CN116836971A - 一种快速且简便地获得正确配对tcr的方法以及获得的tcr - Google Patents
一种快速且简便地获得正确配对tcr的方法以及获得的tcr Download PDFInfo
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- CN116836971A CN116836971A CN202210306873.XA CN202210306873A CN116836971A CN 116836971 A CN116836971 A CN 116836971A CN 202210306873 A CN202210306873 A CN 202210306873A CN 116836971 A CN116836971 A CN 116836971A
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Abstract
本发明提供了获得正确配对的TCR的方法,所述方法通过DNA barcode对单细胞TCR的mRNA和cDNA进行标记,给配对的TCR加上标签,然后再通过克隆筛选找到具有相同标签的TCR进行配对,从而能够在一个反转录体系内实现数百个细胞的配对TCR克隆,实现快速,低成本,并且高通量的获取配对TCR。
Description
技术领域
本发明涉及生物技术领域。更具体地说,本发明涉及快速且简便地获得正确配对TCR的方法以及通过这种方法获得的TCR。
背景技术
T细胞受体(T cell receptor,TCR)是特异性表达于T细胞表面的一种受体分子,它特异性识别抗原提呈细胞上的抗原肽-MHC复合物,进而触发T细胞免疫应答。由于TCR分子决定着T细胞的抗原识别特异性,如果将肿瘤抗原特异性的TCR转入普通T细胞中,能够赋予该T细胞肿瘤抗原的识别能力,经体外活化增殖后再输入患者体内,可以发挥抗肿瘤功效。因此利用TCR基因导入的方法可以方便地获得大量识别特定抗原的T细胞,经TCR基因修饰的T细胞被称为TCR-T,近年来TCR-T已经成为肿瘤免疫治疗中的研究热点,在临床实验中显示了良好的治疗效果。TCR-T细胞疗法(T Cell Receptor-Gene Engineered T Cells),通过筛选和鉴定能够特异性结合靶点抗原的TCR序列,采用基因工程手段将其转入到患者外周血来源的T细胞中(或异源T细胞),再将改造后的T细胞回输至患者体内,使其特异性识别和杀伤表达抗原的肿瘤细胞,从而达到***的目的。TCR-T细胞疗法的技术流程可分为以下几步:第一步:筛选合适的肿瘤特异性抗原,以及对该抗原具有高度特异性的TCRαβ链的基因序列;第二步:从患者体内分离出T细胞;第三步:将上述能够识别肿瘤特异性抗原的TCRαβ链的基因序列装入病毒基因组,构建病毒载体;第四步:病毒转染T细胞,将目标TCR基因序列引入T细胞中,获得能够特异性识别肿瘤抗原的TCR-T细胞;第五步:TCR-T细胞体外培养,大量扩增;第六步:将数量合格的TCR-T细胞输入患者体内,进行治疗。
TCR分子主要由α和β两条链组成,其编码基因V、(D)J、C在T细胞发育过程中经过胚系重排,在胸腺中经过阳性选择和阴性选择过程,成为具有MHC识别限制性。机体成熟T细胞所产生的TCRαβ组成了一个能与成千万种抗原结合的抗原识别受体库(Repertoire)。成功获得肿瘤抗原特异性TCR是TCR-T细胞***的重要前提,肿瘤特异性TCR基因的筛选主要通过获得肿瘤抗原特异性识别的T细胞,然后克隆其TCR基因。与此同时高通量肿瘤特异性TCR鉴定的方法,将大大缩短TCR-T疗法的开放周期,降低成本。目前单细胞测序技术能够从单细胞层面识别肿瘤特异性T细胞克隆,帮助获得肿瘤高亲和力的TCR序列。
TCR筛选技术是TCR相关药物研发最核心的部分。但是目前TCR筛选成本高,耗时长,效率低,尤其是配对TCR全长基因的获取,是整个过程的限速步骤,严重影响了TCR相关药物的研发。
因此,本领域急需能够快速、简便、可靠地获得正确配对TCR全长基因的方法。
发明内容
本发明的目的在于提供一种快速、简便、可靠地获得正确配对TCR全长基因的方法。
本发明还有一目的在于通过本发明的方法提供TCR,从而能够应用于临床。
在第一方面,本发明提供一种获得正确配对的TCR的方法,所述方法包括以下步骤:
1)获得肿瘤细胞激活的T细胞;
2)从步骤1)获得的激活的T细胞获得带有Barcode的单个T细胞的TCR alpha可变区和TCR beta可变区的cDNA;
3)将步骤2)获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中,从而扩增TCR alpha可变区序列和TCR beta可变区序列;
4)从步骤3)获得的TCR alpha可变区序列和TCR beta可变区序列中挑选具有相同细胞Barcode的TCR alpha可变区序列和TCR beta可变区序列,从而获得正确配对的TCR。
在具体的实施方式中,所述步骤1)包括:
1-1.从肿瘤患者样品中分离单个核细胞;
1-2.从步骤1-1得到的单个核细胞中分选出T细胞和肿瘤细胞;
1-3.利用步骤1-2得到的肿瘤细胞刺激T细胞;
1-4.任选检测激活标志物的表达。
在优选的实施方式中,利用步骤1-2得到的肿瘤细胞刺激T细胞包括将步骤1-2得到的肿瘤细胞和T细胞共孵育。
在优选的实施方式中,所述激活标志物包括但不限于CD137、OX40、CD40、ICOS;优选CD137。
在优选的实施方式中,所述步骤1-4还包括任选检测细胞因子的表达。
在优选的实施方式中,所述的细胞因子是IFN-γ。
在具体的实施方式中,所述步骤2)包括以下步骤:
2-1.分选细胞活率在80%以上,优选在85%以上的激活的T细胞,并获得单细胞悬液;
2-2.获得细胞的mRNA;
2-3.利用Barcode引物序列与逆转录酶对获得的mRNA进行逆转录,从而得到带有Barcode的单个细胞的TCR alpha可变区和TCR beta可变区的cDNA;
2-4.任选对获得的cDNA进行纯化和PCR扩增;
2-5.对PCR产物进行环化和线性化。
在具体的实施方式中,所述步骤2-2包括:获得100-2000个,优选100-800个,最优选200-500个激活的T细胞的mRNA。
