪法蛋白质组分析而鉴定的生物标志物候选蛋白质及EV蛋白质的文氏图分析的结果。在根据血清或者细胞培养上清鉴定的EV蛋白质中，356个大肠癌生物标志物候选蛋白质被鉴定为EV蛋白质。
图3是示出SRM目标蛋白质(46)和肽(71)的图。
图4是示出以高度的准确性(AUC＞0.7)所验证的蛋白质的列表的图。
图5-1是示出基于SRM分析的三组(N、C及Cm)之间的血清中肽的相对定量的结果的图。N表示非癌对照患者，C表示没有转移的癌症患者，Cm表示有转移的癌症患者。圆点图是表示内源性肽对SI肽的峰面积比(*表示p<0.05，**表示p<0.01，N.S表示无显著差异)。纵轴表示Area Ratio(面积比)。
图5-2是示出基于SRM分析的三组(N、C及Cm)之间的肽的相对定量的结果的图(图5-1的接续)。
图5-3是示出基于SRM分析的三组(N、C及Cm)之间的肽的相对定量的结果的图(图5-2的接续)。
图5-4是示出基于SRM分析的三组(N、C及Cm)之间的肽的相对定量的结果的图(图5-3的接续)。
图5-5是示出基于SRM分析的三组(N、C及Cm)之间的肽的相对定量的结果的图(图5-4的接续)。
图5-6是示出基于SRM分析的三组(N、C及Cm)之间的肽的相对定量的结果的图(图5-5的接续)。
图6-1是示出目标肽的统计分析的结果的图(其一)。图6-1a示出基于SRM分析的三组(N、C及Cm)之间的肽的相对定量的结果。N表示非癌对照，C表示没有转移的癌症，Cm表示有转移的癌症。圆点图是表示内源性肽对SI肽的峰面积比(*表示p<0.05，**表示p<0.01，N.S表示无显著差异)。纵轴表示Area Ratio(面积比)。图6-1b表示用于识别N和C的ROC曲线分析的结果(1)及用于识别C和Cm的ROC曲线分析的结果(2)。将用于识别的曲线下的面积(AUC)示于各自的曲线图中。纵轴表示sensitivity(灵敏度)，横轴表示1-specificity(特异性)。
图6-2是示出目标肽的统计分析的结果的图(其二)。图6-2c示出基于SRM分析的三组(N、C及Cm)之间的肽的相对定量的结果。N表示非癌对照，C表示没有转移的癌症，Cm表示有转移的癌症。圆点图是表示内源性肽对SI肽的峰面积比(*表示p<0.05，**表示p<0.01，N.S表示无显著差异)。纵轴表示Area Ratio(面积比)。图6-2d示出用于识别N和C的ROC曲线分析的结果(1)及用于识别C和Cm的ROC曲线分析的结果(2)。将用于识别的曲线下的面积(AUC)示于各自的曲线图中。纵轴表示sensitivity(灵敏度)，横轴表示1-specificity(特异性)。
图7-1是示出用于识别N和C的ROC曲线(1)及用于识别C和Cm的ROC曲线(2)的图(其一)。将用于识别的曲线下的面积(AUC)示于各自的曲线图中。纵轴表示sensitivity(灵敏度)，横轴表示1-specificity(特异性)。
图7-2是示出用于识别N和C的ROC曲线(1)及用于识别C和Cm的ROC曲线(2)的图(图7-1的接续)。
图7-3是示出用于识别N和C的ROC曲线(1)及用于识别C和Cm的ROC曲线(2)的图(图7-2的接续)。
图7-4是示出用于识别N和C的ROC曲线(1)及用于识别C和Cm的ROC曲线(2)的图(图7-3的接续)。
图7-5是示出用于识别N和C的ROC曲线(1)及用于识别C和Cm的ROC曲线(2)的图(图7-4的接续)。
图7-6是示出用于识别N和C的ROC曲线(1)及用于识别C和Cm的ROC曲线(2)的图(图7-5的接续)。
图8-1是示出目标肽的组合的ROC曲线分析的结果的图(其一)。在N与C之间评价当将肽进行了组合时的诊断的灵敏度。将曲线下的面积(AUC)、灵敏度(sensitivity)及特异性(specificity)示于各自的曲线图。
图8-2是示出目标肽的组合的ROC曲线分析的结果的图(其二)。在N与C之间评价当将肽进行了组合时的诊断的灵敏度。将曲线下的面积(AUC)、灵敏度(sensitivity)及特异性(specificity)示于各自的曲线图。
图9是示出目标肽的组合的ROC曲线分析的结果的图。在N与C之间评价当将肽进行了组合时的诊断的灵敏度。将曲线下的面积(AUC)、灵敏度(sensitivity)及特异性(specificity)示于各自的曲线图。图9示出了较高的准确性(AUC＞0.9)的组合。其他组合示于图8-1及图8-2。
图10-1是示出目标肽与CEA的灵敏度(sensitivity)的比较结果的图。把将截止值设定为N的最大峰面积时的灵敏度作为样品的比例而算出(虚线表示特异度为100％)。纵轴表示Area Ratio(面积比)。
图10-2是示出目标肽与CEA的灵敏度(sensitivity)的比较结果的图(图10-1的接续)。
图10-3是示出目标肽与CEA的灵敏度(sensitivity)的比较结果的图(图10-2的接续)。
图11是示出目标肽与CEA的灵敏度的比较结果的图。把将截止值(Cutoff)设定为N的最大峰值面积时的灵敏度(sensitivity)作为样品的比例而算出(虚线表示特异性为100％)。在图11中示出了在N和C的识别中的灵敏度为最高的三个肽。关于三个肽的曲线图的纵轴表示Area Ratio(面积比)。灵敏度高于CEA的其他肽在图10-1～10-3中示出。
具体实施方式
下面，详细说明本发明。
本发明是作为用于检测大肠癌(CRC：colorectal cancer)的生物标志物的蛋白质或者其部分肽。另外，本发明是使用该生物标志物来检测大肠癌的方法。进一步，本发明是取得用于使用该生物标志物来诊断大肠癌的辅助数据的方法。通过使用本发明的生物标志物，能够早期检测大肠癌。
本发明中使用的生物标志物能够通过例如蛋白质组分析技术来探索。例如能够利用下面的工序来进行探索。
首先，使用大肠癌患者的组织，通过定量鸟枪法蛋白质组学来探索成为生物标志物的候选的蛋白质。此时，在健康正常人的大肠组织或大肠的良性肿瘤组织和无转移的大肠癌病例的组织和具有转移的大肠癌病例的组织之间进行比较定量，能够将在组织间发现变动的蛋白质锁定作为候选蛋白质。另外，生物标志物候选蛋白质的探索也能够通过使用在现有研究中被认为与大肠癌的发病和进展相关联的蛋白质来进行。能够通过使用了SRM/MRM((Selected reaction monitoring(选择性反应监测)/Multiple reactionmonitoring(多重反应监测))法(Gillette MA et al.，Nat Methods.2013；10：28-34)的定向蛋白质组学法，来进行表达量在组织间的变动的验证，其中SRM/MRM法是针对这些生物标志物候选，选择性地破坏特定质量的母离子，检测所生成的子离子中的特定的离子，由此能够从复杂的样品内高灵敏度地检测出作为目标的来自蛋白质的肽。通过该验证，能够挑选大肠癌生物标志物的最终候选。接下来，确认这些生物标志物候选能否在血液等生物体试样中检测、定量。此时，优选在血中的细胞外囊泡(EV)级分中进行检测、定量。
在本发明的方法中，下面的蛋白质作为用于检测大肠癌的生物标志物而列举：
1.膜联蛋白A11(Annexin A11)(ANXA 11)(序列号1)；
2.膜联蛋白A3(Annexin A3)(ANXA 3)(序列号2)；
3.膜联蛋白A4(Annexin A4)(ANXA 4)(序列号3)；
4.腱生蛋白N(Tanascin-N)(TNN)(序列号4)；
5.转铁蛋白受体蛋白1(Transferrin receptor protein 1)(TFRC)(序列号5)；
6.葡萄糖转运蛋白-1(GLUT-1)(SLC2A1)(序列号6)；
7.补体成分C9(Complement component C9)(C9)(序列号7)；
8.CD88抗原(CD88 antigen)(C5AR1)(序列号8)；
9. 78kDa葡萄糖调节蛋白(78kDa glucose-regulated protein)(HSPA5)(序列号9)；
10.α-1-酸性糖蛋白(α-1-acid glycoprotein)(ORM1)(序列号10)；
11.基质金属蛋白酶9(Matrix metalloproteinase-9)(MMP9)(序列号11)；
12.血管生成素-1(Angiopoietin-1)(ANGPT1)(序列号12)；
13.CD67抗原(CD67 antigen)(CEACAM8)(序列号13)；
14.粘蛋白-5B(Mucin-5B)(MUC5B)(序列号14)；
15.衔接蛋白GRB2(Adapter protein GRB2)(GRB2)(序列号15)
16.膜联蛋白A5(Annexin A5)(ANXA 5)(序列号16)；
17.嗅觉介导素-4(Olfactomedin-4)(OLFM4)(序列号17)；
18.中性氨基酸转运蛋白B(0)(Neutral amino acid transporter B(0))(SLC1A5)(序列号18)；
19.三肽基肽酶1(Tripeptidyl-peptidase 1)(TPP1)(序列号19)；
20.热休克相关70kDa蛋白2(Heat shock-related 70kDa protein 2)(HSPA2)(序列号20)；
21.蛋白酶体亚基α型-5(Proteasome subunit alpha type-5)(PSMA5)(序列号21)；或者
22.中性粒细胞明胶酶相关脂质运载蛋白(Neutrophil gelatinase-associatedlipocalin)(LCN2)(序列号22)。
另外，作为可用作大肠癌检测用的生物标志物的上述蛋白质的部分肽，可列举出：
由通过序列号23或24表示的氨基酸序列构成的肽(膜联蛋白A11的部分肽)，
由通过序列号25或26表示的氨基酸序列构成的肽(膜联蛋白A3的部分肽)，
由通过序列号27或28表示的氨基酸序列构成的肽(膜联蛋白A4的部分肽)，
由通过序列号29或30表示的氨基酸序列构成的肽(肌腱蛋白-N的部分肽)，
由通过序列号31或32表示的氨基酸序列构成的肽(转铁蛋白受体蛋白1的部分肽)，
由通过序列号33或34表示的氨基酸序列构成的肽(葡萄糖转运蛋白-1的部分肽)，
由通过序列号35或36表示的氨基酸序列构成的肽(补体成分C9的部分肽)，
由通过序列号37或38表示的氨基酸序列构成的肽(CD88抗原的部分肽)，
由通过序列号39或40表示的氨基酸序列构成的肽(78kDa葡萄糖调节蛋白的部分肽)，
由通过序列号41或42表示的氨基酸序列构成的肽(α-1-酸性糖蛋白的部分肽)，
由通过序列号43或44表示的氨基酸序列构成的肽(基质金属蛋白酶9的部分肽)
由通过序列号45或46表示的氨基酸序列构成的肽(血管生成素-1的部分肽)，
由通过序列号47或48表示的氨基酸序列构成的肽(CD67抗原的部分肽)，
由通过序列号49或50表示的氨基酸序列构成的肽(粘蛋白-5B的部分肽)，
由通过序列号51或52表示的氨基酸序列构成的肽(衔接蛋白GRB2的部分肽)，
由通过序列号53表示的氨基酸序列构成的肽(膜联蛋白A5的部分肽)；
由通过序列号54表示的氨基酸序列构成的肽(嗅觉介导素-4的部分肽)，
由通过序列号55表示的氨基酸序列构成的肽(中性氨基酸转运蛋白-B(0)的部分肽)，
由通过序列号56表示的氨基酸序列构成的肽(三肽基肽酶1的部分肽)，
由通过序列号57表示的氨基酸序列构成的肽(热休克相关70kDa蛋白2的部分肽)，
由通过序列号58表示的氨基酸序列构成的肽(蛋白酶体亚基α型-5的部分肽)，
由通过序列号59表示的氨基酸序列构成的肽(中性粒细胞明胶酶相关脂质运载蛋白的部分肽)等。
在本发明中，虽然使用上述的蛋白质或该蛋白质的部分肽作为生物标志物，但也包括由在序列号1～59所表示的氨基酸序列中一个或数个氨基酸缺失、置换、添加而得的氨基酸序列构成的蛋白质或者肽，这些蛋白质或者肽也能够在本发明的方法中用作生物标志物。在此，“一个或数个”是指“一个或三个”、“一个或两个”或者“一个”。
肽一般是指分子量1万以下的由氨基酸通过肽键连结而成的物质，作为氨基酸残基的数量，是指数个至50个以下左右。本说明书中，可以使用蛋白质或者其部分肽作为生物标志物，在称为部分肽的情况下是指如下的肽：具有蛋白质所具有的氨基酸序列中的一部分的部分氨基酸序列，分子量没有限制，但优选为1万以下。部分肽也有在基于转录、翻译的表达合成过程中生成的情况，另外，也有在被合成为蛋白质后在生物体内受到消化分解而作为消化分解产物肽生成的情况。
在这些蛋白质和肽中，优选将下面的蛋白质和肽用作大肠癌检测用的生物标志物：ROC分析的结果已明确能够以良好的检测灵敏度和特异度检测大肠癌的葡萄糖转运蛋白-1、转铁蛋白受体蛋白1、膜联蛋白A5、基质金属蛋白酶9、膜联蛋白A4、膜联蛋白A11、腱生蛋白-N、膜联蛋白A3、嗅觉介导素-4、CD88抗原、中性白细胞明胶酶相关脂质运载蛋白或三肽基肽酶1或者它们的部分肽。进一步优选使用膜联蛋白A4或膜联蛋白A11或者它们的部分肽作为用于检测大肠癌的生物标志物。
在本发明中，作为生物标志物，可以使用上述22个蛋白质和部分肽中的至少一个蛋白质及/或者部分肽，但优选使用2种以上，进一步优选使用3种以上、4种以上、5种以上、6种以上，7种类以上、8种以上、9种以上、10种以上、11种以上、12种以上、13种以上、14种以上、15种以上、16种以上、17种以上、18种以上、19种以上、20种以上、21种以上或者22种蛋白质或者部分肽，这样一来，能够以更高的精度检测大肠癌。在该情况下，可以是不同蛋白质的组合，也可以是某一蛋白质与除该蛋白质以外的蛋白质的部分肽或部分肽的组合。另外，也可以仅将多个完整蛋白质作为生物标志物。这样，通过使用多个蛋白质或者部分肽作为生物标志物，能够更准确地检测大肠癌，并且，能够准确地判断大肠癌的发展状态。
例如可列举出：葡萄糖转运蛋白-1与转铁蛋白受体蛋白1的组合、葡萄糖转运蛋白-1与血管生成素-1的组合、基质金属蛋白酶9与转铁蛋白受体蛋白1的组合、转铁蛋白受体蛋白1与中性粒细胞明胶酶相关脂质运载蛋白的组合、转铁蛋白受体蛋白1与血管生成素1的组合、转铁蛋白受体蛋白1与衔接蛋白的组合、78kDa葡萄糖调节蛋白与三肽基肽酶1、腱生蛋白N与中性粒细胞明胶酶相关脂质运载蛋白的组合、转铁蛋白受体蛋白1和中性粒细胞明胶酶相关脂质运载蛋白以及血管生成素-1的组合、转铁蛋白受体蛋白1与嗅觉介导素-4的组合、腱生蛋白-N与血管生成素-1的组合、嗅觉介导素-4与血管生成素-1的组合、腱生蛋白-N与嗅觉介导素-4的组合、嗅觉介导素-4与三肽基肽酶1的组合。也可以是各个蛋白质的部分肽的组合。
另外，通过将上述的蛋白质或者部分肽与以往作为大肠癌的生物标志物而已知的CEA(癌胚抗原)组合起来使用作为用于检测大肠癌的生物标志物，与单独使用相比，能够以更高灵敏度且更高特异性进行检测。
大肠癌患者中，上述蛋白质或者部分肽的表达上升。因此，只要对血液中的这些蛋白质或者部分肽进行定量即可。在此，血液包含血清和血浆。另外，上述蛋白质存在于细胞外囊泡(EV)中。因此，可以将血中存在的EV分离，必要时浓缩后检测EV中的蛋白质或者部分肽。EV能够通过例如超离心分离而分离为EV级分。另外，能够通过EV特异性标志物来进行分离。作为EV的标志物，可列举出CD9、CD63、CD81等，能够利用结合有针对这些标志物的抗体的磁珠等来进行分离。另外，作为其他EV的标志物可举出磷脂酰丝氨酸(PS)，能够利用结合有对磷脂酰丝氨酸具有亲和性的磷脂酰丝氨酸结合蛋白的磁珠等来进行分离。作为磷脂酰丝氨酸结合蛋白，例如具有：含有T细胞免疫球蛋白/粘蛋白结构域的分子4(Tim4)、膜联蛋白A5(Annexin A5)、膜联蛋白V(Annexin V)、乳脂肪球-EGF因子蛋白8(MFG-E8)等。
在本发明的方法中，研究生物体试样中的蛋白质/部分肽图谱，但所使用的生物体试样并不受限制。例如，可举出能够从生物体采集的液体试样例如全血、血清、血浆、尿、唾液等体液。需要说明的是，根据生物体试样的保存状态，生物体试样中的蛋白质、部分蛋白质及部分肽也有时会进一步受到分解，因此生物体试样优选不反复进行冻结融化而使用。另外，也可以在生物体试样中添加蛋白酶抑制剂等。或者，也能够使用FACS(荧光激活细胞分类仪)或者流式细胞仪并以上述标志物为指标来分离外泌体。
从分离出的外泌体提取蛋白质、肽，测定上述蛋白质或者肽即可。
蛋白质或者部分肽可以通过各种方法来检测。下面进行例示，但并不限制于这些方法。
例如，能够通过使用了针对要检测的各个蛋白质或者部分肽的抗体的免疫学测定法来进行测定。作为免疫学测定法，例如，可列举出固相免疫测定法(RIA、EIA、FIA、CLIA等)、斑点印迹法、胶乳凝集法(LA：Latex Agglutination-Turbidimetric Immunoassay)、免疫层析法等。抗体可以在基板上固定化而使用。
其中，从定量性的观点出发，优选作为EIA(Enzyme Immunoassay：酶免疫测定)法的一种的ELISA(Enzyme-Linked ImmunoSorbent Assay：酶联免疫吸附测定)法。在ELISA法中，可以是，向将抗体固相化了的微量滴定板的孔中添加检体，使它们进行抗原抗体反应，再添加酶标记抗体，使它们进行抗原抗体反应，清洗后，使它们与酶基质反应、显色，测定吸光度来检测样品中的标志物蛋白质或者部分肽，并且根据该测定值来算出检体中的蛋白质或者部分肽浓度。另外，也可以使用荧光标记抗体来使它们进行抗原抗体反应后测定荧光。抗原抗体反应可以在4℃～45℃、更优选在20℃～40℃、进一步优选在25℃～38℃下进行，另外，反应时间为10分钟～18小时左右，更优选为10分钟～1小时左右，进一步优选为30分钟～1小时左右。
在免疫学方法中使用的针对标志物蛋白质或者部分肽的抗体可以是单克隆抗体，也可以是多克隆抗体，还能够使用单克隆抗体的Fab、F(ab’)、F(ab’)2等结合活性片段。
本发明还包含一种大肠癌检测用检查试剂或者试剂盒，其包含针对上述22种蛋白质或其部分肽中的一个或者多个的抗体。
也可以通过使用了质谱仪的质谱分析法来对用于检测大肠癌的生物标志物蛋白质或者其部分肽进行分析。质谱分析法特别适用于部分肽的检测。
另外，也可以使用质谱仪进行检测。特别是对于生物标志物蛋白质的部分肽的检测，质谱仪的使用是合适的。质谱仪包括试样导入部、电离室、分析部、检测部、记录部等。作为电离法，可以使用电子碰撞电离(EI)法、化学电离(CI)法、场解吸(FD)法、二次离子质谱(SIMS)法、快速原子轰击(FAB)法、基质辅助激光解吸电离(matrix-assisted laserdesorption ionization：MALDI)法、电喷雾电离(ESI)法等。另外，该分析部还能够使用双聚焦质谱仪、四极杆型质谱仪、飞行时间型质谱仪、傅立叶变换质谱仪、离子回旋加速质谱仪等。为了进行精密的分析，也可以使用将两台质谱仪结合起来的串联质谱仪(MS/MS)。
质谱仪可以单独使用，也可以与液相色谱等分离设备或测定设备等连接，能够通过与高效液相色谱法组合而成的液相色谱法-质谱联用(LC/MS、LC/MS/MS)来进行分析。质谱分析能够通过为了肽的定量性检测而在本领域中广泛使用的LC/MS(LC/MS/MS)系统的、利用了三重四极杆型质谱仪的选择反应监测(SRM：Selected reaction monitoring，SRM)或者多重反应监测(MRM：Multiple reaction monitoring)来进行。通过SRM/MRM，能够同时测定多因子，通过一次测定就能够同时测定数百种蛋白质、肽。
在从受试者采集的样品中大量存在有上述生物标志物蛋白质或者部分肽的情况即为阳性的情况下，可以判断为该受试者患有大肠癌。
在本发明中，也可以将从健康正常人采集的样品作为阴性对象来同时测定。在该情况下，当受试者患有大肠癌时，受试者的检体中的生物标志物蛋白质或者部分肽浓度相比于健康正常人上升，因此当受试者中的生物标志物蛋白质或者部分肽浓度比健康正常人多时，将生物标志物蛋白质或者部分肽判断为阳性，能够判断为受试者患有大肠癌。关于样品中的生物标志物蛋白质或者部分肽是否为阳性，预先对通过SRM/MRM法检测出的生物标志物肽的峰的面积值与作为内部标准的稳定同位素标记肽的峰的面积值之比或者抗体测定值确定截止值，以该截止值为基准，在超过了截止值的情况下，能够判断为是阳性。
截止值例如通过ROC(receiver operating characteristic curve：受试者工作特征曲线)分析来确定。另外，能够通过ROC分析来决定基于本发明的方法的诊断精度(灵敏度以及特异性)。ROC分析对作为试样而从大肠癌患者采集的试样和从健康正常人采集的试样测定生物标志物蛋白质或者部分肽，计算在各截止值的灵敏度和假阳性率(1-特异性)，在将横轴设为1-特异性、将纵轴设为灵敏度的坐标上进行作图。
通过ROC分析来对本发明的方法的测定结果分析了诊断精度的情况下的曲线下面积(AUC：area under the curve)高达0.9以上，灵敏度为70％以上，优选为80％以上，进一步优选为85％以上，进一步优选为90％以上，特异性为70％以上，优选为75％以上，进一步优选为80％以上。通过本发明的方法，能够以非常高的精度检测初期的大肠癌。
实施例
通过下面的实施例来具体说明本发明，但本发明并不受这些实施例限制。
方法
大肠癌患者及健康正常人血清及大肠癌培养细胞
采集大肠癌患者51名和健康正常人26名的血清(在千叶大医学部采集)。各检体血清样品在分析前保存在-80℃。
四个大肠癌细胞株HCT116(ATCC；CCL-247)、DLD-1(ATCC；CCL-221)、SW480(ATCC；CCL-228)和SW620(ATCC；CCL-227)在含有10％牛血清(FBS)及抗生素的RPMI-1640培养基(Gibco Laboratories)中培养。
将各细胞在5％CO2的培养箱中维持在37℃的基础上进行培养，直至细胞增殖到亚融合状态为止，使各细胞增殖到了亚融合状态。然后，将这些培养细胞用不含FBS的培养基清洗，添加新鲜不含FBS的培养基。再在培养箱中培养48小时，然后收集驯化上清培养基，将EV(extracellular vesicle)作为分离用的样品。
EV(extracellular vesicle：细胞外囊泡)的分离
EV的分离通过超离心法来进行。通过在离心管的底铺设蔗糖缓冲剂，能够在EV回收时高效地分离低密度的夹杂物。作为超离心前的处理，将血清100μl以300g离心分离10分钟，除去大的夹杂物。然后，使回收的上清通过0.22μm旋转过滤器(Agilent Technologies，Santa Clara，CA)，以100,000g、90分钟、30％的蔗糖/D2O缓冲剂进行离心分离。回收缓冲剂，再以100,000g、70分钟在超离心分离机中进行两次离心分离，然后回收沉淀于离心管的EV级分。
从EV进行的蛋白质提取及消化
蛋白质提取及蛋白质消化通过相转移溶解剂(PTS：Phase transfer surfactant)方案(Masuda T et al.J.Proteome Res.7,731-740(2008))来进行。EV级分蛋白质在MPEXPTS试剂盒(GL Science，日本)中溶解后，以终浓度5mM加入二硫苏糖醇，在室温下进行30分钟还原反应，再以终浓度20mM加入碘乙酰胺，进行烷基化反应。
然后，对样品添加1％(w/w)胰蛋白酶(蛋白质组学级；Roche Mannheim，德国)，37℃下过夜消化。消化后，加入与液量等量的乙酸乙酯和终浓度1％的三氟乙酸并进行涡旋，将液体中的表面活性剂分离成有机层。离心分离后，回收含有肽的水相，进行基于StageTips(Rappsilber J.et al.，Nat.Protoc.2，1896-1906(2007))的脱盐。
液相色谱(LC)-质谱仪(MS/MS)和蛋白质组的数据分析
消化后的肽通过C18-SCX StageTip色谱柱进行七分级。然后，用连结了LC(UltiMate 3000Nanoflow HPLC system(Dionex，Sunnyveil，CA)的质谱仪(Q-Exactive(Thermo Scientilic，不莱梅，德国))进行分析。样品向质量分析器的导入使用在内径75μm、长度300mm的针中封入1.9μm的C18-AQ树脂的自制分析柱。LC的流动相由缓冲液A(0.1％甲酸和2％乙腈)和B(0.1％甲酸和90％乙腈)构成。将消化后的肽溶解于缓冲液A，装载到捕获柱0.075×20mm，Acclaim PepMap RSLC Nano-Trap Column；Thermo Scientilic)中。纳米LC以280nL/分钟送液，流动相以120分钟、5～35％B的梯度展开。质谱通过Full MS扫描(350～1800m/z、离子累计3×106、分辨率70,000)和MS/MS扫描(Top10前驱体离子、注射时间120ms、分辨率35,000、MS/MS离子选择阈值5×104计数、分离宽度3.0Da)测定。数据文件的分析通过MaxQuant软件(Ver1.5.1.2)进行。在搜索引擎中，对于Andromeda、UniProt人蛋白质数据库，进行了峰值列表的检索。前驱体质量误差范围设定为7ppm，碎片离子质量误差范围设定为0.01Da。鉴定蛋白质和肽以不足相对于反向数据库的1％的假阳性率(FDR 1％>)进行判定。
