CN1331699A - 鉴定抗原基因序列的方法 - Google Patents
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- CN1331699A CN1331699A CN99814733A CN99814733A CN1331699A CN 1331699 A CN1331699 A CN 1331699A CN 99814733 A CN99814733 A CN 99814733A CN 99814733 A CN99814733 A CN 99814733A CN 1331699 A CN1331699 A CN 1331699A
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Abstract
本发明涉及鉴定可能地疫苗抗原的基因序列的方法。还包括了基因序列和由基因序列编码的多肽,以及这种序列在动物中诱导保护性免疫应答的用途。特别地,本发明涉及鉴定支原体,优选猪肺炎支原体的可能地抗原基因序列。在本发明的一个方面,提供了一种鉴定由表达载体中的核苷酸序列翻译的表达蛋白质之方法,所述方法包括与由核苷酸序列翻译的蛋白质共表达的标记的用途。在本发明的又一方面,提供了一种从核苷酸序列样品中鉴定编码疾病治疗性抗原蛋白质的治疗性抗原基因序列的方法。优选地,标记为聚His标签。
Description
本发明涉及鉴定潜在疫苗抗原的基因序列的方法。本发明还包括基因序列和基因序列编码的多肽,以及这些序列在动物体内诱导保护性免疫应答的用途。具体而言,本发明涉及支原体,特别是猪肺炎支原体(Mycoplasma hyopneumoniae)的潜在抗原基因序列的鉴定。
序言
针对病原体的疫苗的成功之处在于鉴定宿主免疫系统易于接受的合适病原体抗原。一旦完成鉴定,就形成了针对病原体的防护的基础。
最近几年,大量的疫苗已经可以由Animal Health公司购买。许多疫苗以灭活的全细胞菌苗为基础。虽然这些疫苗提供了合理水平的功效,但是仍然非常需要改进。因此当前可获得的疫苗可以通过发展不以全细胞或全细胞级分为基础的疫苗得到改进。
产生针对病原体的疫苗的其他方法包括使用各种蛋白质的粗制灭活抗原混合物。然而这些方法的问题在于,虽然它们向宿主呈递多种抗原,但是没有哪一种可以提供合适的全面防护,原因在于宿主免疫系统最易于接受的蛋白质淹没于其它抗原中。此外,由于某些病原体对培养条件的要求苛刻,培养这些病原体是困难的且昂贵的。某些病原体还可能对操作人员有害。因此,不需要培养细胞或细菌生长和加工全细胞或全细胞级分的疫苗将提供更安全、更便宜、且可能更有效的疫苗。
实现更清洁且更特异的疫苗的一种方法是使用重组保护性抗原。通过提供特异抗原,只有那些宿主免疫系统易于接受的抗原可以使用。
这种方法存在鉴定潜在抗原的问题。一种方法是建立基因文库。然而,由文库确定那些可能包含潜在保护性抗原的序列很费时间。
为了对大量的潜在抗原基因高效筛选其提供一定水平疾病防护的功效,首先将它们克隆成表达克隆。在这种方法中,避免了对沉闷且费时的克隆分析和随后的表达亚克隆的需要。加快了发现抗原的整个过程。本申请人现在提供了确保最初调查的克隆表达重组蛋白质的新方法。
先前已经使用对感兴趣蛋白质特异或加富的抗体来对表达文库筛选表达完整或部分特异蛋白质的克隆。
编码蛋白质的DNA序列的鉴定,还使得使用适当表达载体形成重组DNA分子并用那些重组DNA分子转化适当宿主(如原核或真核宿主)成为可能。然后培养经转化宿主,使得宿主能够表达DNA序列并产生期望蛋白质。
以足以引发保护性免疫应答的量施用(如通过注射)产生且随后分离的蛋白质、活性成分或其组合,提供了针对感染的一种免疫方法。已经可以由Animal Health公司购买其疫苗的一种病原体是猪肺炎支原体。此病原体在猪中引起地方性动物肺炎。它很少引起死亡,但是常常导致严重的发病率和降低的生长特性,表现为饲料转变效率显著降低,导致猪体重增加下降。动物出现咳嗽和发烧症状,而且常常有通过机会性微生物而继发感染的倾向。
以提供针对猪肺炎支原体的疫苗为目的的大量尝试还没有取得太大的成功。具体而言,已经证明使用热灭活的或存活的支原体或其提取物在提供防护方面是无效的。以灭活的全细胞菌苗为基础的某些疫苗展示一定水平的功效,但是仍然需要改进。
另外,由于猪肺炎支原体对培养条件的要求苛刻,培养猪肺炎支原体是困难的且昂贵的。因此当前可获得的疫苗可以通过发展不以全细胞或全细胞级分为基础的疫苗得到改进。
相应的,本发明的目的之一是克服或至少减轻先前技术的某些问题。
发明概述
在本发明的一个方面,提供了鉴定由表达载体中核苷酸序列翻译得到的表达蛋白质的方法,该方法包括与由核苷酸序列翻译得到的蛋白质共表达的标记物的使用。
在本发明的优选实施方案中,提供了将聚His标签用于鉴定由表达载体中核苷酸序列编码的表达蛋白质的用途,其中所述聚His标签与所述蛋白质共表达。
在本发明的另一个方法,提供了由核苷酸序列混合物鉴定由核苷酸序列编码的表达蛋白质的方法,该方法包括下列步骤:
提供包含标记物的表达载体;
将来自混合物的核苷酸序列导入表达载体;并
通过测定包含与表达蛋白质共表达的标记物之融合蛋白质的存在,来鉴定由表达载体表达的表达蛋白质。
在一个优选的实施方案中,标记物是聚His标签。
在另一个优选的实施方案中,用表达载体转染用于表达包含表达蛋白质的标记物-融合蛋白质之宿主细胞。优选的标记物融合蛋白质是包含聚His标签和表达蛋白质的聚His标签-融合蛋白质。将表达载体集合导入宿主细胞可以构建基因组或cDNA文库(可以对其进行筛选)。
在另一个优选的实施方案中,所述方法包括使用在融合蛋白质中表达的标记物来纯化表达蛋白质的步骤。优选的标记物是聚His标签。
在另一个方面,提供了由上述方法鉴定的纯化重组蛋白质。优选的重组蛋白质是表达蛋白质。
在另一个方面,提供了编码由上述方法鉴定的重组蛋白质的纯化核苷酸序列。
在本发明的另一个方面,提供了包含标记物,优选用于鉴定由核苷酸序列编码的表达蛋白质的聚His标签的表达载体。
在本发明的还有一个方面,提供了包含表达载体的宿主细胞,该载体包含标记物,优选用于鉴定表达蛋白质的聚His标签。
在本发明的另一个方面,提供了由核苷酸序列样品鉴定编码抗原蛋白质的基因序列的方法,该方法包括下列步骤:
提供包含标记物的表达载体;
将来自样品的核苷酸序列导入表达载体;
通过测定融合蛋白质的存在,来鉴定由表达载体表达的蛋白质的表达,所述融合蛋白包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物;并
鉴定抗原性表达蛋白质。
优选的标记物是聚His标签。
在优选的实施方案中,所述方法进一步包括测定由表达载体编码的抗原蛋白质的基因序列。
在本发明的另一个方面,提供了一种筛选核苷酸序列文库的方法,该方法包括下列步骤:
提供核苷酸序列来源;
提供包含标记物的表达载体;
通过将核苷酸序列导入表达载体来构建文库;
通过测定融合蛋白质的存在,来鉴定由表达载体表达的表达蛋白质,所述融合蛋白质包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物;并
测定表达蛋白质的核苷酸序列。
优选的标记物是聚His标签。
在另一个方面,提供了一种筛选以鉴定表达文库中表达克隆的方法,该方法包括下列步骤:
提供核苷酸来源;
提供包含标记物的表达载体;
将核苷酸序列导入表达载体;
用表达载体转化宿主以构建表达文库;并
通过检测融合蛋白质的表达来找出表达克隆,所述融合蛋白质包含与表达蛋白质共表达的标记物(优选聚His标签)。
在本发明的另一个方面,提供了从核苷酸样品中鉴定编码疾病治疗性抗原蛋白质的治疗性抗原基因序列的方法,该方法包括下列步骤:
提供包含标记物的表达载体;
将来自样品的核苷酸序列导入表达载体;
通过测定融合蛋白质的存在,来鉴定由表达载体表达的抗原表达蛋白质,所述融合蛋白质包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物;
用表达抗原性表达蛋白质的表达载体接种动物;
用疾病攻击动物;
鉴定可提供由接种疫苗诱导的治疗性效果的表达载体和表达蛋白质;并
测定由表达载体编码的抗原的表达蛋白质的基因序列。
优选的标记物是聚His标签。
在本发明的另一个优选实施方案中,提供了对核苷酸序列文库筛选编码支原体(优选猪肺炎支原体)多肽的核苷酸序列的方法,该方法包括下列步骤:
由支原体样品提供核苷酸序列来源;
提供包含标记物的表达载体;
将核苷酸序列导入表达载体;
通过测定融合蛋白质的存在,来鉴定由表达载体表达的支原体表达蛋白质,其中所述融合蛋白质包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物(优选聚His标签);并
测定支原体(优选猪肺炎支原体)表达蛋白质的核苷酸序列。
优选的标记物是聚His标签。
在优选的实施方案中,基因序列编码支原体,优选猪肺炎支原体的抗原多肽。
在本发明的还有一个方面,提供了编码支原体(优选猪肺炎支原体)多肽的核苷酸序列,或其突变体、类似物、衍生物、或其功能活性片段,该核苷酸序列由上述方法鉴定。优选的核苷酸序列包含依照图1-42任一项的序列,或其突变体、类似物、衍生物、或其功能活性片段。更具体的说,核苷酸序列依照SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83,或其突变体、类似物、衍生物、或功能活性片段的序列的序列。
在本发明的还有一个方面,提供了由编码支原体(优选猪肺炎支原体)多肽的核苷酸序列,或其突变体、类似物、衍生物、或功能活性片段编码的多肽,该核苷酸序列由上述方法鉴定。优选的多肽由依照图1-42任一项的序列,或其突变体、类似物、衍生物、或功能活性片段编码的核苷酸序列编码。更具体的说,多肽具有依照SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84的氨基酸序列,或其突变体、类似物、衍生物、或功能活性片段。
在本发明的另一个方面,包括了包含上述多肽或核苷酸序列的组合物。此外,在另一个方面,包括了上述多肽或核苷酸序列的药用组合物和疫苗组合物。
本发明的其它方面包括针对多肽产生的单克隆抗体。
本发明的另一个方面还包括治疗支原体感染的方法,该方法包括施用有效量的由核苷酸序列编码的支原体抗原多肽或编码由上述方法鉴定的多肽的核苷酸序列。优选的抗原多肽由依照图1-42任一项的核苷酸序列,或其突变体、类似物、衍生物、或功能活性片段编码。更具体的说,核苷酸序列依照SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ IDNO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ IDNO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ IDNO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ IDNO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ IDNO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ IDNO:79、SEQ ID NO:81、SEQ ID NO:83的序列,或其突变体、类似物、衍生物、或功能活性片段。
贯穿此说明书的描述和权利要求,术语“包含”及其变化,诸如“包括”,并非意欲排除其它添加剂、成分、组分、或步骤。
附图简述
图1显示克隆pAD612所述的支原体的核苷酸(SEQ ID NO:1)和氨基酸(SEQ ID NO:2)序列。
图2显示克隆pAD633所述的支原体的核苷酸(SEQ ID NO:3)和氨基酸(SEQ ID NO:4)序列。
图3显示克隆pAD639所述的支原体的核苷酸(SEQ ID NO:5)和氨基酸(SEQ ID NO:6)序列。
图4显示克隆pAD640所述的支原体的核苷酸(SEQ ID NO:7)和氨基酸(SEQ ID NO:8)序列。
图5显示克隆pAD641和pAD1033所述的支原体的核苷酸(SEQ IDNO:9)和氨基酸(SEQ ID NO:10)序列。
图6显示克隆pAD653所述的支原体的核苷酸(SEQ ID NO:11)和氨基酸(SEQ ID NO:12)序列。
图7显示克隆pAD657和pAD964所述的支原体的核苷酸(SEQ IDNO:13)和氨基酸(SEQ ID NO:14)序列。
图8显示克隆pAD659和pAD910所述的支原体的核苷酸(SEQ IDNO:15)和氨基酸(SEQ ID NO:16)序列。
图9显示克隆pAD662所述的支原体的核苷酸(SEQ ID NO:17)和氨基酸(SEQ ID NO:18)序列。
图10显示克隆pAD681所述的支原体的核苷酸(SEQ ID NO:19)和氨基酸(SEQ ID NO:20)序列。
图11显示克隆pAD700所述的支原体的核苷酸(SEQ ID NO:21)和氨基酸(SEQ ID NO:22)序列。
图12显示克隆pAD711所述的支原体的核苷酸(SEQ ID NO:23)和氨基酸(SEQ ID NO:24)序列。
图13显示克隆pAD721所述的支原体的核苷酸(SEQ ID NO:25)和氨基酸(SEQ ID NO:26)序列。
图14显示克隆pAD727所述的支原体的核苷酸(SEQ ID NO:27)和氨基酸(SEQ ID NO:28)序列。
图15显示克隆pAD742所述的支原体的核苷酸(SEQ ID NO:29)和氨基酸(SEQ ID NO:30)序列。
图16显示克隆pAD760所述的支原体的核苷酸(SEQ ID NO:31)和氨基酸(SEQ ID NO:32)序列。
图17显示克隆pAD774所述的支原体的核苷酸(SEQ ID NO:33)和氨基酸(SEQ ID NO:34)序列。
图18显示克隆pAD784所述的支原体的核苷酸(SEQ ID NO:35)和氨基酸(SEQ ID NO:36)序列。
图19显示克隆pAD789所述的支原体的核苷酸(SEQ ID NO:37)和氨基酸(SEQ ID NO:38)序列。
图20显示克隆pAD908、pAD981、pAD1013、和pAD1049所述的支原体的核苷酸(SEQ ID NO:39)和氨基酸(SEQ ID NO:40)序列。
图21显示克隆pAD913所述的支原体的核苷酸(SEQ ID NO:41)和氨基酸(SEQ ID NO:42)序列。
图22显示克隆pAD920所述的支原体的核苷酸(SEQ ID NO:43)和氨基酸(SEQ ID NO:44)序列。
图23显示克隆pAD922所述的支原体的核苷酸(SEQ ID NO:45)和氨基酸(SEQ ID NO:46)序列。
图24显示克隆pAD923和pAD925所述的支原体的核苷酸(SEQ IDNO:47)和氨基酸(SEQ ID NO:48)序列。
图25显示克隆pAD950所述的支原体的核苷酸(SEQ ID NO:49)和氨基酸(SEQ ID NO:50)序列。
图26显示克隆pAD951所述的支原体的核苷酸(SEQ ID NO:51)和氨基酸(SEQ ID NO:52)序列。
图27显示克隆pAD977所述的支原体的核苷酸(SEQ ID NO:53)和氨基酸(SEQ ID NO:54)序列。
图28显示克隆pAD983所述的支原体的核苷酸(SEQ ID NO:55)和氨基酸(SEQ ID NO:56)序列。
图29显示克隆pAD984所述的支原体的核苷酸(SEQ ID NO:57)和氨基酸(SEQ ID NO:58)序列。
图30显示克隆pAD994所述的支原体序列30的核苷酸(SEQ ID NO:59)和氨基酸(SEQ ID NO:60)序列。
图31显示克隆pAD1005所述的支原体的核苷酸(SEQ ID NO:61)和氨基酸(SEQ ID NO:62)序列。
图32显示克隆pAD1016所述的支原体的核苷酸(SEQ ID NO:63)和氨基酸(SEQ ID NO:64)序列。
图33显示克隆pAD1020所述的支原体的核苷酸(SEQ ID NO:65)和氨基酸(SEQ ID NO:66)序列。
图34显示克隆pAD1027所述的支原体的核苷酸(SEQ ID NO:67)和氨基酸(SEQ ID NO:68)序列。
图35显示克隆pAD1037所述的支原体的核苷酸(SEQ ID NO:69)和氨基酸(SEQ ID NO:70)序列。
图36显示克隆pAD1038所述的支原体的核苷酸(SEQ ID NO:71)和氨基酸(SEQ ID NO:72)序列。
图37显示克隆pAD1040所述的支原体的核苷酸(SEQ ID NO:73)和氨基酸(SEQ ID NO:74)序列。
图38显示克隆pAD702所述的支原体的核苷酸(SEQ ID NO:75)和氨基酸(SEQ ID NO:76)序列。
图39显示克隆pAD763所述的支原体的核苷酸(SEQ ID NO:77)和氨基酸(SEQ ID NO:78)序列。
图40显示克隆pAD766所述的支原体的核苷酸(SEQ ID NO:79)和氨基酸(SEQ ID NO:80)序列。
图41显示克隆pAD957所述的支原体的核苷酸(SEQ ID NO:81)和氨基酸(SEQ ID NO:82)序列。
图42显示克隆pAD996所述的支原体的核苷酸(SEQ ID NO:83)和氨基酸(SEQ ID NO:84)序列。
发明详述
在本发明的一个方面,提供了一种鉴定由表达载体中核苷酸序列翻译得到的表达蛋白质的方法,该方法包括与由核苷酸序列翻译得到的蛋白质共表达的标记物的使用。
在优选的实施方案中,标记物是可以检测的任何标记物。检测可以通过单克隆抗体或特异性多克隆抗血清进行。标记物优选小的,优选少于30个氨基酸。最优选的标记物是聚His标签。但是标签还可以是下列任一项,诸如:(a)可以由Sigma-Aldrich公司(美国)的质粒载体获得的8个氨基酸的FLAG标签,(b)可以由Amrad有限公司(澳大利亚)的载体获得的6个氨基酸的I-SPY表位标签,(c)9个氨基酸的血凝素表位标签(Clontech Laboratories公司,美国),和(d)12个氨基酸的c-Myc表位标签(Clontech Laboratories公司,美国)。
当标记物是聚His标签时,组氨酸残基的数目优选4-8个组氨酸残基,最优选6个组氨酸残基。假设总共有至少10个氨基酸,且有至少4-8个连续组氨酸残基,则至少4个氨基酸位于聚His标签之前。
为了对大量的潜在抗原基因高效筛选其提供一定水平疾病防护的功效,首先将它们克隆成表达克隆。在这种方法中,避免了对费力且费时的克隆分析和随后的表达亚克隆的需要。加快了发现抗原的整个过程。本申请人现在提供了确保最初调查的克隆表达重组蛋白质的新方法。
当处理基因文库时,情况尤其如此。在任何随机文库中,多数克隆的取向对于表达是不正确的,而且处于表达蛋白质的错误读码框中。在表达载体中使用标记物(优选聚His标签)使得能够有效鉴定表达标记物(优选聚His标签融合蛋白质)的表达克隆。
在不产生重组标记物-融合蛋白质或优选聚His融合蛋白质或仅仅一小段重组蛋白质的克隆中,编码的小标记物或聚His蛋白质在宿主细胞中迅速降解,残余的任何标记物或聚His蛋白质非常微弱的结合在用于菌落筛选的膜上。非生产性克隆由此在筛选中给出阴性结果,而产生含较大标签的重组蛋白质的克隆能够给出阳性信号。由此可以显著降低一般性基因组表达文库的复杂性,并因此能够更快且更有效的筛选并随后利用期望的克隆。
这些表达蛋白质的克隆被认为是潜在的保护性抗原,而且由于用于构建和筛选克隆的策略,最初分离的克隆可以直接用于疫苗,形式可以是用于遗传免疫接种的纯化质粒DNA或纯化标记物-融合重组蛋白质,优选聚His纯化重组融合蛋白质。
当标记物是聚His标签区时,它可以由包含该区的任一来源衍生得到。例如,可以使用编码聚His标签的载体pQE30、pQE31、和pQE32(QiagenPty.公司,澳大利亚)区段。聚His标签的其它来源可以由Novagen公司(美国,pET-1.9-22)获得,或者人工合成聚His标签。
在本发明的另一个方面,提供了由核苷酸序列混合物鉴定由核苷酸序列翻译得到的表达蛋白质的方法,该方法包括下列步骤:
提供包含标记物的表达载体;
将来自混合物的核苷酸序列导入表达载体;并
通过测定包含与表达蛋白质共表达的标记物的融合蛋白质的表达,来鉴定由表达载体表达的表达蛋白质。
优选的标签是聚His标签。下列特定实施例涉及聚His标签标记物和聚His标签融合蛋白质。然而,可以使用可被检测并在表达克隆中与蛋白质共表达的任何标记物。相应的,本发明并不限于聚His标签。该标记物仅仅是例示性的,而不应当以任何方式限制所述发明的通用性。
如上所述,表达蛋白质的鉴定,特别是来自由基因文库衍生的克隆,可以加快编码病原体潜在抗原的核苷酸序列的鉴定。
核苷酸序列混合物可以衍生自任何来源,诸如组织样品、微生物、细胞、或含核酸的任何成分。核酸优选基因组的、线粒体的、重组的、或mRNA。首先可以对DNA样品进行任何方法的片段化处理,诸如超声处理或酶消化(诸如限制酶消化)。或者,核苷酸序列可以衍生自上述DNA序列的片段的PCR。这些序列可以是开放读码框(ORF)。表达载体优选包含聚His标签。如上所述,聚His标签可以衍生自包含聚His标签的任何来源。
聚His标签优选由标准载体(诸如pQE30、pQE31、和pQE32,QiagenPty.有限公司,澳大利亚)衍生得到。这些载体可以编码T5启动子、Lac操纵子、和聚His标签区。可以将该区域克隆到能够表达由核苷酸序列编码的蛋白质的表达载体中。或者,可以人工合成聚His标签。
优选将标记物(优选聚His标签区)克隆到真核表达载体中。最优选的载体是pCI(Promega公司,澳大利亚),且优选产生克隆pCI30、pCI31、和pCI32。
该系列的载体能够克隆在所有3种读码框中位于聚His标签区下游的片段。这些例示性克隆中的巨细胞病毒(CMV)立即早期增强子/启动子、嵌合内含子、和SV40晚期多腺苷酸化信号使得载体能够在细胞培养系统和使用纯化质粒DNA的遗传免疫接种中作为真核表达载体使用。T5启动子能够在原核生物中表达克隆的插入片段。由于这些载体的构建物使得我们能够筛选文库以鉴定表达重组蛋白质的大量克隆,所以它们是重要的。
可以通过技术人员知道的任何方法将来自混合物(诸如基因组DNA的消化物)的核苷酸序列导入或克隆到表达载体中。表达载体可以用于在宿主细胞中表达蛋白质。
在优选的实施方案中,用表达载体转染宿主细胞以表达包含聚His标签和表达蛋白质的聚His融合蛋白质的表达蛋白质。将表达载体导入宿主细胞可以构建基因组或cDNA文库。
宿主细胞可以是有能力接受表达载体的任何细胞。细胞优选是原核的。最优选是大肠杆菌。可以通过测定聚His标签融合蛋白质上聚His标签的存在,来鉴定表达该蛋白质的表达蛋白质(和随后任何克隆或宿主细胞)。
本发明的载体使得能够根据聚His标签一般性筛选所有表达克隆。
聚His标签可以用抗聚His抗体或金属螯合物缀合探针(诸如镍树脂)来鉴定,其中聚His信号通过加入发色底物而显色。
在不产生重组聚His融合蛋白质或仅有短的重组蛋白质的克隆中,编码的小的聚His蛋白质在宿主细胞内快速降解,残余的任何聚His蛋白质非常微弱的结合在用于菌落筛选的膜上。非生产性克隆由此在聚His菌落筛选中给出阴性结果,而产生含较长聚His标签的重组蛋白质的克隆能够给出阳性信号。由此可以显著降低一般性基因组表达文库的复杂性,并因此能够更快且更有效的筛选并随后利用期望的克隆。
在另一个优选实施方案中,本方法包括使用在融合蛋白质中表达的聚His标签纯化表达蛋白质的步骤。
一旦使用聚His标签鉴定了由表达克隆表达的表达蛋白质,则该标签还可以用于纯化任何表达蛋白质。可以使用技术人员知道的任何纯化方法。可以使用亲和层析中含抗聚His抗体的亲和柱。一般而言,借助聚His标签鉴定的表达蛋白质可以通过利用聚His标签的任何方法进行分离。
在另一个方面提供了由上述方法鉴定的纯化重组蛋白质。
一旦分离得到蛋白质,它可以以任意多种方式(包括疫苗试验)以纯化蛋白质的形式使用。蛋白质可以立即鉴定作为潜在抗原。优选地它们是保护性抗原。
在另一个方面,提供了编码由上述方法鉴定的重组蛋白质的纯化核苷酸序列。
由不连续的核苷酸序列(优选重组序列)表达由上述聚His方法鉴定的蛋白质。通过鉴定蛋白质,可以鉴定包含核苷酸序列、表达蛋白质的克隆。
这些克隆可以提供纯化质粒DNA,特别是质粒DNA可能代表潜在的保护性抗原。
在本发明的另一个方面,提供了包含标记物(优选用于鉴定由核苷酸序列编码的表达蛋白质的聚His标签)的表达载体或重组载体。
表达载体可以是产生表达蛋白质的表达的任何载体。它可以是质粒或粘粒。可以将它导入克隆以进一步扩增表达蛋白质,以获得蛋白质用于疫苗接种或鉴定目的。
在本发明的还有一个方面,提供了包含表达载体的宿主,该载体包含标记物,优选用于鉴定由核苷酸序列编码的表达蛋白质的聚His标签。
宿主细胞可以是能够接受表达载体的任何感受态细胞。宿主细胞优选原核的或真核的。
在本发明的另一个方面,提供了由核苷酸序列样品鉴定编码抗原蛋白质的基因序列的方法,该方法包括下列步骤:
提供包含标记物(优选聚His标签)的表达载体;
将来自样品的核苷酸序列导入表达载体;
通过测定融合蛋白质的存在,来鉴定由表达载体表达的蛋白质的表达,所述融合蛋白质包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物;
鉴定抗原蛋白质;并
优选测定由表达载体编码的抗原蛋白质基因的核苷酸序列。
抗原蛋白质的鉴定可以包括鉴定并优选地分离具有聚His标签的融合蛋白质,测定融合蛋白质是否能够引发并非由聚His标签产生的免疫应答。免疫应答的测定可以通过标准体外免疫学测试,使用来自免疫动物的抗血清,进行ELISA或抗体/抗原反应来进行。它们也可以在针对进一步攻击的动物中体内防护试验中进行测试。
在本发明的另一个方面,提供了一种筛选核苷酸序列文库的方法,该方法包括下列步骤:
提供核苷酸序列来源;
提供包含标记物(优选聚His标签)的表达载体;
通过将核苷酸序列导入表达载体来构建文库;
通过测定融合蛋白质的存在,来鉴定由表达载体表达的表达蛋白质,所述融合蛋白质包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物;并
测定表达蛋白质基因的核苷酸序列。
在另一个方面,提供了筛选以鉴定文库中表达克隆的方法,该方法包括下列步骤:
提供核苷酸来源;
提供包含标记物(优选聚His标签)的表达载体;
将核苷酸序列导入表达载体;
用表达载体转化宿主以构建表达文库;并
通过检测包含与表达蛋白质共表达的标记物(优选聚His标签)的融合蛋白质的表达来找出表达克隆。
由表达克隆转录得到的具有聚His标签的表达蛋白质的鉴定进一步鉴定了那些表达开放读码框(ORF)的克隆(宿主细胞)。
相应的,该方法能够鉴定核苷酸序列混合物中的ORF。这种方法有效加速了鉴定编码作为抗原潜在有用的蛋白质的有用核苷酸序列的过程。它快速鉴定可以立即扩增以提供足够核苷酸序列(或质粒DNA或重组蛋白质)样品的克隆,用于诸如遗传疫苗接种或产生针对重组蛋白质的抗体的任一用途。
在本发明的另一个方面,提供了由核苷酸序列样品鉴定编码疾病治疗性抗原蛋白质的治疗性抗原基因序列的方法,该方法包括下列步骤:
提供包含标记物的表达载体;
将来自样品的核苷酸序列导入表达载体;
通过测定融合蛋白质的存在,来鉴定由表达载体表达的抗原表达蛋白质,所述融合蛋白质包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物;
用表达抗原表达蛋白质的表达载体接种动物;
用疾病攻击动物;
鉴定提供由接种疫苗诱导的治疗性效果的表达载体和表达蛋白质;并
测定由表达载体编码的抗原表达蛋白质的基因序列。
本发明的一个优点是提供筛选核苷酸序列文库并将经鉴定的克隆用于DNA疫苗试验以立即评价克隆的疫苗潜力的有效方法。方便的使用经鉴定克隆进行接种疫苗试验的这个方面大大改进了目前可获得的方法。
在本发明的另一个优选实施方案中,提供了对核苷酸序列文库筛选编码支原体(优选猪肺炎支原体)多肽的核苷酸序列的方法,该方法包括下列步骤:
由支原体样品提供核苷酸序列来源;
提供包含标记物(优选聚His标签)的表达载体;
将核苷酸序列导入表达载体;
通过测定包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物(优选聚His标签)的融合蛋白质的存在,来鉴定由表达载体表达的支原体表达蛋白质;并
测定支原体(优选猪肺炎支原体)表达蛋白质基因的核苷酸序列。
来自支原体样品的核苷酸序列来源可以是基因组、线粒体、重组体、或mRNA来源。当来源是基因组时,基因组DNA可以通过技术人员知道的任何方法由支原体分离得到。
支原体优选猪肺炎支原体。鉴定聚His标签-支原体融合蛋白质的方法如上所述。
核苷酸序列优选编码抗原蛋白质。相应的,有进一步的步骤以确定蛋白质是否引发免疫应答。抗原蛋白质优选支原体抗原蛋白质,更优选猪肺炎支原体抗原蛋白质。
术语“免疫应答”指由免疫系统进行的选择性应答,其中特异性抗体和/或细胞毒性细胞是针对外来成分的侵入产生的,一般而言诸如微生物、寄生虫、移植组织、蛋白质、或抗原。
术语“免疫系统”包括能够进行针对外来成分(一般而言诸如微生物、寄生虫、移植组织、蛋白质、或抗原)侵入的特异性防护应答、保护身体免于感染、并在某些情况中引起针对再感染的长期特异性免疫的任何细胞和组织。
在本发明的还有一个方面,提供了编码支原体(优选猪肺炎支原体)多肽的核苷酸序列,及其突变体、类似物、衍生物、或功能活性片段,该核苷酸序列由上述方法鉴定。优选的核苷酸序列包含依照图1-42任一项的序列,或其突变体、类似物、衍生物、或功能活性片段。更具体的说,它包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83,或其突变体、类似物、衍生物、或功能活性片段。
在另一个实施方案中,提供了包含上述核苷酸序列或其突变体、类似物、衍生物、或功能活性片段的重组载体。核苷酸序列优选依照图1-42任一项,或其突变体、类似物、衍生物、或功能活性片段。更具体的说,它包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83,或其突变体、类似物、衍生物、或功能活性片段。
术语“突变体、类似物、或衍生物”指那些可能包含删除、添加、或替换的序列。当这些术语用于蛋白质时,变化基本上不改变蛋白质的活性。当它用于改变的核酸分子时,变化不导致蛋白质编码区读码框的变化,并优选编码无变化、或者生物学功能仅略微降低或升高的蛋白质。
术语“片段”指氨基酸或核酸序列的一部分,比全长少,但是能够与全长序列杂交,并且能够被识别为能够编码氨基酸全长序列的一部分的部分核酸序列。
由于遗传密码的简并性,编码相同或功能等同的氨基酸序列的其它核酸序列包括于本发明的范围的之内。核苷酸序列的这些变化可能包含产生相同或功能上等同的基因产物的不同核苷酸的替换。本发明的范围还包括具有产生功能上等同的基因产物的删除和/或添加的核酸序列。另外,基因产物可能在序列中包含导致变化但仍产生功能活性产物的氨基酸残基的删除、添加、或替换。
在另一个实施方案中,提供了包含上述重组载体的宿主细胞。宿主细胞可以是有能力接受重组载体的任何细胞。优选的宿主细胞是原核生物,更优选大肠杆菌。
在本发明的另一个方面,提供了编码由上述方法鉴定的多肽的氨基酸序列。多肽可以是表达载体的产物,或者是鉴定为与标记物(优选聚His标记物)共表达的表达蛋白质。优选的氨基酸序列由用上述方法鉴定的核苷酸序列翻译得到。
在优选的方面,氨基酸序列编码支原体(优选猪肺炎支原体)多肽。氨基酸序列更优选地包含依照图1-42任一项的序列,或其突变体、类似物、衍生物、或功能片段。更具体的说,它包含SEQ ID NO:2、SEQ IDNO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84,或其突变体、类似物、衍生物、或功能活性片段。
在本发明的还有一个方面,提供了由上述氨基酸序列突变体、类似物、衍生物、或功能活性片段编码的多肽。序列优选编码支原体(优选猪肺炎支原体)多肽,该氨基酸序列由上述方法鉴定。多肽优选由依照图1-42任一项的氨基酸序列,或其突变体、类似物、衍生物、或功能活性片段编码。更具体的说,它包含SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84,或其突变体、类似物、衍生物、或功能活性片段。
可以通过技术人员知道的任何方法分离并纯化多肽。分离方法优选利用用于由核苷酸序列文库鉴定表达蛋白质或表达克隆的聚His标签。
在另一个实施方案中,提供了一种包含上述多肽或核苷酸序列的组合物。组合物可以是包含至少一种上述多肽或核苷酸序列和制药学可接受载体的药用组合物。组合物还可以是包含至少一种上述多肽或核苷酸序列的疫苗组合物。疫苗组合物可能能够引发免疫应答,其中多肽是抗原蛋白质或核苷酸序列,优选支原体的,更优选猪肺炎支原体的。
在本发明的另一个方面,提供了针对上述多肽或由上述方法鉴定的多肽的抗体。抗体优选单克隆抗体。
本领域知道多种方法可以用于产生针对上述多肽表位的抗体。多种宿主动物可以用于在用多肽免疫接种之后产生这些抗体,包括(但不限于)兔、小鼠、山羊、等等。根据宿主物种,可以使用佐剂以提高免疫学应答,包括(但不限于)弗氏(完全和不完全)佐剂。可以使用能够在体外通过细胞系连续产生抗体分子的技术制备单克隆抗体。这些技术包括(但不限于)杂交瘤技术(Kohler和Milstein,自然(Nature)256:495,1975)。针对多肽的抗体可以用于在多种组织、体液、和细胞系中检测抗原性多肽,例如抗原的筛选实验和多肽(优选抗原多肽)的亲和纯化。
疫苗组合物包含纯化的质粒DNA,该DNA分子包含上述核苷酸序列。
本发明的还有一个方面,提供了一种治疗感染的方法,该方法包括施用有效量的由氨基酸序列编码的抗原多肽,该多肽由上述方法鉴定。感染优选支原体感染,更优选猪肺炎支原体感染。
同样优选的是,编码多肽的氨基酸序列由依照图1-42任一项的核苷酸序列,或其突变体、类似物、衍生物、或功能活性片段编码。更具体的说,序列包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ IDNO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ IDNO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ IDNO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ IDNO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ IDNO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ IDNO:79、SEQ ID NO:81、SEQ ID NO:83,或其突变体、类似物、衍生物、或功能活性片段。
同样优选的是,多肽具有依照图1-42任一项的氨基酸序列,或其突变体、类似物、衍生物、或功能活性片段。更具体的说,它包含SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84,或其突变体、类似物、衍生物、或功能活性片段。
本发明还有一个方面,提供了一种治疗感染的方法,该方法包括给需要的动物施用有效量的包含由上述方法鉴定的核苷酸序列的DNA分子。感染优选支原体感染,更优选猪肺炎支原体感染。
核苷酸序列优选依照图1-42任一项,或其突变体、类似物、衍生物、或功能活性片段,更具体的说,SEQ ID NO:1、SEQ ID NO:3、SEQ IDNO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83,或其突变体、类似物、衍生物、或功能活性片段。
可以通过使得细胞能够吸收DNA的注射或局部应用来施用DNA。DNA优选皮下或肌肉内注射到皮肤层中。
在本发明的另一个方面,提供了治疗感染的方法,该方法包括施用有效量的对由上述方法鉴定的多肽特异的抗体。
感染优选支原体感染,更优选猪肺炎支原体感染。
多肽优选由依照图1-42任一项的氨基酸序列,或其突变体、类似物、衍生物、或功能活性片段编码,更具体的说,SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84,或其突变体、类似物、衍生物、或功能活性片段。
现在本发明将由所附实施例和图更全面的描述。应当这样理解,下面的描述仅仅是例示性的,而不应当以任何方式限制所述发明的通用性。
实施例实施例1:在pCI30、pCI31和pCI32中构建基因组文库
通过超声处理或部分限制酶消化,将由猪肺炎支原体分离得到的基因组DNA断裂成合适大小的片段。然后将DNA在琼脂糖凝胶上进行电泳,并由凝胶切下选定大小的部分回收。将载体DNA(pCI30、pCI31和pCI32)用适当的限制酶(SmaI或BamHI)消化,用虾碱性磷酸酶处理除去末端磷酸,然后在琼脂糖凝胶上电泳。切下线性条带并回收DNA。以1∶1和5∶1之间的摩尔比将基因组DNA片段与经切割载体DNA混和,并使用标准连接条件进行连接。将连接混合物用于转化大肠杆菌感受态细胞,并由此产生许多不同的基因组文库实施例2:基因组文库的筛选
将基因组文库铺在含氨苄青霉素(100ug/ml)的Luria-Bertoni(LB)肉汤琼脂上,并于37℃培养过夜。通过将干燥的、无菌硝酸纤维素膜置于菌落上面,然后立即揭下,以菌落面朝上置于含氨苄青霉素和IPTG(1mM)的LB平板上,并于37℃温育4小时,由此完成平板的滤膜复制。然后通过常规方法处理滤膜,以裂解细菌、中和膜、并使蛋白质结合在膜上。然后用抗聚His抗体或用缀合的镍树脂探查干膜,加入发色底物后产生聚His信号。将显色的膜映射回最初的平板,并将阳性菌落转接装有加富生长培养基(添加氨苄青霉素)的96孔深孔板。然后于37℃培养克隆。取出这些板的复制品,对于长期保存置于-70℃。实施例3:选定克隆的序列分析
由每个文库选择一些聚His阳性克隆。制备质粒DNA,并通过自动化双脱氧测序使用与恰好位于载体中插入位点上游的区域同源的引物由插入片段产生DNA序列信息。将产生的序列数据与公开的GenBank数据库进行比较。注意到我们的阳性序列和数据库条目之间的同源性,而且在许多情况中这种信息指示克隆的基因片段可能的功能。根据这种同源性信息,选择有可能代表编码在猪肺炎支原体感染过程中宿主免疫系统易于接受的蛋白质的基因,或由于其它原因被认为有可能提供一定水平的防护作用的一组阳性克隆。这些选定的阳性克隆编码候选保护性抗原。
选择用于进一步研究的某些克隆代表相同基因的不同区段。实施例4:候选保护性抗原的筛选