在具体的实施方式中,所述步骤3)中,携带TCR alpha恒定区的载体具有启动子、TCR alpha恒定区序列和PolyA位点;携带TCR beta恒定区的载体具有启动子、TCR beta恒定区序列和PolyA位点。
在具体的实施方式中,所述步骤4)中,对步骤3)获得的TCR alpha可变区序列和TCR beta可变区序列进行测序并挑选具有相同细胞Barcode的TCR alpha可变区序列和TCRbeta可变区序列,从而获得正确配对的TCR。
在优选的实施方式中,所述测序是进行Sanger测序。
在具体的实施方式中,所述方法还包括以下步骤:
5)任选对获得的TCR进行功能评价。
在第二方面,本发明提供一种TCR,所述TCR采用第一方面所述方法制备得到。
在具体的实施方式中,所述TCR的alpha可变区氨基酸序列选自表2,和/或所述TCR的beta可变区氨基酸序列选自表3。
在优选的实施方式中,所述TCR是表2和表3中所示的TCR-1到TCR-24。
在第三方面,本发明提供第二方面所述的TCR在制备遗传改造的T细胞中的用途。
在第四方面,本发明提供一种遗传改造的T细胞,所述T细胞表达采用第一方面所述方法制备得到的TCR或第二方面所述的TCR。
在第五方面,本发明提供第四方面所述的遗传改造的T细胞在制备***的药物组合物中的用途。
在第六方面,本发明提供一种药物组合物,所述药物组合物包含第四方面所述的遗传改造的T细胞以及药学上可接受的赋形剂。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1显示了实施例1中利用流式细胞仪检测骨髓单个核细胞中CD8+细胞和肿瘤细胞的比例;
图2显示了实施例1分选的CD8+细胞与肿瘤细胞共培养后流式检测肿瘤细胞可以激活CD8+细胞;
图3显示了实施例2电泳检测PCR扩增得到的TCR alpha和TCR beta基因片段;
图4显示了实施例3表达TCR的载体TCR-TRAC-pMax和TCR-TRBC-pMax图谱;
图5显示了实施例3电泳检测载体TCR-TRAC-pMax和TCR-TRBC-pMax线性化基因片段;
图6显示了实施例3电泳检测TCR alpha和TCR beta基因片段连接到表达载体上的阳性克隆;
图7显示了实施例4流式检测敲除TCR的Jurkat细胞;
图8显示了实施例4流式检测配对的TCR质粒电转到细胞中表达情况;
图9显示了实施例4筛选的配对TCR与肿瘤细胞共培养后检测其功能;
图10显示了实施例9TCR富集产物的环化和扩增示意图;
图11显示了实施例9电泳检测PCR扩增得到的TCR alpha和TCR beta完整片段。
具体实施方式
发明人经过广泛而深入的研究,出乎意料地发现了一种全新的获得正确配对的TCR的方法,所述方法通过DNA barcode对单细胞TCR的mRNA和cDNA进行标记,给配对的TCR加上标签,然后再通过克隆筛选找到具有相同标签的TCR进行配对,从而能够在一个反转录体系内实现数百个细胞的配对TCR克隆,实现快速,低成本,并且高通量的获取配对TCR。在此基础上完成了本发明。
DNA Barcode
DNA Barcode是带有不同碱基的DNA片段,是样品的标签。用于区分不同的样品,在该专利中指细胞标签序列。它的作用是特异性的标记单个细胞,使得每个细胞具有不同的barcode。如果测序读取的基因序列具有同样的barcode,则这些基因来源于同一个细胞。
本发明的克隆TCR基因的方法
在本发明中,“克隆TCR基因的方法”与“获得配对TCR的方法”、“本发明的方法”以及“获取全长TCR基因的方法”等具有相同的意义,均是指获取个体的完整TCR基因的方法。
在本领域中,目前主要的克隆TCR基因的方法有以下几种:
1.对T细胞群进行单细胞分选,然后对单细胞进行裂解和反转录,最后使用靶向TCR可变区的多重兼并引物以及靶向TCR恒定区的引物进行单细胞水平PCR,扩增TCR可变区。这是一种快速低成本获取TCR可变区的方法,但是这种方法由于使用的兼并引物在TCR可变区内部,因此扩增得到的区域只是部分区域,不是TCR全长,无法直接应用于下游分子构建和蛋白表达。一般需要进行数据库序列比对,从数据库拿到全长序列后进行基因合成,才能获得全长TCR序列(Rapid Identification and Evaluation of Neoantigen-reactive T-Cell Receptors From Single Cells.J Immunother.2021Jan;44(1):1-8)。
2.对T细胞群进行单细胞分选,然后使用激活剂激活扩增单个T细胞,一般需要扩增两周左右的时间,让单个T细胞变成一个T细胞克隆群,细胞数目达到几百个或者上千个。然后使用5’RACE(rapid amplification of cDNA ends)方法进行TCR可变区全长扩增,扩增后可以得到TCR全长基因,后续直接扩增得到TCR全长基因。但是这种方法耗时很长,很多单细胞不能成功形成多细胞克隆,试剂盒成本很高无法大量扩增TCR基因(Development ofa CD8 co-receptor independent T-cell receptor specific for tumor-associatedantigen MAGE-A4 for next generation T-cell-based immunotherapy.JImmunotherCancer.2021;9(3))。
3.与第二种方法类似,对T细胞群进行单细胞分选,然后直接在单细胞水平进行5‘RACE扩增TCR可变区全长,后续再进行PCR扩增得到TCR全长基因。这种方法虽然比较简单,但是同样因为每个细胞需要一个单独的反应体系,试剂消耗大,试剂盒成本很高,无法大量扩增TCR基因(An immunodominant NP105-113-B*07:02cytotoxic T cell responsecontrols viral replication and is associated with less severe COVID-19disease.Nat Immunol.2022;01;23(1):50-61.)。