LC-SRM/MRM分析
LC-SRM/MRM分析通过以前报告过的方法来进行(Kume,H.et al.,Mol.Cell.Proteomics 13,1471-1484(2014):Muraoka,S.et al.,J.Proteome Res.11,4201-4210(2012):Narumi,R.et al.,J.Proteome Res.11,5311-5322(2012))。将消化肽溶解于含有0.1％三氟乙酸(TFA)的2％乙腈溶液中，然后，使用连结了Nano-flow LCParadigmMS2(Michrom BioResources，Auburn，CA)的三重四极杆质谱仪TSQ-Vantage(Thermo Fisher Scienic，不莱梅，德国)进行分析。样品向质量分析器的导入使用在内径75μm、长度100mm的针中封入了1.9μm的C18-AQ树脂的自制分析柱。LC的流动相由缓冲液A(0.1％甲酸和2％乙腈)和B(0.1％甲酸和90％乙腈)构成。将消化后的肽溶解于缓冲液A，加载到捕获柱(0.075×20mm，Acclaim PepMap RSLC Nano-Trap Column；ThermoScientilic)中。纳米LC以280nL/分钟送液，流动相以60分钟、5～35％B的梯度展开。SRM模式下的分析在下面的条件下进行(Q1峰宽度0.7FWHM，循环时间：1sec，碰撞气体压力1.8mTorr)。碰撞能量按每个SRM跃迁优化，各跃迁的强度以峰值时间宽度5分钟的时间表模式进行测定。
使用了SI-肽的目标肽的定量
基于SRM的目标肽的定量，是将作为内部标准的与目标肽相同序列的稳定同位素标记(SI-肽)混合到由各检测体血清制备的EV中，并根据通过SRM分析而检测出的内源性肽/SI-肽的峰面积比(Peak Area Ratio)算出。与目标肽相同序列的SI-肽的尖峰量通过执行事先分析而调整为接近内源性肽。
基于SRM分析的峰面积比率的统计分析
为了根据通过SRM分析算出的各样品的峰面积比而进行作为目标肽的大肠生物标志物的评价，进行了SRM数据的统计分析。统计分析使用SPSS软件(版本23)(SPSS Inc.，Chicago，IL)。首先，通过算出基于ROC分析(ROC：Receiver Operating Characteristic)的AUC(AUC：Areaunder the Curve，曲线下面积)的值来进行作为各个目标肽单独的生物标志物的评价。并且，还进行组合了多个目标肽的作为多标志物的评价。在目标肽的组合中使用SPSS软件制作逻辑回归模型，进行评价。对于模型有效的肽的组合，同样地利用通过ROC分析算出的AUC的值进行了评价。
结果
CRC相关生物标志物候选蛋白质的探索
图1示出了本发明的生物标志物候选探索的策略。从医学和生物学文献的PubMedDatabase得到CRC生物标志物候选蛋白质的列表。作为检索式，使用“cancer”AND“colorectal”AND“expression”，进行从2003年至2014年为止的数据库上的文献检索，对于大肠癌相关的687个蛋白质，作为CRC生物标志物候选进行列表化。候选蛋白质的选择通过下面的(1)～(4)的基准进行：
(1)通过蛋白质印迹法、ELISA或者免疫组织化学确认了人CRC组织或者血液中的蛋白质表达。
(2)确认了目标蛋白质在癌症中表达上升。(表达上升的蛋白质与表达减少的蛋白质相比更容易检测，因此适于生物标志物)。
(3)与癌症的表达和发展相关联的目标蛋白质的分子功能通过RNAi或者分子的过量表达而在实验上予与确认。
(4)仅通过大规模的分析(例如组学分析)确定的蛋白质除外。
并且，本发明人等以前通过临床检体的目标蛋白质组学，确定了44个CRC生物标志物候选蛋白质(Kume，H.et al.,Mol.Cell.Proteomics 13,1471-1484(2014))。将这些44个蛋白质与上述的生物标志物候选对照，选择将重复的蛋白质除去后的、合计725个蛋白质作为CRC生物标志物候选。
基于鸟枪法蛋白质组学分析的含EV生物标志物候选的选择
为了从所选择的生物标志物候选蛋白质中选择存在于EV的蛋白质，使用从血清和培养细胞上清中制备的EV，通过鸟枪法蛋白质组学分析进行了EV蛋白质的定性分析。将鸟枪法蛋白质组学的工作流程示于图2a。
EV从血清和细胞上清中的制备通过超离心法来进行。血清EV从健康正常人、没有转移的癌症患者、有转移的癌症患者各八人采集。培养细胞上清从四个CRC细胞(HCT116、DLD-1、SW480、SW620)采集。提取出的蛋白质通过使用相关移动溶解剂(PTS)法而消化，然后利用C-18Stage SCX Tip柱进行分级(Masuda,T et al.,J.Proteome Res.7,731-740(2008)；Adachi,J.et al.Anal.Chem.88,7899-7903(2016))。用LC-MS/MS来对分级样品进行分析，通过Mascot数据库检索，分别鉴定702个(血清EV)和4749个(细胞上清EV)蛋白质。通过比较这些EV鉴定蛋白质与生物标志物候选蛋白质，鉴定为356个蛋白质是含有EV蛋白质的生物标志物候选(图2b)。
从在EV中被鉴定的生物标志物候选蛋白质进行的SRM目标肽的选择
为了验证候选蛋白质作为生物标志物是否有效，进行从CRC患者血清采集的EV分级的SRM分析。SRM候选肽从通过鸟枪法蛋白质组学确定的肽中选择。作为候选肽选择的基准，使用下面的(1)～(3)：
(1)所鉴定的蛋白质是蛋白质固有的肽序列，在具有多个的情况下，选择在鸟枪法分析中强度高的目标。
(2)将具有胰蛋白酶切割错误、不适于SRM分析的氨基酸修饰(例如已被氧化的蛋氨酸等)的肽除外。
(3)过于长的肽(>20个氨基酸)被排除在目标肽之外，因为很难合成稳定同位素标记肽(SI-肽)。
满足这些基准的肽在所有已被鉴定的肽中存在3316个肽(346个蛋白质)。
接着，为了从这些候选中选择SRM目标肽，使用由非癌对照的健康正常者(N)池(混合)血清(n＝26)、没有转移的癌症患者(C)的池血清(n＝26)、具有转移的癌症患者(Cm)的池血清(n＝25)制备的EV级分来进行SRM分析。根据鸟枪法分析的结果，每一个肽选择三个或者四个跃迁(前驱体离子与产物离子的对)。SRM分析针对各个池重复测定三次，肽峰面积使用Skyline软件来进行了定量化。选择峰面积在池样品(N对C或者C对Cm)之间显著(p＜0.01)地增加了两倍以上的目标肽作为标志物候选肽。对于检测出了两个以上的候选肽的蛋白质，选择SRM分析中的峰强度高的两个肽。考虑这些基准，选择71个肽(46个蛋白质)作为生物标志物候选的目标肽(图3)。
血清EV级分中的目标肽的基于SRM的定量
对由各个患者的血清制备的EV级分中的37个生物标志物候选肽进行基于SRM的定量分析。各个EV级分由三个组的患者血清(健康正常者(N)，n＝26；无转移癌症患者(C)，n＝26；有转移癌症患者(Cm)，n＝25)的组制备。在各检体中加入等量的SI-peptide作为内部标准，使用Skyline软件来算出相对于内源性的目标肽的峰面积比。对于各个目标肽，通过t检验而进行N、C及Cm组间的定量值的比较，结果在N与C或者C与Cm之间发现了显著的定量值增加(p＜0.05)(图4及图5-1、5-2、5-3、5-4、5-5和5-6)。
基于目标肽的统计分析的生物标志物的评价
为了评价生物标志物的候选肽，对37个肽进行ROC(Receiver OperatingCharacteristic，受试者工作特征曲线)分析。对于各候选肽，评价N与C之间及C与Cm之间的区别。四个肽(三个蛋白质)是灵敏度非常高的蛋白质(AUC＞0.9、图6-1a、b)。而且，22个肽用于识别N与C的灵敏度十分高(0.7-0.9AUC、图7-1、7-2、7-3、7-4、7-5和7-6)。另一方面，11个肽用于识别C与Cm(图6-2c、d)的灵敏度十分高。
接着，通过逻辑回归分析来研究将候选肽进行组合作为多标志物是否可得到更高的灵敏度。针对肽的有意义的组合，使用SPSS软件进行了评价。通过多标志物的ROC曲线的AUC来评价两个以上的肽的逻辑分析的结果。对于候选肽的14个组合，AUC相比于单个标志物的值增加(图8-1及8-2)，特别是对于8个组合，AUC大于0.9(图9)。最高的AUC(0.97)是通过3个肽(转铁蛋白受体蛋白1、中性粒细胞明胶酶相关脂质运载蛋白和血管生成素-1)的组合而得到的。这些结果表明，多个标志物的组合对于改善大肠癌的诊断精度是有效的。
正在广泛使用的肿瘤标志物(癌胚抗原CEA)与目标肽的灵敏度的比较
CEA是当今最为广泛利用的大肠癌的血中标志物之一。但是，CEA的灵敏度对于发现早期阶段患者并不足够，已报告，对阶段2的患者的灵敏度为30％左右。因此，进行新建立的37个生物标志物候选与CEA的比较。因为CEA特异性几乎为100％，因此各目标肽的SRM定量值的截止点设定为100％的特异性。CEA对用于验证的检体组C的灵敏度为38.8％。另一方面，37个目标肽中17个肽(12个蛋白质：膜联蛋白A3、膜联蛋白A4、膜联蛋白A5、膜联蛋白A11、腱生蛋白N、转铁蛋白受体蛋白1、GLUT-1、基质金属蛋白酶-9、嗅觉介导素-4、CD88抗原、三肽基肽酶1、中性粒细胞明胶酶相关脂质运载蛋白)的灵敏度大大超过CEA(图10-1、图10-2和10-3)。特别是膜联蛋白A4及A11的三个肽的灵敏度大于80％(图11)。
产业上的利用可能性
利用本发明的方法，能够早期检测大肠癌。
序列表自由文本
序列号23～59合成
本说明书中引用的全部刊行物、专利及专利申请通过原样引用而编入本说明书中。
序列表
<110> 国立研究开发法人医药基盘健康营养研究所
电化生研株式会社
<120> 用于检测大肠癌的生物标志物
<130> PH-7502-PCT
<150> JP 2017-129941
<151> 2017-06-30
<160> 59
<170> PatentIn version 3.5
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Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
340 345 350
Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser
355 360 365
Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile
370 375 380
Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
385 390 395 400
His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
405 410 415
Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
420 425 430
Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
435 440 445
Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
465 470 475 480
Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495
Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn
500 505 510
Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp
515 520 525
Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
530 535 540
Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
545 550 555 560
Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575
Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
580 585 590
Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn
595 600 605
Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp
610 615 620
Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln
625 630 635 640
Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
645 650 655
Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys
660 665 670
Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro
675 680 685
Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700
Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys
705 710 715 720
Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735
Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
740 745 750
Val Trp Asp Ile Asp Asn Glu Phe
755 760
<210> 6
<211> 492
<212> PRT
<213> 人
<400> 6
Met Glu Pro Ser Ser Lys Lys Leu Thr Gly Arg Leu Met Leu Ala Val
1 5 10 15
Gly Gly Ala Val Leu Gly Ser Leu Gln Phe Gly Tyr Asn Thr Gly Val
20 25 30
Ile Asn Ala Pro Gln Lys Val Ile Glu Glu Phe Tyr Asn Gln Thr Trp
35 40 45
Val His Arg Tyr Gly Glu Ser Ile Leu Pro Thr Thr Leu Thr Thr Leu
50 55 60
Trp Ser Leu Ser Val Ala Ile Phe Ser Val Gly Gly Met Ile Gly Ser
65 70 75 80
Phe Ser Val Gly Leu Phe Val Asn Arg Phe Gly Arg Arg Asn Ser Met
85 90 95
Leu Met Met Asn Leu Leu Ala Phe Val Ser Ala Val Leu Met Gly Phe
100 105 110
Ser Lys Leu Gly Lys Ser Phe Glu Met Leu Ile Leu Gly Arg Phe Ile
115 120 125
Ile Gly Val Tyr Cys Gly Leu Thr Thr Gly Phe Val Pro Met Tyr Val
130 135 140
Gly Glu Val Ser Pro Thr Ala Leu Arg Gly Ala Leu Gly Thr Leu His
145 150 155 160
Gln Leu Gly Ile Val Val Gly Ile Leu Ile Ala Gln Val Phe Gly Leu
165 170 175
Asp Ser Ile Met Gly Asn Lys Asp Leu Trp Pro Leu Leu Leu Ser Ile
180 185 190
Ile Phe Ile Pro Ala Leu Leu Gln Cys Ile Val Leu Pro Phe Cys Pro
195 200 205
Glu Ser Pro Arg Phe Leu Leu Ile Asn Arg Asn Glu Glu Asn Arg Ala
210 215 220
Lys Ser Val Leu Lys Lys Leu Arg Gly Thr Ala Asp Val Thr His Asp
225 230 235 240
Leu Gln Glu Met Lys Glu Glu Ser Arg Gln Met Met Arg Glu Lys Lys
245 250 255
Val Thr Ile Leu Glu Leu Phe Arg Ser Pro Ala Tyr Arg Gln Pro Ile
260 265 270
Leu Ile Ala Val Val Leu Gln Leu Ser Gln Gln Leu Ser Gly Ile Asn
275 280 285
Ala Val Phe Tyr Tyr Ser Thr Ser Ile Phe Glu Lys Ala Gly Val Gln
290 295 300
Gln Pro Val Tyr Ala Thr Ile Gly Ser Gly Ile Val Asn Thr Ala Phe
305 310 315 320
Thr Val Val Ser Leu Phe Val Val Glu Arg Ala Gly Arg Arg Thr Leu
325 330 335
His Leu Ile Gly Leu Ala Gly Met Ala Gly Cys Ala Ile Leu Met Thr
340 345 350
Ile Ala Leu Ala Leu Leu Glu Gln Leu Pro Trp Met Ser Tyr Leu Ser
355 360 365
Ile Val Ala Ile Phe Gly Phe Val Ala Phe Phe Glu Val Gly Pro Gly
370 375 380
Pro Ile Pro Trp Phe Ile Val Ala Glu Leu Phe Ser Gln Gly Pro Arg
385 390 395 400
Pro Ala Ala Ile Ala Val Ala Gly Phe Ser Asn Trp Thr Ser Asn Phe
405 410 415
Ile Val Gly Met Cys Phe Gln Tyr Val Glu Gln Leu Cys Gly Pro Tyr
420 425 430
Val Phe Ile Ile Phe Thr Val Leu Leu Val Leu Phe Phe Ile Phe Thr
435 440 445
Tyr Phe Lys Val Pro Glu Thr Lys Gly Arg Thr Phe Asp Glu Ile Ala
450 455 460
Ser Gly Phe Arg Gln Gly Gly Ala Ser Gln Ser Asp Lys Thr Pro Glu
465 470 475 480
Glu Leu Phe His Pro Leu Gly Ala Asp Ser Gln Val
485 490
<210> 7
<211> 559
<212> PRT
<213> 人
<400> 7
Met Ser Ala Cys Arg Ser Phe Ala Val Ala Ile Cys Ile Leu Glu Ile
1 5 10 15
Ser Ile Leu Thr Ala Gln Tyr Thr Thr Ser Tyr Asp Pro Glu Leu Thr
20 25 30
Glu Ser Ser Gly Ser Ala Ser His Ile Asp Cys Arg Met Ser Pro Trp
35 40 45
Ser Glu Trp Ser Gln Cys Asp Pro Cys Leu Arg Gln Met Phe Arg Ser
50 55 60
Arg Ser Ile Glu Val Phe Gly Gln Phe Asn Gly Lys Arg Cys Thr Asp
65 70 75 80
Ala Val Gly Asp Arg Arg Gln Cys Val Pro Thr Glu Pro Cys Glu Asp
85 90 95
Ala Glu Asp Asp Cys Gly Asn Asp Phe Gln Cys Ser Thr Gly Arg Cys
100 105 110
Ile Lys Met Arg Leu Arg Cys Asn Gly Asp Asn Asp Cys Gly Asp Phe
115 120 125
Ser Asp Glu Asp Asp Cys Glu Ser Glu Pro Arg Pro Pro Cys Arg Asp
130 135 140
Arg Val Val Glu Glu Ser Glu Leu Ala Arg Thr Ala Gly Tyr Gly Ile
145 150 155 160
Asn Ile Leu Gly Met Asp Pro Leu Ser Thr Pro Phe Asp Asn Glu Phe
165 170 175
Tyr Asn Gly Leu Cys Asn Arg Asp Arg Asp Gly Asn Thr Leu Thr Tyr
180 185 190
Tyr Arg Arg Pro Trp Asn Val Ala Ser Leu Ile Tyr Glu Thr Lys Gly
195 200 205
Glu Lys Asn Phe Arg Thr Glu His Tyr Glu Glu Gln Ile Glu Ala Phe
210 215 220
Lys Ser Ile Ile Gln Glu Lys Thr Ser Asn Phe Asn Ala Ala Ile Ser
225 230 235 240
Leu Lys Phe Thr Pro Thr Glu Thr Asn Lys Ala Glu Gln Cys Cys Glu
245 250 255
Glu Thr Ala Ser Ser Ile Ser Leu His Gly Lys Gly Ser Phe Arg Phe
260 265 270
Ser Tyr Ser Lys Asn Glu Thr Tyr Gln Leu Phe Leu Ser Tyr Ser Ser
275 280 285
Lys Lys Glu Lys Met Phe Leu His Val Lys Gly Glu Ile His Leu Gly
290 295 300
Arg Phe Val Met Arg Asn Arg Asp Val Val Leu Thr Thr Thr Phe Val
305 310 315 320
Asp Asp Ile Lys Ala Leu Pro Thr Thr Tyr Glu Lys Gly Glu Tyr Phe
325 330 335
Ala Phe Leu Glu Thr Tyr Gly Thr His Tyr Ser Ser Ser Gly Ser Leu
340 345 350
Gly Gly Leu Tyr Glu Leu Ile Tyr Val Leu Asp Lys Ala Ser Met Lys
355 360 365
Arg Lys Gly Val Glu Leu Lys Asp Ile Lys Arg Cys Leu Gly Tyr His
370 375 380
Leu Asp Val Ser Leu Ala Phe Ser Glu Ile Ser Val Gly Ala Glu Phe
385 390 395 400
Asn Lys Asp Asp Cys Val Lys Arg Gly Glu Gly Arg Ala Val Asn Ile
405 410 415
Thr Ser Glu Asn Leu Ile Asp Asp Val Val Ser Leu Ile Arg Gly Gly
420 425 430
Thr Arg Lys Tyr Ala Phe Glu Leu Lys Glu Lys Leu Leu Arg Gly Thr
435 440 445