进行了一系列动物试验以调查所鉴定的基因的治疗性应用。所有试验遵循相似方案。对来自无猪肺炎支原体群的猪进行3次接种,间隔2周,由6周龄开始。每次接种使用500μg质粒DNA,平均的分成2次注射,每只耳背基部的组织注射1次,使用Biojector 2000无针注射装置和2号注射器。第3次接种后1-2周,通过气管内滴注5ml 0.45μM经过滤的来自患有活性猪肺炎支原体感染的猪的肺匀浆来攻击猪。攻击后3-4周,对猪进行安乐死,切除肺用于肺炎损害评分。通过对受影响肺叶数目的计数和通过估计每个肺叶受疾病影响的百分比来对肺炎损害进行评分。这种评分系统给出疾病状态的两种测量值,叶得分和损害得分。对于每组试验,展示每组的平均结果。展示了具有阳性治疗性效果的那些克隆组的试验结果。没有展示未得出阳性治疗性效果的其它克隆组及其结果。试验1
用包含克隆pAD612-789的一组克隆免疫接种。处理 叶得分 损害得分未接种,未攻击 0 0未接种,攻击 4.4 120接种,攻击 3.25 72.5
含克隆pAD612-789的疫苗具有治疗性效果。经接种的猪中的叶得分比对照低26%,而损害得分低40%。试验2
测试作为疫苗的两个不同组。
第1组:pAD662、774、784、922、和964
第2组:pAD612、633、639、640、641、653、657、659、681、700、711、721、727、742、760、789、908、910、911、913、920、923、925、950、951、966、967、977、981、983、984、994、1001、1005、1013、1016、1020、1027、1033、1037、1038、1040、和1049。处理 叶得分 损害得分未接种,未攻击 0 0接种的载体,攻击 3.4 67接种的第1组,攻击 1.6 38接种的第2组,攻击 2.2 51
在试验2中诱导的疾病的总体严重性比试验1低,指示较温和的攻击。两组克隆都具有治疗性效果。第1组疫苗将叶得分降低了53%,将损害得分降低了43%。第2组疫苗将叶得分降低了35%,将损害得分降低了24%。上述数据是与接种空载体质粒DNA的组进行比较。试验3
第1组:与试验2中第1组的克隆相同
在用biojector(如详述的)标准方法中或通过基因枪递送DNA。对于基因组递送,使用1.6微米的金颗粒,每只猪注射4次,在3次接种时间点每针1.25μg DNA。在耳后和后腿内侧进行接种。递送压力是500psi。处理 叶得分 损害得分未接种,未攻击 0 0接种载体,攻击 4.2 109biojector接种第1组,攻击 2.5 67基因枪接种第1组,攻击 2.2 48.5
这组试验再一次证明,当以裸露DNA递送时,第1组中的5种克隆产生治疗性效果。biojector递送的疫苗将叶得分降低了40%,将损害得分降低了39%。通过基因组递送克隆的相同集合将叶得分降低了48%,将损害得分降低了56%。试验4
在这组试验中使用了试验2中克隆的其它组合。
第3组:pAD653、711、727、920、951、和965
第4组:pAD612、639、640、1005、1020、和1038
第5组:pAD742、760、923、925、994、984、和1037
还包括先前没有用于试验的一组5种其它克隆。
第6组:pAD702、763、766、957、和996处理 叶得分 损害得分未接种,未攻击 0 0接种载体,攻击 5.0 106接种第3组,攻击 2.8 54基因枪接种第3组,攻击 2.6 87接种第4组,攻击 3.0 79接种第5组,攻击 3.6 74接种第6组,攻击 2.75 42.5
所有这些克隆组产生治疗性效果,即它们降低了肺炎损害的严重性。第3组的克隆,当通过biojector递送时,将叶得分降低了44%,将损害得分降低了49%,当通过基因枪递送时,将叶得分降低了48%,将损害得分降低了18%。其它3种实验性疫苗只通过biojector递送。第4组疫苗将叶得分降低了40%,将损害得分降低了25%。第5组疫苗将叶得分降低了28%,将损害得分降低了30%。第6组疫苗将叶得分降低了45%,将损害得分降低了60%。
这一系列试验证明,与未接种或接种空载体的猪相比,本发明中详述的克隆基因的集合能够通过在接种各种重组质粒组合的组中降低肺损害的严重性而产生治疗性效果。根据由此证明的治疗性效果,本发明要求保护42种基因的集合。实施例5:筛选基因组文库以回收完整基因
实施例4中所列克隆只含部分基因序列。由于完整基因有可能编码对设计疫苗有用的其它表位,需要鉴定完整基因的核苷酸序列。通过筛选两个基因组文库来回收实施例4中克隆代表的基因的完整编码区。通过将用限制酶EcoRI和BgIII消化产生的片段连接到用EcoRI和BamHI消化的pUC18载体中来构建基因组文库。通过用连接混合物转化XL2-BlueMRF’感受态细胞(Stratagene),并铺在含100μg/ml氨苄青霉素的Luria-Bertoni琼脂平板上来回收连接产物。将菌落转接到装有丰富生长培养基的96孔板中培养过夜,然后制备各自的质粒DNA。将来自各个菌落的质粒DNA变性,并转到尼龙膜上。然后用洋地黄毒苷标记的、由实施例4中各个克隆中插入的DNA产生的PCR产物探查各个膜。通过限制酶消化、使用基因特异性引物的PCR、和核苷酸测序,鉴定并分析杂交克隆。产生的基因序列详细记录于图1-42。这些序列中的许多代表基因的完整编码序列,但是许多基因只获得了部分序列。
最后,应当这样理解,可以在不偏离此处所述本发明精神的前提下进行各种其它修饰和/或变化。
序列表<210>1<211>1125<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD612<400>1atggcaaaac aagattttta taaaattctg ggagttgaaa aatcagcatc actaacagaa 60ataaaaaaag cttatcgaaa tttagtaaat atttatcatc ctgataaaaa tacaaaaaaa 120tcagctgaag aacaaaaaca agctgaggcc aaatttaaag aaatccagga agcctacgaa 180attttatctg atgaaacaaa gcgaaaacag tacgataaat tcggtcatgc cgcttttgat 240cagcaatttg gtggtgggtc tagtggcttt tcaggatttg attttggcga tattttttca 300agttttacct ctggttttgg ttttggcggc tcacaagaac aaaaatatag tcgtccttta 360aagggcgaaa attttcaagc taaaatttat atcagtttta tcgagtcaat tctcggaaaa 420gaaatctccc agaaattaac aaaatacgat caatgtgata actgtaaggg ttcaggcgct 480aattcttctt ctgatattac aacttgctat aattgtcaag gtcggggaat gcaaactgag 540gtcttaaata tcccgggatt tggtcgggtt cagaacaaaa caacttgttc agtttgttta 600ggttccggga aaaacattac aaaaattgca aagaagtgcc atggaaaaac tatagttgag 660acaaaagagg aagtaactat taaaattccg gccggaatcc aggatggaat gtttatccgc 720gtggccggat ttggtggacc gggacacaaa ggcgggcctt ctggagatct tcatcttgag 780attaatgttc gtcagcataa acattttact agatccggaa atgatattca tgtgaatatg 840ccagtttcaa taattgatat tatcaaccaa aatactgtcg aagttcccaa cccaaccggt 900ttgaaaaaag ttagacttta tgattattat aaatccggtc caattgttaa tgttcttcct 960gctggggctc ctgatccaaa aaatccaaga attattggcg atctcaaggt tcatttaatt 1020ttttatatcc ccgaatttag tccccgtcaa aaagatgagc tcaaccaggt ttttgctcaa 1080atcaatgata aaacaaaggc aaaatgacta aaagaatttc aataa 1125<210>2<211>368<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD612的推定的蛋白质序列<400>2Met Ala Lys Gln Asp Phe Tyr Lys Ile Leu Gly Val Glu Lys Ser Ala1 5 10 15Ser Leu Thr Glu Ile Lys Lys Ala Tyr Arg Asn Leu Val Asn Ile Tyr
20 25 30His Pro Asp Lys Asn Thr Lys Lys Ser Ala Glu Glu Gln Lys Gln Ala
35 40 45Glu Ala Lys Phe Lys Glu Ile Gln Glu Ala Tyr Glu Ile Leu Ser Asp
50 55 60Glu Thr Lys Arg Lys Gln Tyr Asp Lys Phe Gly His Ala Ala Phe Asp65 70 75 80Gln Gln Phe Gly Gly Gly Ser Ser Gly Phe Ser Gly Phe Asp Phe Gly
85 90 95Asp Ile Phe Ser Ser Phe Thr Ser Gly Phe Gly Phe Gly Gly Ser Gln
100 105 110Glu Gln Lys Tyr Ser Arg Pro Leu Lys Gly Glu Asn Phe Gln Ala Lys
115 120 125Ile Tyr Ile Ser Phe Ile Glu Ser Ile Leu Gly Lys Glu Ile Ser Gln
130 135 140Lys Leu Thr Lys Tyr Asp Gln Cys Asp Asn Cys Lys Gly Ser Gly Ala145 150 155 160Asn Ser Ser Ser Asp Ile Thr Thr Cys Tyr Asn Cys Gln Gly Arg Gly
165 170 175Met Gln Thr Glu Val Leu Asn Ile Pro Gly Phe Gly Arg Val Gln Asn
180 185 190Lys Thr Thr Cys Ser Val Cys Leu Gly Ser Gly Lys Asn Ile Thr Lys
195 200 205Ile Ala Lys Lys Cys His Gly Lys Thr Ile Val Glu Thr Lys Glu Glu
210 215 220Val Thr Ile Lys Ile Pro Ala Gly Ile Gln Asp Gly Met Phe Ile Arg225 230 235 240Val Ala Gly Phe Gly Gly Pro Gly His Lys Gly Gly Pro Ser Gly Asp
245 250 255Leu His Leu Glu Ile Asn Val Arg Gln His Lys His Phe Thr Arg Ser
260 265 270Gly Asn Asp Ile His Val Asn Met Pro Val Ser Ile Ile Asp Ile Ile
275 280 285Asn Gln Asn Thr Val Glu Val Pro Asn Pro Thr Gly Leu Lys Lys Val
290 295 300Arg Leu Tyr Asp Tyr Tyr Lys Ser Gly Pro Ile Val Asn Val Leu Pro305 310 315 320Ala Gly Ala Pro Asp Pro Lys Asn Pro Arg Ile Ile Gly Asp Leu Lys
325 330 335Val His Leu Ile Phe Tyr Ile Pro Glu Phe Ser Pro Arg Gln Lys Asp
340 345 350Glu Leu Asn Gln Val Phe Ala Gln Ile Asn Asp Lys Thr Lys Ala Lys
355 360 365<210>3<211>819<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD633<400>3atgataaaag tttccgatgt ttgctttagt tatacaaaca acatggacca gcttgtgctg 60aaaaatatta atgttgtttt tgaaaaaggt aaatattatg caattctagg gcataatggt 120tcaggaaaat caacgttttc taagattctt tcaggaattt ttaaacctca aaaaggtagt 180attgaagttg atggagtttt actaaataag gaaaatttaa cgaaaattag gaaaaaaatt 240ggtataattt ttcaaaaccc agataatcaa tttgttgggg caacggttga agatgacatc 300gctttcagtt tggaaaacat taatgaggat ccaaaaaaaa tgagtcaaat aatcgcaaat 360ttagctgcaa aagtgcaaat ggagtcatat ttagaccgtg agccacaatt tttatctggg 420ggccaaaagc aaaaagtagc aattgcatca gttttagcac taaatcctga gattataatt 480tttgatgaaa taacttcaat gcttgatccc agaggtaaat atgatgttgt taaaattctt 540gatgatctaa gaaaagataa aacaaaaact ttaatttcaa tcacccacaa tatgaatgaa 600gcaattttag ctgatgaaat tattgttttt gcaaatgggg gaattatcgc tcagggggat 660ccaaaattaa ttttaaatga taaaaatatc atcgaaaaag cgaaaattga ctccccattt 720atctataaaa tttccagcgc acttaaatta gttagtccaa cttatgacga aaatgaattg 780ctagagcaac tatgaaaatt aaagcaaaaa acatcgtaa 819<210>4<21l>272<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD633的推定的蛋白质序列<400>4Met Ile Lys Val Ser Asp Val Cys Phe Ser Tyr Thr Asn Asn Met Asp1 5 10 15Gln Leu Val Leu Lys Asn Ile Asn Val Val Phe Glu Lys Gly Lys Tyr
20 25 30Tyr Ala Ile Leu Gly His Asn Gly Ser Gly Lys Ser Thr Phe Ser Lys
35 40 45Ile Leu Ser Gly Ile Phe Lys Pro Gln Lys Gly Ser Ile Glu Val Asp
50 55 60Gly Val Leu Leu Asn Lys Glu Asn Leu Thr Lys Ile Arg Lys Lys Ile65 70 75 80Gly Ile Ile Phe Gln Asn Pro Asp Asn Gln Phe Val Gly Ala Thr Val
85 90 95Glu Asp Asp Ile Ala Phe Ser Leu Glu Asn Ile Asn Glu Asp Pro Lys
100 105 110Lys Met Set Gln Ile Ile Ala Asn Leu Ala Ala Lys Val Gln Met Glu
115 120 125Ser Tyr Leu Asp Arg Glu Pro Gln Phe Leu Ser Gly Gly Gln Lys Gln
130 135 140Lys Val Ala Ile Ala Ser Val Leu Ala Leu Asn Pro Glu Ile Ile Ile145 150 155 160Phe Asp Glu Ile Thr Ser Met Leu Asp Pro Arg Gly Lys Tyr Asp Val
165 170 175Val Lys Ile Leu Asp Asp Leu Arg Lys Asp Lys Thr Lys Thr Leu Ile
180 185 190Ser Ile Thr His Asn Met Asn Glu Ala Ile Leu Ala Asp Glu Ile Ile
195 200 205Val Phe Ala Asn Gly Gly Ile Ile Ala Gln Gly Asp Pro Lys Leu Ile
210 215 220Leu Asn Asp Lys Asn Ile Ile Glu Lys Ala Lys Ile Asp Ser Pro Phe225 230 235 240Ile Tyr Lys Ile Ser Ser Ala Leu Lys Leu Val Ser Pro Thr Tyr Asp
245 250 255Glu Asn Glu Leu Leu Glu Gln Leu Trp Lys Leu Lys Gln Lys Thr Ser
260 265 270<210>5<211>609<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD639<400>5atgtttagtc aaactattta taaatgaaaa agcgaaaatc tcgaaagagt ggtcgtccct 60gaaaaaggac tttcttgcga atataacggt ctttttagca taagaactgg caaaagtttg 120tacgggctgc tacaaaattt agataatgat tattttattg cttacctaag atcaagtgtg 180cttatcttta cctcttccgc atcatccgct ttagtaagag caaagtctta ttgcgaaaac 240gatttttgat tgcataaaaa taatttttta gtcggcctta tcgtctttag tgccggaatt 300tttaaaataa tggatggtag atgagaaaat acctatcttg ttaaatcagg ggatggtttt 360aaccggtttt tacaagaact aaaaagcaaa aaacactata aattagaatg ttttttgctt 420tcaaacttat tttttgtcag cctgagtcta actaatcata tcagaagtct agcccatcca 480gatttaaata attcaactat ttatttaaat gaattatgcc ttgatgatct tagtcaaaaa 540gaaactttag ctctaaaaag tcttggaaat tatgattttg acgatcaaga aaaggaactt 600ttagagatc 609<210>6<211>203<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD639的推定的蛋白质序列<400>6Met Phe Ser Gln Thr Ile Tyr Lys Trp Lys Ser Glu Asn Leu Glu Arg1 5 10 15Val Val Val Pro Glu Lys Gly Leu Ser Cys Glu Tyr Asn Gly Leu Phe
20 25 30Ser Ile Arg Thr Gly Lys Ser Leu Tyr Gly Leu Leu Gln Asn Leu Asp
35 40 45Asn Asp Tyr Phe Ile Ala Tyr Leu Arg Ser Ser Val Leu Ile Phe Thr
50 55 60Ser Ser Ala Ser Ser Ala Leu Val Arg Ala Lys Ser Tyr Cys Glu Asn65 70 75 80Asp Phe Trp Leu His Lys Asn Asn Phe Leu Val Gly Leu Ile Val Phe
85 90 95Ser Ala Gly Ile Phe Lys Ile Met Asp Gly Arg Trp Glu Asn Thr Tyr
100 105 110Leu Val Lys Ser Gly Asp Gly Phe Asn Arg Phe Leu Gln Glu Leu Lys
115 120 125Ser Lys Lys His Tyr Lys Leu Glu Cys Phe Leu Leu Ser Asn Leu Phe
130 135 140Phe Val Ser Leu Ser Leu Thr Asn His Ile Arg Ser Leu Ala His Pro145 150 155 160Asp Leu Asn Asn Ser Thr Ile Tyr Leu Asn Glu Leu Cys Leu Asp Asp
165 170 175Leu Ser Gln Lys Glu Thr Leu Ala Leu Lys Ser Leu Gly Asn Tyr Asp
180 185 190Phe Asp Asp Gln Glu Lys Glu Leu Leu Glu Ile
195 200<210>7<211>471<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD640<400>7gaggtaagca cgattcccgg acgaggcgat attaaactaa caggttcgct taaggatgta 60atgcaagaat cagcccggat tgccctttct tatgttcagt caaaggccaa ggattttggg 120attaattttg attttgaaaa cactttaatt catattcatg tacccgaagg agcaattcca 180aaagatgggc catcagcagg gataactttt gcaacagcaa taatttcagc cctctcgcaa 240aagccggtct cacataatat tgcaatgaca ggggagataa ccttgcgcgg gaaggtttta 300gcaatcggcg gactaaaaga aaagacgatg ggggcctatt taaatgggat taaaattatt 360tttattccca aggcgaacga gaaaaattta gtcgatattc cgcaggaagt aaaagacgtt 420atccagttta ttcccgttga tacttatcaa ccaatttatg attttatttt t 471<210>8<211>157<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD640的推定的蛋白质序列<400>8Glu Val Ser Thr Ile Pro Gly Arg Gly Asp Ile Lys Leu Thr Gly Ser1 5 10 15Leu Lys Asp Val Met Gln Glu Ser Ala Arg Ile Ala Leu Ser Tyr Val
20 25 30Gln Ser Lys Ala Lys Asp Phe Gly Ile Asn Phe Asp Phe Glu Asn Thr
35 40 45Leu Ile His Ile His Val Pro Glu Gly Ala Ile Pro Lys Asp Gly Pro
50 55 60Ser Ala Gly Ile Thr Phe Ala Thr Ala Ile Ile Ser Ala Leu Ser Gln65 70 75 80Lys Pro Val Ser His Asn Ile Ala Met Thr Gly Glu Ile Thr Leu Arg
85 90 95Gly Lys Val Leu Ala Ile Gly Gly Leu Lys Glu Lys Thr Met Gly Ala
100 105 110Tyr Leu Asn Gly Ile Lys Ile Ile Phe Ile Pro Lys Ala Asn Glu Lys
115 120 125Asn Leu Val Asp Ile Pro Gln Glu Val Lys Asp Val Ile Gln Phe Ile
130 135 140Pro Val Asp Thr Tyr Gln Pro Ile Tyr Asp Phe Ile Phe145 150 155<210>9<211>1128<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD641和pAD1033<400>9aaaaaacttt tagatacttt aggggctgaa tttagtccaa aagcacttgt ttcttcgctt 60tcgatctcac aaaagcaatt tatcgaaatc gccaaagctt tatcccaaaa accggaaatt 120atcatttttg atgaaccgac ttcggttcta accgaaaaag atacccaaaa actttatctg 180cttgttgaaa aacttaaaaa acaaggaatt gcaatcgtct gaattaccca tagaatggaa 240gaaattaaga aaacttgtga atttatcact gtgattcgaa acggaatgta tattgaaagt 300aagccaataa atgaatttaa aaacgaagat gagattattt ctttaatggt cggttttgat 360atcgagcagc gctatcccga aaaaacgccg gttagaagta aaaaaccatc gtttttagtt 420agaaatttat caaatgataa agtttctaat atcagttttg aaatcaaacc aggtgaaatt 480ttagtttttt atggccttgt aagttcaggt cgaactgaat tagctagaac tttaattggc 540gatatgcctt atttaaatgg tcatattgaa ctaaatggtc aagaatttcg cccaaaaaat 600attaaggaca gtcttgatca tggaatttat tatctttctg aaaataggaa acaaattggt 660ctaaatgtta atttaccaat taattttaat atcacaattt cttctcttgg ctcaaatcag 720attttttctt tccttccttt tgtctcaaaa gcaaaaataa ctaaaactac aaatcattat 780attaaacaat taaagatcaa aacaacttca caagatacgc cattaacttc tttatcaggt 840ggaaatcaac aaaaagtttc acttgcaaaa gggcttgcaa cccaaccgca agttttcatc 900ctcgatgaac caactcgcgg agtcgatgtt ggcgcaagaa aggaaattta taatttaatt 960caccaattaa aacaagaaaa taaaacaatt atgataattt cttcggatat gcaagaggtt 1020atcggaatcg ctgatcgggt aattacaatg tatgaaggca gaattacaag tgaattagtt 1080ggcccgcaaa ttaccgatca aaatataatg aaatattcac ttaattta 1128<210>10<211>376<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD641和pAD1033的推定的蛋白
质序列<400>10Lys Lys Leu Leu Asp Thr Leu Gly Ala Glu Phe Ser Pro Lys Ala Leu1 5 10 15Val Ser Ser Leu Ser Ile Ser Gln Lys Gln Phe Ile Glu Ile Ala Lys
20 25 30Ala Leu Ser Gln Lys Pro Glu Ile Ile Ile Phe Asp Glu Pro Thr Ser
35 40 45Val Leu Thr Glu Lys Asp Thr Gln Lys Leu Tyr Leu Leu Val Glu Lys
50 55 60Leu Lys Lys Gln Gly Ile Ala Ile Val Trp Ile Thr His Arg Met Glu65 70 75 80Glu Ile Lys Lys Thr Cys Glu Phe Ile Thr Val Ile Arg Asn Gly Met
85 90 95Tyr Ile Glu Ser Lys Pro Ile Asn Glu Phe Lys Asn Glu Asp Glu Ile
100 105 110Ile Ser Leu Met Val Gly Phe Asp Ile Glu Gln Arg Tyr Pro Glu Lys
115 120 125Thr Pro Val Arg Ser Lys Lys Pro Ser Phe Leu Val Arg Asn Leu Ser
130 135 140Asn Asp Lys Val Ser Asn Ile Ser Phe Glu Ile Lys Pro Gly Glu Ile145 150 155 160Leu Val Phe Tyr Gly Leu Val Ser Ser Gly Arg Thr Glu Leu Ala Arg
165 170 175Thr Leu Ile Gly Asp Met Pro Tyr Leu Asn Gly His Ile Glu Leu Asn
180 185 190Gly Gln Glu Phe Arg Pro Lys Asn Ile Lys Asp Ser Leu Asp His Gly
195 200 205Ile Tyr Tyr Leu Ser Glu Asn Arg Lys Gln Ile Gly Leu Asn Val Asn
210 215 220Leu Pro Ile Asn Phe Asn Ile Thr Ile Ser Ser Leu Gly Ser Asn Gln225 230 235 240Ile Phe Ser Phe Leu Pro Phe Val Ser Lys Ala Lys Ile Thr Lys Thr
245 250 255Thr Asn His Tyr Ile Lys Gln Leu Lys Ile Lys Thr Thr Ser Gln Asp
260 265 270Thr Pro Leu Thr Ser Leu Ser Gly Gly Asn Gln Gln Lys Val Ser Leu
275 280 285Ala Lys Gly Leu Ala Thr Gln Pro Gln Val Phe Ile Leu Asp Glu Pro
290 295 300Thr Arg Gly Val Asp Val Gly Ala Arg Lys Glu Ile Tyr Asn Leu Ile305 310 315 320His Gln Leu Lys Gln Glu Asn Lys Thr Ile Met Ile Ile Ser Ser Asp
325 330 335Met Gln Glu Val Ile Gly Ile Ala Asp Arg Val Ile Thr Met Tyr Glu
340 345 350Gly Arg Ile Thr Ser Glu Leu Val Gly Pro Gln Ile Thr Asp Gln Asn
355 360 365Ile Met Lys Tyr Ser Leu Asn Leu
370 375<210>11<211>2394<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD653<400>11acagtcggaa ttaacaaaac cgaaatggat gcaaatacca aaagaatgat gtataatgcc 60gatattactt attcggttca ttctgaatta ggttttgatt atctccggga taatatggtt 120ttttcagcag ctgaaaaagt tcaaagggga ctaaattttt gcctaatcga tgaagtagac 180tcaattttga tcgatgaagc caaaacccct ttgattatca gtggtggcaa aactaacctt 240ccagcccaat atttatccgc gaaccaattt gttaatactc taattgctga agatttttat 300attgatgaag aaactaaggg aattaaatta aatgataaag gaatcgataa ggcaaatgct 360ttttttggcc ttcgtaattt atatgaaatt caaaactcag aaatagttca tcgaattcaa 420aacgcgctga gagccaataa ggtgatgaaa cgcgatgttg aatatattgt ccaggacggc 480aaaattgcct tagttgatca atttactggg cgaattatgg ctggaagatc ttattctgaa 540ggtctccagc aagccctgca agcaaaagag gggcttgaaa ttgaacctga gacaaaaaca 600ctagcaacaa ttacctatca aaattttttt cgccttttta aaaaattatc agggatgacc 660gggactgcca aaaccgaaga acaagaattt atcgatgttt ataatatgcg cgtgaatgtg 720attccgacaa acaaaccgat gattcgtaag gatgaaaaag atgaaatttt tgccactagt 780cacgaaaaaa atcaagctat aatttccgaa gttgaacgtg ttcataaaat ggggcagcca 840attttaattg gaacctcaca agttgttgac tctgaaacgc tttcggagat gctaaaccaa 900aaaggacttt atcatacagt attaaatgca aaacaaaacc aacttgaagc cgaaattatt 960gcccaggcag gacgaaaaaa tgcgattacc atcgcgacaa atatggctgg aagaagaact 1020gatataattt tagagcctgg tgtgactgaa cttgggcggc tttatattct tggaaccgat 1080aaagccgaaa ctagaagaat cgataaccaa ctacgaggtc gctctggacg acaaggtgat 1140gtgggaattt cgcgattttt tatctcactt caggaccaac tttttcggcg ttttaccaat 1200tttgatcaaa tttttggcgc ttatggacaa acaaatgggg caattaaagg aaaatatatt 1260catgcggttt tacttgcagc ccaaaagaaa atcgaaggct ttaacttcga tatgcgcaaa 1320actgtgctta gttatgatga tgttattcgt caacagcgtg atttaattta tgcccaaaga 1380gatattttgc ttcagattga aaattttgac cattatatcc agaagatgat tattcgggct 1440gttgatatca ttttaagcta tgattttata attttaccaa atcaagaaat tcactataaa 1500aatttaataa attttcttaa tgataattta tcaagaatta ctcattttaa ctttgggcaa 1560attggaattg aaaattatcc cattgaacaa cttaatgaat ttttaatcaa acaattagaa 1620actatttatt ttaaacaaat ccaatcagtt ttaaaggaaa atcttggaaa aacctacttt 1680gaatcagaac gttatattat tttatcaaca cttgatagtc agtgacaaaa tcatattgac 1740accattgaca aattaagatc ttctgctaat ttagttcagt attcccagaa aaatccttat 1800caaattttta ccgaggaagc aacaaaaaaa ttcaacattt tagtagcaga atccgcttat 1860caggcaatag tttctttatt taataattca aatgctgaaa aaatagaata tatcaaagca 1920attttgtctg atggaaccgc aatttcttat ccggcagata gccctcaaga aataattgat 1980caaataatcg cctctaacga ggagagaatc gcggctgcaa gaaaagcaaa agaagaaaaa 2040cagcctgaat ttattgaaaa acaacttgct aaactaaaaa ttgaaaaggt tgaatcagga 2100gaggaatttg aactttgaaa aatcggagat agcaaactag ttaacctaaa aaaggaaatg 2160cctcttgatg aaaaacaaaa tattttagta aaaatgcagc aggaacaact tgaaatgatg 2220agcgaggaag aaaaaaacct aatacaagaa caaaatttag agattgtaga gattgaagaa 2280atagaggaag aaattcaaaa tgaaaatccc caaaaagttg aatttgtgga ttttaaaaat 2340gatcctgatg catataataa actgatattc ggtgcggatt atgcagataa ccat 2394<210>12<211>798<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD653的推定的蛋白质序列<400>12Thr Val Gly Ile Asn Lys Thr Glu Met Asp Ala Asn Thr Lys Arg Met1 5 10 15Met Tyr Asn Ala Asp Ile Thr Tyr Ser Val His Ser Glu Leu Gly Phe