4.使用单细胞DNA barcode对T细胞群中的每一个T细胞进行标记,然后使用高通量测序的方法获得配对TCR全长序列信息。对筛选确定的配对TCR进行基因合成得到全长TCR基因。这种方法虽然可以大量低成本的获得配对TCR序列,但是只能得到序列信息,无法直接获得TCR可变区全长基因片段,后续基因合成成本高,无法大量进行TCR的合成(Directidentification of neoantigenspecific TCRs from tumor specimens by high-throughput single-cell sequencing.Journal for ImmunoTherapy of Cancer 2021;9:e002595.)。
与现有技术相比,本发明通过DNA barcode对单细胞TCR的mRNA和cDNA进行标记,给配对的TCR加上标签,然后再通过克隆筛选找到具有相同标签的TCR进行配对,在一个反转录体系内实现数百个细胞的配对TCR克隆,实现快速,低成本,并且高通量的获取配对TCR。在本发明的开发中,巧妙的应用了SMART(switching mechanism at the 5'end ofthe RNA transcript)技术(Takara Bio USA,Inc),在下游开发出了独特的高通量获取配对TCR和筛选配对TCR的技术。本发明人首次实现不依赖基因合成的,快速、低成本的获取正确配对的TCR基因。
SMART技术最早在2001年被开发出来,该技术可以实现全长cDNA文库的构建,即使起始材料是来源于单个细胞的RNA。SMART技术的核心是使用叫做TSO(Template switcholigo)的DNA-RNA复合oligo,TSO通过RNA部分可以结合反转录产生的cDNA末端,然后TSO其余部分可以作为cDNA扩增的恒定模版。
另外,本项目优化了配对TCR的下游功能筛选。首次使用DNA质粒为载体进行高通量配对TCR筛选,大大提高了筛选效率。
通过上述两方面对TCR筛选的优化,使常规需要2-3个月才能完成的流程缩短为3到4周,成本降低为原来的1/5-1/10,解决了TCR序列筛选成本高,耗时长,效率低等问题,为TCR类药物的发展奠定基础,也使得个体化TCR-T作为一种细胞疗法成为可能。
进一步地,本发明人还发现获取单个T细胞的mRNA时,细胞的数量不宜过多。在具体的实施方式中,本发明的方法从获得100-2000个,优选100-800个,最优选200-500个激活的T细胞中获取mRNA。
本发明的优点:
1.本发明首次实现不依赖基因合成的,快速、低成本地获取配对的TCR基因;
2.本发明首次使用DNA质粒为载体进行高通量配对TCR筛选,大大提高了筛选效率;
3.本发明显著降低TCR筛选所需的时间;
4.本发筛选TCR的成本显著降低;
5.本发明为TCR类药物的发展奠定了新的基础;
6.本发明可以应用于完全个体化的TCR-T细胞疗法。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
实施例:
实施例1.骨髓来源T细胞激活
1.分离单个核细胞:使用含有抗凝剂的管子采集AML病人骨髓样本,低温保存。骨髓样本倒入PBMC分离管(达优,Cat#7121012),20℃,800g离心15min后吸取单个核细胞的细胞层。吸取的单个核细胞用RPMI1640完全培养基清洗1-2次,最后用RPMI1640完全培养基重悬细胞备用。取少量分离得到的骨髓单个核细胞细胞加入anti-CD8-PE(Biolegend,344706)、anti-CD33-PE(Biolegend,366606)和anti-CD34-PE(Biolegend,343608)对细胞进行染色,4℃孵育半小时。染色完成后,用PBS清洗细胞后重悬,使用流式细胞仪检测T细胞和肿瘤细胞(CD33或CD34阳性细胞)比例。流式细胞仪检测骨髓单个核细胞中CD8细胞和肿瘤细胞的比例,结果见图1A和1B。
2.T细胞分选:利用美天旎anti-APC Micro Beads(130-090-855)和MS分离柱(130-090-855)从PBMC中先分选出CD8+T细胞。
肿瘤细胞的分选:上一步骤中分选得到的CD8阴性的细胞,用美天旎磁珠和MS分离柱从中分选出CD33+&CD34+肿瘤细胞。
3.激活标志物CD137检测:
3.1细胞共培养:分选得到的CD33+&CD34+肿瘤细胞肿瘤细胞计数后,按照细胞浓度2×105/ml,100ul/孔铺至96孔平底板中。根据效靶比2:1,将CD8+T细胞的密度调整至2×105/ml,按照100ul每孔接种到96孔板中与肿瘤细胞混合共培养。
3.2检测CD137表达:共培养24h后,吸弃上清。剩余的细胞取60ul,加入anti-CD8-APC(BD Pharmingen,555369)、anti-CD137-PE(BD Pharmingen,555956)抗体对细胞进行染色,4℃孵育半小时。染色完成后,用PBS清洗细胞后重悬,使用流式细胞仪检测CD137的表达。共培养后的细胞流式检测结果见图2,从流式结果可以看出分选的CD8+T细胞与肿瘤细胞共培养后能检测到CD137表达上调。
实施例2.使用油包水体系获取单个T细胞TCR alpha和TCR beta可变区序列
1.细胞制备:流式检测到CD137表达上调的T细胞,使用BD FACSjazz cell sorter(BD,655489)分选收集CD137阳性的T细胞,细胞分选流程按照仪器的说明书操作。对分选得到CD137阳性的T细胞进行活性测定和细胞计数,保证细胞活率在85%以上,根据计数将细胞密度调整到1X106/ml,制备成单细胞悬液。
2.单细胞分选:选择300-600个(100-2000个细胞范围内)活性状态良好的T细胞上样,将样品命名为ST。将Master Mix(10x Genomics,Chromium Next GEM Single Cell 5'Library&Gel Bead Kit v1.1,PN-1000165)和细胞悬液共70ul、含有barcode序列的凝胶珠50ul和45ul的油滴分别加入到Chromium Next GEM Chip G的不同小室并且不要产生气泡,经由微流体“双十字”交叉***形成油滴包裹的凝胶珠(Gel Bead-In-EMulsions,GEMs)。