Val Ile Asp Val Thr Asp Phe Val Asn Trp Ala Ser Ser Ile Asn Asp
450 455 460
Ala Pro Val Leu Ile Ser Gln Lys Leu Ser Pro Ile Tyr Asn Leu Val
465 470 475 480
Pro Val Lys Met Lys Asn Ala His Leu Lys Lys Gln Asn Leu Glu Arg
485 490 495
Ala Ile Glu Asp Tyr Ile Asn Glu Phe Ser Val Arg Lys Cys His Thr
500 505 510
Cys Gln Asn Gly Gly Thr Val Ile Leu Met Asp Gly Lys Cys Leu Cys
515 520 525
Ala Cys Pro Phe Lys Phe Glu Gly Ile Ala Cys Glu Ile Ser Lys Gln
530 535 540
Lys Ile Ser Glu Gly Leu Pro Ala Leu Glu Phe Pro Asn Glu Lys
545 550 555
<210> 8
<211> 350
<212> PRT
<213> 人
<400> 8
Met Asp Ser Phe Asn Tyr Thr Thr Pro Asp Tyr Gly His Tyr Asp Asp
1 5 10 15
Lys Asp Thr Leu Asp Leu Asn Thr Pro Val Asp Lys Thr Ser Asn Thr
20 25 30
Leu Arg Val Pro Asp Ile Leu Ala Leu Val Ile Phe Ala Val Val Phe
35 40 45
Leu Val Gly Val Leu Gly Asn Ala Leu Val Val Trp Val Thr Ala Phe
50 55 60
Glu Ala Lys Arg Thr Ile Asn Ala Ile Trp Phe Leu Asn Leu Ala Val
65 70 75 80
Ala Asp Phe Leu Ser Cys Leu Ala Leu Pro Ile Leu Phe Thr Ser Ile
85 90 95
Val Gln His His His Trp Pro Phe Gly Gly Ala Ala Cys Ser Ile Leu
100 105 110
Pro Ser Leu Ile Leu Leu Asn Met Tyr Ala Ser Ile Leu Leu Leu Ala
115 120 125
Thr Ile Ser Ala Asp Arg Phe Leu Leu Val Phe Lys Pro Ile Trp Cys
130 135 140
Gln Asn Phe Arg Gly Ala Gly Leu Ala Trp Ile Ala Cys Ala Val Ala
145 150 155 160
Trp Gly Leu Ala Leu Leu Leu Thr Ile Pro Ser Phe Leu Tyr Arg Val
165 170 175
Val Arg Glu Glu Tyr Phe Pro Pro Lys Val Leu Cys Gly Val Asp Tyr
180 185 190
Ser His Asp Lys Arg Arg Glu Arg Ala Val Ala Ile Val Arg Leu Val
195 200 205
Leu Gly Phe Leu Trp Pro Leu Leu Thr Leu Thr Ile Cys Tyr Thr Phe
210 215 220
Ile Leu Leu Arg Thr Trp Ser Arg Arg Ala Thr Arg Ser Thr Lys Thr
225 230 235 240
Leu Lys Val Val Val Ala Val Val Ala Ser Phe Phe Ile Phe Trp Leu
245 250 255
Pro Tyr Gln Val Thr Gly Ile Met Met Ser Phe Leu Glu Pro Ser Ser
260 265 270
Pro Thr Phe Leu Leu Leu Lys Lys Leu Asp Ser Leu Cys Val Ser Phe
275 280 285
Ala Tyr Ile Asn Cys Cys Ile Asn Pro Ile Ile Tyr Val Val Ala Gly
290 295 300
Gln Gly Phe Gln Gly Arg Leu Arg Lys Ser Leu Pro Ser Leu Leu Arg
305 310 315 320
Asn Val Leu Thr Glu Glu Ser Val Val Arg Glu Ser Lys Ser Phe Thr
325 330 335
Arg Ser Thr Val Asp Thr Met Ala Gln Lys Thr Gln Ala Val
340 345 350
<210> 9
<211> 654
<212> PRT
<213> 人
<400> 9
Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ser Ala Ala
1 5 10 15
Arg Ala Glu Glu Glu Asp Lys Lys Glu Asp Val Gly Thr Val Val Gly
20 25 30
Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Gly Val Phe Lys Asn Gly
35 40 45
Arg Val Glu Ile Ile Ala Asn Asp Gln Gly Asn Arg Ile Thr Pro Ser
50 55 60
Tyr Val Ala Phe Thr Pro Glu Gly Glu Arg Leu Ile Gly Asp Ala Ala
65 70 75 80
Lys Asn Gln Leu Thr Ser Asn Pro Glu Asn Thr Val Phe Asp Ala Lys
85 90 95
Arg Leu Ile Gly Arg Thr Trp Asn Asp Pro Ser Val Gln Gln Asp Ile
100 105 110
Lys Phe Leu Pro Phe Lys Val Val Glu Lys Lys Thr Lys Pro Tyr Ile
115 120 125
Gln Val Asp Ile Gly Gly Gly Gln Thr Lys Thr Phe Ala Pro Glu Glu
130 135 140
Ile Ser Ala Met Val Leu Thr Lys Met Lys Glu Thr Ala Glu Ala Tyr
145 150 155 160
Leu Gly Lys Lys Val Thr His Ala Val Val Thr Val Pro Ala Tyr Phe
165 170 175
Asn Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Thr Ile Ala Gly
180 185 190
Leu Asn Val Met Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala Ile Ala
195 200 205
Tyr Gly Leu Asp Lys Arg Glu Gly Glu Lys Asn Ile Leu Val Phe Asp
210 215 220
Leu Gly Gly Gly Thr Phe Asp Val Ser Leu Leu Thr Ile Asp Asn Gly
225 230 235 240
Val Phe Glu Val Val Ala Thr Asn Gly Asp Thr His Leu Gly Gly Glu
245 250 255
Asp Phe Asp Gln Arg Val Met Glu His Phe Ile Lys Leu Tyr Lys Lys
260 265 270
Lys Thr Gly Lys Asp Val Arg Lys Asp Asn Arg Ala Val Gln Lys Leu
275 280 285
Arg Arg Glu Val Glu Lys Ala Lys Arg Ala Leu Ser Ser Gln His Gln
290 295 300
Ala Arg Ile Glu Ile Glu Ser Phe Tyr Glu Gly Glu Asp Phe Ser Glu
305 310 315 320
Thr Leu Thr Arg Ala Lys Phe Glu Glu Leu Asn Met Asp Leu Phe Arg
325 330 335
Ser Thr Met Lys Pro Val Gln Lys Val Leu Glu Asp Ser Asp Leu Lys
340 345 350
Lys Ser Asp Ile Asp Glu Ile Val Leu Val Gly Gly Ser Thr Arg Ile
355 360 365
Pro Lys Ile Gln Gln Leu Val Lys Glu Phe Phe Asn Gly Lys Glu Pro
370 375 380
Ser Arg Gly Ile Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val
385 390 395 400
Gln Ala Gly Val Leu Ser Gly Asp Gln Asp Thr Gly Asp Leu Val Leu
405 410 415
Leu Asp Val Cys Pro Leu Thr Leu Gly Ile Glu Thr Val Gly Gly Val
420 425 430
Met Thr Lys Leu Ile Pro Arg Asn Thr Val Val Pro Thr Lys Lys Ser
435 440 445
Gln Ile Phe Ser Thr Ala Ser Asp Asn Gln Pro Thr Val Thr Ile Lys
450 455 460
Val Tyr Glu Gly Glu Arg Pro Leu Thr Lys Asp Asn His Leu Leu Gly
465 470 475 480
Thr Phe Asp Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln
485 490 495
Ile Glu Val Thr Phe Glu Ile Asp Val Asn Gly Ile Leu Arg Val Thr
500 505 510
Ala Glu Asp Lys Gly Thr Gly Asn Lys Asn Lys Ile Thr Ile Thr Asn
515 520 525
Asp Gln Asn Arg Leu Thr Pro Glu Glu Ile Glu Arg Met Val Asn Asp
530 535 540
Ala Glu Lys Phe Ala Glu Glu Asp Lys Lys Leu Lys Glu Arg Ile Asp
545 550 555 560
Thr Arg Asn Glu Leu Glu Ser Tyr Ala Tyr Ser Leu Lys Asn Gln Ile
565 570 575
Gly Asp Lys Glu Lys Leu Gly Gly Lys Leu Ser Ser Glu Asp Lys Glu
580 585 590
Thr Met Glu Lys Ala Val Glu Glu Lys Ile Glu Trp Leu Glu Ser His
595 600 605
Gln Asp Ala Asp Ile Glu Asp Phe Lys Ala Lys Lys Lys Glu Leu Glu
610 615 620
Glu Ile Val Gln Pro Ile Ile Ser Lys Leu Tyr Gly Ser Ala Gly Pro
625 630 635 640
Pro Pro Thr Gly Glu Glu Asp Thr Ala Glu Lys Asp Glu Leu
645 650
<210> 10
<211> 201
<212> PRT
<213> 人
<400> 10
Met Ala Leu Ser Trp Val Leu Thr Val Leu Ser Leu Leu Pro Leu Leu
1 5 10 15
Glu Ala Gln Ile Pro Leu Cys Ala Asn Leu Val Pro Val Pro Ile Thr
20 25 30
Asn Ala Thr Leu Asp Gln Ile Thr Gly Lys Trp Phe Tyr Ile Ala Ser
35 40 45
Ala Phe Arg Asn Glu Glu Tyr Asn Lys Ser Val Gln Glu Ile Gln Ala
50 55 60
Thr Phe Phe Tyr Phe Thr Pro Asn Lys Thr Glu Asp Thr Ile Phe Leu
65 70 75 80
Arg Glu Tyr Gln Thr Arg Gln Asp Gln Cys Ile Tyr Asn Thr Thr Tyr
85 90 95
Leu Asn Val Gln Arg Glu Asn Gly Thr Ile Ser Arg Tyr Val Gly Gly
100 105 110
Gln Glu His Phe Ala His Leu Leu Ile Leu Arg Asp Thr Lys Thr Tyr
115 120 125
Met Leu Ala Phe Asp Val Asn Asp Glu Lys Asn Trp Gly Leu Ser Val
130 135 140
Tyr Ala Asp Lys Pro Glu Thr Thr Lys Glu Gln Leu Gly Glu Phe Tyr
145 150 155 160
Glu Ala Leu Asp Cys Leu Arg Ile Pro Lys Ser Asp Val Val Tyr Thr
165 170 175
Asp Trp Lys Lys Asp Lys Cys Glu Pro Leu Glu Lys Gln His Glu Lys
180 185 190
Glu Arg Lys Gln Glu Glu Gly Glu Ser
195 200
<210> 11
<211> 707
<212> PRT
<213> 人
<400> 11
Met Ser Leu Trp Gln Pro Leu Val Leu Val Leu Leu Val Leu Gly Cys
1 5 10 15
Cys Phe Ala Ala Pro Arg Gln Arg Gln Ser Thr Leu Val Leu Phe Pro
20 25 30
Gly Asp Leu Arg Thr Asn Leu Thr Asp Arg Gln Leu Ala Glu Glu Tyr
35 40 45
Leu Tyr Arg Tyr Gly Tyr Thr Arg Val Ala Glu Met Arg Gly Glu Ser
50 55 60
Lys Ser Leu Gly Pro Ala Leu Leu Leu Leu Gln Lys Gln Leu Ser Leu
65 70 75 80
Pro Glu Thr Gly Glu Leu Asp Ser Ala Thr Leu Lys Ala Met Arg Thr
85 90 95
Pro Arg Cys Gly Val Pro Asp Leu Gly Arg Phe Gln Thr Phe Glu Gly
100 105 110
Asp Leu Lys Trp His His His Asn Ile Thr Tyr Trp Ile Gln Asn Tyr
115 120 125
Ser Glu Asp Leu Pro Arg Ala Val Ile Asp Asp Ala Phe Ala Arg Ala
130 135 140
Phe Ala Leu Trp Ser Ala Val Thr Pro Leu Thr Phe Thr Arg Val Tyr
145 150 155 160
Ser Arg Asp Ala Asp Ile Val Ile Gln Phe Gly Val Ala Glu His Gly
165 170 175
Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Ala His Ala Phe
180 185 190
Pro Pro Gly Pro Gly Ile Gln Gly Asp Ala His Phe Asp Asp Asp Glu
195 200 205
Leu Trp Ser Leu Gly Lys Gly Val Val Val Pro Thr Arg Phe Gly Asn
210 215 220
Ala Asp Gly Ala Ala Cys His Phe Pro Phe Ile Phe Glu Gly Arg Ser
225 230 235 240
Tyr Ser Ala Cys Thr Thr Asp Gly Arg Ser Asp Gly Leu Pro Trp Cys
245 250 255
Ser Thr Thr Ala Asn Tyr Asp Thr Asp Asp Arg Phe Gly Phe Cys Pro
260 265 270
Ser Glu Arg Leu Tyr Thr Gln Asp Gly Asn Ala Asp Gly Lys Pro Cys
275 280 285
Gln Phe Pro Phe Ile Phe Gln Gly Gln Ser Tyr Ser Ala Cys Thr Thr
290 295 300
Asp Gly Arg Ser Asp Gly Tyr Arg Trp Cys Ala Thr Thr Ala Asn Tyr
305 310 315 320
Asp Arg Asp Lys Leu Phe Gly Phe Cys Pro Thr Arg Ala Asp Ser Thr
325 330 335
Val Met Gly Gly Asn Ser Ala Gly Glu Leu Cys Val Phe Pro Phe Thr
340 345 350
Phe Leu Gly Lys Glu Tyr Ser Thr Cys Thr Ser Glu Gly Arg Gly Asp
355 360 365
Gly Arg Leu Trp Cys Ala Thr Thr Ser Asn Phe Asp Ser Asp Lys Lys
370 375 380
Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala
385 390 395 400
His Glu Phe Gly His Ala Leu Gly Leu Asp His Ser Ser Val Pro Glu
405 410 415
Ala Leu Met Tyr Pro Met Tyr Arg Phe Thr Glu Gly Pro Pro Leu His
420 425 430
Lys Asp Asp Val Asn Gly Ile Arg His Leu Tyr Gly Pro Arg Pro Glu
435 440 445
Pro Glu Pro Arg Pro Pro Thr Thr Thr Thr Pro Gln Pro Thr Ala Pro
450 455 460
Pro Thr Val Cys Pro Thr Gly Pro Pro Thr Val His Pro Ser Glu Arg
465 470 475 480
Pro Thr Ala Gly Pro Thr Gly Pro Pro Ser Ala Gly Pro Thr Gly Pro
485 490 495
Pro Thr Ala Gly Pro Ser Thr Ala Thr Thr Val Pro Leu Ser Pro Val
500 505 510
Asp Asp Ala Cys Asn Val Asn Ile Phe Asp Ala Ile Ala Glu Ile Gly
515 520 525
Asn Gln Leu Tyr Leu Phe Lys Asp Gly Lys Tyr Trp Arg Phe Ser Glu
530 535 540
Gly Arg Gly Ser Arg Pro Gln Gly Pro Phe Leu Ile Ala Asp Lys Trp
545 550 555 560
Pro Ala Leu Pro Arg Lys Leu Asp Ser Val Phe Glu Glu Arg Leu Ser
565 570 575
Lys Lys Leu Phe Phe Phe Ser Gly Arg Gln Val Trp Val Tyr Thr Gly
580 585 590
Ala Ser Val Leu Gly Pro Arg Arg Leu Asp Lys Leu Gly Leu Gly Ala
595 600 605
Asp Val Ala Gln Val Thr Gly Ala Leu Arg Ser Gly Arg Gly Lys Met
610 615 620
Leu Leu Phe Ser Gly Arg Arg Leu Trp Arg Phe Asp Val Lys Ala Gln
625 630 635 640
Met Val Asp Pro Arg Ser Ala Ser Glu Val Asp Arg Met Phe Pro Gly
645 650 655
Val Pro Leu Asp Thr His Asp Val Phe Gln Tyr Arg Glu Lys Ala Tyr
660 665 670
Phe Cys Gln Asp Arg Phe Tyr Trp Arg Val Ser Ser Arg Ser Glu Leu
675 680 685
Asn Gln Val Asp Gln Val Gly Tyr Val Thr Tyr Asp Ile Leu Gln Cys
690 695 700
Pro Glu Asp
705
<210> 12
<211> 498
<212> PRT
<213> 人
<400> 12
Met Thr Val Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His
1 5 10 15
Ile Gly Cys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg
20 25 30
Tyr Asn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro
35 40 45
Glu His Asp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr
50 55 60
Asn Ala Leu Gln Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser
65 70 75 80
Gln Lys Leu Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp
85 90 95
Leu Gln Lys Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met
100 105 110
Ala Gln Ile Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu
115 120 125
Glu Ile Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys
130 135 140
Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu
145 150 155 160
Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln
165 170 175
Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser
180 185 190
Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu
195 200 205
Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr
210 215 220
Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala
225 230 235 240
Thr Thr Asn Asn Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp
245 250 255
Thr Val His Asn Leu Val Asn Leu Cys Thr Lys Glu Gly Val Leu Leu
260 265 270
Lys Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp
275 280 285
Val Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile
290 295 300
Asn Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn
305 310 315 320
Gly Gly Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp
325 330 335
Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser
340 345 350
Gly Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln
355 360 365
Arg Gln Tyr Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg
370 375 380
Ala Tyr Ser Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn
385 390 395 400
Tyr Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser
405 410 415
Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn
420 425 430
Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp
435 440 445
Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala
450 455 460
Gly Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys
465 470 475 480
Gly Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg Pro Leu
485 490 495
Asp Phe
<210> 13
<211> 349
<212> PRT
<213> 人
<400> 13
Met Gly Pro Ile Ser Ala Pro Ser Cys Arg Trp Arg Ile Pro Trp Gln
1 5 10 15
Gly Leu Leu Leu Thr Ala Ser Leu Phe Thr Phe Trp Asn Pro Pro Thr
20 25 30
Thr Ala Gln Leu Thr Ile Glu Ala Val Pro Ser Asn Ala Ala Glu Gly
35 40 45
Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln Asp Pro Arg Gly
50 55 60
Tyr Asn Trp Tyr Lys Gly Glu Thr Val Asp Ala Asn Arg Arg Ile Ile
65 70 75 80
Gly Tyr Val Ile Ser Asn Gln Gln Ile Thr Pro Gly Pro Ala Tyr Ser
85 90 95
Asn Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Met Arg Asn Val
100 105 110
Thr Arg Asn Asp Thr Gly Ser Tyr Thr Leu Gln Val Ile Lys Leu Asn
115 120 125
Leu Met Ser Glu Glu Val Thr Gly Gln Phe Ser Val His Pro Glu Thr
130 135 140
Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys
145 150 155 160
Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asn Thr Thr Tyr
165 170 175
Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln
180 185 190
Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn
195 200 205
Asp Val Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn
210 215 220
Phe Ser Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro
225 230 235 240
Thr Ile Ser Pro Ser Asp Thr Tyr Tyr His Ala Gly Val Asn Leu Asn
245 250 255
Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ser Gln Tyr Ser Trp Ser
260 265 270
Val Asn Gly Thr Phe Gln Gln Tyr Thr Gln Lys Leu Phe Ile Pro Asn
275 280 285
Ile Thr Thr Lys Asn Ser Gly Ser Tyr Ala Cys His Thr Thr Asn Ser
290 295 300
Ala Thr Gly Arg Asn Arg Thr Thr Val Arg Met Ile Thr Val Ser Asp
305 310 315 320
Ala Leu Val Gln Gly Ser Ser Pro Gly Leu Ser Ala Arg Ala Thr Val
325 330 335
Ser Ile Met Ile Gly Val Leu Ala Arg Val Ala Leu Ile
340 345
<210> 14
<211> 5762
<212> PRT
<213> 人
<400> 14
Met Gly Ala Pro Ser Ala Cys Arg Thr Leu Val Leu Ala Leu Ala Ala
1 5 10 15
Met Leu Val Val Pro Gln Ala Glu Thr Gln Gly Pro Val Glu Pro Ser
20 25 30
Trp Glu Asn Ala Gly His Thr Met Asp Gly Gly Ala Pro Thr Ser Ser
35 40 45
Pro Thr Arg Arg Val Ser Phe Val Pro Pro Val Thr Val Phe Pro Ser
50 55 60
Leu Ser Pro Leu Asn Pro Ala His Asn Gly Arg Val Cys Ser Thr Trp
65 70 75 80
Gly Asp Phe His Tyr Lys Thr Phe Asp Gly Asp Val Phe Arg Phe Pro
85 90 95
Gly Leu Cys Asn Tyr Val Phe Ser Glu His Cys Arg Ala Ala Tyr Glu
100 105 110
Asp Phe Asn Val Gln Leu Arg Arg Gly Leu Val Gly Ser Arg Pro Val
115 120 125
Val Thr Arg Val Val Ile Lys Ala Gln Gly Leu Val Leu Glu Ala Ser
130 135 140
Asn Gly Ser Val Leu Ile Asn Gly Gln Arg Glu Glu Leu Pro Tyr Ser
145 150 155 160
Arg Thr Gly Leu Leu Val Glu Gln Ser Gly Asp Tyr Ile Lys Val Ser
165 170 175
Ile Arg Leu Val Leu Thr Phe Leu Trp Asn Gly Glu Asp Ser Ala Leu
180 185 190
Leu Glu Leu Asp Pro Lys Tyr Ala Asn Gln Thr Cys Gly Leu Cys Gly
195 200 205
Asp Phe Asn Gly Leu Pro Ala Phe Asn Glu Phe Tyr Ala His Asn Ala
210 215 220
Arg Leu Thr Pro Leu Gln Phe Gly Asn Leu Gln Lys Leu Asp Gly Pro
225 230 235 240
Thr Glu Gln Cys Pro Asp Pro Leu Pro Leu Pro Ala Gly Asn Cys Thr
245 250 255
Asp Glu Glu Gly Ile Cys His Arg Thr Leu Leu Gly Pro Ala Phe Ala
260 265 270
Glu Cys His Ala Leu Val Asp Ser Thr Ala Tyr Leu Ala Ala Cys Ala
275 280 285
Gln Asp Leu Cys Arg Cys Pro Thr Cys Pro Cys Ala Thr Phe Val Glu
290 295 300
Tyr Ser Arg Gln Cys Ala His Ala Gly Gly Gln Pro Arg Asn Trp Arg
305 310 315 320
Cys Pro Glu Leu Cys Pro Arg Thr Cys Pro Leu Asn Met Gln His Gln
325 330 335
Glu Cys Gly Ser Pro Cys Thr Asp Thr Cys Ser Asn Pro Gln Arg Ala
340 345 350
Gln Leu Cys Glu Asp His Cys Val Asp Gly Cys Phe Cys Pro Pro Gly
355 360 365
Thr Val Leu Asp Asp Ile Thr His Ser Gly Cys Leu Pro Leu Gly Gln
370 375 380
Cys Pro Cys Thr His Gly Gly Arg Thr Tyr Ser Pro Gly Thr Ser Phe
385 390 395 400
Asn Thr Thr Cys Ser Ser Cys Thr Cys Ser Gly Gly Leu Trp Gln Cys
405 410 415
Gln Asp Leu Pro Cys Pro Gly Thr Cys Ser Val Gln Gly Gly Ala His
420 425 430
Ile Ser Thr Tyr Asp Glu Lys Leu Tyr Asp Leu His Gly Asp Cys Ser
435 440 445
Tyr Val Leu Ser Lys Lys Cys Ala Asp Ser Ser Phe Thr Val Leu Ala
450 455 460
Glu Leu Arg Lys Cys Gly Leu Thr Asp Asn Glu Asn Cys Leu Lys Ala
465 470 475 480
Val Thr Leu Ser Leu Asp Gly Gly Asp Thr Ala Ile Arg Val Gln Ala
485 490 495
Asp Gly Gly Val Phe Leu Asn Ser Ile Tyr Thr Gln Leu Pro Leu Ser
500 505 510
Ala Ala Asn Ile Thr Leu Phe Thr Pro Ser Ser Phe Phe Ile Val Val
515 520 525
Gln Thr Gly Leu Gly Leu Gln Leu Leu Val Gln Leu Val Pro Leu Met
530 535 540
Gln Val Phe Val Arg Leu Asp Pro Ala His Gln Gly Gln Met Cys Gly
545 550 555 560
Leu Cys Gly Asn Phe Asn Gln Asn Gln Ala Asp Asp Phe Thr Ala Leu
565 570 575
Ser Gly Val Val Glu Ala Thr Gly Ala Ala Phe Ala Asn Thr Trp Lys
580 585 590
Ala Gln Ala Ala Cys Ala Asn Ala Arg Asn Ser Phe Glu Asp Pro Cys
595 600 605
Ser Leu Ser Val Glu Asn Glu Asn Tyr Ala Arg His Trp Cys Ser Arg
610 615 620
Leu Thr Asp Pro Asn Ser Ala Phe Ser Arg Cys His Ser Ile Ile Asn
625 630 635 640
Pro Lys Pro Phe His Ser Asn Cys Met Phe Asp Thr Cys Asn Cys Glu
645 650 655
Arg Ser Glu Asp Cys Leu Cys Ala Ala Leu Ser Ser Tyr Val His Ala
660 665 670
Cys Ala Ala Lys Gly Val Gln Leu Ser Asp Trp Arg Asp Gly Val Cys
675 680 685
Thr Lys Tyr Met Gln Asn Cys Pro Lys Ser Gln Arg Tyr Ala Tyr Val
690 695 700
Val Asp Ala Cys Gln Pro Thr Cys Arg Gly Leu Ser Glu Ala Asp Val
705 710 715 720
Thr Cys Ser Val Ser Phe Val Pro Val Asp Gly Cys Thr Cys Pro Ala
725 730 735
Gly Thr Phe Leu Asn Asp Ala Gly Ala Cys Val Pro Ala Gln Glu Cys
740 745 750
Pro Cys Tyr Ala His Gly Thr Val Leu Ala Pro Gly Glu Val Val His
755 760 765
Asp Glu Gly Ala Val Cys Ser Cys Thr Gly Gly Lys Leu Ser Cys Leu
770 775 780
Gly Ala Ser Leu Gln Lys Ser Thr Gly Cys Ala Ala Pro Met Val Tyr
785 790 795 800
Leu Asp Cys Ser Asn Ser Ser Ala Gly Thr Pro Gly Ala Glu Cys Leu
805 810 815
Arg Ser Cys His Thr Leu Asp Val Gly Cys Phe Ser Thr His Cys Val
820 825 830
Ser Gly Cys Val Cys Pro Pro Gly Leu Val Ser Asp Gly Ser Gly Gly
835 840 845
Cys Ile Ala Glu Glu Asp Cys Pro Cys Val His Asn Glu Ala Thr Tyr
850 855 860
Lys Pro Gly Glu Thr Ile Arg Val Asp Cys Asn Thr Cys Thr Cys Arg
865 870 875 880
Asn Arg Arg Trp Glu Cys Ser His Arg Leu Cys Leu Gly Thr Cys Val
885 890 895
Ala Tyr Gly Asp Gly His Phe Ile Thr Phe Asp Gly Asp Arg Tyr Ser
900 905 910
Phe Glu Gly Ser Cys Glu Tyr Ile Leu Ala Gln Asp Tyr Cys Gly Asp
915 920 925
Asn Thr Thr His Gly Thr Phe Arg Ile Val Thr Glu Asn Ile Pro Cys
930 935 940
Gly Thr Thr Gly Thr Thr Cys Ser Lys Ala Ile Lys Leu Phe Val Glu
945 950 955 960
Ser Tyr Glu Leu Ile Leu Gln Glu Gly Thr Phe Lys Ala Val Ala Arg
965 970 975
Gly Pro Gly Gly Asp Pro Pro Tyr Lys Ile Arg Tyr Met Gly Ile Phe
980 985 990
Leu Val Ile Glu Thr His Gly Met Ala Val Ser Trp Asp Arg Lys Thr
995 1000 1005
Ser Val Phe Ile Arg Leu His Gln Asp Tyr Lys Gly Arg Val Cys
1010 1015 1020
Gly Leu Cys Gly Asn Phe Asp Asp Asn Ala Ile Asn Asp Phe Ala
1025 1030 1035
Thr Arg Ser Arg Ser Val Val Gly Asp Ala Leu Glu Phe Gly Asn
1040 1045 1050
Ser Trp Lys Leu Ser Pro Ser Cys Pro Asp Ala Leu Ala Pro Lys
1055 1060 1065
Asp Pro Cys Thr Ala Asn Pro Phe Arg Lys Ser Trp Ala Gln Lys
1070 1075 1080
Gln Cys Ser Ile Leu His Gly Pro Thr Phe Ala Ala Cys Arg Ser
1085 1090 1095
Gln Val Asp Ser Thr Lys Tyr Tyr Glu Ala Cys Val Asn Asp Ala
1100 1105 1110
Cys Ala Cys Asp Ser Gly Gly Asp Cys Glu Cys Phe Cys Thr Ala
1115 1120 1125
Val Ala Ala Tyr Ala Gln Ala Cys His Asp Ala Gly Leu Cys Val
1130 1135 1140
Ser Trp Arg Thr Pro Asp Thr Cys Pro Leu Phe Cys Asp Phe Tyr
1145 1150 1155
Asn Pro His Gly Gly Cys Glu Trp His Tyr Gln Pro Cys Gly Ala
1160 1165 1170
Pro Cys Leu Lys Thr Cys Arg Asn Pro Ser Gly His Cys Leu Val
1175 1180 1185
Asp Leu Pro Gly Leu Glu Gly Cys Tyr Pro Lys Cys Pro Pro Ser
1190 1195 1200
Gln Pro Phe Phe Asn Glu Asp Gln Met Lys Cys Val Ala Gln Cys
1205 1210 1215
Gly Cys Tyr Asp Lys Asp Gly Asn Tyr Tyr Asp Val Gly Ala Arg
1220 1225 1230
Val Pro Thr Ala Glu Asn Cys Gln Ser Cys Asn Cys Thr Pro Ser
1235 1240 1245
Gly Ile Gln Cys Ala His Ser Leu Glu Ala Cys Thr Cys Thr Tyr
1250 1255 1260
Glu Asp Arg Thr Tyr Ser Tyr Gln Asp Val Ile Tyr Asn Thr Thr
1265 1270 1275
Asp Gly Leu Gly Ala Cys Leu Ile Ala Ile Cys Gly Ser Asn Gly
1280 1285 1290
Thr Ile Ile Arg Lys Ala Val Ala Cys Pro Gly Thr Pro Ala Thr
1295 1300 1305
Thr Pro Phe Thr Phe Thr Thr Ala Trp Val Pro His Ser Thr Thr
1310 1315 1320
Ser Pro Ala Leu Pro Val Ser Thr Val Cys Val Arg Glu Val Cys
1325 1330 1335
Arg Trp Ser Ser Trp Tyr Asn Gly His Arg Pro Glu Pro Gly Leu
1340 1345 1350
Gly Gly Gly Asp Phe Glu Thr Phe Glu Asn Leu Arg Gln Arg Gly
1355 1360 1365
Tyr Gln Val Cys Pro Val Leu Ala Asp Ile Glu Cys Arg Ala Ala
1370 1375 1380
Gln Leu Pro Asp Met Pro Leu Glu Glu Leu Gly Gln Gln Val Asp
1385 1390 1395
Cys Asp Arg Met Arg Gly Leu Met Cys Ala Asn Ser Gln Gln Ser
1400 1405 1410
Pro Pro Leu Cys His Asp Tyr Glu Leu Arg Val Leu Cys Cys Glu
1415 1420 1425
Tyr Val Pro Cys Gly Pro Ser Pro Ala Pro Gly Thr Ser Pro Gln
1430 1435 1440
Pro Ser Leu Ser Ala Ser Thr Glu Pro Ala Val Pro Thr Pro Thr
1445 1450 1455
Gln Thr Thr Ala Thr Glu Lys Thr Thr Leu Trp Val Thr Pro Ser
1460 1465 1470
Ile Arg Ser Thr Ala Ala Leu Thr Ser Gln Thr Gly Ser Ser Ser
1475 1480 1485
Gly Pro Val Thr Val Thr Pro Ser Ala Pro Gly Thr Thr Thr Cys
1490 1495 1500
Gln Pro Arg Cys Gln Trp Thr Glu Trp Phe Asp Glu Asp Tyr Pro
1505 1510 1515
Lys Ser Glu Gln Leu Gly Gly Asp Val Glu Ser Tyr Asp Lys Ile
1520 1525 1530