20 25 30Asp Tyr Leu Arg Asp Asn Met Val Phe Ser Ala Ala Glu Lys Val Gln
35 40 45Arg Gly Leu Asn Phe Cys Leu Ile Asp Glu Val Asp Ser Ile Leu Ile
50 55 60Asp Glu Ala Lys Thr Pro Leu Ile Ile Ser Gly Gly Lys Thr Asn Leu65 70 75 80Pro Ala Gln Tyr Leu Ser Ala Asn Gln Phe Val Asn Thr Leu Ile Ala
85 90 95Glu Asp Phe Tyr Ile Asp Glu Glu Thr Lys Gly Ile Lys Leu Asn Asp
100 105 110Lys Gly Ile Asp Lys Ala Asn Ala Phe Phe Gly Leu Arg Asn Leu Tyr
115 120 125Glu Ile Gln Asn Ser Glu Ile Val His Arg Ile Gln Asn Ala Leu Arg
130 135 140Ala Asn Lys Val Met Lys Arg Asp Val Glu Tyr Ile Val Gln Asp Gly145 150 155 160Lys Ile Ala Leu Val Asp Gln Phe Thr Gly Arg Ile Met Ala Gly Arg
165 170 175Ser Tyr Ser Glu Gly Leu Gln Gln Ala Leu Gln Ala Lys Glu Gly Leu
180 185 190Glu Ile Glu Pro Glu Thr Lys Thr Leu Ala Thr Ile Thr Tyr Gln Asn
195 200 205Phe Phe Arg Leu Phe Lys Lys Leu Ser Gly Met Thr Gly Thr Ala Lys
210 215 220Thr Glu Glu Gln Glu Phe Ile Asp Val Tyr Asn Met Arg Val Asn Val225 230 235 240Ile Pro Thr Asn Lys Pro Met Ile Arg Lys Asp Glu Lys Asp Glu Ile
245 250 255Phe Ala Thr Ser His Glu Lys Asn Gln Ala Ile Ile Ser Glu Val Glu
260 265 270Arg Val His Lys Met Gly Gln Pro Ile Leu Ile Gly Thr Ser Gln Val
275 280 285Val Asp Ser Glu Thr Leu Ser Glu Met Leu Asn Gln Lys GlyLeu Tyr
290 295 300His Thr Val Leu Asn Ala Lys Gln Asn Gln Leu Glu Ala Glu Ile Ile305 310 315 320Ala Gln Ala Gly Arg Lys Asn Ala Ile Thr Ile Ala Thr Asn Met Ala
325 330 335Gly Arg Arg Thr Asp Ile Ile Leu Glu Pro Gly Val Thr Glu Leu Gly
340 345 350Arg Leu Tyr Ile Leu Gly Thr Asp Lys Ala Glu Thr Arg Arg Ile Asp
355 360 365Asn Gln Leu Arg Gly Arg Ser Giy Arg Gln Gly Asp Val Gly Ile Ser
370 375 380Arg Phe Phe Ile Ser Leu Gln Asp Gln Leu Phe Arg Arg Phe Thr Asn385 390 395 400Phe Asp Gln Ile Phe Gly Ala Tyr Gly Gln Thr Asn Gly Ala Ile Lys
405 410 415Gly Lys Tyr Ile His Ala Val Leu Leu Ala Ala Gln Lys Lys Ile Glu
420 425 430Gly Phe Asn Phe Asp Met Arg Lys Thr Val Leu Ser Tyr Asp Asp Val
435 440 445Ile Arg Gln Gln Arg Asp Leu Ile Tyr Ala Gln Arg Asp Ile Leu Leu
450 455 460Gln Ile Glu Asn Phe Asp His Tyr Ile Gln Lys Met Ile Ile Arg Ala465 470 475 480Val Asp Ile Ile Leu Ser Tyr Asp Phe Ile Ile Leu Pro Asn Gln Glu
485 490 495Ile His Tyr Lys Asn Leu Ile Asn Phe Leu Asn Asp Asn Leu Ser Arg
500 505 510Ile Thr His Phe Asn Phe Gly Gln Ile Gly Ile Glu Asn Tyr Pro Ile
515 520 525Glu Gln Leu Asn Glu Phe Leu Ile Lys Gln Leu Glu Thr Ile Tyr Phe
530 535 540Lys Gln Ile Gln Ser Val Leu Lys Glu Asn Leu Gly Lys Thr Tyr Phe545 550 555 560Glu Ser Glu Arg Tyr Ile Ile Leu Ser Thr Leu Asp Ser Gln Trp Gln
565 570 575Asn His Ile Asp Thr Ile Asp Lys Leu Arg Ser Ser Ala Asn Leu Val
580 585 590Gln Tyr Ser Gln Lys Asn Pro Tyr Gln Ile Phe Thr Glu Glu Ala Thr
595 600 605Lys Lys Phe Asn Ile Leu Val Ala Glu Ser Ala Tyr Gln Ala Ile Val
610 615 620Ser Leu Phe Asn Asn Ser Asn Ala Glu Lys Ile Glu Tyr Ile Lys Ala625 630 635 640Ile Leu Ser Asp Gly Thr Ala Ile Ser Tyr Pro Ala Asp Ser Pro Gln
645 650 655Glu Ile Ile Asp Gln Ile Ile Ala Ser Asn Glu Glu Arg Ile Ala Ala
660 665 670Ala Arg Lys Ala Lys Glu Glu Lys Gln Pro Glu Phe Ile Glu Lys Gln
675 680 685Leu Ala Lys Leu Lys Ile Glu Lys Val Glu Ser Gly Glu Glu Phe Glu
690 695 700Leu Trp Lys Ile Gly Asp Ser Lys Leu Val Asn Leu Lys Lys Glu Met705 710 715 720Pro Leu Asp Glu Lys Gln Asn Ile Leu Val Lys Met Gln Gln Glu Gln
725 730 735Leu Glu Met Met Ser Glu Glu Glu Lys Asn Leu Ile Gln Glu Gln Asn
740 745 750Leu Glu Ile Val Glu Ile Glu Glu Ile Glu Glu Glu Ile Gln Asn Glu
755 760 765Asn Pro Gln Lys Val Glu Phe Val Asp Phe Lys Asn Asp Pro Asp Ala
770 775 780Tyr Asn Lys Leu Ile Phe Gly Ala Asp Tyr Ala Asp Asn His785 790 795<210>13<211>1410<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD657和pAD964<400>13atgatttcgt atttttattc aaaatcagca ccgcagtgtc ttaaaactga aaatcccaga 60ttttgttata aactaaataa aaatttagta aaatttcaaa aagaattaga tctattaaaa 120cagaaaaaac ttgcaccaaa agaatatgaa agccagttta gtgatctaaa agaaaaattt 180ttagcttatg aagttaatat aaaaaaacat tatcaggcaa aaaaatccta taaattacgt 240gctatttgag atcggattca aaaatattga catacaagct ttaacagatc ccattttgat 300tttgaagcat tttctaaaaa tgttgaatat aaacaaatag gaaataaacg tcataaaatt 360gttgcccgga tcaaaaattt aaacctttcc tttgtcaatc cagcaaatcc cgagattaga 420aatatcgtga ttcgtaatgc ctcaattgat ttttatgaag gcgaaattca tgccttaatt 480ggcgagtctg gttcaggaaa atcagtaatt acttcttgtc tttatggtct tgtcggtcaa 540aacggggtaa ttgaatcagg tgaaatcaaa ctttttaaca atccagtgca aaattttgat 600tttcgtgctt gagaactttc aaattatcgg ggaaaagtta tttcagctgt cttccagaat 660ccgatgtcaa ctttaaatcc aacaaaaaaa ataggcatcc aaattatgga aggaatgtta 720ttaaacaaga ttgttaaaac gaaaaaagaa gcctatgaaa aagcactttt atatcttaga 780atgaccaaaa ttgctaaccc ggaaatggtt atgaaattat atccccatga gctttcagga 840ggtatgattc aaagaattgt gatctcagca attttatcac ttgaacctaa aattatcgtt 900atggatgaac caacaacagc tttggataca accgtgcaag ctttagttct tgatattatc 960cgcgatctcc aaaaaagact aaaaattaca attattttca ttactcacga ccttggagtt 1020gtcgcttctc ttgcaactta tatctcaatc atgtatgctg gtcaagttgt cgaggaaggt 1080acaagagatg aaattctttt aaatccaaga catccatata cttgagggct aattacttca 1140atgcctgatg tcaataaagg cgaacgactt cagtcaattc gcggggttgt tccttcttct 1200ttaaattcaa ttgttggcga tgcttttgca gttagaaacg attatgcctt agaacaagat 1260ttttttattg aacctaaatt ttacagaata agtccaactc accgagtcaa atcagcttta 1320cttgatccaa aagcaccaaa agttgtccca ccaaaaatta tttaccaaaa atgactgcaa 1380tttgcaaaga tgaggcaaga aaatggaaga 1410<210>14<211>470<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD657和pAD964的推定的蛋白
质序列<400>14Met Ile Ser Tyr Phe Tyr Ser Lys Ser Ala Pro Gln Cys Leu Lys Thr1 5 10 15Glu Asn Pro Arg Phe Cys Tyr Lys Leu Asn Lys Asn Leu Val Lys Phe
20 25 30Gln Lys Glu Leu Asp Leu Leu Lys Gln Lys Lys Leu Ala Pro Lys Glu
35 40 45Tyr Glu Ser Gln Phe Ser Asp Leu Lys Glu Lys Phe Leu Ala Tyr Glu
50 55 60Val Asn Ile Lys Lys His Tyr Gln Ala Lys Lys Ser Tyr Lys Leu Arg65 70 75 80Ala Ile Trp Asp Arg Ile Gln Lys Tyr Trp His Thr Ser Phe Asn Arg
85 90 95Ser His Phe Asp Phe Glu Ala Phe Ser Lys Asn Val Glu Tyr Lys Gln
100 105 110Ile Gly Asn Lys Arg His Lys Ile Val Ala Arg Ile Lys Asn Leu Asn
115 120 125Leu Ser Phe Val Asn Pro Ala Asn Pro Glu Ile Arg Asn Ile Val Ile
130 135 140Arg Asn Ala Ser Ile Asp Phe Tyr Glu Gly Glu Ile His Ala Leu Ile145 150 155 160Gly Glu Ser Gly Ser Gly Lys Ser Val Ile Thr Ser Cys Leu Tyr Gly
165 170 175Leu Val Gly Gln Asn Gly Val Ile Glu Set Gly Glu Ile Lys Leu Phe
180 185 190Asn Asn Pro Val Gln Asn Phe Asp Phe Arg Ala Trp Glu Leu Ser Asn
195 200 205Tyr Arg Gly Lys Val Ile Ser Ala Val Phe Gln Asn Pro Met Ser Thr
210 215 220Leu Asn Pro Thr Lys Lys Ile Gly Ile Gln Ile Met Glu Gly Met Leu225 230 235 240Leu Asn Lys Ile Val Lys Thr Lys Lys Glu Ala Tyr Glu Lys Ala Leu
245 250 255Leu Tyr Leu Arg Met Thr Lys Ile Ala Asn Pro Glu Met Val Met Lys
260 265 270
Leu Tyr Pro His Glu Leu Ser Gly Gly Met Ile Gln Arg Ile Val Ile
275 280 285
Ser Ala Ile Leu Ser Leu Glu Pro Lys Ile Ile Val Met Asp Glu Pro
290 295 300Thr Thr Ala Leu Asp Thr Thr Val Gln Ala Leu Val Leu Asp Ile Ile305 310 315 320Arg Asp Leu Gln Lys Arg Leu Lys Ile Thr Ile Ile Phe Ile Thr His
325 330 335Asp Leu Gly Val Val Ala Ser Leu Ala Thr Tyr Ile Ser Ile Met Tyr
340 345 350Ala Gly Gln Val Val Glu Glu Gly Thr Arg Asp Glu Ile Leu Leu Asn
355 360 365Pro Arg His Pro Tyr Thr Trp Gly Leu Ile Thr Ser Met Pro Asp Val
370 375 380Asn Lys Gly Glu Arg Leu Gln Ser Ile Arg Gly Val Val Pro Ser Ser385 390 395 400Leu Asn Ser Ile Val Gly Asp Ala Phe Ala Val Arg Asn Asp Tyr Ala
405 410 415Leu Glu Gln Asp Phe Phe Ile Glu Pro Lys Phe Tyr Arg Ile Ser Pro
420 425 430Thr His Arg Val Lys Ser Ala Leu Leu Asp Pro Lys Ala Pro Lys Val
435 440 445Val Pro Pro Lys Ile Ile Tyr Gln Lys Trp Leu Gln Phe Ala Lys Met
450 455 460Arg Gln Glu Asn Gly Arg465 470<210>15<211>1332<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD659和pAD910<400>15atgaaaaata ttgaaaaaag tgaaataatt atctcccttg ttgatgttga taaagaattt 60ggtgataaaa aagttttaga tcaaataaat ttggacatta aacgaggaga ttttgtcaca 120cttttagggc cctcagggtc cgggaagaca acaattttac gtttaattgg tggttttgaa 180tgaactactc gcggcgaaat caaatttaat ggcatcgata taaaagacgt tccggcacat 240aaacgtgata cagctacaat ttttcaagat tatgcacttt ttccacattt atcagttcgt 300ggaaatattg aatttggtct taaattaaaa agaattaaaa aaaaggcaga agaaattccg 360gatgtagtct ggaaaaaatt tgagcactta aagaaaaaat gacaggataa gcaaaagcga 420aagattaaag agttaaaaat tttacaggct catttagaaa aactgcttga aaatccacag 480ttagatatta aaaaacgtaa aaaattacag gataaattag atgattctga ttttagatat 540tcaaattgag aaaattatct aacatccaaa tcagaaagtt ttaaaaaaaa atacctaacc 600cgaaagatca caaaacagga aattaataaa gaaattaccg atattattga ccttgttggt 660ctaactggaa atgaaaatcg agcaatttcg gaattatcag gaggaatgaa acaacgcgta 720gcacttgcaa gatcgcttgt aattgagcct gaaattgtcc tacttgatga acctttatca 780gctttagata caaaaattag gcaaaaaatg caagtttttc taaaaaaaat tcaacaaaaa 840cttggcctaa cttttatttt tgttactcat gatcaagatg aagccttgca attatcagat 900aaaatcgcca taatccgtaa tggaaaaatc gcccaatacg atgaaccaaa acaaatttat 960gactatccag ttaataaatg ggtggctaat tttattggtg attctaattt ttttcaggca 1020aaatacatta aaaaaaatca ggtcgaaatt cttggtctta aattatatac aattcatgat 1080gagtttatcc caggccaaaa attagattgc ctgattcgtc cagaagatat cgatattgac 1140ctaaattcag gctattttaa aggaaaagtt atccaaaata tttataaagg ttcatactat 1200tcacttgata tcaaagtaga aaatacaata attaatgtcg aaactaacga tttttatgac 1260ctcgagactc aagtttttct aaaatgagat gatgatgcta ttcatttaat ggagatggaa 1320aatgctgaaa tt 1332<210>16<211>444<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD659和pAD910的推定的蛋白
质序列<400>16Met Lys Asn Ile Glu Lys Ser Glu Ile Ile Ile Ser Leu Val Asp Val1 5 10 15Asp Lys Glu Phe Gly Asp Lys Lys Val Leu Asp Gln Ile Asn Leu Asp
20 25 30Ile Lys Arg Gly Asp PheVal Thr Leu Leu Gly Pro Ser Gly Ser Gly
35 40 45Lys Thr Thr Ile Leu Arg Leu Ile Gly Gly Phe Glu Trp Thr Thr Arg
50 55 60Gly Glu Ile Lys Phe Asn Gly Ile Asp Ile Lys Asp Val Pro Ala His65 70 75 80Lys Arg Asp Thr Ala Thr Ile Phe Gln Asp Tyr Ala Leu Phe Pro His
85 90 95Leu Ser Val Arg Gly Asn Ile Glu Phe Gly Leu Lys Leu Lys Arg Ile
100 105 110Lys Lys Lys Ala Glu Glu Ile Pro Asp Val Val Trp Lys Lys Phe Glu
115 120 125His Leu Lys Lys Lys Trp Gln Asp Lys Gln Lys Arg Lys Ile Lys Glu
130 135 140Leu Lys Ile Leu Gln Ala His Leu Glu Lys Leu Leu Glu Asn Pro Gln145 150 155 160Leu Asp Ile Lys Lys Arg Lys Lys Leu Gln Asp Lys Leu Asp Asp Ser
165 170 175Asp Phe Arg Tyr Ser Asn Trp Glu Asn Tyr Leu Thr Ser Lys Ser Glu
180 185 190Ser Phe Lys Lys Lys Tyr Leu Thr Arg Lys Ile Thr Lys Gln Glu Ile
195 200 205Asn Lys Glu Ile Thr Asp Ile Ile Asp Leu Val Gly Leu Thr Gly Asn
210 215 220Glu Asn Arg Ala Ile Ser Glu Leu Ser Gly Gly Met Lys Gln Arg Val225 230 235 240Ala Leu Ala Arg Ser Leu Val Ile Glu Pro Glu Ile Val Leu Leu Asp
245 250 255Glu Pro Leu Ser Ala Leu Asp Thr Lys Ile Arg Gln Lys Met Gln Val
260 265 270Phe Leu Lys Lys Ile Gln Gln Lys Leu Gly Leu Thr Phe Ile Phe Val
275 280 285Thr His Asp Gln Asp Glu Ala Leu Gln Leu Ser Asp Lys Ile Ala Ile
290 295 300Ile Arg Asn Gly Lys Ile Ala Gln Tyr Asp Glu Pro Lys Gln Ile Tyr305 310 315 320Asp Tyr Pro Val Asn Lys Trp Val Ala Asn Phe Ile Gly Asp Ser Asn
325 330 335Phe Phe Gln Ala Lys Tyr Ile Lys Lys Asn Gln Val Glu Ile Leu Gly
340 345 350Leu Lys Leu Tyr Thr Ile His Asp Glu Phe Ile Pro Gly Gln Lys Leu
355 360 365Asp Cys Leu Ile Arg Pro Glu Asp Ile Asp Ile Asp Leu Asn Ser Gly
370 375 380Tyr Phe Lys Gly Lys Val Ile Gln Asn Ile Tyr Lys Gly Ser Tyr Tyr385 390 395 400Ser Leu Asp Ile Lys Val Glu Asn Thr Ile Ile Asn Val Glu Thr Asn
405 410 415Asp Phe Tyr Asp Leu Glu Thr Gln Val Phe Leu Lys Trp Asp Asp Asp
420 425 430Ala Ile His Leu Met Glu Met Glu Asn Ala Glu Ile
435 440<210>17<211>1590<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD662<400>17gatattaaat tgaaaaaaac taatattcta tcactaaaaa aaataaaaaa agtttatggt 60cctgtaattg ctctttctga tgtgactttt gttgttccaa aaggggaaat aactagccta 120gttggtgaaa atggcgcggg aaaatcgaca cttttaaaaa ttttatcagg agtgattcct 180gctggacaat atgaaggtga tctaattttt gaagataaaa ttatggcttt tgcaaataca 240aaagcctccg aacgtgtcgg aattgcaata attcatcaag aactttcaat ttcaccttat 300ttatcaattt gcgagaacat gtatatcggt aattatccga ctaaatttgg caaagttaac 360tgaaataaga tgatttccga atgcaaaaaa tatctagaaa tggtcggtct tgatgaagat 420ccaacaacaa ttgctggctc tctttcgatt gcaaaacagc aaatggttga gatcgcaaaa 480gcactttcaa aaaatgcaaa actactaatt ttagatgaac cgacttcctc tttaaatgat 540gaaaatgctt ttcgcttact tgatattatg aaaagtttaa aaagtaaagg aattacttcg 600atttttgtct cccataaatt aaatgaagtc aaatatgtct ctgataatat tgttgtaatc 660cgcgatggta aattcatttc ccagtataat aaaaatgaag aaataattga tgaaaaccgg 720ctaattcagg acattgttgg ccggccttta aagtccaaat ttcctcctag ggatttagat 780cgaaaaatcg gggaaattat ttttgagatc aaagatatag ttattcctca tgctagtatt 840gcaaattata atgttgtcaa aaatgcttcc cttgatgtta aacaaggcga aattgtcgga 900atttccggac ttgttggatc gggtcgaacc gaattaatgc tttcactttt tgggcagtat 960tataacaaac cttcaagtgg caaagttttc tataaaggta aagaagtaaa atttactaac 1020acaaaacagg caatcaaatc gggaattatg tatgcttccg aagatcgaaa aaatgttggt 1080ctaatccaaa ttttttcgat tcaaaataat atcacttccg ccgctttgca tttattttca 1140aaatgaggaa ttctaaataa aaataaggaa ataattaatg cccaaaaact aaaaaaagat 1200gtaagtatta aaacaaaaaa tattctaaat aatgtcgaat ccctttctgg gggaaatcag 1260caaaaagttg taattgccaa agctttaagc accaaatttg accttctaat tatcgatgag 1320ccaacaaaag gtattgatgt tggctcaaaa tacgaaattt ataaaatttt actagacctt 1380tcatcacaag gtaaaacaat tattgtaatc tcttcggaaa ttgaagaact tttaggaatc 1440accgatcacc tttattttgt gactggaaaa ctttttggtc aaaatcaaga ctgactttcc 1500cagtttttgc atctttttta taactttttt gaccaaaagg attaccataa ggacttccaa 1560attttgagac ataatcttgg tgaagcgagt 1590<210>18<211>530<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD662的推定的蛋白质序列<400>18Asp Ile Lys Leu Lys Lys Thr Asn Ile Leu Ser Leu Lys Lys Ile Lys1 5 10 15Lys Val Tyr Gly Pro Val Ile Ala Leu Ser Asp Val Thr Phe Val Val
20 25 30Pro Lys Gly Glu Ile Thr Ser Leu Val Gly Glu Asn Gly Ala Gly Lys
35 40 45Ser Thr Leu Leu Lys Ile Leu Ser Gly Val Ile Pro Ala Gly Gln Tyr
50 55 60Glu Gly Asp Leu Ile Phe Glu Asp Lys Ile Met Ala Phe Ala Asn Thr65 70 75 80Lys Ala Ser Glu Arg Val Gly Ile Ala Ile Ile His Gln Glu Leu Ser
85 90 95Ile Ser Pro Tyr Leu Ser Ile Cys Glu Asn Met Tyr Ile Gly Asn Tyr
100 105 110Pro Thr Lys Phe Gly Lys Val Asn Trp Asn Lys Met Ile Ser Glu Cys
115 120 125Lys Lys Tyr Leu Glu Met Val Gly Leu Asp Glu Asp Pro Thr Thr Ile
130 135 140Ala Gly Ser Leu Ser Ile Ala Lys Gln Gln Met Val Glu Ile Ala Lys145 150 155 160Ala Leu Ser Lys Asn Ala Lys Leu Leu Ile Leu Asp Glu Pro Thr Ser
165 170 175Ser Leu Asn Asp Glu Asn Ala Phe Arg Leu Leu Asp Ile Met Lys Ser
180 185 190Leu Lys Ser Lys Gly Ile Thr Ser Ile Phe Val Ser His Lys Leu Asn
195 200 205Glu Val Lys Tyr Val Ser Asp Asn Ile Val Val Ile Arg Asp Gly Lys
210 215 220Phe Ile Ser Gln Tyr Asn Lys Asn Glu Glu Ile Ile Asp Glu Asn Arg225 230 235 240Leu Ile Gln Asp Ile Val Gly Arg Pro Leu Lys Ser Lys Phe Pro Pro
245 250 255Arg Asp Leu Asp Arg Lys Ile Gly Glu Ile Ile Phe Glu Ile Lys Asp
260 265 270Ile Val Ile Pro His Ala Ser Ile Ala Asn Tyr Asn Val Val Lys Asn
275 280 285Ala Ser Leu Asp Val Lys Gln Gly Glu Ile Val Gly Ile Ser Gly Leu
290 295 300Val Gly Ser Gly Arg Thr Glu Leu Met Leu Ser Leu Phe Gly Gln Tyr305 310 315 320Tyr Asn Lys Pro Ser Ser Gly Lys Val Phe Tyr Lys Gly Lys Glu Val
325 330 335Lys Phe Thr Asn Thr Lys Gln Ala Ile Lys Ser Gly Ile Met Tyr Ala
340 345 350Ser Glu Asp Arg Lys Asn Val Gly Leu Ile Gln Ile Phe Ser Ile Gln
355 360 365Asn Asn Ile Thr Ser Ala Ala Leu His Leu Phe Ser Lys Trp Gly Ile
370 375 380Leu Asn Lys Asn Lys Glu Ile Ile Asn Ala Gln Lys Leu Lys Lys Asp385 390 395 400Val Ser Ile Lys Thr Lys Asn Ile Leu Asn Asn Val Glu Ser Leu Ser
405 410 415Gly Gly Asn Gln Gln Lys Val Val Ile Ala Lys Ala Leu Ser Thr Lys
420 425 430Phe Asp Leu Leu Ile Ile Asp Glu Pro Thr Lys Gly Ile Asp Val Gly
435 440 445Ser Lys Tyr Glu Ile Tyr Lys Ile Leu Leu Asp Leu Ser Ser Gln Gly
450 455 460Lys Thr Ile Ile Val Ile Ser Ser Glu Ile Glu Glu Leu Leu Gly Ile465 470 475 480Thr Asp His Leu Tyr Phe Val Thr Gly Lys Leu Phe Gly Gln Asn Gln
485 490 495Asp Trp Leu Ser Gln Phe Leu His Leu Phe Tyr Asn Phe Phe Asp Gln
500 505 510Lys Asp Tyr His Lys Asp Phe Gln Ile Leu Arg His Asn Leu Gly Glu
515 520 525Ala Ser