3.反转录:收集GEMs,凝胶珠在每个油滴内自动溶解释放大量Barcode引物序列,同时细胞裂解释放出mRNA。因为beads已带上接头,其中有一段是poly dT序列,在细胞裂解释放的核酸中,只有mRNA带有polyA的尾巴,于是这段beads上面poly dT就可以从众多的裂解产物里捕获到mRNA。同时Master Mix中带有反转录试剂,当mRNA被捕获后,在逆转录酶的作用下发生逆转录反应,产生用于测序的带有Barcode和UMI信息的cDNA一链。收集混合裂解后的产物在PCR仪上进行反转录:53℃45min;85℃5min;4℃保存。
4.反转录产物纯化
4.1将纯化磁珠取出后上下颠倒8-10次并且多次震荡,直到所有磁珠都充分悬浮。
4.2在反转录产物中加入200ul的磁珠,并在室温下孵育5min。放置在磁性分离器2min,吸出上清液将EP管继续留在磁性分离器上。
4.3每管加入300ul 80%的乙醇清洗30s,无需震荡并保持EP管在磁性分离器上30s。吸出上清液。重复该步骤一次。
4.4最后一次尽可能吸尽上清液室温下风干2min,过干可能会降低产量。
4.5将EP管从磁性分离器上取下来,加入35.5ul洗脱溶液,室温下静置2min。
4.6将EP管放在磁性分离器上1-2min直到磁珠被磁铁吸附,吸取35ul上清到新的EP管中,尽量不要吸到磁珠。
5.cDNA扩增和纯化
5.1在冰上预先配制以下试剂:
扩增master mix | 50ul |
ddH2O | 8ul |
cDNA additive | 5ul |
引物mix | 2ul |
将配制好的65ul的cDNA扩增mix全部加入到上一步骤纯化的35ul产物中。
5.2PCR扩增条件:
5.3cDNA产物纯化:
5.3.1将纯化磁珠从冰箱取出后上下颠倒8-10次并且多次震荡,直到所有磁珠都充分悬浮。
5.3.2在cDNA产物中加入0.6X的磁珠60ul,并在室温下孵育5min。放置在磁性分离器上2min,吸出上清液,将EP管继续留在磁性分离器上。
5.3.3每管加入200ul 80%的乙醇清洗30s,无需震荡并保持EP管在磁性分离器上。吸出上清液,重复此步骤两次,最后一次尽可能吸尽上清液。
5.3.4室温下风干2min,过干可能会降低产量。
5.3.5加入45.5ul洗脱溶液混匀,室温下静置5min,将EP管放在磁性分离器上1-2min直到磁珠被磁铁吸附,吸取45ul上清到新的EP管中,尽量不要吸到磁珠。
6.TCR富集
6.1取纯化后的cDNA 2ul加入到33ul的ddH2O,得到总体积为35u样品备用。
在冰上预先配制以下试剂:
扩增master mix | 50ul |
ddH2O | 5ul |
cDNA additive | 5ul |
T细胞mix1 | 5ul |
将以上配制好的65ul扩增mix全部加入到备用的35ul样品中。
6.2PCR扩增条件:
6.3TCR富集产物纯化:
6.3.1将纯化磁珠从冰箱取出后上下颠倒8-10次并且多次震荡,直到所有磁珠都充分悬浮。
6.3.2在一轮TCR产物中加入0.8X的磁珠80ul,并在室温下孵育5min。放置在磁性分离器上2min,吸出上清液,将EP管继续留在磁性分离器上。
6.3.3每管加入200ul 80%的乙醇清洗30s,无需震荡并保持EP管在磁性分离器上。吸出上清液,重复此步骤两次,最后一次尽可能吸尽上清液。
6.3.4室温下风干2min,过干可能会降低产量。
6.3.5加入35.5ul洗脱溶液混匀,室温下静置5min,将EP管放在磁性分离器上1-2min直到磁珠被磁铁吸附,吸取45ul上清到新的EP管中,尽量不要吸到磁珠。
7.PCR分别扩增TCR alpha和TCR beta可变区
7.1对富集后的TCR产物分别扩增TCR alpha和TCR beta可变区
PCR扩增体系:50ul
Taq mix 2X | 25ul |
ddH2O | 22ul |
二轮TCR富集产物 | 1ul |
F-R1 | 1ul |
TCR alpha-R/TCR beta-R | 1ul |
其中F-R1引物序列为:ccactttgcctttctctccacaggGGTACCAATGATACGGCGACCACCGAGATCT(SEQ ID NO:5)
TCR alpha-R引物序列为:AGTCTCTCAGCTGGTACACG(SEQ ID NO:6)
TCR beta-R引物序列为:TCTGATGGCTCAAACACAGC(SEQ ID NO:7)
PCR扩增条件:
琼脂糖凝胶电泳检测PCR产物,结果见图3。从电泳结果可以看出,TCR alpha和TCRbeta可变区扩增后形成单一的条带。
7.2PCR产物纯化,按照试剂盒提供的标准操作流程进行(天根生化科技有限公司,DP219-03)操作。
7.2.1将单一的DNA条带从琼脂糖凝胶中切下(尽量切除多余部分)放入干净的离心管中,称取重量。
7.2.2向凝胶块中加入3倍体积溶胶液PE(如果凝胶重0.1g,其体积可视为100ul,则加入300ul溶胶液PE)。室温15℃-25℃溶胶5-10min,期间不断温和地上下翻转离心管,以确保胶块充分溶解。
7.2.3将上一步所得溶液加入一个吸附柱CA5中(吸附柱放入收集管中),室温放置2min,12000rpm离心30-60s,倒掉收集管中的废液,将吸附柱CA5放入收集管中。
7.2.4向吸附柱CA5中加入600ul漂洗液PW,12000rpm离心30-60s,倒掉收集管中的废液,将吸附柱CA5放入收集管中。
7.2.5重复操作步骤7.2.4。
7.2.6将吸附柱CA5放回收集管中,12000rpm离心2min,尽量除尽漂洗液。将吸附柱CA5置于室温放置数分钟,彻底地晾干,以防止残留的漂洗液影响下一步实验。
7.2.7将吸附柱CA5放到一个干净的离心管中,向吸附膜中间位置悬空滴加适量洗脱缓冲液TB或提前预热的ddH2O,室温放置2min。12000rpm离心2min收集DNA溶液。纯化回收后的产物分别标记为:TCR-TRAV和TCR-TRBV,共2个产物。该产物携带5‘端barcode序列和其他元件,TCR可变区全长以及部分的TCR恒定区序列。
注:TCR富集产物包含一部分恒定区序列,作为重组所用的同源序列。
实施例3.配对全长TCR序列获得