Arg Ala Ala Gly Gly His Leu Cys Gln Gln Pro Lys Asp Ile Glu
1535 1540 1545
Cys Gln Ala Glu Ser Phe Pro Asn Trp Thr Leu Ala Gln Val Gly
1550 1555 1560
Gln Lys Val His Cys Asp Val His Phe Gly Leu Val Cys Arg Asn
1565 1570 1575
Trp Glu Gln Glu Gly Val Phe Lys Met Cys Tyr Asn Tyr Arg Ile
1580 1585 1590
Arg Val Leu Cys Cys Ser Asp Asp His Cys Arg Gly Arg Ala Thr
1595 1600 1605
Thr Pro Pro Pro Thr Thr Glu Leu Glu Thr Ala Thr Thr Thr Thr
1610 1615 1620
Thr Gln Ala Leu Phe Ser Thr Pro Gln Pro Thr Ser Ser Pro Gly
1625 1630 1635
Leu Thr Arg Ala Pro Pro Ala Ser Thr Thr Ala Val Pro Thr Leu
1640 1645 1650
Ser Glu Gly Leu Thr Ser Pro Arg Tyr Thr Ser Thr Leu Gly Thr
1655 1660 1665
Ala Thr Thr Gly Gly Pro Thr Thr Pro Ala Gly Ser Thr Glu Pro
1670 1675 1680
Thr Val Pro Gly Val Ala Thr Ser Thr Leu Pro Thr Arg Ser Ala
1685 1690 1695
Leu Pro Gly Thr Thr Gly Ser Leu Gly Thr Trp Arg Pro Ser Gln
1700 1705 1710
Pro Pro Thr Leu Ala Pro Thr Thr Met Ala Thr Ser Arg Ala Arg
1715 1720 1725
Pro Thr Gly Thr Ala Ser Thr Ala Ser Lys Glu Pro Leu Thr Thr
1730 1735 1740
Ser Leu Ala Pro Thr Leu Thr Ser Glu Leu Ser Thr Ser Gln Ala
1745 1750 1755
Glu Thr Ser Thr Pro Arg Thr Glu Thr Thr Met Ser Pro Leu Thr
1760 1765 1770
Asn Thr Thr Thr Ser Gln Gly Thr Thr Arg Cys Gln Pro Lys Cys
1775 1780 1785
Glu Trp Thr Glu Trp Phe Asp Val Asp Phe Pro Thr Ser Gly Val
1790 1795 1800
Ala Gly Gly Asp Met Glu Thr Phe Glu Asn Ile Arg Ala Ala Gly
1805 1810 1815
Gly Lys Met Cys Trp Ala Pro Lys Ser Ile Glu Cys Arg Ala Glu
1820 1825 1830
Asn Tyr Pro Glu Val Ser Ile Asp Gln Val Gly Gln Val Leu Thr
1835 1840 1845
Cys Ser Leu Glu Thr Gly Leu Thr Cys Lys Asn Glu Asp Gln Thr
1850 1855 1860
Gly Arg Phe Asn Met Cys Phe Asn Tyr Asn Val Arg Val Leu Cys
1865 1870 1875
Cys Asp Asp Tyr Ser His Cys Pro Ser Thr Pro Ala Thr Ser Ser
1880 1885 1890
Thr Ala Thr Pro Ser Ser Thr Pro Gly Thr Thr Trp Ile Leu Thr
1895 1900 1905
Lys Pro Thr Thr Thr Ala Thr Thr Thr Ala Ser Thr Gly Ser Thr
1910 1915 1920
Ala Thr Pro Thr Ser Thr Leu Arg Thr Ala Pro Pro Pro Lys Val
1925 1930 1935
Leu Thr Thr Thr Ala Thr Thr Pro Thr Val Thr Ser Ser Lys Ala
1940 1945 1950
Thr Pro Ser Ser Ser Pro Gly Thr Ala Thr Ala Leu Pro Ala Leu
1955 1960 1965
Arg Ser Thr Ala Thr Thr Pro Thr Ala Thr Ser Val Thr Pro Ile
1970 1975 1980
Pro Ser Ser Ser Leu Gly Thr Thr Trp Thr Arg Leu Ser Gln Thr
1985 1990 1995
Thr Thr Pro Thr Ala Thr Met Ser Thr Ala Thr Pro Ser Ser Thr
2000 2005 2010
Pro Glu Thr Ala His Thr Ser Thr Val Leu Thr Ala Thr Ala Thr
2015 2020 2025
Thr Thr Gly Ala Thr Gly Ser Val Ala Thr Pro Ser Ser Thr Pro
2030 2035 2040
Gly Thr Ala His Thr Thr Lys Val Pro Thr Thr Thr Thr Thr Gly
2045 2050 2055
Phe Thr Ala Thr Pro Ser Ser Ser Pro Gly Thr Ala Leu Thr Pro
2060 2065 2070
Pro Val Trp Ile Ser Thr Thr Thr Thr Pro Thr Thr Arg Gly Ser
2075 2080 2085
Thr Val Thr Pro Ser Ser Ile Pro Gly Thr Thr His Thr Ala Thr
2090 2095 2100
Val Leu Thr Thr Thr Thr Thr Thr Val Ala Thr Gly Ser Met Ala
2105 2110 2115
Thr Pro Ser Ser Ser Thr Gln Thr Ser Gly Thr Pro Pro Ser Leu
2120 2125 2130
Thr Thr Thr Ala Thr Thr Ile Thr Ala Thr Gly Ser Thr Thr Asn
2135 2140 2145
Pro Ser Ser Thr Pro Gly Thr Thr Pro Ile Pro Pro Val Leu Thr
2150 2155 2160
Thr Thr Ala Thr Thr Pro Ala Ala Thr Ser Asn Thr Val Thr Pro
2165 2170 2175
Ser Ser Ala Leu Gly Thr Thr His Thr Pro Pro Val Pro Asn Thr
2180 2185 2190
Met Ala Thr Thr His Gly Arg Ser Leu Pro Pro Ser Ser Pro His
2195 2200 2205
Thr Val Arg Thr Ala Trp Thr Ser Ala Thr Ser Gly Ile Leu Gly
2210 2215 2220
Thr Thr His Ile Thr Glu Pro Ser Thr Val Thr Ser His Thr Leu
2225 2230 2235
Ala Ala Thr Thr Gly Thr Thr Gln His Ser Thr Pro Ala Leu Ser
2240 2245 2250
Ser Pro His Pro Ser Ser Arg Thr Thr Glu Ser Pro Pro Ser Pro
2255 2260 2265
Gly Thr Thr Thr Pro Gly His Thr Thr Ala Thr Ser Arg Thr Thr
2270 2275 2280
Ala Thr Ala Thr Pro Ser Lys Thr Arg Thr Ser Thr Leu Leu Pro
2285 2290 2295
Ser Ser Pro Thr Ser Ala Pro Ile Thr Thr Val Val Thr Met Gly
2300 2305 2310
Cys Glu Pro Gln Cys Ala Trp Ser Glu Trp Leu Asp Tyr Ser Tyr
2315 2320 2325
Pro Met Pro Gly Pro Ser Gly Gly Asp Phe Asp Thr Tyr Ser Asn
2330 2335 2340
Ile Arg Ala Ala Gly Gly Ala Val Cys Glu Gln Pro Leu Gly Leu
2345 2350 2355
Glu Cys Arg Ala Gln Ala Gln Pro Gly Val Pro Leu Arg Glu Leu
2360 2365 2370
Gly Gln Val Val Glu Cys Ser Leu Asp Phe Gly Leu Val Cys Arg
2375 2380 2385
Asn Arg Glu Gln Val Gly Lys Phe Lys Met Cys Phe Asn Tyr Glu
2390 2395 2400
Ile Arg Val Phe Cys Cys Asn Tyr Gly His Cys Pro Ser Thr Pro
2405 2410 2415
Ala Thr Ser Ser Thr Ala Met Pro Ser Ser Thr Pro Gly Thr Thr
2420 2425 2430
Trp Ile Leu Thr Glu Leu Thr Thr Thr Ala Thr Thr Thr Glu Ser
2435 2440 2445
Thr Gly Ser Thr Ala Thr Pro Ser Ser Thr Pro Gly Thr Thr Trp
2450 2455 2460
Ile Leu Thr Glu Pro Ser Thr Thr Ala Thr Val Thr Val Pro Thr
2465 2470 2475
Gly Ser Thr Ala Thr Ala Ser Ser Thr Gln Ala Thr Ala Gly Thr
2480 2485 2490
Pro His Val Ser Thr Thr Ala Thr Thr Pro Thr Val Thr Ser Ser
2495 2500 2505
Lys Ala Thr Pro Phe Ser Ser Pro Gly Thr Ala Thr Ala Leu Pro
2510 2515 2520
Ala Leu Arg Ser Thr Ala Thr Thr Pro Thr Ala Thr Ser Phe Thr
2525 2530 2535
Ala Ile Pro Ser Ser Ser Leu Gly Thr Thr Trp Thr Arg Leu Ser
2540 2545 2550
Gln Thr Thr Thr Pro Thr Ala Thr Met Ser Thr Ala Thr Pro Ser
2555 2560 2565
Ser Thr Pro Glu Thr Val His Thr Ser Thr Val Leu Thr Thr Thr
2570 2575 2580
Ala Thr Thr Thr Gly Ala Thr Gly Ser Val Ala Thr Pro Ser Ser
2585 2590 2595
Thr Pro Gly Thr Ala His Thr Thr Lys Val Leu Thr Thr Thr Thr
2600 2605 2610
Thr Gly Phe Thr Ala Thr Pro Ser Ser Ser Pro Gly Thr Ala Arg
2615 2620 2625
Thr Leu Pro Val Trp Ile Ser Thr Thr Thr Thr Pro Thr Thr Arg
2630 2635 2640
Gly Ser Thr Val Thr Pro Ser Ser Ile Pro Gly Thr Thr His Thr
2645 2650 2655
Pro Thr Val Leu Thr Thr Thr Thr Thr Thr Val Ala Thr Gly Ser
2660 2665 2670
Met Ala Thr Pro Ser Ser Ser Thr Gln Thr Ser Gly Thr Pro Pro
2675 2680 2685
Ser Leu Thr Thr Thr Ala Thr Thr Ile Thr Ala Thr Gly Ser Thr
2690 2695 2700
Thr Asn Pro Ser Ser Thr Pro Gly Thr Thr Pro Ile Pro Pro Val
2705 2710 2715
Leu Thr Thr Thr Ala Thr Thr Pro Ala Ala Thr Ser Ser Thr Val
2720 2725 2730
Thr Pro Ser Ser Ala Leu Gly Thr Thr His Thr Pro Pro Val Pro
2735 2740 2745
Asn Thr Thr Ala Thr Thr His Gly Arg Ser Leu Ser Pro Ser Ser
2750 2755 2760
Pro His Thr Val Arg Thr Ala Trp Thr Ser Ala Thr Ser Gly Thr
2765 2770 2775
Leu Gly Thr Thr His Ile Thr Glu Pro Ser Thr Gly Thr Ser His
2780 2785 2790
Thr Pro Ala Ala Thr Thr Gly Thr Thr Gln His Ser Thr Pro Ala
2795 2800 2805
Leu Ser Ser Pro His Pro Ser Ser Arg Thr Thr Glu Ser Pro Pro
2810 2815 2820
Ser Pro Gly Thr Thr Thr Pro Gly His Thr Arg Ala Thr Ser Arg
2825 2830 2835
Thr Thr Ala Thr Ala Thr Pro Ser Lys Thr Arg Thr Ser Thr Leu
2840 2845 2850
Leu Pro Ser Ser Pro Thr Ser Ala Pro Ile Thr Thr Val Val Thr
2855 2860 2865
Met Gly Cys Glu Pro Gln Cys Ala Trp Ser Glu Trp Leu Asp Tyr
2870 2875 2880
Ser Tyr Pro Met Pro Gly Pro Ser Gly Gly Asp Phe Asp Thr Tyr
2885 2890 2895
Ser Asn Ile Arg Ala Ala Gly Gly Ala Val Cys Glu Gln Pro Leu
2900 2905 2910
Gly Leu Glu Cys Arg Ala Gln Ala Gln Pro Gly Val Pro Leu Arg
2915 2920 2925
Glu Leu Gly Gln Val Val Glu Cys Ser Leu Asp Phe Gly Leu Val
2930 2935 2940
Cys Arg Asn Arg Glu Gln Val Gly Lys Phe Lys Met Cys Phe Asn
2945 2950 2955
Tyr Glu Ile Arg Val Phe Cys Cys Asn Tyr Gly His Cys Pro Ser
2960 2965 2970
Thr Pro Ala Thr Ser Ser Thr Ala Thr Pro Ser Ser Thr Pro Gly
2975 2980 2985
Thr Thr Trp Ile Leu Thr Glu Gln Thr Thr Ala Ala Thr Thr Thr
2990 2995 3000
Ala Thr Thr Gly Ser Thr Ala Ile Pro Ser Ser Thr Pro Gly Thr
3005 3010 3015
Ala Pro Pro Pro Lys Val Leu Thr Ser Thr Ala Thr Thr Pro Thr
3020 3025 3030
Ala Thr Ser Ser Lys Ala Thr Ser Ser Ser Ser Pro Arg Thr Ala
3035 3040 3045
Thr Thr Leu Pro Val Leu Thr Ser Thr Ala Thr Lys Ser Thr Ala
3050 3055 3060
Thr Ser Phe Thr Pro Ile Pro Ser Phe Thr Leu Gly Thr Thr Gly
3065 3070 3075
Thr Leu Pro Glu Gln Thr Thr Thr Pro Met Ala Thr Met Ser Thr
3080 3085 3090
Ile His Pro Ser Ser Thr Pro Glu Thr Thr His Thr Ser Thr Val
3095 3100 3105
Leu Thr Thr Lys Ala Thr Thr Thr Arg Ala Thr Ser Ser Met Ser
3110 3115 3120
Thr Pro Ser Ser Thr Pro Gly Thr Thr Trp Ile Leu Thr Glu Leu
3125 3130 3135
Thr Thr Ala Ala Thr Thr Thr Ala Ala Thr Gly Pro Thr Ala Thr
3140 3145 3150
Pro Ser Ser Thr Pro Gly Thr Thr Trp Ile Leu Thr Glu Pro Ser
3155 3160 3165
Thr Thr Ala Thr Val Thr Val Pro Thr Gly Ser Thr Ala Thr Ala
3170 3175 3180
Ser Ser Thr Arg Ala Thr Ala Gly Thr Leu Lys Val Leu Thr Ser
3185 3190 3195
Thr Ala Thr Thr Pro Thr Val Ile Ser Ser Arg Ala Thr Pro Ser
3200 3205 3210
Ser Ser Pro Gly Thr Ala Thr Ala Leu Pro Ala Leu Arg Ser Thr
3215 3220 3225
Ala Thr Thr Pro Thr Ala Thr Ser Val Thr Ala Ile Pro Ser Ser
3230 3235 3240
Ser Leu Gly Thr Ala Trp Thr Arg Leu Ser Gln Thr Thr Thr Pro
3245 3250 3255
Thr Ala Thr Met Ser Thr Ala Thr Pro Ser Ser Thr Pro Glu Thr
3260 3265 3270
Val His Thr Ser Thr Val Leu Thr Thr Thr Thr Thr Thr Thr Arg
3275 3280 3285
Ala Thr Gly Ser Val Ala Thr Pro Ser Ser Thr Pro Gly Thr Ala
3290 3295 3300
His Thr Thr Lys Val Pro Thr Thr Thr Thr Thr Gly Phe Thr Ala
3305 3310 3315
Thr Pro Ser Ser Ser Pro Gly Thr Ala Leu Thr Pro Pro Val Trp
3320 3325 3330
Ile Ser Thr Thr Thr Thr Pro Thr Thr Arg Gly Ser Thr Val Thr
3335 3340 3345
Pro Ser Ser Ile Pro Gly Thr Thr His Thr Ala Thr Val Leu Thr
3350 3355 3360
Thr Thr Thr Thr Thr Val Ala Thr Gly Ser Met Ala Thr Pro Ser
3365 3370 3375
Ser Ser Thr Gln Thr Ser Gly Thr Pro Pro Ser Leu Thr Thr Thr
3380 3385 3390
Ala Thr Thr Ile Thr Ala Thr Gly Ser Thr Thr Asn Pro Ser Ser
3395 3400 3405
Thr Pro Gly Thr Thr Pro Ile Pro Pro Val Leu Thr Thr Thr Ala
3410 3415 3420
Thr Thr Pro Ala Ala Thr Ser Ser Thr Val Thr Pro Ser Ser Ala
3425 3430 3435
Leu Gly Thr Thr His Thr Pro Pro Val Pro Asn Thr Thr Ala Thr
3440 3445 3450
Thr His Gly Arg Ser Leu Pro Pro Ser Ser Pro His Thr Val Arg
3455 3460 3465
Thr Ala Trp Thr Ser Ala Thr Ser Gly Ile Leu Gly Thr Thr His
3470 3475 3480
Ile Thr Glu Pro Ser Thr Val Thr Ser His Thr Pro Ala Ala Thr
3485 3490 3495
Thr Ser Thr Thr Gln His Ser Thr Pro Ala Leu Ser Ser Pro His
3500 3505 3510
Pro Ser Ser Arg Thr Thr Glu Ser Pro Pro Ser Pro Gly Thr Thr
3515 3520 3525
Thr Pro Gly His Thr Arg Gly Thr Ser Arg Thr Thr Ala Thr Ala
3530 3535 3540
Thr Pro Ser Lys Thr Arg Thr Ser Thr Leu Leu Pro Ser Ser Pro
3545 3550 3555
Thr Ser Ala Pro Ile Thr Thr Val Val Thr Thr Gly Cys Glu Pro
3560 3565 3570
Gln Cys Ala Trp Ser Glu Trp Leu Asp Tyr Ser Tyr Pro Met Pro
3575 3580 3585
Gly Pro Ser Gly Gly Asp Phe Asp Thr Tyr Ser Asn Ile Arg Ala
3590 3595 3600
Ala Gly Gly Ala Val Cys Glu Gln Pro Leu Gly Leu Glu Cys Arg
3605 3610 3615
Ala Gln Ala Gln Pro Gly Val Pro Leu Arg Glu Leu Gly Gln Val
3620 3625 3630
Val Glu Cys Ser Leu Asp Phe Gly Leu Val Cys Arg Asn Arg Glu
3635 3640 3645
Gln Val Gly Lys Phe Lys Met Cys Phe Asn Tyr Glu Ile Arg Val
3650 3655 3660
Phe Cys Cys Asn Tyr Gly His Cys Pro Ser Thr Pro Ala Thr Ser