530<210>19<211>852<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD681<400>19atcgaaacca tcaaaattga gctgggtgag cagctcgaat tttttgaaaa aaataataaa 60ttagtcgaaa aacaacgact aaaagaccga gtcaataatg atattgactc gctttctgaa 120ttcggaattt gttcaggaat tgagaattat gcccgccata ttgacggacg ccaaaaaggc 180gaaaaaccat ttagtttact agattattta ccccaagacg gcctaatttt tattgatgaa 240tcccatatta tgatcagcca aattaagggc atgtatgaag gtgatcgaag ccgaaaacaa 300accttggttg actatggtta tcgactacct tcagctcttg ataatcggcc cttaaaactc 360agtgaatttg agaaatatca acaggcaaaa atttatgttt cagccacacc ggccagctat 420gaaattgata aaacaaatgg cgaaattgtc tcgcaaatta tcagaccaac tggactaatt 480gatccagaaa tagtaattga atctaccaaa aatcaaatgg agaaaatttt tcagtatttg 540ctaaaacaga aggaaaaaaa agaaagaagt ctcattttaa ctaccacaaa acgactggcc 600gaagaaatca gcaagtatct ccaggaagaa aaattacaaa atgtctatta tttgcactca 660gaaatgacga cttttgagcg cgatgaaatc ataattaagc ttcgaaaagg aatttatgat 720gcaattgtcg ggataaattt acttcgtgaa ggcgttgata tcccggaagt ttctttgatt 780tttgttcttg aagccggtct tgtttctttt ttgcgatccg catgcgagct cggtaccccg 840ggtcgacctg ca 852<210>20<211>284<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD681的推定的蛋白质序列<400>20Ile Glu Thr Ile Lys Ile Glu Leu Gly Glu Gln Leu Glu Phe Phe Glu1 5 10 15Lys Asn Asn Lys Leu Val Glu Lys Gln Arg Leu Lys Asp Arg Val Asn
20 25 30Asn Asp Ile Asp Ser Leu Set Glu Phe Gly Ile Cys Ser Gly Ile Glu
35 40 45Asn Tyr Ala Arg His Ile Asp Gly Arg Gln Lys Gly Glu Lys Pro Phe
50 55 60Ser Leu Leu Asp Tyr Leu Pro Gln Asp Gly Leu Ile Phe Ile Asp Glu65 70 75 80Ser His Ile Met Ile Ser Gln Ile Lys Gly Met Tyr Glu Gly Asp Arg
85 90 95Ser Arg Lys Gln Thr Leu Val Asp Tyr Gly Tyr Arg Leu Pro Ser Ala
100 105 110Leu Asp Asn Arg Pro Leu Lys Leu Ser Glu Phe Glu Lys Tyr Gln Gln
115 120 125Ala Lys Ile Tyr Val Ser Ala Thr Pro Ala Ser Tyr Glu Ile Asp Lys
130 135 140Thr Asn Gly Glu Ile Val Ser Gln Ile Ile Arg Pro Thr Gly Leu Ile145 150 155 160Asp Pro Glu Ile Val Ile Glu Ser Thr Lys Asn Gln Met Glu Lys Ile
165 170 175Phe Gln Tyr Leu Leu Lys Gln Lys Glu Lys Lys Glu Arg Ser Leu Ile
180 185 190Leu Thr Thr Thr Lys Arg Leu Ala Glu Glu Ile Ser Lys Tyr Leu Gln
195 200 205Glu Glu Lys Leu Gln Asn Val Tyr Tyr Leu His Ser Glu Met Thr Thr
210 215 220Phe Glu Arg Asp Glu Ile Ile Ile Lys Leu Arg Lys Gly Ile Tyr Asp225 230 235 240Ala Ile Val Gly Ile Asn Leu Leu Arg Glu Gly Val Asp Ile Pro Glu
245 250 255Val Ser Leu Ile Phe Val Leu Glu Ala Gly Leu Val Ser Phe Leu Arg
260 265 270Ser Ala Cys Glu Leu Gly Thr Pro Gly Arg Pro Ala
275 280<210>21<211>843<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD700<400>21atgcccaaat taaaccgact tagagcccga tttgtccaga ttcaaaacat tgaaaaaatg 60acaaatgtaa tggaaatgat tgcgaatgca aaaattccaa aaataaaaaa caagtttaaa 120attgttcaag aatattttga aaatttagat tatatttttc aaaatattct tgcaaattta 180tctaagaagg ttgaggaatt aactaatgct gattccaaaa aaaatcttta tattattttt 240ggatcaaatt taggtttttg tggtgccctt aataatttaa tcttaaaaaa tgttgtccca 300caacttcaga aaaatgatga aattatcgtc tttggtgaaa aaatttataa ttttttgtca 360ataaattact ctaatttaat tattaaattt tttctaaata ttgaagaaac taattttagt 420gaaccaattt tagaaatctc aaactttgta aatcaatcga tttttgagag aaaatataaa 480aaaattttta tttgttataa caaatttatc agtattatac attcaagccc agagatgcaa 540aatttatttg atttcaaaaa aaatactata aaatacggtg gttacgggat tgagtttgaa 600ccaaatgcta ctgaggtttt taaaaaatta atgccctttt atataaaatc catccttgaa 660aaacttttta tcgaatccaa attagttgag acttcaacta gacgaacatc aatggaaagt 720gcactgaaaa tgccagttga aattttgcat aagtttagaa acagaaataa ttccagtcgt 780ccagccatga ttaccccaga aattattgag attattagtg gtaaaatgtt gaaaaagtta 840ggt 843<210>22<211>281<212>PRT<213>人工序列<220><223>人工序列的描述:克隆PAD700的推定的蛋白质序列<400>22Met Pro Lys Leu Asn Arg Leu Arg Ala Arg Phe Val Gln Ile Gln Asn1 5 10 15Ile Glu Lys Met Thr Asn Val Met Glu Met Ile Ala Asn Ala Lys Ile
20 25 30Pro Lys Ile Lys Asn Lys Phe Lys Ile Val Gln Glu Tyr Phe Glu Asn
35 40 45Leu Asp Tyr Ile Phe Gln Asn Ile Leu Ala Asn Leu Ser Lys Lys Val
50 55 60Glu Glu Leu Thr Asn Ala Asp Ser Lys Lys Asn Leu Tyr Ile Ile Phe65 70 75 80Gly Ser Asn Leu Gly Phe Cys Gly Ala Leu Asn Asn Leu Ile Leu Lys
85 90 95Asn Val Val Pro Gln Leu Gln Lys Asn Asp Glu Ile Ile Val Phe Gly
100 105 110Glu Lys Ile Tyr Asn Phe Leu Ser Ile Asn Tyr Ser Asn Leu Ile Ile
115 120 125Lys Phe Phe Leu Asn Ile Glu Glu Thr Asn Phe Ser Glu Pro Ile Leu
130 135 140Glu Ile Ser Asn Phe Val Asn Gln Ser Ile Phe Glu Arg Lys Tyr Lys145 150 155 160Lys Ile Phe Ile Cys Tyr Asn Lys Phe Ile Ser Ile Ile His Ser Ser
165 170 175Pro Glu Met Gln Asn Leu Phe Asp Phe Lys Lys Asn Thr Ile Lys Tyr
180 185 190Gly Gly Tyr Gly Ile Glu Phe Glu Pro Asn Ala Thr Glu Val Phe Lys
195 200 205Lys Leu Met Pro Phe Tyr Ile Lys Ser Ile Leu Glu Lys Leu Phe Ile
210 215 220Glu Ser Lys Leu Val Glu Thr Ser Thr Arg Arg Thr Ser Met Glu Ser225 230 235 240Ala Leu Lys Met Pro Val Glu Ile Leu His Lys Phe Arg Asn Arg Asn
245 250 255Asn Ser Ser Arg Pro Ala Met Ile Thr Pro Glu Ile Ile Glu Ile Ile
260 265 270Ser Gly Lys Met Leu Lys Lys Leu Gly
275 280<210>23<211>615<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD711<400>23tcaggtatgt caaaaaatat taaggaaatt tcaatcctac cccttaaatt aaatcctgct 60ggaattatgc ctgtaatttt tgccttaatt atcgtttcac tgccgacact ttttagcgga 120tttcttgata gaaatacctc agcagttcgg aattgaatag ataataatat gcaaatttat 180cacccaatcg gtcttatcat ttttattgtt tttaatgtct ccttttcaat aataatgtcc 240ttacaacaat cccgagttga taaaattgca caggattttg ccaaaaattc aacttttatc 300cctgggattc gcccaggaga acagactgaa gattatttaa tttcagtggt tttgcgactt 360tcagttttca gtgcgattta tcttaccttt ttaggaattc tccaacctgt tgaaattatg 420ttaggtcttc cttcggcaat cacaatttca ggaacttcga taataatttt agcaacaact 480acacttgaaa cgatttcgca gatcaaagcc cgttatgatg cacaaaaagt tctaaaacaa 540agtaaaaaga tccgcaaaaa tttacaagtt cgaaaaaatt ctccttctat tgattcaaat 600caggatcttt tatgg 615<210>24<211>205<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD711的推定的蛋白质序列<400>24Ser Gly Met Ser Lys Asn Ile Lys Glu Ile Ser Ile Leu Pro Leu Lys1 5 10 15Leu Asn Pro Ala Gly Ile Met Pro Val Ile Phe Ala Leu Ile Ile Val
20 25 30Ser Leu Pro Thr Leu Phe Ser Gly Phe Leu Asp Arg Asn Thr Ser Ala
35 40 45Val Arg Asn Trp Ile Asp Asn Asn Met Gln Ile Tyr His Pro Ile Gly
50 55 60Leu Ile Ile Phe Ile Val Phe Asn Val Ser Phe Ser Ile Ile Met Ser65 70 75 80Leu Gln Gln Ser Arg Val Asp Lys Ile Ala Gln Asp Phe Ala Lys Asn
85 90 95Ser Thr Phe Ile Pro Gly Ile Arg Pro Gly Glu Gln Thr Glu Asp Tyr
100 105 110Leu Ile Ser Val Val Leu Arg Leu Ser Val Phe Ser Ala Ile Tyr Leu
115 120 125Thr Phe Leu Gly Ile Leu Gln Pro Val Glu Ile Met Leu Gly Leu Pro
130 135 140Ser Ala Ile Thr Ile Ser Gly Thr Ser Ile Ile Ile Leu Ala Thr Thr145 150 155 160Thr Leu Glu Thr Ile Ser Gln Ile Lys Ala Arg Tyr Asp Ala Gln Lys
165 170 175Val Leu Lys Gln Ser Lys Lys Ile Arg Lys Asn Leu Gln Val Arg Lys
180 185 190Asn Ser Pro Ser Ile Asp Ser Asn Gln Asp Leu Leu Trp
195 200 205<210>25<211>648<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD721<400>25atcgccgaag aagtttcaag tttttctccg tttgaccgac ttttattttt taggatgtta 60gatactgcaa ctgcaggtga tattttcacc tacttttcac cagaaattca gaccaaatta 120gtactaagtt taccaaatga gctaatcaat aaattacttg atgaacttta tgttgatgaa 180attgtcgaac ttcttgatga agtccctgat aatgttgcca aaagaatttt gcgcaacatt 240gacattgata ctcgtaaaca aataaatcaa cttttgcagt ataccgacga tcaaattggc 300gcttttatgt cagttgatat cgtctatctt tttaaagatt cgacttgtca tcaagcactt 360gaaaaaatta gaaactataa agatatctcc gaattagtgc attattatta tgtcgttgat 420caaaacaaga aaataatcgg ggcaactact ttagaagata ttgtcttttc tgatcctaat 480actcagatca aagaaattgt ttttcaagtc ccttttcttg ttacactgat aaaaaagatt 540atgccgccga agtttttgcc caaaatgatt tttccagtac tcccggttgt taataccagc 600cagaaactaa tccgaatggt tccagttgat gatattatcc gatattgt 648<210>26<211>216<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD721的推定的蛋白质序列<400>26Ile Ala Glu Glu Val Ser Ser Phe Ser Pro Phe Asp Arg Leu Leu Phe1 5 10 15Phe Arg Met Leu Asp Thr Ala Thr Ala Gly Asp Ile Phe Thr Tyr Phe
20 25 30Ser Pro Glu Ile Gln Thr Lys Leu Val Leu Ser Leu Pro Asn Glu Leu
35 40 45Ile Asn Lys Leu Leu Asp Glu Leu Tyr Val Asp Glu Ile Val Glu Leu
50 55 60Leu Asp Glu Val Pro Asp Asn Val Ala Lys Arg Ile Leu Arg Asn Ile65 70 75 80Asp Ile Asp Thr Arg Lys Gln Ile Asn Gln Leu Leu Gln Tyr Thr Asp
85 90 95Asp Gln Ile Gly Ala Phe Met Ser Val Asp Ile Val Tyr Leu Phe Lys
100 105 110Asp Ser Thr Cys His Gln Ala Leu Glu Lys Ile Arg Asn Tyr Lys Asp
115 120 125Ile Ser Glu Leu Val His Tyr Tyr Tyr Val Val Asp Gln Asn Lys Lys
130 135 140Ile Ile Gly Ala Thr Thr Leu Glu Asp Ile Val Phe Ser Asp Pro Asn145 150 155 160Thr Gln Ile Lys Glu Ile Val Phe Gln Val Pro Phe Leu Val Thr Leu
165 170 175Ile Lys Lys Ile Met Pro Pro Lys Phe Leu Pro Lys Met Ile Phe Pro
180 185 190Val Leu Pro Val Val Asn Thr Ser Gln Lys Leu Ile Arg Met Val Pro
195 200 205Val Asp Asp Ile Ile Arg Tyr Cys
210 215<210>27<211>837<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD727<400>27tcatttggtt caggatttaa tttagcaatt gattttagtg gcggaactaa ctttttaatt 60gaaagctcaa attcaagtta tgatttaatt acaaaggaaa aagccgaaaa aataattagt 120tttcttgatt cacaaaatat aaacaagtca aattcaacaa ttttgcttaa tccattaaat 180gaaaatggaa atatttttaa tcttgaaatt aaaacaaaac ttgatctagc aacaaaaatt 240gcctcattaa atacggcaat ccagaataat ttttctaata ttcgaatgac aaattattca 300atttcgaatg aagaagctca aaaattaatt ttcaatgcaa ttctttcagt tggaatcgca 360cttatttttg taactatttt tacgctaatt aggtttaaat ggactttttc gcttgcaata 420attttctcac ttctttttaa tgttttaatg gttttgctag caattattat tacacggatc 480gaaatatcgc agaatttagt tgttgcaatt cttactttaa ttggttatac agtaaatgat 540acaatcgtgg tttttgatag agtaaaagca agattttcag aaataaatca tgaaaatgtt 600tataaatttg ataaaattaa agaaatttcc ttacaagcaa ttagagaaac agcaaaaagg 660tcggtatata catccttgac aaccatttta acaattgttg ttttaatgat tttttatgaa 720tcaattgata ttgtctttag cctgacgatg ttaattggtg tgataattgg aacatattcc 780tccttattta tcgcaacccg catttggatt attcttgaat catcccgtaa tcgtaaa 837<210>28<211>279<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD727的推定的蛋白质序列<400>28Ser Phe Gly Ser Gly Phe Asn Leu Ala Ile Asp Phe Ser Gly Gly Thr 1 5 10 15Asn Phe Leu Ile Glu Ser Ser Asn Ser Ser Tyr Asp Leu Ile Thr Lys
20 25 30Glu Lys Ala Glu Lys Ile Ile Ser Phe Leu Asp Ser Gln Asn Ile Asn
35 40 45Lys Ser Asn Ser Thr Ile Leu Leu Asn Pro Leu Asn Glu Asn Gly Asn
50 55 60Ile Phe Asn Leu Glu Ile Lys Thr Lys Leu Asp Leu Ala Thr Lys Ile65 70 75 80Ala Ser Leu Asn Thr Ala Ile Gln Asn Asn Phe Ser Asn Ile Arg Met
85 90 95Thr Asn Tyr Ser Ile Ser Asn Glu Glu Ala Gln Lys Leu Ile Phe Asn
100 105 110Ala Ile Leu Ser Val Gly Ile Ala Leu Ile Phe Val Thr Ile Phe Thr
115 120 125Leu Ile Arg Phe Lys Trp Thr Phe Ser Leu Ala Ile Ile Phe Ser Leu
130 135 140Leu Phe Asn Val Leu Met Val Leu Leu Ala Ile Ile Ile Thr Arg Ile145 150 155 160Glu Ile Ser Gln Asn Leu Val Val Ala Ile Leu Thr Leu Ile Gly Tyr
165 170 175Thr Val Asn Asp Thr Ile Val Val Phe Asp Arg Val Lys Ala Arg Phe
180 185 190Ser Glu Ile Asn His Glu Asn Val Tyr Lys Phe Asp Lys Ile Lys Glu
195 200 205Ile Ser Leu Gln Ala Ile Arg Glu Thr Ala Lys Arg Ser Val Tyr Thr
210 215 220Ser Leu Thr Thr Ile Leu Thr Ile Val Val Leu Met Ile Phe Tyr Glu225 230 235 240Ser Ile Asp Ile Val Phe Ser Leu Thr Met Leu Ile Gly Val Ile Ile
245 250 255Gly Thr Tyr Ser Ser Leu Phe Ile Ala Thr Arg Ile Trp Ile Ile Leu
260 265 270Glu Ser Ser Arg Asn Arg Lys
275<210>29<211>2523<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD742<400>29atgaagttaa taaaaattga aattgaaggt tttaaatcct ttgctgaacc tgtaagtatt 60aaatttgatg gttcaattgt tggaataatt gggccaaatg gctctggaaa atccaatata 120aatgatgcaa ttaaatgagt tttaggcgaa aaatcagtta aacaattacc gggcccaaat 180atggatgatg gcatttttgc tggctcaaaa acaggtatga tcgcaattga aaaaattgaa 240atcaggacaa aagagcttga aaaacaagta aaacagcttg aaaaacaagc agaaaatgca 300aaaatttacc tcgaaaaatc aaagcaatta gaatctgttg aagtcggctt aattgtctct 360gatattaaaa agtaccaaac agaattagat caagtccagg aaaaattaaa tgatctaaaa 420tttcaagaac ctaaatttat cagcgaaatt gaagcaaatg agaaaataat tattacaaat 480acacaaaaaa ggtcagaaat tgaagctgaa atcaacacta aaaaccgcga aattcaccgc 540ctaaaagagc aaattaatac cctaaattta gcttatgcaa aagcaactca acttcaagaa 600atgattttat caagtgaaat tagtgtaaat tttgagcaaa aaatggctgc tttgcgccaa 660aaatatagtc ttataagcgc gcaaaaagac aattttgcaa aattaatcag ccaaaacaag 720ctcaaaaaac tagaaattga agaaaaactt aatacattta ggacacaaaa gggtgaaatt 780gaaaggaatt tatacagtct aaattccgaa aaaattatta gtcaaactag aatctccgag 840ctaaaaaagt ccttagaatc aatgtctttt ttgccaaagg ggacaaaaat cattatcgaa 900aatagctttc tttttcctgg atattgtggg cttgtctctg atttaatcaa aattttccca 960aaatatacag gcgcaattga agccgcattg ggtccaactt taaaacaaat tgttgttgac 1020caacctgaaa ccgcagtttc agcaattaat tttctaaaga aaaattatgc aggaagcgca 1080acatttatcc ccctttcaac attaaaaccg cgatttattc ccgatttata tcttgaacat 1140ctaaattcac aaaaaggttt tataaattta gctagcaatt tagtcgattt tgaaaaaaaa 1200tacaagattt tagcggattt tttactagga gggattattg ttgctgatac aattgattca 1260gcaaatcgaa tcgcaaactt ccttaaccac aaaaacatga tcgtaacttt agacggcgat 1320gttattagaa ctagcgggat aatttctggg ggtcataaga taaaaaatga ttcttccttt 1380tcaattcagt ataaaatcga tgaactaaca aataatttga atttttttga agaaaaaatt 1440caagaattta aagttaaatc taatgaattt gaacagttaa ttacaagaga atctgtattt 1500ttacagcaaa ttaatattaa tcttaatgat ttagagcaaa aatttagtaa ttctgaaaat 1560gaattaatcg aaattaaagc tcaaaatgaa ggtcttgaag agagtctaaa tcaaaaagat 1620gacctaaatt taagtctaaa tcgaacttta aaagaaaaaa ttgaacttga aaatgtggtt 1680ttagaacttg aaaatcaatg caaaatttta aaaactgaaa aaaaacagct cgataatcaa 1740atttcagaac ttacagtttt agttcaagaa cttaaccaaa aacagcgaaa aatcaacgca 1800gatcttaacc aaaatcaaaa ttacaaagac aaatatgaat ttttaattac aaatttacga 1860aataatttat cccaaaaata cagtctaact tttgaaggcg cagctcaaaa atatgaactt 1920gaaattccag aaaaagatgc tcgcgaattt gttaatagtc taaatttaga gattaaagcg 1980cttggaaatg ttaatttaga tgcaattaat gactttgaaa caacgagtca aagactcgaa 2040aaactaaaaa aaagtcaaaa tgaacttgaa actgccaggt caaaaatttt agaagttatc 2100tcggatttag ataaaattat cattggaaaa acccaggaaa ttgtcgatct agttaattcc 2160gagtttaacc ttgttttcca gaatatgttt ggtgggggaa gtgcaaaaat ttattttagt 2220gataaaaacg atattttaaa ttcggggatt gaaataagtg cccaaccacc tggaaaaact 2280atcaaaaata ttaggctttt ttctgggggc gaaaaggcaa ttattgcaat ttcacttttg 2340ttttcaatta ttaaggcaag accaattccg ctttgcattc ttgatgaagt tgaagctgcc 2400cttgatgagt caaatgtgat cagatatgtg gaatttcgaa agcagttaaa acaaaaaacg 2460cagtttttga tcatcaccca tcggcacgga acgatgtccc gagttgatca acttttagga 2520atc 2523<210>30<211>84l<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD742的推定的蛋白质序列<400>30Met Lys Leu Ile Lys Ile Glu Ile Glu Gly Phe Lys Ser Phe Ala Glu1 5 10 15Pro Val Ser Ile Lys Phe Asp Gly Ser Ile Val Gly Ile Ile Gly Pro
20 25 30Asn Gly Ser Gly Lys Ser Asn Ile Asn Asp Ala Ile Lys Trp Val Leu
35 40 45Gly Glu Lys Ser Val Lys Gln Leu Pro Gly Pro Asn Met Asp Asp Gly
50 55 60Ile Phe Ala Gly Ser Lys Thr Gly Met Ile Ala Ile Glu Lys Ile Glu65 70 75 80Ile Arg Thr Lys Glu Leu Glu Lys Gln Val Lys Gln Leu Glu Lys Gln
85 90 95Ala Glu Asn Ala Lys Ile Tyr Leu Glu Lys Ser Lys Gln Leu Glu Ser
100 105 110Val Glu Val Gly Leu Ile Val Ser Asp Ile Lys Lys Tyr Gln Thr Glu
115 120 125Leu Asp Gln Val Gln Glu Lys Leu Asn Asp Leu Lys Phe Gln Glu Pro
130 135 140Lys Phe Ile Ser Glu Ile Glu Ala Asn Glu Lys Ile Ile Ile Thr Asn145 150 155 160Thr Gln Lys Arg Ser Glu Ile Glu Ala Glu Ile Asn Thr Lys Asn Arg
165 170 175Glu Ile His Arg Leu Lys Glu Gln Ile Asn Thr Leu Asn Leu Ala Tyr
180 185 190Ala Lys Ala Thr Gln Leu Gln Glu Met Ile Leu Ser Ser Glu Ile Ser
195 200 205Val Asn Phe Glu Gln Lys Met Ala Ala Leu Arg Gln Lys Tyr Ser Leu
210 215 220Ile Ser Ala Gln Lys Asp Asn Phe Ala Lys Leu Ile Ser Gln Asn Lys225 230 235 240Leu Lys Lys Leu Glu Ile Glu Glu Lys Leu Asn Thr Phe Arg Thr Gln
245 250 255Lys Gly Glu Ile Glu Arg Asn Leu Tyr Ser Leu Asn Ser Glu Lys Ile
260 265 270Ile Ser Gln Thr Arg Ile Ser Glu Leu Lys Lys Ser Leu Glu Ser Met
275 280 285Ser Phe Leu Pro Lys Gly Thr Lys Ile Ile Ile Glu Asn Ser Phe Leu
290 295 300Phe Pro Gly Tyr Cys Gly Leu Val Ser Asp Leu Ile Lys Ile Phe Pro305 310 315 320Lys Tyr Thr Gly Ala Ile Glu Ala Ala Leu Gly Pro Thr Leu Lys Gln
325 330 335Ile Val Val Asp Gln Pro Glu Thr Ala Val Ser Ala Ile Asn Phe Leu
340 345 350Lys Lys Asn Tyr Ala Gly Ser Ala Thr Phe Ile Pro Leu Ser Thr Leu
355 360 365Lys Pro Arg Phe Ile Pro Asp Leu Tyr Leu Glu His Leu Asn Ser Gln
370 375 380Lys Gly Phe Ile Asn Leu Ala Ser Asn Leu Val Asp Phe Glu Lys Lys385 390 395 400Tyr Lys Ile Leu Ala Asp Phe Leu Leu Gly Gly Ile Ile Val Ala Asp
405 410 415Thr Ile Asp Ser Ala Asn Arg Ile Ala Asn Phe Leu Asn His Lys Asn
420 425 430Met Ile Val Thr Leu Asp Gly Asp Val Ile Arg Thr Ser Gly Ile Ile
435 440 445Ser Gly Gly His Lys Ile Lys Asn Asp Ser Ser Phe Ser Ile Gln Tyr
450 455 460Lys Ile Asp Glu Leu Thr Asn Asn Leu Asn Phe Phe Glu Glu Lys Ile465 470 475 480Gln Glu Phe Lys Val Lys Ser Asn Glu Phe Glu Gln Leu Ile Thr Arg
485 490 495Glu Ser Val Phe Leu Gln Gln Ile Asn Ile Asn Leu Asn Asp Leu Glu
500 505 510Gln Lys Phe Ser Asn Ser Glu Asn Glu Leu Ile Glu Ile Lys Ala Gln
515 520 525Asn Glu Gly Leu Glu Glu Ser Leu Asn Gln Lys Asp Asp Leu Asn Leu