1.连接TCR-pMax载体:该载体携带CMV启动子,TCR alpha和TCR beta恒定区序列和polyA位点。恒定区使用人TCR alpha和TCR beta恒定区。载体中人TCR alpha恒定区命名为TRAC(核酸序列见Seq ID No.1,氨基酸序列见Seq ID No.2)。载体中人的TCR beta恒定区命名为TRBC(核酸序列见Seq ID No.3,氨基酸序列见Seq ID No.4),其中TCR-TRAC-pMax和TCR-TRBC-pMax载体图谱见图4,从图谱可以看出TCR-TRAC-pMax和TCR-TRBC-pMax载体中除了包含基因表达所需要的启动子、还包含TCR alpha和TCR beta的恒定区序列和相应的酶切位点,便于实施例2中富集的TCR产物快速克隆到该载体中。
人的TCR alpha恒定区核酸序列Seq ID No.1
Catatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc
人的TCR alpha恒定区氨基酸序列Seq ID No.2
HIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
人的TCR beta恒定区核酸序列Seq ID No.3
Gacctgaacaaggtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtgtgcctggccacaggcttcttccctgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacggacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacccgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggctttacctcggtgtcctaccagcaaggggtcctgtctgccaccatcctctatgagatcctgctagggaaggccaccctgtatgctgtgctggtcagcgcccttgtgttgatggccatggtcaagagaaaggatttc
人的TCR beta恒定区氨基酸序列Seq ID No.4
DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
通过重组的方法将纯化后的TCR alpha富集产物重组到TCR-TRAC-pMax、纯化后的TCR beta富集产物重组到TCR-TRBC-pMax载体,最终的TCR序列具有完整的恒定区和TCR表达框,可以高效表达TCR亚基。具体步骤为:
1.1制备线性化载体:利用BamHI(NEB:R3136S)和KpnI(NEB:R3142S)将TCR-TRAC-pMax、TCR-TRBC-pMax线性化并纯化。TCR-TRAC-pMax、TCR-TRBC-pMax线性化后进行琼脂糖凝胶电泳检测,结果见图5。
1.2重组反应:
50℃反应20min
2.转化大肠杆菌
2.1在冰上解冻感受态细胞DH5α(上海唯地生物科技有限公司),一般转化产物的体积不超过感受态体积的1/10。
2.2取10ul重组产物,加入到100ul感受态细胞中,轻弹管壁数下混匀,在冰上放置20min。
2.3 42℃热激90s,冰上放置2min,加600ul无双抗LB培养基(上海生工),37℃摇床220rpm复苏1h,4500rpm离心5min。吸去600ul上清,剩余的菌液全部涂含Amp平板,37℃微生物培养箱培养过夜。
3.菌检
第二天从转化TCR alpha和TCR beta基因片段的平板上分别挑选300个单菌落于96孔深孔板中37℃摇床摇2h,深孔板每孔放入1ml含Amp抗性LB培养基。每个取2ul菌液PCR初步鉴定阳性克隆。
PCR菌检体系:20ul
Taq mix 2X | 10ul |
ddH2O | 7ul |
菌液 | 2ul |
ll25-JJ-F1 | 0.5ul |
ll25-JJ-R1 | 0.5ul |
其中ll25-JJ-F1引物序列(菌检引物)为:taggcacctattggtcttac
ll25-JJ-R1引物序列(菌检引物)为:tcactgcattctagttgtgg
PCR菌检条件:
琼脂糖凝胶电泳检测菌检结果见图6,从电泳结果可以看出***序列长度在750bp的条带为阳性克隆,筛选得到的阳性克隆的剩余菌液使用无内毒素质粒小提中量试剂盒(天根生化科技有限公司,DP118-02)抽提质粒。
4.Sanger测序
对初步鉴定出来的阳性克隆送相应的质粒sanger测序验证。TCR alpha共送测192个克隆,分别按照TCR alpha1-192进行标记。TCR beta送测192个克隆,分别按照TCR beta1-192进行标记。其中测序引物序列为:taggcacctattggtcttac。
5.序列比对,挑选配对TCR序列
通过sanger测序得到TCR alpha和TCR beta可变区的完整序列以及单个细胞的barcode序列。完整序列通过DNA序列分析软件分析得到TCR-alpha和TCR-beta可变区序列,可以判定具有同一个细胞barcode的TCR-alpha和TCR-beta克隆为一对TCR即配对TCR。通过这种方法在所有挑选的克隆中获得配对的TCR序列,将配对TCR alpha和TCR beta质粒找出并标记。共筛选到110对配对的TCR质粒,然后将配对的TCR alpha和TCR beta质粒混合到一起重新按照TCRab1-110进行标记。其中表1展示了其中的24对具有相同barcode的配对TCR。进一步使用IMGT数据库(https://www.imgt.org)分别分析不同TCR-alpha和TCR-beta可变区对应的可变区类型,结果见表2和表3。
表1.24对具有相同barcode的配对TCR序列
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表2.TCR-alpha可变区对应的可变区类型
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表3.TCR-beta可变区对应的可变区类型
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实施例4.高通量TCR功能评价