3665 3670 3675
Ser Thr Ala Thr Pro Ser Ser Thr Pro Gly Thr Thr Trp Ile Leu
3680 3685 3690
Thr Lys Leu Thr Thr Thr Ala Thr Thr Thr Glu Ser Thr Gly Ser
3695 3700 3705
Thr Ala Thr Pro Ser Ser Thr Pro Gly Thr Thr Trp Ile Leu Thr
3710 3715 3720
Glu Pro Ser Thr Thr Ala Thr Val Thr Val Pro Thr Gly Ser Thr
3725 3730 3735
Ala Thr Ala Ser Ser Thr Gln Ala Thr Ala Gly Thr Pro His Val
3740 3745 3750
Ser Thr Thr Ala Thr Thr Pro Thr Val Thr Ser Ser Lys Ala Thr
3755 3760 3765
Pro Phe Ser Ser Pro Gly Thr Ala Thr Ala Leu Pro Ala Leu Arg
3770 3775 3780
Ser Thr Ala Thr Thr Pro Thr Ala Thr Ser Phe Thr Ala Ile Pro
3785 3790 3795
Ser Ser Ser Leu Gly Thr Thr Trp Thr Arg Leu Ser Gln Thr Thr
3800 3805 3810
Thr Pro Thr Ala Thr Met Ser Thr Ala Thr Pro Ser Ser Thr Pro
3815 3820 3825
Glu Thr Ala His Thr Ser Thr Val Leu Thr Thr Thr Ala Thr Thr
3830 3835 3840
Thr Arg Ala Thr Gly Ser Val Ala Thr Pro Ser Ser Thr Pro Gly
3845 3850 3855
Thr Ala His Thr Thr Lys Val Pro Thr Thr Thr Thr Thr Gly Phe
3860 3865 3870
Thr Val Thr Pro Ser Ser Ser Pro Gly Thr Ala Arg Thr Pro Pro
3875 3880 3885
Val Trp Ile Ser Thr Thr Thr Thr Pro Thr Thr Ser Gly Ser Thr
3890 3895 3900
Val Thr Pro Ser Ser Val Pro Gly Thr Thr His Thr Pro Thr Val
3905 3910 3915
Leu Thr Thr Thr Thr Thr Thr Val Ala Thr Gly Ser Met Ala Thr
3920 3925 3930
Pro Ser Ser Ser Thr Gln Thr Ser Gly Thr Pro Pro Ser Leu Ile
3935 3940 3945
Thr Thr Ala Thr Thr Ile Thr Ala Thr Gly Ser Thr Thr Asn Pro
3950 3955 3960
Ser Ser Thr Pro Gly Thr Thr Pro Ile Pro Pro Val Leu Thr Thr
3965 3970 3975
Thr Ala Thr Thr Pro Ala Ala Thr Ser Ser Thr Val Thr Pro Ser
3980 3985 3990
Ser Ala Leu Gly Thr Thr His Thr Pro Pro Val Pro Asn Thr Thr
3995 4000 4005
Ala Thr Thr His Gly Arg Ser Leu Ser Pro Ser Ser Pro His Thr
4010 4015 4020
Val Arg Thr Ala Trp Thr Ser Ala Thr Ser Gly Thr Leu Gly Thr
4025 4030 4035
Thr His Ile Thr Glu Pro Ser Thr Gly Thr Ser His Thr Pro Ala
4040 4045 4050
Ala Thr Thr Gly Thr Thr Gln His Ser Thr Pro Ala Leu Ser Ser
4055 4060 4065
Pro His Pro Ser Ser Arg Thr Thr Glu Ser Pro Pro Ser Pro Gly
4070 4075 4080
Thr Thr Thr Pro Gly His Thr Thr Ala Thr Ser Arg Thr Thr Ala
4085 4090 4095
Thr Ala Thr Pro Ser Lys Thr Arg Thr Ser Thr Leu Leu Pro Ser
4100 4105 4110
Ser Pro Thr Ser Ala Pro Ile Thr Thr Val Val Thr Thr Gly Cys
4115 4120 4125
Glu Pro Gln Cys Ala Trp Ser Glu Trp Leu Asp Tyr Ser Tyr Pro
4130 4135 4140
Met Pro Gly Pro Ser Gly Gly Asp Phe Asp Thr Tyr Ser Asn Ile
4145 4150 4155
Arg Ala Ala Gly Gly Ala Val Cys Glu Gln Pro Leu Gly Leu Glu
4160 4165 4170
Cys Arg Ala Gln Ala Gln Pro Gly Val Pro Leu Gly Glu Leu Gly
4175 4180 4185
Gln Val Val Glu Cys Ser Leu Asp Phe Gly Leu Val Cys Arg Asn
4190 4195 4200
Arg Glu Gln Val Gly Lys Phe Lys Met Cys Phe Asn Tyr Glu Ile
4205 4210 4215
Arg Val Phe Cys Cys Asn Tyr Gly His Cys Pro Ser Thr Pro Ala
4220 4225 4230
Thr Ser Ser Thr Ala Met Pro Ser Ser Thr Pro Gly Thr Thr Trp
4235 4240 4245
Ile Leu Thr Glu Leu Thr Thr Thr Ala Thr Thr Thr Ala Ser Thr
4250 4255 4260
Gly Ser Thr Ala Thr Pro Ser Ser Thr Pro Gly Thr Ala Pro Pro
4265 4270 4275
Pro Lys Val Leu Thr Ser Pro Ala Thr Thr Pro Thr Ala Thr Ser
4280 4285 4290
Ser Lys Ala Thr Ser Ser Ser Ser Pro Arg Thr Ala Thr Thr Leu
4295 4300 4305
Pro Val Leu Thr Ser Thr Ala Thr Lys Ser Thr Ala Thr Ser Val
4310 4315 4320
Thr Pro Ile Pro Ser Ser Thr Leu Gly Thr Thr Gly Thr Leu Pro
4325 4330 4335
Glu Gln Thr Thr Thr Pro Val Ala Thr Met Ser Thr Ile His Pro
4340 4345 4350
Ser Ser Thr Pro Glu Thr Thr His Thr Ser Thr Val Leu Thr Thr
4355 4360 4365
Lys Ala Thr Thr Thr Arg Ala Thr Ser Ser Thr Ser Thr Pro Ser
4370 4375 4380
Ser Thr Pro Gly Thr Thr Trp Ile Leu Thr Glu Leu Thr Thr Ala
4385 4390 4395
Ala Thr Thr Thr Ala Ala Thr Gly Pro Thr Ala Thr Pro Ser Ser
4400 4405 4410
Thr Pro Gly Thr Thr Trp Ile Leu Thr Glu Leu Thr Thr Thr Ala
4415 4420 4425
Thr Thr Thr Ala Ser Thr Gly Ser Thr Ala Thr Pro Ser Ser Thr
4430 4435 4440
Pro Gly Thr Thr Trp Ile Leu Thr Glu Pro Ser Thr Thr Ala Thr
4445 4450 4455
Val Thr Val Pro Thr Gly Ser Thr Ala Thr Ala Ser Ser Thr Gln
4460 4465 4470
Ala Thr Ala Gly Thr Pro His Val Ser Thr Thr Ala Thr Thr Pro
4475 4480 4485
Thr Val Thr Ser Ser Lys Ala Thr Pro Ser Ser Ser Pro Gly Thr
4490 4495 4500
Ala Thr Ala Leu Pro Ala Leu Arg Ser Thr Ala Thr Thr Pro Thr
4505 4510 4515
Ala Thr Ser Phe Thr Ala Ile Pro Ser Ser Ser Leu Gly Thr Thr
4520 4525 4530
Trp Thr Arg Leu Ser Gln Thr Thr Thr Pro Thr Ala Thr Met Ser
4535 4540 4545
Thr Ala Thr Pro Ser Ser Thr Pro Glu Thr Val His Thr Ser Thr
4550 4555 4560
Val Leu Thr Ala Thr Ala Thr Thr Thr Gly Ala Thr Gly Ser Val
4565 4570 4575
Ala Thr Pro Ser Ser Thr Pro Gly Thr Ala His Thr Thr Lys Val
4580 4585 4590
Pro Thr Thr Thr Thr Thr Gly Phe Thr Ala Thr Pro Ser Ser Ser
4595 4600 4605
Pro Gly Thr Ala Leu Thr Pro Pro Val Trp Ile Ser Thr Thr Thr
4610 4615 4620
Thr Pro Thr Thr Thr Thr Pro Thr Thr Ser Gly Ser Thr Val Thr
4625 4630 4635
Pro Ser Ser Ile Pro Gly Thr Thr His Thr Ala Arg Val Leu Thr
4640 4645 4650
Thr Thr Thr Thr Thr Val Ala Thr Gly Ser Met Ala Thr Pro Ser
4655 4660 4665
Ser Ser Thr Gln Thr Ser Gly Thr Pro Pro Ser Leu Thr Thr Thr
4670 4675 4680
Ala Thr Thr Ile Thr Ala Thr Gly Ser Thr Thr Asn Pro Ser Ser
4685 4690 4695
Thr Pro Gly Thr Thr Pro Ile Thr Pro Val Leu Thr Ser Thr Ala
4700 4705 4710
Thr Thr Pro Ala Ala Thr Ser Ser Lys Ala Thr Ser Ser Ser Ser
4715 4720 4725
Pro Arg Thr Ala Thr Thr Leu Pro Val Leu Thr Ser Thr Ala Thr
4730 4735 4740
Lys Ser Thr Ala Thr Ser Phe Thr Pro Ile Pro Ser Ser Thr Leu
4745 4750 4755
Trp Thr Thr Trp Thr Val Pro Ala Gln Thr Thr Thr Pro Met Ser
4760 4765 4770
Thr Met Ser Thr Ile His Thr Ser Ser Thr Pro Glu Thr Thr His
4775 4780 4785
Thr Ser Thr Val Leu Thr Thr Thr Ala Thr Met Thr Arg Ala Thr
4790 4795 4800
Asn Ser Thr Ala Thr Pro Ser Ser Thr Leu Gly Thr Thr Arg Ile
4805 4810 4815
Leu Thr Glu Leu Thr Thr Thr Ala Thr Thr Thr Ala Ala Thr Gly
4820 4825 4830
Ser Thr Ala Thr Leu Ser Ser Thr Pro Gly Thr Thr Trp Ile Leu
4835 4840 4845
Thr Glu Pro Ser Thr Ile Ala Thr Val Met Val Pro Thr Gly Ser
4850 4855 4860
Thr Ala Thr Ala Ser Ser Thr Leu Gly Thr Ala His Thr Pro Lys
4865 4870 4875
Val Val Thr Thr Met Ala Thr Met Pro Thr Ala Thr Ala Ser Thr
4880 4885 4890
Val Pro Ser Ser Ser Thr Val Gly Thr Thr Arg Thr Pro Ala Val
4895 4900 4905
Leu Pro Ser Ser Leu Pro Thr Phe Ser Val Ser Thr Val Ser Ser
4910 4915 4920
Ser Val Leu Thr Thr Leu Arg Pro Thr Gly Phe Pro Ser Ser His
4925 4930 4935
Phe Ser Thr Pro Cys Phe Cys Arg Ala Phe Gly Gln Phe Phe Ser
4940 4945 4950
Pro Gly Glu Val Ile Tyr Asn Lys Thr Asp Arg Ala Gly Cys His
4955 4960 4965
Phe Tyr Ala Val Cys Asn Gln His Cys Asp Ile Asp Arg Phe Gln
4970 4975 4980
Gly Ala Cys Pro Thr Ser Pro Pro Pro Val Ser Ser Ala Pro Leu
4985 4990 4995
Ser Ser Pro Ser Pro Ala Pro Gly Cys Asp Asn Ala Ile Pro Leu
5000 5005 5010
Arg Gln Val Asn Glu Thr Trp Thr Leu Glu Asn Cys Thr Val Ala
5015 5020 5025
Arg Cys Val Gly Asp Asn Arg Val Val Leu Leu Asp Pro Lys Pro
5030 5035 5040
Val Ala Asn Val Thr Cys Val Asn Lys His Leu Pro Ile Lys Val
5045 5050 5055
Ser Asp Pro Ser Gln Pro Cys Asp Phe His Tyr Glu Cys Glu Cys
5060 5065 5070
Ile Cys Ser Met Trp Gly Gly Ser His Tyr Ser Thr Phe Asp Gly
5075 5080 5085
Thr Ser Tyr Thr Phe Arg Gly Asn Cys Thr Tyr Val Leu Met Arg
5090 5095 5100
Glu Ile His Ala Arg Phe Gly Asn Leu Ser Leu Tyr Leu Asp Asn
5105 5110 5115
His Tyr Cys Thr Ala Ser Ala Thr Ala Ala Ala Ala Arg Cys Pro
5120 5125 5130
Arg Ala Leu Ser Ile His Tyr Lys Ser Met Asp Ile Val Leu Thr
5135 5140 5145
Val Thr Met Val His Gly Lys Glu Glu Gly Leu Ile Leu Phe Asp
5150 5155 5160
Gln Ile Pro Val Ser Ser Gly Phe Ser Lys Asn Gly Val Leu Val
5165 5170 5175
Ser Val Leu Gly Thr Thr Thr Met Arg Val Asp Ile Pro Ala Leu
5180 5185 5190
Gly Val Ser Val Thr Phe Asn Gly Gln Val Phe Gln Ala Arg Leu
5195 5200 5205
Pro Tyr Ser Leu Phe His Asn Asn Thr Glu Gly Gln Cys Gly Thr
5210 5215 5220
Cys Thr Asn Asn Gln Arg Asp Asp Cys Leu Gln Arg Asp Gly Thr
5225 5230 5235
Thr Ala Ala Ser Cys Lys Asp Met Ala Lys Thr Trp Leu Val Pro
5240 5245 5250
Asp Ser Arg Lys Asp Gly Cys Trp Ala Pro Thr Gly Thr Pro Pro
5255 5260 5265
Thr Ala Ser Pro Ala Ala Pro Val Ser Ser Thr Pro Thr Pro Thr
5270 5275 5280
Pro Cys Pro Pro Gln Pro Leu Cys Asp Leu Met Leu Ser Gln Val
5285 5290 5295
Phe Ala Glu Cys His Asn Leu Val Pro Pro Gly Pro Phe Phe Asn
5300 5305 5310
Ala Cys Ile Ser Asp His Cys Arg Gly Arg Leu Glu Val Pro Cys
5315 5320 5325
Gln Ser Leu Glu Ala Tyr Ala Glu Leu Cys Arg Ala Arg Gly Val
5330 5335 5340
Cys Ser Asp Trp Arg Gly Ala Thr Gly Gly Leu Cys Asp Leu Thr
5345 5350 5355
Cys Pro Pro Thr Lys Val Tyr Lys Pro Cys Gly Pro Ile Gln Pro
5360 5365 5370
Ala Thr Cys Asn Ser Arg Asn Gln Ser Pro Gln Leu Glu Gly Met
5375 5380 5385
Ala Glu Gly Cys Phe Cys Pro Glu Asp Gln Ile Leu Phe Asn Ala
5390 5395 5400
His Met Gly Ile Cys Val Gln Ala Cys Pro Cys Val Gly Pro Asp
5405 5410 5415
Gly Phe Pro Lys Phe Pro Gly Glu Arg Trp Val Ser Asn Cys Gln
5420 5425 5430
Ser Cys Val Cys Asp Glu Gly Ser Val Ser Val Gln Cys Lys Pro
5435 5440 5445
Leu Pro Cys Asp Ala Gln Gly Gln Pro Pro Pro Cys Asn Arg Pro
5450 5455 5460
Gly Phe Val Thr Val Thr Arg Pro Arg Ala Glu Asn Pro Cys Cys
5465 5470 5475
Pro Glu Thr Val Cys Val Cys Asn Thr Thr Thr Cys Pro Gln Ser
5480 5485 5490
Leu Pro Val Cys Pro Pro Gly Gln Glu Ser Ile Cys Thr Gln Glu
5495 5500 5505
Glu Gly Asp Cys Cys Pro Thr Phe Arg Cys Arg Pro Gln Leu Cys
5510 5515 5520
Ser Tyr Asn Gly Thr Phe Tyr Gly Val Gly Ala Thr Phe Pro Gly
5525 5530 5535
Ala Leu Pro Cys His Met Cys Thr Cys Leu Ser Gly Asp Thr Gln
5540 5545 5550
Asp Pro Thr Val Gln Cys Gln Glu Asp Ala Cys Asn Asn Thr Thr
5555 5560 5565
Cys Pro Gln Gly Phe Glu Tyr Lys Arg Val Ala Gly Gln Cys Cys
5570 5575 5580
Gly Glu Cys Val Gln Thr Ala Cys Leu Thr Pro Asp Gly Gln Pro
5585 5590 5595
Val Gln Leu Asn Glu Thr Trp Val Asn Ser His Val Asp Asn Cys
5600 5605 5610
Thr Val Tyr Leu Cys Glu Ala Glu Gly Gly Val His Leu Leu Thr
5615 5620 5625
Pro Gln Pro Ala Ser Cys Pro Asp Val Ser Ser Cys Arg Gly Ser
5630 5635 5640
Leu Arg Lys Thr Gly Cys Cys Tyr Ser Cys Glu Glu Asp Ser Cys
5645 5650 5655
Gln Val Arg Ile Asn Thr Thr Ile Leu Trp His Gln Gly Cys Glu
5660 5665 5670
Thr Glu Val Asn Ile Thr Phe Cys Glu Gly Ser Cys Pro Gly Ala
5675 5680 5685
Ser Lys Tyr Ser Ala Glu Ala Gln Ala Met Gln His Gln Cys Thr
5690 5695 5700
Cys Cys Gln Glu Arg Arg Val His Glu Glu Thr Val Pro Leu His
5705 5710 5715
Cys Pro Asn Gly Ser Ala Ile Leu His Thr Tyr Thr His Val Asp
5720 5725 5730
Glu Cys Gly Cys Thr Pro Phe Cys Val Pro Ala Pro Met Ala Pro
5735 5740 5745
Pro His Thr Arg Gly Phe Pro Ala Gln Glu Ala Thr Ala Val
5750 5755 5760
<210> 15
<211> 217
<212> PRT
<213> 人
<400> 15
Met Glu Ala Ile Ala Lys Tyr Asp Phe Lys Ala Thr Ala Asp Asp Glu
1 5 10 15
Leu Ser Phe Lys Arg Gly Asp Ile Leu Lys Val Leu Asn Glu Glu Cys
20 25 30
Asp Gln Asn Trp Tyr Lys Ala Glu Leu Asn Gly Lys Asp Gly Phe Ile
35 40 45