530 535 540Ser Leu Asn Arg Thr Leu Lys Glu Lys Ile Glu Leu Glu Asn Val Val545 550 555 560Leu Glu Leu Glu Asn Gln Cys Lys Ile Leu Lys Thr Glu Lys Lys Gln
565 570 575Leu Asp Asn Gln Ile Ser Glu Leu Thr Val Leu Val Gln Glu Leu Asn
580 585 590Gln Lys Gln Arg Lys Ile Asn Ala Asp Leu Asn Gln Asn Gln Asn Tyr
595 600 605Lys Asp Lys Tyr Glu Phe Leu Ile Thr Asn Leu Arg Asn Asn Leu Ser
610 615 620Gln Lys Tyr Ser Leu Thr Phe Glu Gly Ala Ala Gln Lys Tyr Glu Leu625 630 635 640Glu Ile Pro Glu Lys Asp Ala Arg Glu Phe Val Asn Ser Leu Asn Leu
645 650 655Glu Ile Lys Ala Leu Gly Asn Val Asn Leu Asp Ala Ile Asn Asp Phe
660 665 670Glu Thr Thr Ser Gln Arg Leu Glu Lys Leu Lys Lys Ser Gln Asn Glu
675 680 685Leu Glu Thr Ala Arg Ser Lys Ile Leu Glu Val Ile Ser Asp Leu Asp
690 695 700Lys Ile Ile Ile Gly Lys Thr Gln Glu Ile Val ASp Leu Val Asn Ser705 710 715 720Glu Phe Asn Leu Val Phe Gln Asn Met Phe Gly Gly Gly Ser Ala Lys
725 730 735Ile Tyr Phe Ser Asp Lys Asn Asp Ile Leu Asn Ser Gly Ile Glu Ile
740 745 750Ser Ala Gln Pro Pro Gly Lys Thr Ile Lys Asn Ile Arg Leu Phe Ser
755 760 765Gly Gly Glu Lys Ala Ile Ile Ala Ile Ser Leu Leu Phe Ser Ile Ile
770 775 780Lys Ala Arg Pro Ile Pro Leu Cys Ile Leu Asp Glu Val Glu Ala Ala785 790 795 800Leu Asp Glu Ser Asn Val Ile Arg Tyr Val Glu Phe Arg Lys Gln Leu
805 810 815Lys Gln Lys Thr Gln Phe Leu Ile Ile Thr His Arg His Gly Thr Met
820 825 830Ser Arg Val Asp Gln Leu Leu Gly Ile
835 840<210>31<211>1698<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD760<400>31atgaatagaa aaaaaaccga aaaatcaaaa attagttcaa aagatagtaa aaaattaata 60attcaagcta tccaagatgt ggcaaaaaat agcgaattaa atctggaagc agttattgat 120atttttcagg aagcaattga atttgtaata acaaagaaaa ttgacccaga tgcgcaaata 180aaaattgaag ctgatcttga acaattgagt tttaaagttt ttaatacaaa cgggattgtt 240gttgaagaaa attattttga tgatcttaca gatgaggaaa aagttaacga tcttgtttct 300tttattttac tatcaaaggc aaaagaaact gatcctgaga ttcaggttga tgatattttt 360tcaattgaaa ttaatcttga aagttttgaa cattgacttt ttatggcaat tatgcacgct 420tttaagcaaa aaatttctga aattgtccga aataatgttt ataacaaata tttatcgctt 480aaaaataacg tagttttggc cactgttact aataaaattg ctgctggtta tattttcgaa 540attgacgatg ataaagtttc tgcctttatg ccaagccatt atgcaagtgg acaaaattta 600aaaataggca ctaaacatga agttgtaatt gaaaatgtat caaaaaatac aaaacagtcg 660caggttgtga tatcctcaaa atcagttcaa cttgtcaaga aaaaaataat cgatgcaatc 720cctgaactac agtcaaaatt tcttgaaatc acttcaattg cacggattcc aggggaaaga 780tgtaaagtcg caattcgcag aaatgaagat gccgaagctg ataatatttc tgaaatcggc 840tcaattgtag gagcaactgg ctcaagagtt cttgcaattt ctcaagaact tcaaggtgaa 900aaaattgagg tgattaaata tgatgataat attgtcaaat ttattgttaa tgcgatgtcg 960ccttcaaaag ttatttgcgt aaaagagttc aaaataggtc ataaattacg tcgttttatc 1020gtagttgttc ctgattttca acatagttta gccattggaa aaaacggttc aaatgttaaa 1080ctagtagcag atctaacacg ttgtcaagtg caaattatcc cgtattcaag cgcgctaaaa 1140gatcaaaatt ttaaaattga atgaaatgga aatattaaag acattcaaga actaaacagt 1200cttaacaatg agtatattca tcgccagcag ggtagaattt atcaaaatca tcggaattca 1260tatggtcaag gtaataataa ttttgactta attttacagc aattcgaatc tgatattcgc 1320gagttagaaa aaccttatgg gattgaaaac gagtttatac caaaaaatga gcaaaaacag 1380gtaagaagtc accaagaatt tccaaaaaac aagaataatc tagcaaaagc agcaactaaa 1440tcgcgtaatt ttaacaaaag ccagaatatc cgcgaaaata tttcaaaaga ttttgattat 1500ggctttgaaa acgagaaaga ttctaattct ttccaaaata ttagtcaaag atcttttttt 1560gatgcagatt cactttttga ttcggctcta aatgaggcaa ttagtgaaaa cgagttaatc 1620gataaaattc accaagaaga ggaaaaaaaa caagaattat tattacaaga gaaggaaaaa 1680tgagcaaaaa atgaagct 1698<210>32<211>566<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD760的推定的蛋白质序列<400>32Met Asn Arg Lys Lys Thr Glu Lys Ser Lys Ile Ser Ser Lys Asp Ser1 5 10 15Lys Lys Leu Ile Ile Gln Ala Ile Gln Asp Val Ala Lys Asn Ser Glu
20 25 30Leu Asn Leu Glu Ala Val Ile Asp Ile Phe Gln Glu Ala Ile Glu Phe
35 40 45Val Ile Thr Lys Lys Ile Asp Pro Asp Ala Gln Ile Lys Ile Glu Ala
50 55 60Asp Leu Glu Gln Leu Ser Phe Lys Val Phe Asn Thr Asn Gly Ile Val65 70 75 80Val Glu Glu Asn Tyr Phe Asp Asp Leu Thr Asp Glu Glu Lys Val Asn
85 90 95Asp Leu Val Ser Phe Ile Leu Leu Ser Lys Ala Lys Glu Thr Asp Pro
100 105 110Glu Ile Gln Val Asp Asp Ile Phe Ser Ile Glu Ile Asn Leu Glu Ser
115 120 125Phe Glu His Trp Leu Phe Met Ala Ile Met His Ala Phe Lys Gln Lys
130 135 140Ile Ser Glu Ile Val Arg Asn Asn Val Tyr Asn Lys Tyr Leu Ser Leu145 150 155 160Lys Asn Asn Val Val Leu Ala Thr Val Thr Asn Lys Ile Ala Ala Gly
165 170 175Tyr Ile Phe Glu Ile Asp Asp Asp Lys Val Ser Ala Phe Met Pro Ser
180 185 190His Tyr Ala Ser Gly Gln Asn Leu Lys Ile Gly Thr Lys His Glu Val
195 200 205Val Ile Glu Asn Val Ser Lys Asn Thr Lys Gln Ser Gln Val Val Ile
210 215 220Ser Ser Lys Ser Val Gln Leu Val Lys Lys Lys Ile Ile Asp Ala Ile225 230 235 240Pro Glu Leu Gln Ser Lys Phe Leu Glu Ile Thr Ser Ile Ala Arg Ile
245 250 255Pro Gly Glu Arg Cys Lys Val Ala Ile Arg Arg Asn Glu Asp Ala Glu
260 265 270Ala Asp Asn Ile Ser Glu Ile Gly Ser Ile Val Gly Ala Thr Gly Ser
275 280 285Arg Val Leu Ala Ile Ser Gln Glu Leu Gln Gly Glu Lys Ile Glu Val
290 295 300Ile Lys Tyr Asp Asp Asn Ile Val Lys Phe Ile Val Asn Ala Met Ser305 310 315 320Pro Ser Lys Val Ile Cys Val Lys Glu Phe Lys Ile Gly His Lys Leu
325 330 335Arg Arg Phe Ile Val Val Val Pro Asp Phe Gln His Ser Leu Ala Ile
340 345 350Gly Lys Asn Gly Ser Asn Val Lys Leu Val Ala Asp Leu Thr Arg Cys
355 360 365Gln Val Gln Ile Ile Pro Tyr Ser Ser Ala Leu Lys Asp Gln Asn Phe
370 375 380Lys Ile Glu Trp Asn Gly Asn Ile Lys Asp Ile Gln Glu Leu Asn Ser385 390 395 400Leu Asn Asn Glu Tyr Ile His Arg Gln Gln Gly Arg Ile Tyr Gln Asn
405 410 415His Arg Asn Ser Tyr Gly Gln Gly Asn Asn Asn Phe Asp Leu Ile Leu
420 425 430Gln Gln Phe Glu Ser Asp Ile Arg Glu Leu Glu Lys Pro Tyr Gly Ile
435 440 445Glu Asn Glu Phe Ile Pro Lys Asn Glu Gln Lys Gln Val Arg Ser His
450 455 460Gln Glu Phe Pro Lys Asn Lys Asn Asn Leu Ala Lys Ala Ala Thr Lys465 470 475 480Ser Arg Asn Phe Asn Lys Ser Gln Asn Ile Arg Glu Asn Ile Ser Lys
485 490 495Asp Phe Asp Tyr Gly Phe Glu Asn Glu Lys Asp Ser Asn Ser Phe Gln
500 505 510Asn Ile Ser Gln Arg Ser Phe Phe Asp Ala Asp Ser Leu Phe Asp Ser
515 520 525Ala Leu Asn Glu Ala Ile Ser Glu Asn Glu Leu Ile Asp Lys Ile His
530 535 540Gln Glu Glu Glu Lys Lys Gln Glu Leu Leu Leu Gln Glu Lys Glu Lys545 550 555 560Trp Ala Lys Asn Glu Ala
565<210>33<211>1860<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD774<400>33gaattctacc tttgatcacc taatgcaaat cgggtacatt ttgcaattta taaagatccg 60gaagacaaaa ttccggctga aattattgtg atgtcaaaaa ataatgatgt ttggttttgc 120caaattaatg cctcttttaa tggatattcc tataatttat taattgagca tcacgattta 180aaaataactg aggcacttga tccttatgcc tttagtattg cgccttttga ttgaaaaaaa 240aatgaaagtc caaaagcata tttaattgac attttttccg aaaaaactgg aaaaaatcct 300tcaaaattag aaggatttaa caaaaatccg caaattgatg ctcaaattta tcagctgcac 360attcgagatt tttcatctat tagtaaaaaa acagaaaata aaggtacttt tatcggagcg 420ctagaaaatg atgtttttag ttatttaaat agcttaaaat ttaatttttt acaattatta 480ccaatccact cttgttataa tttcagccaa aaaaacgcta gcatcctcca caaaggggat 540ggaaacggtc attttagcac ttataattgg ggttatgacc caattggtta cttttcgata 600aattcaagtt attcaacaga tccaatggat ccatatctgc gaatttttga gtttaaaaaa 660tttgttgact ccgcccataa aaataagatc ggaattgttc ttgatgttga ttttagtcac 720acctttaaaa attcaattct tgaggatgta gctcacgggc atttttaccg ggatgaagca 780gctgttttac ctgccggatt tccgccactt gatacacgaa aaccaatggc atttaggcta 840attttggatt ccttaatttt ttttactaaa tattataaag ttgatggatt tcgctttaat 900ttagcatctt ttttagataa aaaagcaatt acagttattg ccagtgaact aaaaaaagtt 960aatccaaata ttcttttata tggtgatttt tcaaatccta gtgacctacc aagcagaaat 1020cgacttgaaa aagggaaaac aggaaatagt tttaactttg gatatttaaa cgatacaatc 1080caaacagcaa ttatcgggag cggaaatccg cgtgataaag gtttaatttt atcaaaaact 1140agtaaaaaat tcgctgctta tgtttcttca attccgggga acattgcaaa ttttgacttc 1200caaaatttgc catattcaaa aaaaaaatac gacctttttg caaacgatat cagcctaaat 1260cttgcctatc ttacttgtta taatggcccg actttagccg ataaaattct tagtgcgaca 1320acgcgaattg gaaaaagaga atttcttgaa atctaccggc aagccttaat gatggtcaat 1380tttgtccaag gaaaaatttc acttagtgct gggactgaat ttgctttttc aagaatttgt 1440gatttttctg ggggcagtta tcaaaattgc tatcctaatt taaacataaa aagaccgcct 1500ttttcgtttt tagcgggcaa atatcttgat tttcattctg ataaaaccac agatttcaca 1560aatggattga attttgaaat tcttaaaaac aatgaaatca aagagaaaat ctttgatttt 1620cttgccgaaa ttaatcaatt tcgccaaaat tctccatttt ttcggcttga tacaaaccaa 1680aaaatcaaaa aacagctaaa atttgaaact gttgataaca ataaaggatt aattatcttt 1740aaaattctgc taaaaataaa gtgattaaag ttattcataa tttttcacat ctttcttatg 1800aatatgattt taaaaatttt aatattcttt ttagctcaaa gattaaagtt attcctaatt 1860<210>34<211>620<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD774的推定的蛋白质序列<400>34Glu Phe Tyr Leu Trp Ser Pro Asn Ala Asn Arg Val His Phe Ala Ile1 5 10 15Tyr Lys Asp Pro Glu Asp Lys Ile Pro Ala Glu Ile Ile Val Met Ser
20 25 30Lys Asn Asn Asp Val Trp Phe Cys Gln Ile Asn Ala Ser Phe Asn Gly
35 40 45Tyr Ser Tyr Asn Leu Leu Ile Glu His His Asp Leu Lys Ile Thr Glu
50 55 60Ala Leu Asp Pro Tyr Ala Phe Ser Ile Ala Pro Phe Asp Trp Lys Lys65 70 75 80Asn Glu Ser Pro Lys Ala Tyr Leu Ile Asp Ile Phe Ser Glu Lys Thr
85 90 95Gly Lys Asn Pro Ser Lys Leu Glu Gly Phe Asn Lys Asn Pro Gln Ile
100 105 110Asp Ala Gln Ile Tyr Gln Leu His Ile Arg Asp Phe Ser Ser Ile Ser
115 120 125Lys Lys Thr Glu Asn Lys Gly Thr Phe Ile Gly Ala Leu Glu Asn Asp
130 135 140Val Phe Ser Tyr Leu Asn Ser Leu Lys Phe Asn Phe Leu Gln Leu Leu145 150 155 160Pro Ile His Ser Cys Tyr Asn Phe Ser Gln Lys Asn Ala Ser Ile Leu
165 170 175His Lys Gly Asp Gly Asn Gly His Phe Ser Thr Tyr Asn Trp Gly Tyr
180 185 190Asp Pro Ile Gly Tyr Phe Ser Ile Asn Ser Ser Tyr Ser Thr Asp Pro
195 200 205Met Asp Pro Tyr Leu Arg Ile Phe Glu Phe Lys Lys Phe Val Asp Ser
210 215 220Ala His Lys Asn Lys Ile Gly Ile Val Leu Asp Val Asp Phe Ser His225 230 235 240Thr Phe Lys Asn Ser Ile Leu Glu Asp Val Ala His Gly His Phe Tyr
245 250 255Arg Asp Glu Ala Ala Val Leu Pro Ala Gly Phe Pro Pro Leu Asp Thr
260 265 270Arg Lys Pro Met Ala Phe Arg Leu Ile Leu Asp Ser Leu Ile Phe Phe
275 280 285Thr Lys Tyr Tyr Lys Val Asp Gly Phe Arg Phe Asn Leu Ala Ser Phe
290 295 300Leu Asp Lys Lys Ala Ile Thr Val Ile Ala Ser Glu Leu Lys Lys Val305 310 315 320Asn Pro Asn Ile Leu Leu Tyr Gly Asp Phe Ser Asn Pro Ser Asp Leu
325 330 335Pro Ser Arg Asn Arg Leu Glu Lys Gly Lys Thr Gly Asn Ser Phe Asn
340 345 350Phe Gly Tyr Leu Asn Asp Thr Ile Gln Thr Ala Ile Ile Gly Ser Gly
355 360 365Asn Pro Arg Asp Lys Gly Leu Ile Leu Ser Lys Thr Ser Lys Lys Phe
370 375 380Ala Ala Tyr Val Ser Ser Ile Pro Gly Asn Ile Ala Asn Phe Asp Phe385 390 395 400Gln Asn Leu Pro Tyr Ser Lys Lys Lys Tyr Asp Leu Phe Ala Asn Asp
405 410 415Ile Ser Leu Asn Leu Ala Tyr Leu Thr Cys Tyr Asn Gly Pro Thr Leu
420 425 430Ala Asp Lys Ile Leu Ser Ala Thr Thr Arg Ile Gly Lys Arg Glu Phe
435 440 445Leu Glu Ile Tyr Arg Gln Ala Leu Met Met Val Asn Phe Val Gln Gly
450 455 460Lys Ile Set Leu Set Ala Gly Thr Glu Phe Ala Phe Ser Arg Ile Cys465 470 475 480Asp Phe Ser Gly Gly Ser Tyr Gln Asn Cys Tyr Pro Asn Leu Asn Ile
485 490 495Lys Arg Pro Pro Phe Ser Phe Leu Ala Gly Lys Tyr Leu Asp Phe His
500 505 510Ser Asp Lys Thr Thr Asp Phe Thr Asn Gly Leu Asn Phe Glu Ile Leu
515 520 525Lys Asn Asn Glu Ile Lys Glu Lys Ile Phe Asp Phe Leu Ala Glu Ile
530 535 540Asn Gln Phe Arg Gln Asn Ser Pro Phe Phe Arg Leu Asp Thr Asn Gln545 550 555 560Lys Ile Lys Lys Gln Leu Lys Phe Glu Thr Val Asp Asn Asn Lys Gly
565 570 575Leu Ile Ile Phe Lys Ile Leu Leu Lys Ile Lys Trp Leu Lys Leu Phe
580 585 590Ile Ile Phe His Ile Phe Leu Met Asn Met Ile Leu Lys Ile Leu Ile
595 600 605Phe Phe Leu Ala Gln Arg Leu Lys Leu Phe Leu Ile
610 615 620<210>35<211>897<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD784<400>35gcttttttgt ttgttgatgg tcggtatatt gaaaaagctg aaaaagatgc taaaaattgt 60caggtttttt tacctaccaa gcaaatcttg aagatttttt caagaaaacc cgtatcaaaa 120aatcggcatt gatctgaata tttaactatt gaccaatttg ataaaataag aagttgattt 180ccaaatgccg attttgttaa gttacaagcc caactttttc gaattataaa aacagaagaa 240gaaatcaaaa atatcgaaaa agctgttgaa atctcactcg cggcttataa taaaatattt 300ccaaaaatca aaccgggaat gacggagaaa agtatcgatg tcaacctaaa ttatcaaatg 360aagcttttag gagccgaaaa agaatccttt gattcaataa ttgcaactgg ttctaattcg 420gcaatgccgc attgaagggc gagtgaaacc gaaattttag ataatgatct tttaaaaatt 480gattttggtg cgctttttaa cggttattgc gctgatatta caagaacttc ttatcttgga 540cagattagtg aaaaaaaatt agaaattttg gaaatagtaa aaaaagctgc tgaaattggt 600agaaaaaaag ttgctcctgg ggttaaagcc agcgaaattg accttgcttg ccggaatttt 660atcaccgaac aaggctatgg aaaatatttt attcactcaa ctggccacgg ggttggtatt 720gatatccatg aattgccagt tgttagttca actagccaga caattttaga gcccggaatg 780gtaataactg ttgaacccgg aatttatatc cctggacttg gaggcgcaag aattgaggat 840gttgttttag taactgaaag tggttttcgt accttgtcac gaaaaggtga aagaatt 897<210>36<211>299<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD784的推定的蛋白质序列<400>36Ala Phe Leu Phe Val Asp Gly Arg Tyr Ile Glu Lys Ala Glu Lys Asp1 5 10 15Ala Lys Asn Cys Gln Val Phe Leu Pro Thr Lys Gln Ile Leu Lys Ile
20 25 30Phe Ser Arg Lys Pro Val Ser Lys Asn Arg His Trp Ser Glu Tyr Leu
35 40 45Thr Ile Asp Gln Phe Asp Lys Ile Arg Ser Trp Phe Pro Asn Ala Asp
50 55 60Phe Val Lys Leu Gln Ala Gln Leu Phe Arg Ile Ile Lys Thr Glu Glu65 70 75 80Glu Ile Lys Asn Ile Glu Lys Ala Val Glu Ile Ser Leu Ala Ala Tyr
85 90 95Asn Lys Ile Phe Pro Lys Ile Lys Pro Gly Met Thr Glu Lys Ser Ile
100 105 110Asp Val Asn Leu Asn Tyr Gln Met Lys Leu Leu Gly Ala Glu Lys Glu
115 120 125Ser Phe Asp Ser Ile Ile Ala Thr Gly Ser Asn Ser Ala Met Pro His
130 135 140Trp Arg Ala Ser Glu Thr Glu Ile Leu Asp Asn Asp Leu Leu Lys Ile145 150 155 160Asp Phe Gly Ala Leu Phe Asn Gly Tyr Cys Ala Asp Ile Thr Arg Thr
165 170 175Ser Tyr Leu Gly Gln Ile Ser Glu Lys Lys Leu Glu Ile Leu Glu Ile
180 185 190Val Lys Lys Ala Ala Glu Ile Gly Arg Lys Lys Val Ala Pro Gly Val
195 200 205Lys Ala Ser Glu Ile Asp Leu Ala Cys Arg Asn Phe Ile Thr Glu Gln
210 215 220Gly Tyr Gly Lys Tyr Phe Ile His Ser Thr Gly His Gly Val Gly Ile225 230 235 240Asp Ile His Glu Leu Pro Val Val Ser Ser Thr Ser Gln Thr Ile Leu
245 250 255Glu Pro Gly Met Val Ile Thr Val Glu Pro Gly Ile Tyr Ile Pro Gly
260 265 270Leu Gly Gly Ala Arg Ile Glu Asp Val Val Leu Val Thr Glu Ser Gly
275 280 285Phe Arg Thr Leu Ser Arg Lys Gly Glu Arg Ile
290 295<210>37<211>633<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD789<400>37gatcttgtat ttaaagtaga aaattctgaa aatcaattac aagatttaga tggaactttt 60tctttaatta gtattaaaaa tttaaactat aaattggaag atagagtttt atttaataat 120ttaaatttag aagttcaaaa aggtaaaaaa tatttactaa aaggagctaa cgggtctgga 180aagtccacat tttcaaggat tttattaggc attgagaagg aatttgaagg tcaaatttta 240ataaataaca aatacgatat aaaaaaaata aatcctgatt ctataaataa ccatattaat 300tatgtataca acaattcaga cttaattaat gcatcaactc tagaaaatat ttcgcttttg 360gaaccgaaaa caaaagatga gattaggccg ttattagaaa aggtaaattt tgaaaacctt 420gatttagaca agaaaattga ttctgatgtt tgattatttt tccactgggc aaatccaaaa 480aatccccttg cacgctcact ttattctcca aaagaaattt taataattga cgaaggtctt 540tccaacttag accaagaaag ttatgttaaa atcatatctg aacttattgc ggataaaaat 600ttaacattaa ttttcattac ccctcacttt gat 633<210>38<211>211<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD789的推定的蛋白质序列<400>38Asp Leu Val Phe Lys Val Glu Asn Ser Glu Asn Gln Leu Gln Asp Leu1 5 10 15Asp Gly Thr Phe Ser Leu Ile Ser Ile Lys Asn Leu Asn Tyr Lys Leu
20 25 30Glu Asp Arg Val Leu Phe Asn Asn Leu Asn Leu Glu Val Gln Lys Gly
35 40 45Lys Lys Tyr Leu Leu Lys Gly Ala Asn Gly Ser Gly Lys Ser Thr Phe
50 55 60Ser Arg Ile Leu Leu Gly Ile Glu Lys Glu Phe Glu Gly Gln Ile Leu65 70 75 80Ile Asn Asn Lys Tyr Asp Ile Lys Lys Ile Asn Pro Asp Ser Ile Asn
85 90 95Asn His Ile Asn Tyr Val Tyr Asn Asn Ser Asp Leu Ile Asn Ala Ser
100 105 110Thr Leu Glu Asn Ile Ser Leu Leu Glu Pro Lys Thr Lys Asp Glu Ile
115 120 125Arg Pro Leu Leu Glu Lys Val Asn Phe Glu Asn Leu Asp Leu Asp Lys
130 135 140Lys Ile Asp Ser Asp Val Trp Leu Phe Phe His Trp Ala Asn Pro Lys145 150 155 160Asn Pro Leu Ala Arg Ser Leu Tyr Ser Pro Lys Glu Ile Leu Ile Ile
165 170 175Asp Glu Gly Leu Ser Asn Leu Asp Gln Glu Ser Tyr Val Lys Ile Ile
180 185 190Ser Glu Leu Ile Ala Asp Lys Asn Leu Thr Leu Ile Phe Ile Thr Pro
195 200 205His Phe Asp
210<210>39<211>1791<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD908,