1.效应细胞构建和测试
使用带有NFAT-luciferase的慢病毒感染Jurkat细胞(中国科学院细胞库),并挑取单克隆培养得到Jurkat-NFAT-luciferase的reporter细胞系(TCR reconstitution inJurkat reporter cells facilitates the identification of novel tumor antigensby cDNA expression cloning.Int J Cancer.2002May 1;99(1):7-13)。使用CRISPR/Cas9电转同时敲除TCR基因,选择未敲除的细胞做阴性对照。具体步骤为:
1.1取一定量的Jurkat-NFAT-luciferase细胞,1200rpm离心5min后用培养基opti-MEM洗2次备用。每1X106细胞用25ul的opti-MEM重悬细胞后备用。
1.2冰上准备Cas9和sgRNA:每1X106细胞加入10ul Cas9(500ng/ul)蛋白(南京金斯瑞提供)、2.5ul的TRAC-sgRNA和2.5ul的TRBC-sgRNA)(400ng/ul南京金斯瑞提供),两者常温下孵育10min,加入提前准备好的细胞开始电转。
1.3电转条件:BTX-ECM830的1mm电击杯,电压为250V,脉冲时间1ms。
1.4电转结束迅速在电击杯里加入1ml培养基,然后将细胞转入培养孔里培养。
1.5电转敲除48h后,取一定体积的细胞,加入anti-CD3-APC抗体(BD Pharmingen,555335)对细胞进行染色,4℃孵育半小时。染色完成后,用PBS清洗细胞后重悬,使用流式细胞仪检测TCR敲除的情况。Jurkat细胞中敲除TCR结果见图7,从流式结果可以看出Jurkat细胞中的TCR已全部敲除,CD3不再表达。通过该步骤制备得到的效应细胞命名为:Jurkat-KO-ER。
2.配对的TCR质粒电转进效应细胞
根据电转最优条件,选取110对配对的TCR质粒中序列完整的24对配对的TCR,分别命名为TCRab-1-24,电转进Jurkat-KO-ER。电转流程为:
2.1取Jurkat-KO-ER细胞,1200rpm离心5min后用培养基opti-MEM洗2次备用。
2.2每1X106的Jurkat-KO-ER细胞加入配对的TCR质粒4ug(TCR alpha和TCR beta质粒各2ug),补足培养基opti-MEM至100ul。
2.3将质粒和细胞混合物100ul加入到96孔电转板(BTX,45-0450),电压为260V,脉冲时间1ms。电转后将细胞转移到新的96孔板中,将细胞继续培养。
3.筛选有功能的TCR
3.1流式检测细胞中TCR的表达:配对TCR质粒电转进Jurkat-KO-ER的细胞24h后,取一定体积的细胞,加入anti-CD3-APC抗体(BD Pharmingen,555335)对细胞进行染色,4℃孵育半小时。染色完成后,用PBS清洗细胞后重悬,使用流式细胞仪检测TCR细胞的表达情况。电转的TCR在Jurkat-KO-ER细胞的表达的代表性结果见图8。从图8可以看出,4对代表性的配对的TCR电转到细胞内以后,CD3恢复表达。
3.2细胞铺板:将实施例1中获取的CD33/CD34阳性肿瘤细胞按照细胞浓度1×106/ml,50ul/孔铺至96孔平底不透明白板中。将电转了不同TCR的Jurkat-KO-ER的细胞按照效靶比1:1即细胞浓度1×106/ml,50ul/孔加入到肿瘤细胞中混匀。将96孔板放置于37℃,5%CO2细胞培养箱中培养。
3.3酶标仪检测:细胞共培养24h后测定细胞中荧光素酶活性(相对光单位,RLU),来检测每个TCR的功能。具体步骤为:每孔加入2X的D-luciferin底物(Thermo FisherScientific:88293)100ul混匀避光显色5min,在酶标仪用化学发光模式检测荧光强度。由图9可见,TCR克隆:TCRab-2,TCRab-13和TCRab-16能够特异性的识别肿瘤细胞,可以做进一步筛选评价。
实施例5.TCR alpha和beta克隆至mRNA载体
以TCRab 22,TCRab 33和TCRab 66为模板设计引物分别扩增3个TCR基因序列,PCR扩增条带经琼脂糖凝胶电泳检测后分别进行胶回收纯化得到DNA片段。利用一步法快速克隆试剂盒(翌圣生物科技有限公司:10911ES20)分别将3个TCR基因序列重组进mRNA载体得到重组产物。将3个重组产物分别转入大肠杆菌DH5α(上海唯地生物科技有限公司)菌株,第二天挑取中等大小的白色菌落在含Amp抗生素的液体LB培养基中摇过夜,第二天抽提质粒,酶切鉴定正确后送苏州金唯智生物科技有限公司测序验证,测序验证3个载体均构建正确。得到载体分别为TCRab-22-m、TCRab-33-m和TCRab-66-m。
实施例6.mRNA制备
使用试剂盒制备mRNA(近岸蛋白)。
实施例7.mRNA TCR-T制备和功能评价
1.T细胞激活和mRNA电转:将人外周血单个核细胞(PBMCs,购自上海妙顺生物)稀释到1×106/ml,按照细胞与磁珠1:3的比例使用抗人的CD3/CD28磁珠(Thermo FisherScientific)激活T细胞,同时培养基中添加300IU/ml的IL-2(stemcell:78036)。取一定量激活的T细胞1200rpm离心5min后用培养基opti-MEM洗2次备用。每1X10^6细胞用25ul的opti-MEM重悬细胞后备用。冰上融合mRNA:每1X10^6细胞加入1ug的mRNA,开始电转。电转条件:BTX-ECM830的1mm电击杯,电压为250V,脉冲时间1ms。电转结束迅速在电击杯里加入1ml的T细胞培养基,然后将细胞转入培养孔里培养。
2.电转24h后,取一定体积的细胞,加入抗小鼠TCR恒定区的抗体对细胞进行染色,4℃孵育半小时。染色完成后,用PBS清洗细胞后重悬,使用流式细胞仪检测TCR表达的情况。通过该步骤制备得到了mRNA TCR-T。
3.体外功能评价
TCR-T与病人的肿瘤细胞共培养,未电转的T细胞作为对照细胞。
4.动物体内功能评价。
1)病人原代肿瘤细胞皮下建模;
2)TCR-T细胞回输;
3)功能评价;
在不同时间点量取肿瘤大小。
实施例8.