Pro Lys Asn Tyr Ile Glu Met Lys Pro His Pro Trp Phe Phe Gly Lys
50 55 60
Ile Pro Arg Ala Lys Ala Glu Glu Met Leu Ser Lys Gln Arg His Asp
65 70 75 80
Gly Ala Phe Leu Ile Arg Glu Ser Glu Ser Ala Pro Gly Asp Phe Ser
85 90 95
Leu Ser Val Lys Phe Gly Asn Asp Val Gln His Phe Lys Val Leu Arg
100 105 110
Asp Gly Ala Gly Lys Tyr Phe Leu Trp Val Val Lys Phe Asn Ser Leu
115 120 125
Asn Glu Leu Val Asp Tyr His Arg Ser Thr Ser Val Ser Arg Asn Gln
130 135 140
Gln Ile Phe Leu Arg Asp Ile Glu Gln Val Pro Gln Gln Pro Thr Tyr
145 150 155 160
Val Gln Ala Leu Phe Asp Phe Asp Pro Gln Glu Asp Gly Glu Leu Gly
165 170 175
Phe Arg Arg Gly Asp Phe Ile His Val Met Asp Asn Ser Asp Pro Asn
180 185 190
Trp Trp Lys Gly Ala Cys His Gly Gln Thr Gly Met Phe Pro Arg Asn
195 200 205
Tyr Val Thr Pro Val Asn Arg Asn Val
210 215
<210> 16
<211> 320
<212> PRT
<213> 人
<400> 16
Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp
1 5 10 15
Glu Arg Ala Asp Ala Glu Thr Leu Arg Lys Ala Met Lys Gly Leu Gly
20 25 30
Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala
35 40 45
Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp
50 55 60
Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu
65 70 75 80
Ile Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu Leu
85 90 95
Lys His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu
100 105 110
Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val
115 120 125
Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp
130 135 140
Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn
145 150 155 160
Arg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala
165 170 175
Gln Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu
180 185 190
Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys
195 200 205
Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr
210 215 220
Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val
225 230 235 240
Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr
245 250 255
Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val
260 265 270
Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu Phe
275 280 285
Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr
290 295 300
Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp
305 310 315 320
<210> 17
<211> 510
<212> PRT
<213> 人
<400> 17
Met Arg Pro Gly Leu Ser Phe Leu Leu Ala Leu Leu Phe Phe Leu Gly
1 5 10 15
Gln Ala Ala Gly Asp Leu Gly Asp Val Gly Pro Pro Ile Pro Ser Pro
20 25 30
Gly Phe Ser Ser Phe Pro Gly Val Asp Ser Ser Ser Ser Phe Ser Ser
35 40 45
Ser Ser Arg Ser Gly Ser Ser Ser Ser Arg Ser Leu Gly Ser Gly Gly
50 55 60
Ser Val Ser Gln Leu Phe Ser Asn Phe Thr Gly Ser Val Asp Asp Arg
65 70 75 80
Gly Thr Cys Gln Cys Ser Val Ser Leu Pro Asp Thr Thr Phe Pro Val
85 90 95
Asp Arg Val Glu Arg Leu Glu Phe Thr Ala His Val Leu Ser Gln Lys
100 105 110
Phe Glu Lys Glu Leu Ser Lys Val Arg Glu Tyr Val Gln Leu Ile Ser
115 120 125
Val Tyr Glu Lys Lys Leu Leu Asn Leu Thr Val Arg Ile Asp Ile Met
130 135 140
Glu Lys Asp Thr Ile Ser Tyr Thr Glu Leu Asp Phe Glu Leu Ile Lys
145 150 155 160
Val Glu Val Lys Glu Met Glu Lys Leu Val Ile Gln Leu Lys Glu Ser
165 170 175
Phe Gly Gly Ser Ser Glu Ile Val Asp Gln Leu Glu Val Glu Ile Arg
180 185 190
Asn Met Thr Leu Leu Val Glu Lys Leu Glu Thr Leu Asp Lys Asn Asn
195 200 205
Val Leu Ala Ile Arg Arg Glu Ile Val Ala Leu Lys Thr Lys Leu Lys
210 215 220
Glu Cys Glu Ala Ser Lys Asp Gln Asn Thr Pro Val Val His Pro Pro
225 230 235 240
Pro Thr Pro Gly Ser Cys Gly His Gly Gly Val Val Asn Ile Ser Lys
245 250 255
Pro Ser Val Val Gln Leu Asn Trp Arg Gly Phe Ser Tyr Leu Tyr Gly
260 265 270
Ala Trp Gly Arg Asp Tyr Ser Pro Gln His Pro Asn Lys Gly Leu Tyr
275 280 285
Trp Val Ala Pro Leu Asn Thr Asp Gly Arg Leu Leu Glu Tyr Tyr Arg
290 295 300
Leu Tyr Asn Thr Leu Asp Asp Leu Leu Leu Tyr Ile Asn Ala Arg Glu
305 310 315 320
Leu Arg Ile Thr Tyr Gly Gln Gly Ser Gly Thr Ala Val Tyr Asn Asn
325 330 335
Asn Met Tyr Val Asn Met Tyr Asn Thr Gly Asn Ile Ala Arg Val Asn
340 345 350
Leu Thr Thr Asn Thr Ile Ala Val Thr Gln Thr Leu Pro Asn Ala Ala
355 360 365
Tyr Asn Asn Arg Phe Ser Tyr Ala Asn Val Ala Trp Gln Asp Ile Asp
370 375 380
Phe Ala Val Asp Glu Asn Gly Leu Trp Val Ile Tyr Ser Thr Glu Ala
385 390 395 400
Ser Thr Gly Asn Met Val Ile Ser Lys Leu Asn Asp Thr Thr Leu Gln
405 410 415
Val Leu Asn Thr Trp Tyr Thr Lys Gln Tyr Lys Pro Ser Ala Ser Asn
420 425 430
Ala Phe Met Val Cys Gly Val Leu Tyr Ala Thr Arg Thr Met Asn Thr
435 440 445
Arg Thr Glu Glu Ile Phe Tyr Tyr Tyr Asp Thr Asn Thr Gly Lys Glu
450 455 460
Gly Lys Leu Asp Ile Val Met His Lys Met Gln Glu Lys Val Gln Ser
465 470 475 480
Ile Asn Tyr Asn Pro Phe Asp Gln Lys Leu Tyr Val Tyr Asn Asp Gly
485 490 495
Tyr Leu Leu Asn Tyr Asp Leu Ser Val Leu Gln Lys Pro Gln
500 505 510
<210> 18
<211> 541
<212> PRT
<213> 人
<400> 18
Met Val Ala Asp Pro Pro Arg Asp Ser Lys Gly Leu Ala Ala Ala Glu
1 5 10 15
Pro Thr Ala Asn Gly Gly Leu Ala Leu Ala Ser Ile Glu Asp Gln Gly
20 25 30
Ala Ala Ala Gly Gly Tyr Cys Gly Ser Arg Asp Gln Val Arg Arg Cys
35 40 45
Leu Arg Ala Asn Leu Leu Val Leu Leu Thr Val Val Ala Val Val Ala
50 55 60
Gly Val Ala Leu Gly Leu Gly Val Ser Gly Ala Gly Gly Ala Leu Ala
65 70 75 80
Leu Gly Pro Glu Arg Leu Ser Ala Phe Val Phe Pro Gly Glu Leu Leu
85 90 95
Leu Arg Leu Leu Arg Met Ile Ile Leu Pro Leu Val Val Cys Ser Leu
100 105 110
Ile Gly Gly Ala Ala Ser Leu Asp Pro Gly Ala Leu Gly Arg Leu Gly
115 120 125
Ala Trp Ala Leu Leu Phe Phe Leu Val Thr Thr Leu Leu Ala Ser Ala
130 135 140
Leu Gly Val Gly Leu Ala Leu Ala Leu Gln Pro Gly Ala Ala Ser Ala
145 150 155 160
Ala Ile Asn Ala Ser Val Gly Ala Ala Gly Ser Ala Glu Asn Ala Pro
165 170 175
Ser Lys Glu Val Leu Asp Ser Phe Leu Asp Leu Ala Arg Asn Ile Phe
180 185 190
Pro Ser Asn Leu Val Ser Ala Ala Phe Arg Ser Tyr Ser Thr Thr Tyr
195 200 205
Glu Glu Arg Asn Ile Thr Gly Thr Arg Val Lys Val Pro Val Gly Gln
210 215 220
Glu Val Glu Gly Met Asn Ile Leu Gly Leu Val Val Phe Ala Ile Val
225 230 235 240
Phe Gly Val Ala Leu Arg Lys Leu Gly Pro Glu Gly Glu Leu Leu Ile
245 250 255
Arg Phe Phe Asn Ser Phe Asn Glu Ala Thr Met Val Leu Val Ser Trp
260 265 270
Ile Met Trp Tyr Ala Pro Val Gly Ile Met Phe Leu Val Ala Gly Lys
275 280 285
Ile Val Glu Met Glu Asp Val Gly Leu Leu Phe Ala Arg Leu Gly Lys
290 295 300
Tyr Ile Leu Cys Cys Leu Leu Gly His Ala Ile His Gly Leu Leu Val
305 310 315 320
Leu Pro Leu Ile Tyr Phe Leu Phe Thr Arg Lys Asn Pro Tyr Arg Phe
325 330 335
Leu Trp Gly Ile Val Thr Pro Leu Ala Thr Ala Phe Gly Thr Ser Ser
340 345 350
Ser Ser Ala Thr Leu Pro Leu Met Met Lys Cys Val Glu Glu Asn Asn
355 360 365
Gly Val Ala Lys His Ile Ser Arg Phe Ile Leu Pro Ile Gly Ala Thr
370 375 380
Val Asn Met Asp Gly Ala Ala Leu Phe Gln Cys Val Ala Ala Val Phe
385 390 395 400
Ile Ala Gln Leu Ser Gln Gln Ser Leu Asp Phe Val Lys Ile Ile Thr
405 410 415
Ile Leu Val Thr Ala Thr Ala Ser Ser Val Gly Ala Ala Gly Ile Pro
420 425 430
Ala Gly Gly Val Leu Thr Leu Ala Ile Ile Leu Glu Ala Val Asn Leu
435 440 445
Pro Val Asp His Ile Ser Leu Ile Leu Ala Val Asp Trp Leu Val Asp
450 455 460
Arg Ser Cys Thr Val Leu Asn Val Glu Gly Asp Ala Leu Gly Ala Gly
465 470 475 480
Leu Leu Gln Asn Tyr Val Asp Arg Thr Glu Ser Arg Ser Thr Glu Pro
485 490 495
Glu Leu Ile Gln Val Lys Ser Glu Leu Pro Leu Asp Pro Leu Pro Val
500 505 510
Pro Thr Glu Glu Gly Asn Pro Leu Leu Lys His Tyr Arg Gly Pro Ala
515 520 525
Gly Asp Ala Thr Val Ala Ser Glu Lys Glu Ser Val Met
530 535 540
<210> 19
<211> 563
<212> PRT
<213> 人
<400> 19
Met Gly Leu Gln Ala Cys Leu Leu Gly Leu Phe Ala Leu Ile Leu Ser
1 5 10 15
Gly Lys Cys Ser Tyr Ser Pro Glu Pro Asp Gln Arg Arg Thr Leu Pro
20 25 30
Pro Gly Trp Val Ser Leu Gly Arg Ala Asp Pro Glu Glu Glu Leu Ser
35 40 45
Leu Thr Phe Ala Leu Arg Gln Gln Asn Val Glu Arg Leu Ser Glu Leu
50 55 60
Val Gln Ala Val Ser Asp Pro Ser Ser Pro Gln Tyr Gly Lys Tyr Leu
65 70 75 80
Thr Leu Glu Asn Val Ala Asp Leu Val Arg Pro Ser Pro Leu Thr Leu
85 90 95
His Thr Val Gln Lys Trp Leu Leu Ala Ala Gly Ala Gln Lys Cys His
100 105 110
Ser Val Ile Thr Gln Asp Phe Leu Thr Cys Trp Leu Ser Ile Arg Gln
115 120 125
Ala Glu Leu Leu Leu Pro Gly Ala Glu Phe His His Tyr Val Gly Gly
130 135 140
Pro Thr Glu Thr His Val Val Arg Ser Pro His Pro Tyr Gln Leu Pro
145 150 155 160
Gln Ala Leu Ala Pro His Val Asp Phe Val Gly Gly Leu His Arg Phe
165 170 175
Pro Pro Thr Ser Ser Leu Arg Gln Arg Pro Glu Pro Gln Val Thr Gly
180 185 190
Thr Val Gly Leu His Leu Gly Val Thr Pro Ser Val Ile Arg Lys Arg
195 200 205
Tyr Asn Leu Thr Ser Gln Asp Val Gly Ser Gly Thr Ser Asn Asn Ser
210 215 220
Gln Ala Cys Ala Gln Phe Leu Glu Gln Tyr Phe His Asp Ser Asp Leu
225 230 235 240
Ala Gln Phe Met Arg Leu Phe Gly Gly Asn Phe Ala His Gln Ala Ser
245 250 255
Val Ala Arg Val Val Gly Gln Gln Gly Arg Gly Arg Ala Gly Ile Glu
260 265 270
Ala Ser Leu Asp Val Gln Tyr Leu Met Ser Ala Gly Ala Asn Ile Ser
275 280 285
Thr Trp Val Tyr Ser Ser Pro Gly Arg His Glu Gly Gln Glu Pro Phe
290 295 300
Leu Gln Trp Leu Met Leu Leu Ser Asn Glu Ser Ala Leu Pro His Val
305 310 315 320
His Thr Val Ser Tyr Gly Asp Asp Glu Asp Ser Leu Ser Ser Ala Tyr
325 330 335
Ile Gln Arg Val Asn Thr Glu Leu Met Lys Ala Ala Ala Arg Gly Leu
340 345 350
Thr Leu Leu Phe Ala Ser Gly Asp Ser Gly Ala Gly Cys Trp Ser Val
355 360 365
Ser Gly Arg His Gln Phe Arg Pro Thr Phe Pro Ala Ser Ser Pro Tyr
370 375 380
Val Thr Thr Val Gly Gly Thr Ser Phe Gln Glu Pro Phe Leu Ile Thr
385 390 395 400
Asn Glu Ile Val Asp Tyr Ile Ser Gly Gly Gly Phe Ser Asn Val Phe
405 410 415
Pro Arg Pro Ser Tyr Gln Glu Glu Ala Val Thr Lys Phe Leu Ser Ser
420 425 430
Ser Pro His Leu Pro Pro Ser Ser Tyr Phe Asn Ala Ser Gly Arg Ala
435 440 445
Tyr Pro Asp Val Ala Ala Leu Ser Asp Gly Tyr Trp Val Val Ser Asn
450 455 460
Arg Val Pro Ile Pro Trp Val Ser Gly Thr Ser Ala Ser Thr Pro Val
465 470 475 480
Phe Gly Gly Ile Leu Ser Leu Ile Asn Glu His Arg Ile Leu Ser Gly
485 490 495
Arg Pro Pro Leu Gly Phe Leu Asn Pro Arg Leu Tyr Gln Gln His Gly
500 505 510
Ala Gly Leu Phe Asp Val Thr Arg Gly Cys His Glu Ser Cys Leu Asp
515 520 525
Glu Glu Val Glu Gly Gln Gly Phe Cys Ser Gly Pro Gly Trp Asp Pro
530 535 540
Val Thr Gly Trp Gly Thr Pro Asn Phe Pro Ala Leu Leu Lys Thr Leu
545 550 555 560
Leu Asn Pro
<210> 20
<211> 639
<212> PRT
<213> 人
<400> 20
Met Ser Ala Arg Gly Pro Ala Ile Gly Ile Asp Leu Gly Thr Thr Tyr
1 5 10 15
Ser Cys Val Gly Val Phe Gln His Gly Lys Val Glu Ile Ile Ala Asn
20 25 30
Asp Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr
35 40 45
Glu Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val Ala Met Asn Pro
50 55 60
Thr Asn Thr Ile Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe Glu
65 70 75 80
Asp Ala Thr Val Gln Ser Asp Met Lys His Trp Pro Phe Arg Val Val
85 90 95
Ser Glu Gly Gly Lys Pro Lys Val Gln Val Glu Tyr Lys Gly Glu Thr
100 105 110
Lys Thr Phe Phe Pro Glu Glu Ile Ser Ser Met Val Leu Thr Lys Met