pAD981,pADl013,和pAD1049<400>39cttgaaataa tcaaatatgg atcaaaagaa tctctaaatt actgactaat ttctgaaagc 60gggaaaagat atgactattt tcgaccaatt gaaggaattc ttaataaaat tcagagaaaa 120ttttgggaga catcaagcga agatttacga ctttgattta agaaaatgat gtcagaattt 180ccttgtagta gttgcaaagg agcccggctt aacaagtatg cgcttgccgt tttcattgaa 240aagtataata tctttcaatt atcccaactt tcaattaaag atttaataac tttttttaga 300aatttaaaat taactgaatt tgacggaaaa atttctactt taattctcga tgaaattaaa 360tcacgactgt catttttagc aaatgttggt cttgaatatt taactttaaa tagatcaacg 420gcaaccttat caggaggcga atcccaacga attaggcttg caagccaggt tggatatcaa 480ctaaccggaa ttctttatgt tcttgatgaa ccttcaattg gcctacatca aaaagataat 540gacaaattaa ttgcgacact gaaaaaaatg gttgaaattg gtaatagttt aattgtagtc 600gagcatgatt ttgagacaat tttagctgct gattatattg ttgatatcgg gccaaaagct 660ggtgaaaacg gtggtttttt ggttgctgca ggatcaatta aggatattga aaatgaacca 720aaatcactta ccggccaatt tttaactgga aaattggaaa ttccagtacc aaaaaaacga 780cgggctggca atggcaaatt tataattatt gaaaaagctg ctgaaaataa tttaaaaaaa 840attaatgtca acattcctct aggcaaattt gttgttgtca ctggtgtttc tggatctgga 900aagtcgacat tagttaatca aattatcgta aatgcgattg ccaaaaatct tggaacaact 960aatattcgca ttgggaaaaa tgtgaggaaa ttaaagggct ttttaatatt gataagttga 1020ttgcaatcaa tcaaagtcaa tcggacgaac cctagatcaa atcccgcaac ttatacctct 1080gtttttgatg atatccgtga ggtttttgcc aatactgagc aggcaagagc gcttggtttt 1140tcaaagtcaa aattttcctt taatctgcaa actgggcggt gtgataaatg ccaaggagac 1200gggcaaatta aaattgaaat gtattttatg cctgatattt atgttttatg tgatcactgc 1260caaggaaaaa gatacaagcc ggatgtccta caaattcgtt tttatggaaa aacaatcgcg 1320gatattcttg atttaacagt ttcagaagca cttgaatttt tccataattg gcctaaaata 1380atcgcaaaat tacaaaccct agctgatgtt ggtcttggtt atataaaact tggccaatca 1440gcaataactt tatcaggagg agaagctcaa cgaattaaat tagccacttt tttacaaaaa 1500aaacctaaag gaaaatcact ttttgtactt gacgagccaa caactggact ccataattat 1560gatgttgcta atttaattaa agtgctaaat cgaatagtcg ataacggtga tagcataatt 1620gtaatcgagc ataatttaga ggtaattaaa gttgctgact atattattga tttaggccca 1680aacggcgggg ataacggggg ccaaatagtt gcaaaaggaa caccagaagc tgtagcaaaa 1740gttagtgaat catatactgg cgcttattta aaaacaattt taaatataaa a 1791<210>40<211>597<212>PRT<213>人工序列<220><223>人工序列的描述:克隆克隆pAD908,pAD981,pAD1013和pAD1049的推定的蛋白质序列<400>40Leu Glu Ile Ile Lys Tyr Gly Ser Lys Glu Ser Leu Asn Tyr Trp Leu 1 5 10 15Ile Ser Glu Ser Gly Lys Arg Tyr Asp Tyr Phe Arg Pro Ile Glu Gly
20 25 30Ile Leu Asn Lys Ile Gln Arg Lys Phe Trp Glu Thr Ser Ser Glu Asp
35 40 45Leu Arg Leu Trp Phe Lys Lys Met Met Set Glu Phe Pro Cys Set Set
50 55 60Cys Lys Gly Ala Arg Leu Asn Lys Tyr Ala Leu Ala Val Phe Ile Glu65 70 75 80Lys Tyr Asn Ile Phe Gln Leu Ser Gln Leu Ser Ile Lys Asp Leu Ile
85 90 95Thr Phe Phe Arg Asn Leu Lys Leu Thr Glu Phe Asp Gly Lys Ile Ser
100 105 110Thr Leu Ile Leu Asp Glu Ile Lys Ser Arg Leu Ser Phe Leu Ala Asn
115 120 125Val Gly Leu Glu Tyr Leu Thr Leu Asn Arg Ser Thr Ala Thr Leu Ser
130 135 140Gly Gly Glu Ser Gln Arg Ile Arg Leu Ala Ser Gln Val Gly Tyr Gln145 150 155 160Leu Thr Gly Ile Leu Tyr Val Leu Asp Glu Pro Ser Ile Gly Leu His
165 170 175Gln Lys Asp Asn Asp Lys Leu Ile Ala Thr Leu Lys Lys Met Val Glu
180 185 190Ile Gly Asn Ser Leu Ile Val Val Glu His Asp Phe Glu Thr Ile Leu
195 200 205Ala Ala Asp Tyr Ile Val Asp Ile Gly Pro Lys Ala Gly Glu Asn Gly
210 215 220Gly Phe Leu Val Ala Ala Gly Ser Ile Lys Asp Ile Glu Asn Glu Pro225 230 235 240Lys Ser Leu Thr Gly Gln Phe Leu Thr Gly Lys Leu Glu Ile Pro Val
245 250 255Pro Lys Lys Arg Arg Ala Gly Asn Gly Lys Phe Ile Ile Ile Glu Lys
260 265 270Ala Ala Glu Asn Asn Leu Lys Lys Ile Asn Val Asn Ile Pro Leu Gly
275 280 285Lys Phe Val Val Val Thr Gly Val Ser Gly Ser Gly Lys Ser Thr Leu
290 295 300Val Asn Gln Ile Ile Val Asn Ala Ile Ala Lys Asn Leu Gly Thr Thr305 310 315 320Asn Ile Arg Ile Gly Lys Asn Val Arg Lys Leu Lys Gly Phe Leu Ile
325 330 335Leu Ile Ser Trp Leu Gln Ser Ile Lys Val Asn Arg Thr Asn Pro Arg
340 345 350Ser Asn Pro Ala Thr Tyr Thr Ser Val Phe Asp Asp Ile Arg Glu Val
355 360 365Phe Ala Asn Thr Glu Gln Ala Arg Ala Leu Gly Phe Ser Lys Ser Lys
370 375 380Phe Ser Phe Asn Leu Gln Thr Gly Arg Cys Asp Lys Cys Gln Gly Asp385 390 395 400Gly Gln Ile Lys Ile Glu Met Tyr Phe Met Pro Asp Ile Tyr Val Leu
405 410 415Cys Asp His Cys Gln Gly Lys Arg Tyr Lys Pro Asp Val Leu Gln Ile
420 425 430Arg Phe Tyr Gly Lys Thr Ile Ala Asp Ile Leu Asp Leu Thr Val Ser
435 440 445Glu Ala Leu Glu Phe Phe His Asn Trp Pro Lys Ile Ile Ala Lys Leu
450 455 460Gln Thr Leu Ala Asp Val Gly Leu Gly Tyr Ile Lys Leu Gly Gln Ser465 470 475 480Ala Ile Thr Leu Ser Gly Gly Glu Ala Gln Arg Ile Lys Leu Ala Thr
485 490 495Phe Leu Gln Lys Lys Pro Lys Gly Lys Ser Leu Phe Val Leu Asp Glu
500 505 510Pro Thr Thr Gly Leu His Asn Tyr Asp Val Ala Asn Leu Ile Lys Val
515 520 525Leu Asn Arg Ile Val Asp Asn Gly Asp Ser Ile Ile Val Ile Glu His
530 535 540Asn Leu Glu Val Ile Lys Val Ala Asp Tyr Ile Ile Asp Leu Gly Pro545 550 555 560Asn Gly Gly Asp Asn Gly Gly Gln Ile Val Ala Lys Gly Thr Pro Glu
565 570 575Ala Val Ala Lys Val Ser Glu Ser Tyr Thr Gly Ala Tyr Leu Lys Thr
580 585 590Ile Leu Asn Ile Lys
595<210>41<211>900<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD913<400>41atgaaaatta aagcaaaaac catcgtaaaa atttatgatc aaaaattacc atcagaatta 60aaagcccttg ataaagtaac tactgaaata aatcagggcg agtttattgc aataattggc 120caaactggtt caggaaaaac aacttttatt cagcatatga atgcactttt gctaccagat 180caaggcgaaa ttgagtatct ctattttgat tcaaaaaatc aagaaaaaaa attagttgtt 240caaaaaccgc gtttttttag aaaaaaacta aaatttatta atgaaattcg tcggcgtgtg 300ggcgtcgttt ttcagtttgc tgaatatcag ctttttgagc aaacaattga aaaagacatc 360atatttgggg ctgtttcaat gggaactcca aaaaatgagg caaaaaaaat tgccgcagaa 420ataattgaat tagttggtct tgatcaaagt tttttacaaa aatcaccttt tgaactttca 480ggtggccaga aacgccgagt tgcaattgcc ggaattttag caatggatcc tgatattatt 540ttttttgatg aacccacggc cggacttgat ccccaaggaa cgctaaaaat gcttgaaatt 600cttgatactt tatataaaaa gggcaagaca atcattctgg caactcatga tcttgatagt 660gttttagaat gaacaaaacg ttgtattttt tttaaagatg gtagaattat ttatgatggt 720gatacttatt caattttagc aaataataaa tttttaattg aaaataagat gttaccaact 780aatttactca attttcgcga aaaattaatc aaaattggtt atccaatttc taatgttaga 840tcagtatctg agttaatcag tgaaattaat atgctaattc aaaaggaaac aaatgcagat 900<210>42<211>300<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD913的推定的蛋白质序列<400>42Met Lys Ile Lys Ala Lys Thr Ile Val Lys Ile Tyr Asp Gln Lys Leu1 5 10 15Pro Ser Glu Leu Lys Ala Leu Asp Lys Val Thr Thr Glu Ile Asn Gln
20 25 30Gly Glu Phe Ile Ala Ile Ile Gly Gln Thr Gly Ser Gly Lys Thr Thr
35 40 45Phe Ile Gln His Met Asn Ala Leu Leu Leu Pro Asp Gln Gly Glu Ile
50 55 60Glu Tyr Leu Tyr Phe Asp Ser Lys Asn Gln Glu Lys Lys Leu Val Val65 70 75 80Gln Lys Pro Arg Phe Phe Arg Lys Lys Leu Lys Phe Ile Asn Glu Ile
85 90 95Arg Arg Arg Val Gly Val Val Phe Gln Phe Ala Glu Tyr Gln Leu Phe
100 105 110Glu Gln Thr Ile Glu Lys Asp Ile Ile Phe Gly Ala Val Ser Met Gly
115 120 125Thr Pro Lys Asn Glu Ala Lys Lys Ile Ala Ala Glu Ile Ile Glu Leu
130 135 140Val Gly Leu Asp Gln Ser Phe Leu Gln Lys Ser Pro Phe Glu Leu Ser145 150 155 160Gly Gly Gln Lys Arg Arg Val Ala Ile Ala Gly Ile Leu Ala Met Asp
165 170 175Pro Asp Ile Ile Phe Phe Asp Glu Pro Thr Ala Gly Leu Asp Pro Gln
180 185 190Gly Thr Leu Lys Met Leu Glu Ile Leu Asp Thr Leu Tyr Lys Lys Gly
195 200 205Lys Thr Ile Ile Leu Ala Thr His Asp Leu Asp Ser Val Leu Glu Trp
210 215 220Thr Lys Arg Cys Ile Phe Phe Lys Asp Gly Arg Ile Ile Tyr Asp Gly225 230 235 240Asp Thr Tyr Ser Ile Leu Ala Asn Asn Lys Phe Leu Ile Glu Asn Lys
245 250 255Met Leu Pro Thr Asn Leu Leu Asn Phe Arg Glu Lys Leu Ile Lys Ile
260 265 270Gly Tyr Pro Ile Ser Asn Val Arg Ser Val Ser Glu Leu Ile Ser Glu
275 280 285Ile Asn Met Leu Ile Gln Lys Glu Thr Asn Ala Asp
290 295 300<210>43<211>489<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD920<400>43ttaaaatccc gtttttatca aaaagtaaat tcgcagatag acgttaaaaa aaacacagat 60caagaaaaag ataaaaaaac tgagcccgaa aaaattaatt tttatactct gaaaaaagta 120atttttcctg aaagtttgct tgagattgat gattatgcct tttatgtcga tagtgcaaat 180ttagatcaaa atgaaaaaat tcaagaactt gatttttcaa aagcaatgaa attaaggagg 240attgggagtt tcgcttttca aggaaacaat ataaaaacgc tcgttttacc cccttctatt 300acatcaattg gaaaacaagc ttttgcaaaa aacagtttag aaacagtaga tttttcccag 360gcaacaaaac tagaaacaat tgaacctggt gccttttttg ataataaaat caccgaactt 420gatttgtcaa aaaatttgat tttagccgaa attttcaggg tagttttgag acaaatcaaa 480ttatcaagt 489<210>44<211>163<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD920的推定的蛋白质序列<400>44Leu Lys Ser Arg Phe Tyr Gln Lys Val Asn Ser Gln Ile Asp Val Lys1 5 10 15Lys Asn Thr Asp Gln Glu Lys Asp Lys Lys Thr Glu Pro Glu Lys Ile
20 25 30Asn Phe Tyr Thr Leu Lys Lys Val Ile Phe Pro Glu Ser Leu Leu Glu
35 40 45Ile Asp Asp Tyr Ala Phe Tyr Val Asp Ser Ala Asn Leu Asp Gln Asn
50 55 60Glu Lys Ile Gln Glu Leu Asp Phe Ser Lys Ala Met Lys Leu Arg Arg65 70 75 80Ile Gly Ser Phe Ala Phe Gln Gly Asn Asn Ile Lys Thr Leu Val Leu
85 90 95Pro Pro Ser Ile Thr Ser Ile Gly Lys Gln Ala Phe Ala Lys Asn Ser
100 105 110Leu Glu Thr Val Asp Phe Ser Gln Ala Thr Lys Leu Glu Thr Ile Glu
115 120 125Pro Gly Ala Phe Phe Asp Asn Lys Ile Thr Glu Leu Asp Leu Ser Lys
130 135 140Asn Leu Ile Leu Ala Glu Ile Phe Arg Val Val Leu Arg Gln Ile Lys145 150 155 160Leu Ser Ser<210>45<211>1101<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD922<400>45actgatcaga tcttaatttt ccatttggcc aaaactttag accaaaaata tcttgaaatt 60gaccttgaaa tgctcgaaaa aggtaacttt gaatttcaag atttcattaa tttctggcaa 120tctcggattg aaaaaataga agaaaattta gcgcaaattt ctaccgataa aatcacagag 180gcaaaaatca acgaattttt taattcttat ttgctttatt ttgaaaaatt acaaaaatta 240tttagctcat catataatct tggctatgaa aatgtggcca aattatatga ttatttctat 300gaagtccaaa aaatttaccg acaaaaacag caagcaaaag tcgaatttga ctaccgcagt 360gctaaaaaag attatgaaga ccagctaaaa aaaataaagc aagaaaaagc ttttttcatt 420aaaacattaa atgtaaaagc gcttaattta aaaaaagagg cccaactcga gattgacaaa 480ttcaccgctc aaaacaattt gttgacttcc tatattgacg aatttaatta tgaatataaa 540attgcaaata acaaagcgct agtaacaaca gatctaaaaa attattcatt ttttaaaaaa 600caagcaataa tcaataagga aattgccaaa tttcttgata ggagaaatat tttgttactt 660gaaaaaaacc ttttttcctt tcttaatatt tctgagattg aaaaattatt tgaaattatg 720aataatttca aaaaaagtca aattgaaaag tataaaagtt tgactttcga taaaaaagat 780gaaaaaaatt atacaaatac aaaattattt agtcaattaa tccgtaccga aattatcatt 840ttggatattc aaggtttaaa agaaattgcc caaaatcgta aaaaaactta tcaagaaaaa 900gtaaattttc aaacaaaatt tctccaattt aaaaataaat attcatataa taaaaaaaga 960agtagtccgc aagccgaaaa tctagaaaaa ctcaatgaat taaaagaaaa actggcccaa 1020aaagaagcaa tttatgaaga agaaaaagac ctttttatta gaaaatatac ctcttggaaa 1080acaaaaccag agcaaaaaaa t 1101<210>46<211>367<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD922的推定的蛋白质序列<400>46Thr Asp Gln Ile Leu Ile Phe His Leu Ala Lys Thr Leu Asp Gln Lys1 5 10 15Tyr Leu Glu Ile Asp Leu Glu Met Leu Glu Lys Gly Asn Phe Glu Phe
20 25 30Gln Asp Phe Ile Asn Phe Trp Gln Ser Arg Ile Glu Lys Ile Glu Glu
35 40 45Asn Leu Ala Gln Ile Ser Thr Asp Lys Ile Thr Glu Ala Lys Ile Asn
50 55 60Glu Phe Phe Asn Ser Tyr Leu Leu Tyr Phe Glu Lys Leu Gln Lys Leu65 70 75 80Phe Ser Ser Ser Tyr Asn Leu Gly Tyr Glu Asn Val Ala Lys Leu Tyr
85 90 95Asp Tyr Phe Tyr Glu Val Gln Lys Ile Tyr Arg Gln Lys Gln Gln Ala
100 105 110Lys Val Glu Phe Asp Tyr Arg Ser Ala Lys Lys Asp Tyr Glu Asp Gln
115 120 125Leu Lys Lys Ile Lys Gln Glu Lys Ala Phe Phe Ile Lys Thr Leu Asn
130 135 140Val Lys Ala Leu Asn Leu Lys Lys Glu Ala Gln Leu Glu Ile Asp Lys145 150 155 160Phe Thr Ala Gln Asn Asn Leu Leu Thr Ser Tyr Ile Asp Glu Phe Asn
165 170 175Tyr Glu Tyr Lys Ile Ala Asn Asn Lys Ala Leu Val Thr Thr Asp Leu
180 185 190Lys Asn Tyr Ser Phe Phe Lys Lys Gln Ala Ile Ile Asn Lys Glu Ile
195 200 205Ala Lys Phe Leu Asp Arg Arg Asn Ile Leu Leu Leu Glu Lys Asn Leu
210 215 220Phe Ser Phe Leu Asn Ile Ser Glu Ile Glu Lys Leu Phe Glu Ile Met225 230 235 240Asn Asn Phe Lys Lys Ser Gln Ile Glu Lys Tyr Lys Ser Leu Thr Phe
245 250 255Asp Lys Lys Asp Glu Lys Asn Tyr Thr Asn Thr Lys Leu Phe Ser Gln
260 265 270Leu Ile Arg Thr Glu Ile Ile Ile Leu Asp Ile Gln Gly Leu Lys Glu
275 280 285Ile Ala Gln Asn Arg Lys Lys Thr Tyr Gln Glu Lys Val Asn Phe Gln
290 295 300Thr Lys Phe Leu Gln Phe Lys Asn Lys Tyr Ser Tyr Asn Lys Lys Arg305 310 315 320Ser Ser Pro Gln Ala Glu Asn Leu Glu Lys Leu Asn Glu Leu Lys Glu
325 330 335Lys Leu Ala Gln Lys Glu Ala Ile Tyr Glu Glu Glu Lys Asp Leu Phe
340 345 350Ile Arg Lys Tyr Thr ser Trp Lys Thr Lys Pro Glu Gln Lys Asn
355 360 365<210>47<211>438<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD923和pAD925<400>47tataatttaa aaaaagaaac taatttgagg aaatttttaa tgtcaaaaaa atctaaaaat 60tcaagcattg aatttgatgc tattgttgtc ggtggcggcc atgctgggat cgaagcagtt 120tatgcattat taaaaaaaaa gttaaaagtt gttctaataa ctcttgataa gaaaaaatta 180gcttcaatgc cttgtaatcc cgcaattggt gggccagcaa aaggaattat aactcgcgag 240atcgatgccc ttggaggagt tcagggaaaa ttttcagatt tagcaatgat ccaaattaaa 300tatttaaatg aatcaaaagg tcctgccgtt ttagcaatta gagcccaaat tgataaggaa 360aaatattcaa aattaatatt aaaggatttg aaaaaacagg aaaatttatt aattatcgag 420gatttggtta gtgaactc 438<210>48<211>146<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD923和pAD925的推定的蛋白
质序列<400>48Tyr Asn Leu Lys Lys Glu Thr Asn Leu Arg Lys Phe Leu Met Ser Lys1 5 10 15Lys Ser Lys Asn Ser Ser Ile Glu Phe Asp Ala Ile Val Val Gly Gly
20 25 30Gly His Ala Gly Ile Glu Ala Val Tyr Ala Leu Leu Lys Lys Lys Leu
35 40 45Lys Val Val Leu Ile Thr Leu Asp Lys Lys Lys Leu Ala Ser Met Pro
50 55 60Cys Asn Pro Ala Ile Gly Gly Pro Ala Lys Gly Ile Ile Thr Arg Glu65 70 75 80Ile Asp Ala Leu Gly Gly Val Gln Gly Lys Phe Ser Asp Leu Ala Met
85 90 95Ile Gln Ile Lys Tyr Leu Asn Glu Ser Lys Gly Pro Ala Val Leu Ala
100 105 110Ile Arg Ala Gln Ile Asp Lys Glu Lys Tyr Ser Lys Leu Ile Leu Lys
115 120 125Asp Leu Lys Lys Gln Glu Asn Leu Leu Ile Ile Glu Asp Leu Val Ser
130 135 140Glu Leu145<210>49<211>1074<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD950<400>49caaattgtac aatcagaacc agaaatttta aatcaaaaat tttttttatg taaaaaaata 60ctacaggaac aaaaattaat tagtttttgc gaacaaaaat tagaaaaagc aaagaaaaat 120aaccaatttg aactcgccaa cgaatatcac aaagcgctta ttgcactgaa aaaaactaaa 180attgaacaac aaaatattga gcttaacaac ctaaaaaata ttgattttct ttattatagt 240gaaattggcg agaataattt agtaattagt tttgcttttt atcgcaatgg tgttttttta 300tctaataaaa attttattat tgatattata cttaattata cagaagtttt aattaatttt 360ttaaataatt attataaaat taatatttat cccgatgagt tagtagttaa aaatttttgg 420cctaaaaatg ctgaattttt agacccaaaa attaatatca aaattggaaa aagcttaaaa 480tataagcata ttttaaacac tttagcaaaa aatcaccaag attttatcag ccataatttt 540gaccaagaaa ttaagaaaaa aattaaaaat cagaaaattt tagaactagt taaaaccagt 600ttaaaaattg aaaatgttga aaaaattatg gcaattgact gctcaaattt agagtcaaac 660taccccacaa ctggaattat tttctatata aacggaatat atgagcgaaa ttacaataga 720tttttcaatt ataggggaac aaaaaaaggt gatacaaatt atatgagaca gggttttgaa 780aaatatatta aaaatccaaa atttctaaaa cctgatttga ttttagtaga tggaggaatt 840caacaaatta atttaattat agaaatttta agaaaaaatc actttgaaat tccgattttt 900ggaatggtaa aaaataaaag gcataaaact gaaaaaatta ttgacttaaa tggtaaaaaa 960attaacctag ctcaagaagt tcttaatttc tttgctttaa ttcaagaaaa tgtcgattta 1020tttgttaagg aaaaaatgaa gaaaaaacaa ataaaaagtt tattttctaa ggaa 1074<210>50<211>358<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD950的推定的蛋白质序列<400>50Gln Ile Val Gln Ser Glu Pro Glu Ile Leu Asn Gln Lys Phe Phe Leu1 5 10 15Cys Lys Lys Ile Leu Gln Glu Gln Lys Leu Ile Ser Phe Cys Glu Gln
20 25 30Lys Leu Glu Lys Ala Lys Lys Asn Asn Gln Phe Glu Leu Ala Asn Glu
35 40 45Tyr His Lys Ala Leu Ile Ala Leu Lys Lys Thr Lys Ile Glu Gln Gln
50 55 60Asn Ile Glu Leu Asn Asn Leu Lys Asn Ile Asp Phe Leu Tyr Tyr Ser65 70 75 80Glu Ile Gly Glu Asn Asn Leu Val Ile Ser Phe Ala Phe Tyr Arg Asn
85 90 95Gly Val Phe Leu Ser Asn Lys Asn Phe Ile Ile Asp Ile Ile Leu Asn
100 105 110Tyr Thr Glu Val Leu Ile Asn Phe Leu Asn Asn Tyr Tyr Lys Ile Asn
115 120 125Ile Tyr Pro Asp Glu Leu Val Val Lys Asn Phe Trp Pro Lys Asn Ala
130 135 140Glu Phe Leu Asp Pro Lys Ile Asn Ile Lys Ile Gly Lys Ser Leu Lys145 150 155 160Tyr Lys His Ile Leu Asn Thr Leu Ala Lys Asn His Gln Asp Phe Ile
165 170 175Ser His Asn Phe Asp Gln Glu Ile Lys Lys Lys Ile Lys Asn Gln Lys
180 185 190Ile Leu Glu Leu Val Lys Thr Ser Leu Lys Ile Glu Asn Val Glu Lys
195 200 205Ile Met Ala Ile Asp Cys Ser Asn Leu Glu Ser Asn Tyr Pro Thr Thr
210 215 220Gly Ile Ile Phe Tyr Ile Asn Gly Ile Tyr Glu Arg Asn Tyr Asn Arg225 230 235 240Phe Phe Asn Tyr Arg Gly Thr Lys Lys Gly Asp Thr Asn Tyr Met Arg
245 250 255Gln Gly Phe Glu Lys Tyr Ile Lys Asn Pro Lys Phe Leu Lys Pro Asp
260 265 270Leu Ile Leu Val Asp Gly Gly Ile Gln Gln Ile Asn Leu Ile Ile Glu
275 280 285Ile Leu Arg Lys Asn His Phe Glu Ile Pro Ile Phe Gly Met Val Lys
290 295 300Asn Lys Arg His Lys Thr Glu Lys Ile Ile Asp Leu Asn Gly Lys Lys305 310 315 320Ile Asn Leu Ala Gln Glu Val Leu Asn Phe Phe Ala Leu Ile Gln Glu
325 330 335Asn Val Asp Leu Phe Val Lys Glu Lys Met Lys Lys Lys Gln Ile Lys
340 345 350Ser Leu Phe Ser Lys Glu
355<210>51<211>282<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD951<400>51tcgaatataa ctgacaaaac aggaaaactt ttaaaaattt ctaacaataa aaatacttta 60atttttaaag ttgttggagt ttttgatcct gaaaaagatg acgaaaatat tgctattttt 120aacaataata ttgaaaaata ttctagtgaa ttacttccaa tagctgctgt ggtttatttt 180gatcatgata atttatataa taatattaat gaatttttaa ataaatatag caaaccgggc 240gttagtcgtt attactcgac aaatggcggc cggatcaaat tc 282<210>52<211>94<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD951的推定的蛋白质序列<400>52Ser Asn Ile Thr Asp Lys Thr Gly Lys Leu Leu Lys Ile Ser Asn Asn1 5 10 15Lys Asn Thr Leu Ile Phe Lys Val Val Gly Val Phe Asp Pro Glu Lys
20 25 30Asp Asp Glu Asn Ile Ala Ile Phe Asn Asn Asn Ile Glu Lys Tyr Ser