参考实施例2和实施例3技术流程,选择细胞上样量为1500个细胞,经过PCR扩增和连接TCR-pMax载体后,TCR alpha和TCR beta分别挑选了共300个克隆进行测序和序列比对。经过序列分析发现,在挑选的克隆中只有两对配对的TCR,筛选到的配对的TCR占挑取的TCR总克隆的0.67%,获得配对TCR的效率大大降低。因此需要挑选更多的克隆才可能获得更多的配对TCR,大大增加了工作量和成本。
实施例9.获取TCR全长
由于上述实施例获得的TCR alpha和TCR beta全长序列5‘端包含各种barcode序列和一些恒定元件,可能会影响后续TCR的表达。因此本发明重新设计了凝胶珠负载的oligo序列,增加了TCR富集产物的环化和扩增步骤,将5’端的元件连接到TCR基因片段的3‘端,从而不再影响所克隆TCR的表达,可以直接进行下游应用(示意图见图10)。由于cDNA扩增产物只包含部分的TCR恒定区,因此在环化步骤本发明添加了包含其余TCR恒定区的基因片段进行重组环化。基本步骤如下:
1.扩增产物环化
1.1参考实施例1步骤,将扩增的cDNA放于PCR管中,再添加另一个基因片段:TCR恒定区片段(通过PCR获得),用TE Buffer补充至50ul。置于PCR仪上95℃反应3min。反应结束后立即将PCR管转移至冰上,冰浴2min。
1.2按照下表配制配制反应体系:
组分 | 体积 |
Universal Splint Buffer | 19.5ul |
DNA Ligase | 0.5ul |
将上述反应液加入步骤1.1反应后的PCR管中混匀后,瞬时离心将反应液收集至管底。37℃反应15min。反应结束后取出PCR管立即进入下步反应。
1.3酶切消化,取出Digestion Enzyme置于冰上自然融解,混合均匀,瞬时离心。取2ul的Digestion Enzyme加入步骤1.2反应后的PCR管中混匀后,涡旋震荡瞬时离心将反应液收集至管底。37℃反应15min。反应结束后,向每个反应中加入7ul Digestion StopBuffer混匀后,使用移液器轻轻吹打混合均匀,瞬时离心使全部反应液置于PCR管底部。
1.4环化产物纯化
1.4.1取出DNA Clean Beads充分震荡混匀,室温恢复半个小时再使用。
1.4.2吸取170ul DNA Clean Beads至步骤6.3的产物中,用移液器轻轻吹打混匀,室温孵育10min。
1.4.3孵育结束后瞬时离心,将1.5mLEP管置于磁力架,静置5min至液体澄清,用移液器小心吸弃上清。
1.4.4保持1.5ml EP管置于磁力架上,沿离心管侧壁加入500ul新鲜配制的80%乙醇漂洗磁珠及管壁,小心吸取并丢弃上清。重复该步骤一次。
1.4.5将离心管瞬时离心后放置于磁力架上,使用10ul吸头移去少量残留乙醇,注意勿吸到磁珠。
1.4.6打开离心管管盖,并于室温静置约5min,至乙醇挥发完全。
1.4.7移出离心管,加入22ul TE Buffer进行DNA洗脱,用移液器轻轻吹打至混匀,室温孵育5min。
1.4.8瞬时离心,将1.5ml EP管置于磁力架上静置2min至液体完全澄清,使用移液器小心转移20ul上清液转移到新的1.5ml EP管中,既得到纯化的产物。
2.全长TCR富集
2.1对富集后的TCR产物分别扩增TCR-alpha和TCR-beta全长。扩增结果见图11。PCR的正向和反向引物在TSO序列内部(见图10)。
PCR扩增体系:50ul
Taq mix 2X | 25ul |
ddH2O | 18ul |
环化产物 | 5ul |
TSO-F | 1ul |
TSO-R | 1ul |
PCR扩增条件:
2.2TCR全长纯化,按照试剂盒提供的标准操作流程进行(天根生化科技有限公司,DP219-03)操作。
2.2.1将单一的DNA条带从琼脂糖凝胶中切下(尽量切除多余部分)放入干净的离心管中,称取重量。
2.2.2向凝胶块中加入3倍体积溶胶液PE(如果凝胶重0.1g,其体积可视为100ul,则加入300ul溶胶液PE)。室温15℃-25℃溶胶5-10min,期间不断温和地上下翻转离心管,以确保胶块充分溶解。
2.2.3将上一步所得溶液加入一个吸附柱CA5中(吸附柱放入收集管中),室温放置2min,12000rpm离心30-60s,倒掉收集管中的废液,将吸附柱CA5放入收集管中。
2.2.4向吸附柱CA5中加入600ul漂洗液PW,12000rpm离心30-60s,倒掉收集管中的废液,将吸附柱CA5放入收集管中。
2.2.5重复操作步骤2.2.4。
2.2.6将吸附柱CA5放回收集管中,12000rpm离心2min,尽量除尽漂洗液。将吸附柱CA5置于室温放置数分钟,彻底地晾干,以防止残留的漂洗液影响下一步实验。
2.2.7将吸附柱CA5放到一个干净的离心管中,向吸附膜中间位置悬空滴加适量洗脱缓冲液TB或提前预热的ddH2O,室温放置2min。12000rpm离心2min收集DNA溶液。纯化回收后的产物分别标记为:TCR-TRA和TCR-TRB,共2个产物。然后将两个产物分别连接到空的pMax载体,pMax载体本身具有CMV启动子和poly A位点,因此可以直接获得TCR全长表达框。参考实施例3进行配对TCR的鉴定。参考实施例4-7进行下游应用。本实施例获得的TCRalpha和TCR beta全长序列5‘端没有各种barcode序列和恒定元件,可以直接应用于下游筛选和功能测试。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 立凌生物制药(苏州)有限公司
<120> 一种快速且简便地获得正确配对TCR的方法以及获得的TCR
<130> P2022-0593
<160> 175
<170> PatentIn version 3.5
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catatccaga accctgaccc tgccgtgtac cagctgagag actctaaatc cagtgacaag 60
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attattccag aagacacctt cttccccagc ccagaaagtt cctgtgatgt caagctggtc 300
gagaaaagct ttgaaacaga tacgaaccta aactttcaaa acctgtcagt gattgggttc 360
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gagctgagct ggtgggtgaa tgggaaggag gtgcacagtg gggtcagcac ggacccgcag 180
cccctcaagg agcagcccgc cctcaatgac tccagatact gcctgagcag ccgcctgagg 240
gtctcggcca ccttctggca gaacccccgc aaccacttcc gctgtcaagt ccagttctac 300
gggctctcgg agaatgacga gtggacccag gatagggcca aacccgtcac ccagatcgtc 360
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tactggtacc gacagaggct ggggcagggc ctggagtttt taatttactt ccaaggcaac 300
agtgcaccag acaaatcagg gctgcccagt gatcgcttct ctgcagagag gactggggaa 360
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gtgactatac taacagcatg tttgattatt tcctatggta caaaaaatac cctgctgaag 360
gtcctacatt cctgatatct ataagttcca ttaaggataa aaatgaagat ggaagattca 420
ctgttttctt aaacaaaagt gccaagcacc tctctctgca cattgtgccc tcccagcctg 480
gagactctgc agtgtacttc tgtgcagcaa gcgtgggttc tgggggttac cagaaagtta 540
cctttggaac tggaacaaag ctccaagtca tccca 575
<210> 12
<211> 494