115 120 125
Lys Glu Ile Ala Glu Ala Tyr Leu Gly Gly Lys Val His Ser Ala Val
130 135 140
Ile Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys
145 150 155 160
Asp Ala Gly Thr Ile Thr Gly Leu Asn Val Leu Arg Ile Ile Asn Glu
165 170 175
Pro Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Gly Cys Ala
180 185 190
Gly Gly Glu Lys Asn Val Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe
195 200 205
Asp Val Ser Ile Leu Thr Ile Glu Asp Gly Ile Phe Glu Val Lys Ser
210 215 220
Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Met
225 230 235 240
Val Ser His Leu Ala Glu Glu Phe Lys Arg Lys His Lys Lys Asp Ile
245 250 255
Gly Pro Asn Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg
260 265 270
Ala Lys Arg Thr Leu Ser Ser Ser Thr Gln Ala Ser Ile Glu Ile Asp
275 280 285
Ser Leu Tyr Glu Gly Val Asp Phe Tyr Thr Ser Ile Thr Arg Ala Arg
290 295 300
Phe Glu Glu Leu Asn Ala Asp Leu Phe Arg Gly Thr Leu Glu Pro Val
305 310 315 320
Glu Lys Ala Leu Arg Asp Ala Lys Leu Asp Lys Gly Gln Ile Gln Glu
325 330 335
Ile Val Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Ile Gln Lys Leu
340 345 350
Leu Gln Asp Phe Phe Asn Gly Lys Glu Leu Asn Lys Ser Ile Asn Pro
355 360 365
Asp Glu Ala Val Ala Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu Ile
370 375 380
Gly Asp Lys Ser Glu Asn Val Gln Asp Leu Leu Leu Leu Asp Val Thr
385 390 395 400
Pro Leu Ser Leu Gly Ile Glu Thr Ala Gly Gly Val Met Thr Pro Leu
405 410 415
Ile Lys Arg Asn Thr Thr Ile Pro Thr Lys Gln Thr Gln Thr Phe Thr
420 425 430
Thr Tyr Ser Asp Asn Gln Ser Ser Val Leu Val Gln Val Tyr Glu Gly
435 440 445
Glu Arg Ala Met Thr Lys Asp Asn Asn Leu Leu Gly Lys Phe Asp Leu
450 455 460
Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu Val Thr
465 470 475 480
Phe Asp Ile Asp Ala Asn Gly Ile Leu Asn Val Thr Ala Ala Asp Lys
485 490 495
Ser Thr Gly Lys Glu Asn Lys Ile Thr Ile Thr Asn Asp Lys Gly Arg
500 505 510
Leu Ser Lys Asp Asp Ile Asp Arg Met Val Gln Glu Ala Glu Arg Tyr
515 520 525
Lys Ser Glu Asp Glu Ala Asn Arg Asp Arg Val Ala Ala Lys Asn Ala
530 535 540
Leu Glu Ser Tyr Thr Tyr Asn Ile Lys Gln Thr Val Glu Asp Glu Lys
545 550 555 560
Leu Arg Gly Lys Ile Ser Glu Gln Asp Lys Asn Lys Ile Leu Asp Lys
565 570 575
Cys Gln Glu Val Ile Asn Trp Leu Asp Arg Asn Gln Met Ala Glu Lys
580 585 590
Asp Glu Tyr Glu His Lys Gln Lys Glu Leu Glu Arg Val Cys Asn Pro
595 600 605
Ile Ile Ser Lys Leu Tyr Gln Gly Gly Pro Gly Gly Gly Ser Gly Gly
610 615 620
Gly Gly Ser Gly Ala Ser Gly Gly Pro Thr Ile Glu Glu Val Asp
625 630 635
<210> 21
<211> 241
<212> PRT
<213> 人
<400> 21
Met Phe Leu Thr Arg Ser Glu Tyr Asp Arg Gly Val Asn Thr Phe Ser
1 5 10 15
Pro Glu Gly Arg Leu Phe Gln Val Glu Tyr Ala Ile Glu Ala Ile Lys
20 25 30
Leu Gly Ser Thr Ala Ile Gly Ile Gln Thr Ser Glu Gly Val Cys Leu
35 40 45
Ala Val Glu Lys Arg Ile Thr Ser Pro Leu Met Glu Pro Ser Ser Ile
50 55 60
Glu Lys Ile Val Glu Ile Asp Ala His Ile Gly Cys Ala Met Ser Gly
65 70 75 80
Leu Ile Ala Asp Ala Lys Thr Leu Ile Asp Lys Ala Arg Val Glu Thr
85 90 95
Gln Asn His Trp Phe Thr Tyr Asn Glu Thr Met Thr Val Glu Ser Val
100 105 110
Thr Gln Ala Val Ser Asn Leu Ala Leu Gln Phe Gly Glu Glu Asp Ala
115 120 125
Asp Pro Gly Ala Met Ser Arg Pro Phe Gly Val Ala Leu Leu Phe Gly
130 135 140
Gly Val Asp Glu Lys Gly Pro Gln Leu Phe His Met Asp Pro Ser Gly
145 150 155 160
Thr Phe Val Gln Cys Asp Ala Arg Ala Ile Gly Ser Ala Ser Glu Gly
165 170 175
Ala Gln Ser Ser Leu Gln Glu Val Tyr His Lys Ser Met Thr Leu Lys
180 185 190
Glu Ala Ile Lys Ser Ser Leu Ile Ile Leu Lys Gln Val Met Glu Glu
195 200 205
Lys Leu Asn Ala Thr Asn Ile Glu Leu Ala Thr Val Gln Pro Gly Gln
210 215 220
Asn Phe His Met Phe Thr Lys Glu Glu Leu Glu Glu Val Ile Lys Asp
225 230 235 240
Ile
<210> 22
<211> 198
<212> PRT
<213> 人
<400> 22
Met Pro Leu Gly Leu Leu Trp Leu Gly Leu Ala Leu Leu Gly Ala Leu
1 5 10 15
His Ala Gln Ala Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro
20 25 30
Leu Ser Lys Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln
35 40 45
Gly Lys Trp Tyr Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu
50 55 60
Asp Lys Asp Pro Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu
65 70 75 80
Asp Lys Ser Tyr Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys
85 90 95
Asp Tyr Trp Ile Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe
100 105 110
Thr Leu Gly Asn Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val
115 120 125
Arg Val Val Ser Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys
130 135 140
Lys Val Ser Gln Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg
145 150 155 160
Thr Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser
165 170 175
Lys Ser Leu Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile
180 185 190
Asp Gln Cys Ile Asp Gly
195
<210> 23
<211> 13
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 23
Gly Thr Ile Thr Asp Ala Pro Gly Phe Asp Pro Leu Arg
1 5 10
<210> 24
<211> 9
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 24
Ser Glu Thr Asp Leu Leu Asp Ile Arg
1 5
<210> 25
<211> 16
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 25
Gly Ala Gly Thr Asn Glu Asp Ala Leu Ile Glu Ile Leu Thr Thr Arg
1 5 10 15
<210> 26
<211> 13
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 26
Ser Asp Thr Ser Gly Asp Tyr Glu Ile Thr Leu Leu Lys
1 5 10
<210> 27
<211> 16
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 27
Gly Ala Gly Thr Asp Glu Gly Cys Leu Ile Glu Ile Leu Ala Ser Arg
1 5 10 15
<210> 28
<211> 12
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 28
Asp Glu Gly Asn Tyr Leu Asp Asp Ala Leu Val Arg
1 5 10
<210> 29
<211> 8
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 29
Glu Glu Gln Asn Ile Ile Phe Arg
1 5
<210> 30
<211> 9
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 30
Ala Gln Thr Glu Ile Asp Gly Pro Lys
1 5
<210> 31
<211> 10
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 31
Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg
1 5 10
<210> 32
<211> 13
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 32
Val Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys
1 5 10
<210> 33
<211> 8
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 33
Val Thr Ile Leu Glu Leu Phe Arg
1 5
<210> 34
<211> 10
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 34
Thr Phe Asp Glu Ile Ala Ser Gly Phe Arg
1 5 10
<210> 35
<211> 11
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 35
Leu Ser Pro Ile Tyr Asn Leu Val Pro Val Lys
1 5 10
<210> 36
<211> 12
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 36
Ala Ile Glu Asp Tyr Ile Asn Glu Phe Ser Val Arg
1 5 10
<210> 37
<211> 7
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 37
Ser Leu Pro Ser Leu Leu Arg
1 5
<210> 38
<211> 10
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 38
Asn Val Leu Thr Glu Glu Ser Val Val Arg
1 5 10
<210> 39
<211> 8
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 39
Val Leu Glu Asp Ser Asp Leu Lys
1 5
<210> 40
<211> 14
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 40
Ser Asp Ile Asp Glu Ile Val Leu Val Gly Gly Ser Thr Arg
1 5 10
<210> 41
<211> 15
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 41
Tyr Val Gly Gly Gln Glu His Phe Ala His Leu Leu Ile Leu Arg
1 5 10 15
<210> 42
<211> 14
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 42
Glu Gln Leu Gly Glu Phe Tyr Glu Ala Leu Asp Cys Leu Arg
1 5 10
<210> 43
<211> 12
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 43
Gln Ser Thr Leu Val Leu Phe Pro Gly Asp Leu Arg
1 5 10
<210> 44
<211> 9
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 44
Phe Gln Thr Phe Glu Gly Asp Leu Lys
1 5
<210> 45
<211> 14
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 45
Leu Glu Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys
1 5 10
<210> 46
<211> 9
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 46
Glu Asn Leu Gln Gly Leu Val Thr Arg
1 5
<210> 47
<211> 14
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 47
Glu Val Leu Leu Leu Val His Asn Leu Pro Gln Asp Pro Arg
1 5 10
<210> 48
<211> 9
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 48
Leu Phe Ile Pro Asn Ile Thr Thr Lys
1 5
<210> 49
<211> 16
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 49
Ala Ala Gly Gly Ala Val Cys Glu Gln Pro Leu Gly Leu Glu Cys Arg
1 5 10 15
<210> 50
<211> 19
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 50
Val Cys Gly Leu Cys Gly Asn Phe Asp Asp Asn Ala Ile Asn Asp Phe
1 5 10 15
Ala Thr Arg
<210> 51
<211> 7
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 51
Tyr Phe Leu Trp Val Val Lys
1 5
<210> 52
<211> 7
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 52
Asn Gln Gln Ile Phe Leu Arg
1 5
<210> 53
<211> 9
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 53
Ser Glu Ile Asp Leu Phe Asn Ile Arg
1 5
<210> 54
<211> 12
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 54
Val Gln Ser Ile Asn Tyr Asn Pro Phe Asp Gln Lys
1 5 10
<210> 55
<211> 12
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 55
Gly Pro Ala Gly Asp Ala Thr Val Ala Ser Glu Lys
1 5 10
<210> 56
<211> 14
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 56
Leu Phe Gly Gly Asn Phe Ala His Gln Ala Ser Val Ala Arg
1 5 10
<210> 57
<211> 11
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 57
Asn Ala Leu Glu Ser Tyr Thr Tyr Asn Ile Lys
1 5 10
<210> 58
<211> 14
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 58
Pro Phe Gly Val Ala Leu Leu Phe Gly Gly Val Asp Glu Lys
1 5 10
<210> 59
<211> 7
<212> PRT
<213> 人工的
<220>
<223> 合成的
<400> 59
Glu Leu Thr Ser Glu Leu Lys
1 5

Claims (4)

1.一种用于检测大肠癌的大肠癌生物标志物，其特征在于，是下面的编号4及编号12的蛋白质的组合或下面的编号4及编号12的蛋白质与下面的其余10个蛋白质中的至少一个蛋白质的组合、或者下面的编号18的部分肽与编号7或者编号8的部分肽的组合、或下面的编号18的部分肽及编号7或者编号8的部分肽与下面的其余15个部分肽中的至少一个部分肽的组合：
作为上述蛋白质，为：
1.通过序列号1表示的膜联蛋白A11(ANXA 11)；
2.通过序列号2表示的膜联蛋白A3(ANXA 3)；
3.通过序列号3表示的膜联蛋白A4(ANXA 4)；
4.通过序列号4表示的腱生蛋白N(TNN)；
5.通过序列号5表示的转铁蛋白受体蛋白1(TFRC)；
6.通过序列号6表示的葡萄糖转运蛋白-1即GLUT-1(SLC2A1)；
7.通过序列号8表示的CD88抗原(C5AR1)；
8.通过序列号11表示的基质金属蛋白酶9(MMP9)；
9.通过序列号16表示的膜联蛋白A5(ANXA 5)；
10.通过序列号17表示的嗅觉介导素-4(OLFM4)；
11.通过序列号19表示的三肽基肽酶1(TPP1)；或者
12.通过序列号22表示的中性粒细胞明胶酶相关脂质运载蛋白(LCN2)，
作为上述部分肽，为：
1～2.作为膜联蛋白A11的部分肽的、由通过序列号23或24表示的氨基酸序列构成的肽；
3～4.作为膜联蛋白A3的部分肽的、由通过序列号25或26表示的氨基酸序列构成的肽；
5～6.作为膜联蛋白A4的部分肽的、由通过序列号27或28表示的氨基酸序列构成的肽；
7～8.作为肌腱蛋白-N的部分肽的、由通过序列号29或30表示的氨基酸序列构成的肽；
9～10.作为转铁蛋白受体蛋白1的部分肽的、由通过序列号31或32表示的氨基酸序列构成的肽；
11.作为葡萄糖转运蛋白-1的部分肽的、由通过序列号34表示的氨基酸序列构成的肽；
12～13.作为CD88抗原的部分肽的、由通过序列号37或38表示的氨基酸序列构成的肽；
14.作为基质金属蛋白酶9的部分肽的、由通过序列号44表示的氨基酸序列构成的肽；
15.作为膜联蛋白A5的部分肽的、由通过序列号53表示的氨基酸序列构成的肽；
16.作为嗅觉介导素-4的部分肽的、由通过序列号54表示的氨基酸序列构成的肽；
17.作为三肽基肽酶1的部分肽的、由通过序列号56表示的氨基酸序列构成的肽；
18.作为中性粒细胞明胶酶相关脂质运载蛋白的部分肽的、由通过序列号59表示的氨基酸序列构成的肽。
2.一种用于检测大肠癌的大肠癌生物标志物，其特征在于，是将权利要求1所述的大肠癌生物标志物和CEA组合而成的。
3.一种针对生物体试样中的权利要求1或2所述的大肠癌生物标志物的抗体在制备用于检测大肠癌的试剂盒中的应用。
4.一种大肠癌的检测试剂盒，其特征在于，包含针对生物体试样中的权利要求1或2所述的大肠癌生物标志物的抗体。
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