35 40 45Ser Glu Leu Leu Pro Ile Ala Ala Val Val Tyr Phe Asp His Asp Asn
50 55 60Leu Tyr Asn Asn Ile Asn Glu Phe Leu Asn Lys Tyr Ser Lys Pro Gly65 70 75 80Val Ser Arg Tyr Tyr Ser Thr Asn Gly Gly Arg Ile Lys Phe
85 90<210>53<211>438<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD977<400>53attttaatta ataattcaat tgaatataag gaattagacc caaaccagtt aagaaaacat 60attgcactaa caacaaatga aaacataatt ttcgaagaca ctttggcaaa caacataact 120ttatgagata aaaatcccga tttagatttg ctaaattctt taataaaaaa gtataaaatt 180gataattttt caaaaccaga aactgaaatt agctcaaaaa atttatctga gggcgaaaaa 240caaaaagttg cattggccag attagagtac aaaaatttag atatttgatg tttagatgaa 300gctcttgata acattttcaa ggaagatgct tttgaaattt acagtgattt actttcaaaa 360ccgaataaaa caatttttat cgcaagtcac cacattcctg aaaaaataaa accgatgttt 420gaccaaataa ttgaaatt 438<210>54<211>146<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD977的推定的蛋白质序列<400>54Ile Leu Ile Asn Asn Ser Ile Glu Tyr Lys Glu Leu Asp Pro Asn Gln1 5 10 15Leu Arg Lys His Ile Ala Leu Thr Thr Asn Glu Asn Ile Ile Phe Glu
20 25 30Asp Thr Leu Ala Asn Asn Ile Thr Leu Trp Asp Lys Asn Pro Asp Leu
35 40 45Asp Leu Leu Asn Ser Leu Ile Lys Lys Tyr Lys Ile Asp Asn Phe Ser
50 55 60Lys Pro Glu Thr Glu Ile Ser Ser Lys Asn Leu Ser Glu Gly Glu Lys65 70 75 80Gln Lys Val Ala Leu Ala Arg Leu Glu Tyr Lys Asn Leu Asp Ile Trp
85 90 95Cys Leu Asp Glu Ala Leu Asp Asn Ile Phe Lys Glu Asp Ala Phe Glu
100 105 110Ile Tyr Ser Asp Leu Leu Ser Lys Pro Asn Lys Thr Ile Phe Ile Ala
115 120 125Ser His His Ile Pro Glu Lys Ile Lys Pro Met Phe Asp Gln Ile Ile
130 135 140Glu Ile145<210>55<211>400<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD983<400>55tcaacgggct gccaaattga acctgacaaa ccgctggtaa aaaaatgggt tatgggtgtt 60ttatttaatt atagctttta ttattcagga attctaagca tagttttagg atttttttct 120tctgaaataa caattttctt tcttcaaaca gcaggggctg atattaatgt tccagtttga 180ggccatctaa taattgggac agttttttgt atttttttca ctagccttaa ttatatttca 240ataaaaacat caggatgaat tgcgcttgca tcaacaattt taaaatttat tcctttagta 300tttgcagttt ttgcaggaat tctatttcca aaaacttata atgccggcgg ttctaatgcc 360tttgttcaaa cagctcaaat agttttaatt ttgcaaaatt 400<210>56<211>133<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD983的推定的蛋白质序列<400>56Ser Thr Gly Cys Gln Ile Glu Pro Asp Lys Pro Leu Val Lys Lys Trp1 5 10 15Val Met Gly Val Leu Phe Asn Tyr Ser Phe Tyr Tyr Ser Gly Ile Leu
20 25 30Ser Ile Val Leu Gly Phe Phe Ser Ser Glu Ile Thr Ile Phe Phe Leu
35 40 45Gln Thr Ala Gly Ala Asp Ile Asn Val Pro Val Trp Gly His Leu Ile
50 55 60Ile Gly Thr Val Phe Cys Ile Phe Phe Thr Ser Leu Asn Tyr Ile Ser65 70 75 80Ile Lys Thr Ser Gly Trp Ile Ala Leu Ala Ser Thr Ile Leu Lys Phe
85 90 95Ile Pro Leu Val Phe Ala Val Phe Ala Gly Ile Leu Phe Pro Lys Thr
100 105 110Tyr Asn Ala Gly Gly Ser Asn Ala Phe Val Gln Thr Ala Gln Ile Val
115 120 125Leu Ile Leu Gln Asn130<210>57<211>522<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD984<400>57ttaaaaagtg aaaaccaaaa agaaacagca aatttaaata ctacttttac tcaaacaatt 60agtaaaaaag atatcgaaat aaccaattta agaaatgaaa ttggcaaatt tcttgatgaa 120aaagataaaa tgcgaagtga cattcttgca aatgatgatg agataaaggc gatgaggagt 180gaaatttctc aactaaaaga agcaaatgcc aacctacaaa atgtcaagtt agaagaaatt 240tcaaatttaa aactagaaca taaggacgaa attaatgaaa aagaccgtaa aattagttat 300ttagagaata aatttaacga cttggaagaa gaaaaaaata attcaattca aaatgctgta 360agtcaaaaaa cacgagaaat aaaggaaaaa attgaaaaag agctagaaat taaatgggga 420agaaaaaata aaacaggaaa aatcagattt aagaggaaaa ttttaaggag caaattaata 480aaccaggaga aaaaattcaa gattagaaga agaattaaat ta 522<210>58<211>174<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD984的推定的蛋白质序列<400> 58Leu Lys Ser Glu Asn Gln Lys Glu Thr Ala Asn Leu Asn Thr Thr Phe1 5 10 15Thr Gln Thr Ile Ser Lys Lys Asp Ile Glu Ile Thr Asn Leu Arg Asn
20 25 30Glu Ile Gly Lys Phe Leu Asp Glu Lys Asp Lys Met Arg Ser Asp Ile
35 40 45Leu Ala Asn Asp Asp Glu Ile Lys Ala Met Arg Ser Glu Ile Ser Gln
50 55 60Leu Lys Glu Ala Asn Ala Asn Leu Gln Asn Val Lys Leu Glu Glu Ile65 70 75 80Ser Asn Leu Lys Leu Glu His Lys Asp Glu Ile Asn Glu Lys Asp Arg
85 90 95Lys Ile Ser Tyr Leu Glu Asn Lys Phe Asn Asp Leu Glu Glu Glu Lys
100 105 110Asn Asn Ser Ile Gln Asn Ala Val Ser Gln Lys Thr Arg Glu Ile Lys
115 120 125Glu Lys Ile Glu Lys Glu Leu Glu Ile Lys Trp Gly Arg Lys Asn Lys
130 135 140Thr Gly Lys Ile Arg Phe Lys Arg Lys Ile Leu Arg Ser Lys Leu Ile145 150 155 160Asn Gln Glu Lys Lys Phe Lys Ile Arg Arg Arg Ile Lys Leu
165 170<210>59<211>633<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD994<400>59attaaacact tttttaaaag atttgaaatg tataaacgat tagttcagga attctttcct 60aaattagatt ttgaaaattt agaaaaatac gtaaatttaa ttgaatttag taataaaaac 120tttaatttaa ccgctttttc tggtgatatt ctttgaaaag agggaatttt tgagtcaatt 180tttacaatga atttcattgt tggtttagta aataataaag aaaataaaaa attaaaaatt 240ttggatattg gggctggatc aggttttcct tcaattcctt ttttgattac aaacccagaa 300attgagctaa caatttctga gtcaatgcaa aaaagatgcc agtttttaaa ggatgtttct 360gaaaaattag atttgaaatt caatttaatt tgcaaaccag ttcaagaaat taatccacaa 420aaatttgata taataactgc cagagcagtg gcaaatttgg aaaagcttga gaaaattaca 480aaaaaaattc attttccaaa aacgctttta gcttttatta aagggcccaa agtttttaat 540gaagttcaaa attgtaaaaa ttgtaattat aaaatcatta aagttaataa taatataaat 600aaaaaaattt ttatcgcatt taaacaagtt tct 633<210>60<211>211<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD994的推定的蛋白质序列<400>60Ile Lys His Phe Phe Lys Arg Phe Glu Met Tyr Lys Arg Leu Val Gln1 5 10 15Glu Phe Phe Pro Lys Leu Asp Phe Glu Asn Leu Glu Lys Tyr Val Asn
20 25 30Leu Ile Glu Phe Ser Asn Lys Asn Phe Asn Leu Thr Ala Phe Ser Gly
35 40 45Asp Ile Leu Trp Lys Glu Gly Ile Phe Glu Ser Ile Phe Thr Met Asn
50 55 60Phe Ile Val Gly Leu Val Asn Asn Lys Glu Asn Lys Lys Leu Lys Ile65 70 75 80Leu Asp Ile Gly Ala Gly Ser Gly Phe Pro Ser Ile Pro Phe Leu Ile
85 90 95Thr Asn Pro Glu Ile Glu Leu Thr Ile Ser Glu Ser Met Gln Lys Arg
100 105 110Cys Gln Phe Leu Lys Asp Val Ser Glu Lys Leu Asp Leu Lys Phe Asn
115 120 125Leu Ile Cys Lys Pro Val Gln Glu Ile Asn Pro Gln Lys Phe Asp Ile
130 135 140Ile Thr Ala Arg Ala Val Ala Asn Leu Glu Lys Leu Glu Lys Ile Thr145 150 155 160Lys Lys Ile His Phe Pro Lys Thr Leu Leu Ala Phe Ile Lys Gly Pro
165 170 175Lys Val Phe Asn Glu Val Gln Asn Cys Lys Asn Cys Asn Tyr Lys Ile
180 185 190Ile Lys Val Asn Asn Asn Ile Asn Lys Lys Ile Phe Ile Ala Phe Lys
195 200 205Gln Val Ser
210<210>61<211>537<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD1005<400>61atgaaaaaat tattagttat tttgcttgat aaattccagg atattgaact tacaactttt 60atttccctga ttaaaaaagc agaaattttt acagatattg aattttttaa ccctaaaaat 120aataaattag taataggtca attcggagtt gtatcaattc aagcacataa tcactggaaa 180tcagatgact ttgatgctgt ttttattccg gggggttttg cggcccaatt attccgcaag 240gattcaaaat caattcaact tgtgagcgag ttttttgcgc aaaacaaaca tatttttgcc 300atttgtgatg caccaaatgc aatttttgaa ctaaaattag cagaaaatta tcaatttagt 360tcatatccaa accaacataa ttccaaaatt agactaagac aagattcgtt agtaactatt 420gaccgcaatt atatttcggc aagaaatgca gcaagttcgg cagattttgc tttcgttgta 480attgaaaagc tgggatcaaa agagttagct caaaaaatta gaaatggatt ttatctt 537<210>62<211>179<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD1005的推定的蛋白质序列<400>62Met Lys Lys Leu Leu Val Ile Leu Leu Asp Lys Phe Gln Asp Ile Glu1 5 10 15Leu Thr Thr Phe Ile Ser Leu Ile Lys Lys Ala Glu Ile Phe Thr Asp
20 25 30Ile Glu Phe Phe Asn Pro Lys Asn Asn Lys Leu Val Ile Gly Gln Phe
35 40 45Gly Val Val Ser Ile Gln Ala His Asn His Trp Lys Ser Asp Asp Phe
50 55 60Asp Ala Val Phe Ile Pro Gly Gly Phe Ala Ala Gln Leu Phe Arg Lys65 70 75 80Asp Ser Lys Ser Ile Gln Leu Val Ser Glu Phe Phe Ala Gln Asn Lys
85 90 95His Ile Phe Ala Ile Cys Asp Ala Pro Asn Ala Ile Phe Glu Leu Lys
100 105 110Leu Ala Glu Asn Tyr Gln Phe Ser Ser Tyr Pro Asn Gln His Asn Ser
115 120 125Lys Ile Arg Leu Arg Gln Asp Ser Leu Val Thr Ile Asp Arg Asn Tyr
130 135 140Ile Ser Ala Arg Asn Ala Ala Ser Ser Ala Asp Phe Ala Phe Val Val145 150 155 160Ile Glu Lys Leu Gly Ser Lys Glu Leu Ala Gln Lys Ile Arg Asn Gly
165 170 175Phe Tyr Leu<210>63<211>486<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD1016<400>63ttaatcgttt ttgcttatat gatgttggtt gtaatgaatt gaggttttgc ctctgccgga 60cttaacggta aagcgggaat aagtggttat ttaggtcact tttttccaaa tgctaatgaa 120gccccaggaa ccgttgtaaa tcaagcagtt aactggggta tcacaattgg tcgtggaatt 180ggatcagttc ttgttggttg attaattgtg aaaatttcgc ataaatatac agtaattttg 240tctttatttt ttatgctttt tggaattatt gccccttatt caccaactta tgccgggttt 300ataattctta gaacaatttt tgcaattggc ggaacaatgc agattatttt aattcaacca 360gttgtctcaa attatttaaa tcaaaggcaa aaagctgtta tttcacaagt tttccccttt 420tttttatcca attggaacaa taattacgct tattcctttt gcaggaatta tttggtcaag 480agctca 486<210>64<211>162<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD1016的推定的蛋白质序列<400>64Leu Ile Val Phe Ala Tyr Met Met Leu Val Val Met Asn Trp Gly Phe1 5 10 15Ala Ser Ala Gly Leu Asn Gly Lys Ala Gly Ile Ser Gly Tyr Leu Gly
20 25 30His Phe Phe Pro Asn Ala Asn Glu Ala Pro Gly Thr Val Val Asn Gln
35 40 45Ala Val Asn Trp Gly Ile Thr Ile Gly Arg Gly Ile Gly Ser Val Leu
50 55 60Val Gly Trp Leu Ile Val Lys Ile Ser His Lys Tyr Thr Val Ile Leu65 70 75 80Ser Leu Phe Phe Met Leu Phe Gly Ile Ile Ala Pro Tyr Ser Pro Thr
85 90 95Tyr Ala Gly Phe Ile Ile Leu Arg Thr Ile Phe Ala Ile Gly Gly Thr
100 105 110Met Gln Ile Ile Leu Ile Gln Pro Val Val Ser Asn Tyr Leu Asn Gln
115 120 125Arg Gln Lys Ala Val Ile Ser Gln Val Phe Pro Phe Phe Leu Ser Asn
130 135 140Trp Asn Asn Asn Tyr Ala Tyr Ser Phe Cys Arg Asn Tyr Leu Val Lys145 150 155 160Ser Ser<210>65<211>456<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD1020<400>65tctgatcgtt ttccagttac aagtttagaa aaattatttc aatttgtcgc taattcagcg 60cctatttttg aaaaattcaa aaaagcaaaa gaaaaatttt atcaagcaaa atttgggact 120aagatgaata gttgagaccg tttagttcca ttagttgaga caaaaaataa ttattctgtt 180gaagatgcac aaaaaatagt gcttggggca atcaaaccat taggtcaaga atataaagat 240gttgtagaaa aagcatttag ccaaagatga attgactatc attatgttga ttcaaaaaga 300tctggtgctt attcaattgg gggttcatat gggcttgaaa aaaaatatat tttaatgaat 360tatgacttta ctataaacgc agttcatact ttagcacatg aattaggtca ttcgctccat 420tcttattatt ctgataaaaa ccagaattat cataat 456<210>66<211>152<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD1020的推定的蛋白质序列<400>66Ser Asp Arg Phe Pro Val Thr Ser Leu Glu Lys Leu Phe Gln Phe Val1 5 10 15Ala Asn Ser Ala Pro Ile Phe Glu Lys Phe Lys Lys Ala Lys Glu Lys
20 25 30Phe Tyr Gln Ala Lys Phe Gly Thr Lys Met Asn Ser Trp Asp Arg Leu
35 40 45Val Pro Leu Val Glu Thr Lys Asn Asn Tyr Ser Val Glu Asp Ala Gln
50 55 60Lys Ile Val Leu Gly Ala Ile Lys Pro Leu Gly Gln Glu Tyr Lys Asp65 70 75 80Val Val Glu Lys Ala Phe Ser Gln Arg Trp Ile Asp Tyr His Tyr Val
85 90 95Asp Ser Lys Arg Ser Gly Ala Tyr Ser Ile Gly Gly Ser Tyr Gly Leu
100 105 110Glu Lys Lys Tyr Ile Leu Met Asn Tyr Asp Phe Thr Ile Asn Ala Val
115 120 125His Thr Leu Ala His Glu Leu Gly His Ser Leu His Ser Tyr Tyr Ser
130 135 140Asp Lys Asn Gln Asn Tyr His Asn145 150<210>67<211>576<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD1027<400>67gaattaatta gggaaaattt atcacttgca aaatcatttt atgttgataa aaataataat 60ccttggatat caacaacaaa aaattttgaa aacttatttg attatgtaca aagcgagcat 120ctaattaata ctaataaaat aaaaaattat atcacaaaca taaattttaa aatcaaaaaa 180aatagtgaaa tacctgcttt agaacttaat aatttgctaa aagatgataa aattcggctt 240gaaataaatg ttgatatctc aaagtgagtc caacaaaaac taattaaaat tttaagtttt 300aagtttgatt gggacctaaa accagacctg aatcagtatg cccggatttt tgcacaaaat 360ctacccgagc caaaatctga ggtattctta ctaagaaaag atgaaaattc agcagcgtga 420actagtaaaa aactagtaaa tataataaat aaaattaagg gatttaacaa tggattagac 480ccagaaaatc ctgatttaag gttagttagc caactttatt tacttgattt tggcaaaatt 540ggtgatgaaa atgctataga aaattttaaa gggatt 576<210>68<211>192<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD1027的推定的蛋白质序列<400>68Glu Leu Ile Arg Glu Asn Leu Ser Leu Ala Lys Ser Phe Tyr Val Asp1 5 10 15Lys Asn Asn Asn Pro Trp Ile Ser Thr Thr Lys Asn Phe Glu Asn Leu
20 25 30Phe Asp Tyr Val Gln Ser Glu His Leu Ile Asn Thr Asn Lys Ile Lys
35 40 45Asn Tyr Ile Thr Asn Ile Asn Phe Lys Ile Lys Lys Asn Ser Glu Ile
50 55 60Pro Ala Leu Glu Leu Asn Asn Leu Leu Lys Asp Asp Lys Ile Arg Leu65 70 75 80Glu Ile Asn Val Asp Ile Ser Lys Trp Val Gln Gln Lys Leu Ile Lys
85 90 95Ile Leu Ser Phe Lys Phe Asp Trp Asp Leu Lys Pro Asp Leu Asn Gln
100 105 110Tyr Ala Arg Ile Phe Ala Gln Asn Leu Pro Glu Pro Lys Ser Glu Val
115 120 125Phe Leu Leu Arg Lys Asp Glu Asn Ser Ala Ala Trp Thr Ser Lys Lys
130 135 140Leu Val Asn Ile Ile Asn Lys Ile Lys Gly Phe Asn Asn Gly Leu Asp145 150 155 160Pro Glu Asn Pro Asp Leu Arg Leu Val Ser Gln Leu Tyr Leu Leu Asp
165 170 175Phe Gly Lys Ile Gly Asp Glu Asn Ala Ile Glu Asn Phe Lys Gly Ile
180 185 190<210>69<211>351<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD1037<400>69catatgttaa ttgaagtttt aataattcac taccgtcaag ttcagtatgg ccaaagtatt 60aaaaaatcag taatttataa cttaataaca accctgattt tagtgccgat tattacagtt 120ggcgcctttt tgaaccgttt ttttattaaa acaggctgac taataccatt ttttaatgtt 180tctggcgggg caattttaag ttttgttgtc ataattgagt tagttccaga atttatccat 240ttaagaaata acccttcttt tcagtgacat ttttctcttt ttttgtttgc tttaggaatt 300attttagcct taattatttt aatttacatg aacattaagc gccgtagatc c 351<210>70<211>117<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD1037的推定的蛋白质序列<400>70His Met Leu Ile Glu Val Leu Ile Ile His Tyr Arg Gln Val Gln Tyr 1 5 10 15Gly Gln Ser Ile Lys Lys Ser Val Ile Tyr Asn Leu Ile Thr Thr Leu
20 25 30Ile Leu Val Pro Ile Ile Thr Val Gly Ala Phe Leu Asn Arg Phe Phe
35 40 45Ile Lys Thr Gly Trp Leu Ile Pro Phe Phe Asn Val Ser Gly Gly Ala
50 55 60Ile Leu Ser Phe Val Val Ile Ile Glu Leu Val Pro Glu Phe Ile His65 70 75 80Leu Arg Asn Asn Pro Ser Phe Gln Trp His Phe Ser Leu Phe Leu Phe
85 90 95Ala Leu Gly Ile Ile Leu Ala Leu Ile Ile Leu Ile Tyr Met Asn Ile
100 105 110Lys Arg Arg Arg Ser
115<210>71<211>1776<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD1038<400>71gaattcgaaa aacgaattaa ggcaattttg caagaaattg agcaaaattc cgatcaagtt 60attattttta ttgatgaaat tcaccttcta attggaacag gatcttctgg gactgattca 120atggattttg ccaatatcct aaaaccaatt atggctcgcg gacagattaa attaatcggg 180gctaccacaa attccgaata tcgcttatat atcgaaaaag atggcgccct tgaaagaaga 240atgcaaaaag tagaaatttt agagccttca gttattgata caattaatat tttacgggga 300attaaggaaa ggctagaaaa tttccatcaa gtaaaaatta aggattctgc tcttgttttt 360gctacaaaag cggcaaatcg ttacattttt gaccgctttc tacctgataa agctatcgat 420ttagtcgatg aagctgctgc ttctttaaaa gttgaaatca actaccaacc agaaaaactt 480gaaaaagcaa agcgcgagct aatttattta aaaatggaag aaattaactc gcaaaaacaa 540gataattcag aattaaaatc caaaattgaa aatcttgaaa atgaagtaaa aaaattacaa 600gatcaatggg atcaatcaaa aaaatcagcc tctgaaatcg ctagcttatc ccaggaactt 660gaaaaactaa aatatcaaca aaattactta atggaacaag gagactacca aaaagccgcc 720gagattaaat acggaaaaat tcccaaaata agtaaaaaat taggcgaaat taaagcaaga 780aggcaggaaa tttccaatgt tctagacgaa agtcagatcg caaaggttgt ctctaattga 840acaaaaattc cgattgaaaa acttttagaa tcagaaattc aaaaatattt gaatttagaa 900aaaaatttag caaaatcgct taagggtcaa aatcaggcaa ttaaggctgt ttcagatgcg 960attttgcggt ttaaagctaa aattaatgat gaatcccgcc caatttcatc atttttcttt 1020atgggaccaa ctggggtggg aaaaactgaa cttgctagag ctttagctct taatttattt 1080aataataaaa accaaataat ccgtcttgat atgtcagaat atatggaaaa acatagtgtt 1140tcaaagctaa ttggggctcc tccgggttat attggttttg aacaaggtgg taatctaaca 1200aataaagtaa gactaaatcc ttattcgatt attttgcttg atgaaattga aaaagctcat 1260ccggaagtaa tcaacatttt tttacaaatt cttgataatg gtgaaattgt tgatagtaag 1320tcacaaaaag taaattttcg caatacaatt ataattatga cctcaaatat cggtgctaat 1380aaaattcttg agggtaaaaa gatgaatgaa attgaggcaa aaaaggaact tttaagatat 1440ttaaagccag aatttctcaa ccgaattgat gaaattatcg tatttaatcc tttaaattat 1500gatataattt ttgaaattat tgaacttgaa ctaaaggatt tgcaaaatcg tctaaaggaa 1560aataattttg agattgaatt tgaaaaatca gtcaaaaatt gaattttaga gtttggatat 1620gataaaaatt ttggtgccag gccaattaag cgctttatta agaaagaaat tgaaaatttt 1680gttgccaaaa aaatagtggc cgaagaaatt ttaaaagata aaaaatacaa tttatctttt 1740aaaaatgata aattgcatct taatgaaagc gaaaat 1776<210>72<211>592<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD1038的推定的蛋白质序列<400>72Glu Phe Glu Lys Arg Ile Lys Ala Ile Leu Gln Glu Ile Glu Gln Asn1 5 10 15Ser Asp Gln Val Ile Ile Phe Ile Asp Glu Ile His Leu Leu Ile Gly
20 25 30Thr Gly Ser Ser Gly Thr Asp Ser Met Asp Phe Ala Asn Ile Leu Lys
35 40 45Pro Ile Met Ala Arg Gly Gln Ile Lys Leu Ile Gly Ala Thr Thr Asn
50 55 60Ser Glu Tyr Arg Leu Tyr Ile Glu Lys Asp Gly Ala Leu Glu Arg Arg65 70 75 80Met Gln Lys Val Glu Ile Leu Glu Pro Ser Val Ile Asp Thr Ile Asn
85 90 95Ile Leu Arg Gly Ile Lys Glu Arg Leu Glu Asn Phe His Gln Val Lys
100 105 110Ile Lys Asp Ser Ala Leu Val Phe Ala Thr Lys Ala Ala Asn Arg Tyr
115 120 125Ile Phe Asp Arg Phe Leu Pro Asp Lys Ala Ile Asp Leu Val Asp Glu
130 135 140Ala Ala Ala Ser Leu Lys Val Glu Ile Asn Tyr Gln Pro Glu Lys Leu145 150 155 160Glu Lys Ala Lys Arg Glu Leu Ile Tyr Leu Lys Met Glu Glu Ile Asn
165 170 175Ser Gln Lys Gln Asp Asn Ser Glu Leu Lys Ser Lys Ile Glu Asn Leu
180 185 190Glu Asn Glu Val Lys Lys Leu Gln Asp Gln Trp Asp Gln Ser Lys Lys
195 200 205Ser Ala Ser Glu Ile Ala Ser Leu Ser Gln Glu Leu Glu Lys Leu Lys
210 215 220Tyr Gln Gln Asn Tyr Leu Met Glu Gln Gly Asp Tyr Gln Lys Ala Ala225 230 235 240Glu Ile Lys Tyr Gly Lys Ile Pro Lys Ile Ser Lys Lys Leu Gly Glu
245 250 255Ile Lys Ala Arg Arg Gln Glu Ile Ser Asn Val Leu Asp Glu Ser Gln
260 265 270Ile Ala Lys Val Val Ser Asn Trp Thr Lys Ile Pro Ile Glu Lys Leu
275 280 285Leu Glu Ser Glu Ile Gln Lys Tyr Leu Asn Leu Glu Lys Asn Leu Ala
290 295 300Lys Ser Leu Lys Gly Gln Asn Gln Ala Ile Lys Ala Val Ser Asp Ala305 310 315 320Ile Leu Arg Phe Lys Ala Lys Ile Asn Asp Glu Ser Arg Pro Ile Ser
325 330 335Ser Phe Phe Phe Met Gly Pro Thr Gly Val Gly Lys Thr Glu Leu Ala
340 345 350Arg Ala Leu Ala Leu Asn Leu Phe Asn Asn Lys Asn Gln Ile Ile Arg
355 360 365Leu Asp Met Set Glu Tyr Met Glu Lys His Ser Val Ser Lys Leu Ile
370 375 380Gly Ala Pro Pro Gly Tyr Ile Gly Phe Glu Gln Gly Gly Asn Leu Thr385 390 395 400Asn Lys Val Arg Leu Asn Pro Tyr Ser Ile Ile Leu Leu Asp Glu Ile
405 410 415Glu Lys Ala His Pro Glu Val Ile Asn Ile Phe Leu Gln Ile Leu Asp
420 425 430Asn Gly Glu Ile Val Asp Ser Lys Ser Gln Lys Val Asn Phe Arg Asn
435 440 445Thr Ile Ile Ile Met Thr Ser Asn Ile Gly Ala Asn Lys Ile Leu Glu
450 455 460Gly Lys Lys Met Asn Glu Ile Glu Ala Lys Lys Glu Leu Leu Arg Tyr465 470 475 480Leu Lys Pro Glu Phe Leu Asn Arg Ile Asp Glu Ile Ile Val Phe Asn
485 490 495Pro Leu Asn Tyr Asp Ile Ile Phe Glu Ile Ile Glu Leu Glu Leu Lys
500 505 510Asp Leu Gln Asn Arg Leu Lys Glu Asn Asn Phe Glu Ile Glu Phe Glu
515 520 525Lys Ser Val Lys Asn Trp Ile Leu Glu Phe Gly Tyr Asp Lys Asn Phe
530 535 540Gly Ala Arg Pro Ile Lys Arg Phe Ile Lys Lys Glu Ile Glu Asn Phe545 550 555 560Val Ala Lys Lys Ile Val Ala Glu Glu Ile Leu Lys Asp Lys Lys Tyr
565 570 575Asn Leu Ser Phe Lys Asn Asp Lys Leu His Leu Asn Glu Ser G1u Asn580 585 590<210>73<211>796<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD1040<400>73atggtaaaat ctacaaaaca tttcaaattt atcctttgaa attgattata tttgattttt 60acgatttttt ttaaaattta tctcattgtt gctccttatt ttatttttac ttttattcta 120aatgaaaatt taactttttt ttgggtagcc acaacatctt ttttaggggt tagaattttt 180aatatctttt tagattttat gaatcaagca tattttaaag ggtttttgat ctttcataag 240atgaaacttg ccgaaaaaat aacaaatttt ttggaaaaaa cgacttacaa aaaatataac 300gaaaattcaa gtgggtttta ctattcggaa attgaaaata caatagaaaa aagcgtttca 360caattttatg caaatttatt gtcctttttg caaactcttt ccataatttt gatgacttta 420ggtttatttt tttatataaa ctggatttta gcgttaatta ttgtcggtgt tataaccttt 480tttgtaatta caacttcttt actatctaaa aaattaacca aacttcaatc cgcaaaattg 540caagcaattt cggattttaa caattcttta agcacttatc ttttaacttt gccgcaatta 600aaaaccttaa attctgatga taaattcgaa tttataatta ataaaagaaa caagaaaaat 660tgaataacta gagaaaaata tggtatattt tccgacttaa tttcattttt taatgaatat 720tccaataatt ttttctccgc aataatcaca attggaattg cattttggac actttattat 780aaaaataata atagct 796<210>74<211>265<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD1040的推定的蛋白质序列<400>74Met Val Lys Ser Thr Lys His Phe Lys Phe Ile Leu Trp Asn Trp Leu1 5 10 15Tyr Leu Ile Phe Thr Ile Phe Phe Lys Ile Tyr Leu Ile Val Ala Pro
20 25 30Tyr Phe Ile Phe Thr Phe Ile Leu Asn Glu Asn Leu Thr Phe Phe Trp
35 40 45Val Ala Thr Thr Ser Phe Leu Gly Val Arg Ile Phe Asn Ile Phe Leu
50 55 60Asp Phe Met Asn Gln Ala Tyr Phe Lys Gly Phe Leu Ile Phe His Lys65 70 75 80Met Lys Leu Ala Glu Lys Ile Thr Asn Phe Leu Glu Lys Thr Thr Tyr
85 90 95Lys Lys Tyr Asn Glu Asn Ser Ser Gly Phe Tyr Tyr Ser Glu Ile Glu
100 105 110Asn Thr Ile Glu Lys Ser Val Ser Gln Phe Tyr Ala Asn Leu Leu Ser
115 120 125Phe Leu Gln Thr Leu Ser Ile Ile Leu Met Thr Leu Gly Leu Phe Phe
130 135 140Tyr Ile Asn Trp Ile Leu Ala Leu Ile Ile Val Gly Val Ile Thr Phe145 150 155 160Phe Val Ile Thr Thr Ser Leu Leu Ser Lys Lys Leu Thr Lys Leu Gln
165 170 175Ser Ala Lys Leu Gln Ala Ile Ser Asp Phe Asn Asn Ser Leu Ser Thr
180 185 190Tyr Leu Leu Thr Leu Pro Gln Leu Lys Thr Leu Asn Ser Asp Asp Lys
195 200 205Phe Glu Phe Ile Ile Asn Lys Arg Asn Lys Lys Asn Trp Ile Thr Arg
210 215 220Glu Lys Tyr Gly Ile Phe Ser Asp Leu Ile Ser Phe Phe Asn Glu Tyr225 230 235 240Ser Asn Asn Phe Phe Ser Ala Ile Ile Thr Ile Gly Ile Ala Phe Trp
245 250 255Thr Leu Tyr Tyr Lys Asn Asn Asn Ser
260 265<210>75<211>453<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD702<400>75tcttatgaga aaaaatattt acctttgcta atagtccctg gaatttttgg cgctatttta 60ttttttcttt ttattaaaac acttttagac tataaagcaa tcaaaaaatc tgttatttat 120tttcgttccc agttgcaaaa taatgcaaat cgacttgaaa tgccaccaat gattccatga 180cttgtaaaaa aagtgaatca aaaagaggta aatgctatct gacttagcgg ctttactttg 240tttgcaacaa ttatgatggg cttaacttac tgagtgttat taaaatatta tccggagaaa 300aatattcaaa attctgccga atatataact gcaatggcag taaatggcgc tttgtttata 360gttatgctaa tttatgattt aatgcttcgt cggcgtttgg gaaatattga agcaattttt 420ggtcccattt atcataaaag ttttgatata ggt 453<210>76<211>151<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD702的推定的蛋白质序列<400>76Ser Tyr Glu Lys Lys Tyr Leu Pro Leu Leu Ile Val Pro Gly Ile Phe1 5 10 15Gly Ala Ile Leu Phe Phe Leu Phe Ile Lys Thr Leu Leu Asp Tyr Lys
20 25 30Ala Ile Lys Lys Ser Val Ile Tyr Phe Arg Ser Gln Leu Gln Asn Asn
35 40 45Ala Asn Arg Leu Glu Met Pro Pro Met Ile Pro Trp Leu Val Lys Lys
50 55 60Val Asn Gln Lys Glu Val Asn Ala Ile Trp Leu Ser Gly Phe Thr Leu65 70 75 80Phe Ala Thr Ile Met Met Gly Leu Thr Tyr Trp Val Leu Leu Lys Tyr
85 90 95Tyr Pro Glu Lys Asn Ile Gln Asn Ser Ala Glu Tyr Ile Thr Ala Met
100 105 110Ala Val Asn Gly Ala Leu Phe Ile Val Met Leu Ile Tyr Asp Leu Met
115 120 125Leu Arg Arg Arg Leu Gly Asn Ile Glu Ala Ile Phe Gly Pro Ile Tyr
130 135 140His Lys Ser Phe Asp Ile Gly145 150<210>77<211>99<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD763<400>77gatctttgtg ttatttttgt taataaaacc aagtttaaaa gtcatttccc ctggtttgtc 60agtggtttta acatcataat aatagacaaa ataattctt 99<210>78<211>33<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD763的推定的蛋白质序列<400>78Asp Leu Cys Val Ile Phe Val Asn Lys Thr Lys Phe Lys Ser His Phe1 5 10 15Pro Trp Phe Val Ser Gly Phe Asn Ile Ile Ile Ile Asp Lys Ile Ile
20 25 30Leu<210>79<211>489<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD766<400>79gatcaacaaa aaccacaacc aaaagaagaa aaagaagaaa aacaagaaaa agaagaaaaa 60aaaccgccga tagttcaagg tcctagtcca aaaccacaaa agattgaaaa tatcggtctt 120gttaatgatt tttataaata caagtttaac gataaaattc ataaatttga accgactgag 180tattataaaa atacagcaaa tttttctcag ggtggccttt atagtgcaaa tttgctcgaa 240ttagaaaagg aaataaagaa acaagatccg gataatccta aaatatttta tgttcaaaga 300cgaattgata ttggtggttt tctaacaaaa ggcacacttt taccatttca acccgcaaat 360cttgagaata atttatcaag cctttcgctt tttgatagat attcccaatt tctgaggagc 420ggcagattcg ataacaatta ttatataatt ggatccgata aggttgagga atttgatagg 480ttgaaaaga 489<210>80<211>163<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD766的推定的蛋白质序列<400>80Asp Gln Gln Lys Pro Gln Pro Lys Glu Glu Lys Glu Glu Lys Gln Glu1 5 10 15Lys Glu Glu Lys Lys Pro Pro Ile Val Gln Gly Pro Ser Pro Lys Pro
20 25 30Gln Lys Ile Glu Asn Ile Gly Leu Val Asn Asp Phe Tyr Lys Tyr Lys
35 40 45Phe Asn Asp Lys Ile His Lys Phe Glu Pro Thr Glu Tyr Tyr Lys Asn
50 55 60Thr Ala Asn Phe Ser Gln Gly Gly Leu Tyr Ser Ala Asn Leu Leu Glu65 70 75 80Leu Glu Lys Glu Ile Lys Lys Gln Asp Pro Asp Asn Pro Lys Ile Phe
85 90 95Tyr Val Gln Arg Arg Ile Asp Ile Gly Gly Phe Leu Thr Lys Gly Thr
100 105 110Leu Leu Pro Phe Gln Pro Ala Asn Leu Glu Asn Asn Leu Ser Ser Leu
115 120 125Ser Leu Phe Asp Arg Tyr Ser Gln Phe Leu Arg Ser Gly Arg Phe Asp
130 135 140Asn Asn Tyr Tyr Ile Ile Gly Ser Asp Lys Val Glu Glu Phe Asp Arg145 150 155 160Leu Lys Arg<210>81<211>474<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD957<400>81agctatttta gtattataag ccctttgttt ttggctgttt cttgcacaaa cattataatt 60agcaaatctg aattatcaaa aatagaaagt aatattttta attttataat aaacgaaaat 120gaaaaaaatt taactagatt taccgcaact ttagtaaaaa aaactaataa taacttgact 180tttgtcagta cttttcattc gctaaattca ataaaaaaca atatacagca acaagttttt 240gatatttttt tacaacaatt tagtgtgaaa aatttagaaa ctaaattaaa atcaaaaatt 300agagttgaat atgaaaataa agaaaaagat atcatagttt tttcgctaga tataaaggaa 360cccttattgt tgagaatttc ggattctatt gattttcagg ttctagagga ttttacaaac 420acaaaaaata gcctatttag cttaaggttt ctcaccgatc ttaaacgaag attt 474<210>82<211>158<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD957的推定的蛋白质序列<400>82Set Tyr Phe Ser Ile Ile Ser Pro Leu Phe Leu Ala Val Ser Cys Thr 1 5 10 15Asn Ile Ile Ile Ser Lys Ser Glu Leu Ser Lys Ile Glu Ser Asn Ile
20 25 30Phe Asn Phe Ile Ile Asn Glu Asn Glu Lys Asn Leu Thr Arg Phe Thr
35 40 45Ala Thr Leu Val Lys Lys Thr Asn Asn Asn Leu Thr Phe Val Ser Thr
50 55 60Phe His Ser Leu ASn Ser Ile Lys Asn Asn Ile Gln Gln Gln Val Phe65 70 75 80Asp Ile Phe Leu Gln Gln Phe Ser Val Lys Asn Leu Glu Thr Lys Leu
85 90 95Lys Ser Lys Ile Arg Val Glu Tyr Glu Asn Lys Glu Lys Asp Ile Ile
100 105 110Val Phe Ser Leu Asp Ile Lys Glu Pro Leu Leu Leu Arg Ile Ser Asp
115 120 125Ser Ile Asp Phe Gln Val Leu Glu Asp Phe Thr Asn Thr Lys Asn Ser
130 135 140Leu Phe Ser Leu Arg Phe Leu Thr Asp Leu Lys Arg Arg Phe145 150 155<210>83<211>123<212>DNA<213>人工序列<220><223>人工序列的描述:克隆pAD996<400>83agctatctat taattatgat gccgcagggg gaacctatca cactactggt taaggtcatt 60acgctagcgg ttatgcactc agatgaaaat gctgaaaggt acataaactc tgatgatccg 120atc 123<210>84<211>41<212>PRT<213>人工序列<220><223>人工序列的描述:克隆pAD996的推定的蛋白质序列<400>84Ser Tyr Leu Leu Ile Met Met Pro Gln Gly Glu Pro Ile Thr Leu Leu1 5 10 15Val Lys Val Ile Thr Leu Ala Val Met His Ser Asp Glu Asn Ala Glu
20 25 30Arg Tyr Ile Asn Ser Asp Asp Pro Ile
35 40
Claims (40)
1.一种鉴定由表达载体中核苷酸序列翻译得到的表达蛋白质的方法,该方法包括与由核苷酸序列翻译得到的蛋白质共表达标记物。
2.一种鉴定由来自核苷酸序列混合物的核苷酸序列翻译得到的表达蛋白质的方法,该方法包括下列步骤:
提供包含标记物的表达载体;
将来自混合物的核苷酸序列导入表达载体;并
通过测定包含与表达蛋白质共表达的标记物之融合蛋白质的存在,来鉴定由表达载体表达的表达蛋白质。
3.权利要求1或2的方法,其中标记物是可检测标记物。
4.权利要求1-3任一项的方法,其中标记物少于30个氨基酸并选自下组:
(a)聚His标签;
(b)FLAG标签;
(c)I-SPY表位标签;
(d)血凝素标签;和
(e)c-Myc表位标签。
5.权利要求1-4任一项的方法,其中标记物是聚His标签。
6.权利要求1-5任一项的方法,其中聚His标签包含4-8个连续组氨酸残基。
7.权利要求6的方法,其中聚His标签包含6个连续组氨酸残基。
8.权利要求2-7任一项的方法,其中表达载体是真核表达载体。
9.权利要求2-8任一项的方法,其中进一步用表达载体转染宿主细胞以表达表达蛋白质。
10.权利要求1-9任一项的方法,其中表达蛋白质通过抗标记物抗体或金属螯合物缀合探针进行鉴定。
11.权利要求10的方法,其中抗标记物抗体是抗聚His抗体。
12.权利要求1-11任一项的方法,进一步包括通过利用与表达蛋白质共表达的标记物来纯化表达蛋白质,该方法包括通过标记物来选择表达蛋白质。
13.一种由权利要求1-12任一项的方法鉴定的纯化重组蛋白质。
14.一种编码权利要求13的重组蛋白质的纯化核苷酸序列。
15.一种筛选以鉴定文库中表达克隆的方法,该方法包括:
提供核苷酸序列来源;
提供包含标记物的表达载体;
将核苷酸序列导入表达载体;
用表达载体转化宿主细胞以构建表达文库;并
通过检测包含与表达蛋白质共表达的标记物之融合蛋白质的表达,来找出表达克隆。
16.一种筛选核苷酸序列文库以鉴定表达的核苷酸序列的方法,该方法包括下列步骤:
提供核苷酸序列来源;
提供包含标记物的表达载体;
通过将核苷酸序列导入表达载体来构建文库;
通过测定包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物之融合蛋白质的存在,来鉴定由表达载体表达的表达蛋白质;并
测定表达蛋白质的核苷酸序列。
17.一种由核苷酸序列样品鉴定抗原基因序列的方法,该方法包括下列步骤:
提供包含标记物的表达载体;
将来自样品的核苷酸序列导入表达载体;
通过测定包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物之融合蛋白质的存在,来鉴定由蛋白质载体表达的抗原表达蛋白质;
鉴定抗原表达蛋白质;并
测定由表达载体编码的抗原表达蛋白质的基因序列。
18.一种由核苷酸样品鉴定编码疾病治疗性抗原蛋白质的治疗性抗原基因序列的方法,该方法包括下列步骤:
提供包含标记物的表达载体;
将来自样品的核苷酸序列导入表达载体;
通过测定包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物之融合蛋白质的存在,来鉴定由表达载体表达的抗原表达蛋白质;
用表达抗原表达蛋白质的表达载体接种动物;
用疾病攻击动物;
鉴定提供由接种疫苗诱导的治疗性效果的表达载体和表达蛋白质;并
测定由表达载体编码的抗原表达蛋白质的基因序列。
19.权利要求15-18任一项的方法,其中文库是翻译成支原体多肽的核苷酸序列文库,该方法包括下列步骤:
由支原体样品提供核苷酸序列来源;
提供包含标记物的表达载体;
将核苷酸序列导入表达载体;
通过测定包含与由核苷酸序列翻译得到的表达蛋白质共表达的标记物之融合蛋白质的存在,来鉴定由表达载体表达的支原体表达蛋白质;并
测定支原体表达蛋白质的核苷酸序列。
20.权利要求19的方法,其中支原体是猪肺炎支原体。
21.权利要求15-20任一项的方法,其中标记物是聚His标签。
22.一种由权利要求16-21任一项的方法鉴定的核苷酸序列。
23.一种具有依照图1-42任一项的序列,或SEQ ID NO:1、SEQ IDNO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83的支原体核苷酸序列或其突变体、类似物、衍生物、或功能活性片段。
24.一种包含权利要求22或23的核苷酸序列的重组载体。
25.一种包含权利要求24的重组载体的宿主细胞。
26.一种编码由权利要求1-12任一项的方法鉴定的蛋白质之氨基酸序列。
27.一种具有依照图1-42任一项的序列,或SEQ ID NO:2、SEQ IDNO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84的支原体氨基酸序列,或其突变体、类似物、衍生物、或功能活性片段。
28.一种由权利要求26的氨基酸序列编码的分离的和纯化的多肽。
29.一种由权利要求27的氨基酸序列编码的分离的和纯化的多肽。
30.一种由权利要求22或23的核苷酸序列翻译得到的分离的和纯化的多肽。
31.一种对权利要求13、28、29、或30任一项的多肽特异的抗体。
32.权利要求31的抗体,该抗体是单克隆抗体。
33.一种治疗感染的方法,该方法包括施用有效量的权利要求28-30任一项的抗原多肽。
34.一种治疗感染方法,该方法包括对需要的动物施用有效量的包含权利要求22或23的核苷酸序列的DNA分子。
35.权利要求34的方法,其中DNA分子通过权利要求24的重组载体进行施用。
36.一种治疗感染方法,该方法包括施用有效量的对权利要求31或32的多肽特异的抗体。
37.权利要求33-36任一项的方法,其中感染是支原体感染。
38.权利要求37的方法,其中感染是猪肺炎支原体感染。
39.权利要求34或35的方法,其中核苷酸序列包含依照图1-42任一项的序列,或SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ IDNO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ IDNO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ IDNO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ IDNO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ IDNO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ IDNO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ IDNO:79、SEQ ID NO:81、SEQ ID NO:83,或其突变体、类似物、衍生物、或功能活性片段。
40.权利要求33或36的方法,其中多肽由依照图1-42任一项的氨基酸序列,或SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ IDNO:8、SEO ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ IDNO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ IDNO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ IDNO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ IDNO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ IDNO:48、SEQ ID NO:50、SEO ID NO:52、SEQ ID NO:54、SEO IDNO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ IDNO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ IDNO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ IDNO:80、SEQ ID NO:82、SEQ ID NO:84,或其突变体、类似物、衍生物、或功能活性片段编码。
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AUPP7273A AUPP727398A0 (en) | 1998-11-20 | 1998-11-20 | Methods of identifying antigen gene sequences |
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CN112979769A (zh) * | 2021-03-08 | 2021-06-18 | 珠海丽禾医疗诊断产品有限公司 | 一种氨基酸序列、蛋白质及其制备方法和应用 |
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US20030176644A1 (en) * | 2001-02-07 | 2003-09-18 | Devon Byrd | Compositions and methods for molecular biology |
BR0312274A (pt) | 2002-06-28 | 2007-06-19 | Univ Iowa State Res Found Inc | polipeptìdeos imunogênicos de mycoplasma hyopneumoniae |
US20090028891A1 (en) * | 2004-11-11 | 2009-01-29 | Queensland University Of Technology | Chlamydia Antigens and Uses Thereof |
CN101851661B (zh) * | 2010-05-10 | 2015-09-09 | 其昌達生物高科技(上海)有限公司 | 非诊断目的的在a-peg-b列阵下加快检测支原体时间的方法 |
WO2015123728A1 (en) * | 2014-02-18 | 2015-08-27 | University Of Technology, Sydney | Mycoplasma surface proteins and uses thereof |
BR102014014727B1 (pt) * | 2014-06-16 | 2018-04-03 | Ouro Fino Saúde Animal Ltda | COMPLEXO DE POLIPROTEÍNAS IMUNOGÊNICAS DE M. hyopneumoniae, GENE SINTÉTICO CODIFICANTE DO COMPLEXO DE POLIPROTEÍNAS IMUNOGÊNICAS DE M. hyopneumoniae, COMPOSIÇÃO ANTIGÊNICA, PROCESSO DE OBTENÇÃO DE UM COMPLEXO DE POLIPROTEÍNAS IMUNOGÊNICAS DE M. hyopneumoniae |
ES2926548T3 (es) | 2016-10-31 | 2022-10-26 | Univ Zuerich | Colección de etiquetas y métodos para la detección de proteínas, preferentemente por espectrometría de masas |
CN113185588A (zh) * | 2021-04-28 | 2021-07-30 | 华中农业大学 | 一种牛支原体MbovP701蛋白及其制备方法与应用 |
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EP0359919B1 (en) * | 1988-06-29 | 1996-02-28 | Ml Technology Ventures, L.P. | Recombinant mycoplasma hyopneumoniae antigen and uses therefor |
WO1993003157A1 (en) * | 1991-07-29 | 1993-02-18 | Dana Farber Cancer Institute | Plasmids for the rapid preparation of modified proteins |
US5703057A (en) * | 1995-04-07 | 1997-12-30 | Board Of Regents The University Of Texas System | Expression library immunization |
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1999
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- 1999-11-19 MX MXPA01005011A patent/MXPA01005011A/es not_active IP Right Cessation
- 1999-11-19 NZ NZ511623A patent/NZ511623A/xx unknown
- 1999-11-19 CN CN99814733A patent/CN1331699A/zh active Pending
- 1999-11-19 CA CA002351736A patent/CA2351736A1/en not_active Abandoned
- 1999-11-19 KR KR1020017006157A patent/KR100810699B1/ko not_active IP Right Cessation
- 1999-11-19 EP EP99957754A patent/EP1140978A4/en not_active Withdrawn
- 1999-11-19 HU HU0104519A patent/HUP0104519A3/hu unknown
- 1999-11-19 JP JP2000583942A patent/JP2002535604A/ja active Pending
- 1999-11-19 BR BR9915066-2A patent/BR9915066A/pt not_active Application Discontinuation
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2001
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Cited By (1)
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CN112979769A (zh) * | 2021-03-08 | 2021-06-18 | 珠海丽禾医疗诊断产品有限公司 | 一种氨基酸序列、蛋白质及其制备方法和应用 |
Also Published As
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WO2000031115A1 (en) | 2000-06-02 |
KR20010081018A (ko) | 2001-08-25 |
CA2351736A1 (en) | 2000-06-02 |
HUP0104519A3 (en) | 2004-10-28 |
EP1140978A1 (en) | 2001-10-10 |
BR9915066A (pt) | 2002-01-15 |
MXPA01005011A (es) | 2002-04-24 |
EP1140978A4 (en) | 2006-03-01 |
NZ511623A (en) | 2003-04-29 |
HUP0104519A2 (hu) | 2002-03-28 |
ZA200103677B (en) | 2002-05-07 |
US6759516B2 (en) | 2004-07-06 |
US20020068289A1 (en) | 2002-06-06 |
AUPP727398A0 (en) | 1998-12-17 |
JP2002535604A (ja) | 2002-10-22 |
KR100810699B1 (ko) | 2008-03-07 |
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