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 12
actgctcaga attcccacat ttaccttttc ttatatggga cagtgatcct gatctggtaa 60
agctcccatc ctgccctgac cctgccatgg gcaccaggct cctcttctgg gtggccttct 120
gtctcctggg ggcatatcac acaggagctg gagtctccca gtcccccagt aacaaggtca 180
cagagaaggg aaaggatgta gagctcaggt gtgatccaat ttcaggtcat actgcccttt 240
actggtaccg acagaggctg gggcagggcc tggagttttt aatttacttc caaggcaaca 300
gtgcaccaga caaatcaggg ctgcccagtg atcgcttctc tgcagagagg actggggaat 360
ccgtctccac tctgacgatc cagcgcacac agcaggagga ctcggccgtg tatctctgtg 420
ccagcagagg gggcaggggg ctctctgggg ccaacgtcct gactttcggg gccggcagca 480
ggctgaccgt gctg 494
<210> 13
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 13
actgctcaga attccc 16
<210> 14
<211> 444
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 14
caccagggtc taggtttggt actgattttc ttatatggga cactaaagat gaactattct 60
ccaggcttag tatctctgat actcttactg cttggaagaa cccgtggaga ttcagtgacc 120
cagatggaag ggccagtgac tctctcagaa gaggccttcc tgactataaa ctgcacgtac 180
acagccacag gatacccttc ccttttctgg tatgtccaat atcctggaga aggtctacag 240
ctcctcctga aagccacgaa ggctgatgac aagggaagca acaaaggttt tgaagccaca 300
taccgtaaag aaaccacttc tttccacttg gagaaaggct cagttcaagt gtcagactca 360
gcggtgtact tctgtgctct gagtgataaa actggaggct tcaaaactat ctttggagca 420
ggaacaagac tatttgttaa agca 444
<210> 15
<211> 481
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 15
caccagggtc taggttcaac gaatcatttc ttatatggga agggcctcat ctaggtgaag 60
gctccacctg ccccaccctg ccatggccac caggctcctc tgctgtgtgg ttctttgtct 120
cctgggagaa gagcttatag atgctagagt cacccagaca ccaagggaca aggtgacaga 180
gatgggacaa gaagtaacaa tgagatgtca gccaatttta ggccacaata ctgttttctg 240
gtacagacag accatgatgc aaggactgga gttgctggct tacttccgca accgggctcc 300
tctagatgat tcggggatgc cgaaggatcg attctcagca gagatgcctg atgcaacttt 360
agccactctg aagatccagc cctcagaacc cagggactca gctgtgtatt tttgtgctag 420
tgctattagc gggagcacag atacgcagta ttttggccca ggcacccggc tgacagtgct 480
c 481
<210> 16
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 16
caccagggtc taggtt 16
<210> 17
<211> 574
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 17
gtctcgtgta cgcaccaatt aggttttttc ttatatgggg ttcatatgta aaatgaaggg 60
tctgtggaag gacatgaata aagcacagga ggttgaagtc agatttgcag ctttctaggc 120
aggagataag acaatctgca tcttcacagg agggatggcc atgctcctgg gggcatcagt 180
gctgattctg tggcttcagc cagactgggt aaacagtcaa cagaagaatg atgaccagca 240
agttaagcaa aattcaccat ccctgagcgt ccaggaagga agaatttcta ttctgaactg 300
tgactatact aacagcatgt ttgattattt cctatggtac aaaaaatacc ctgctgaagg 360
tcctacattc ctgatatcta taagttccat taaggataaa aatgaagatg gaagattcac 420
tgttttctta aacaaaagtg ccaagcacct ctctctgcac attgtgccct cccagcctgg 480
agactctgca gtgtacttct gtgcagcaag cgtgggttct gggggttacc agaaagttac 540
ctttggaact ggaacaaagc tccaagtcat ccca 574
<210> 18
<211> 489
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 18
gtctcgtgta cgcacctgta aaccaatttc ttatatgggg atcctgatct ggtaaagctc 60
ccatcctgcc ctgaccctgc catgggcacc aggctcctct tctgggtggc cttctgtctc 120
ctgggggcat atcacacagg agctggagtc tcccagtccc ccagtaacaa ggtcacagag 180
aagggaaagg atgtagagct caggtgtgat ccaatttcag gtcatactgc cctttactgg 240
taccgacaga ggctggggca gggcctggag tttttaattt acttccaagg caacagtgca 300
ccagacaaat cagggctgcc cagtgatcgc ttctctgcag agaggactgg ggaatccgtc 360
tccactctga cgatccagcg cacacagcag gaggactcgg ccgtgtatct ctgtgccagc 420
agagggggca gggggctctc tggggccaac gtcctgactt tcggggccgg cagcaggctg 480
accgtgctg 489
<210> 19
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 19
gtctcgtgta cgcacc 16
<210> 20
<211> 489
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 20
ctcgggagtc agataaacgc ttaagttttc ttatatgggg ataactggag gttgcaggtc 60
aatgactgat cttaattggg aagaacaagg atgacatcca ttcgagctgt atttatattc 120
ctgtggctgc agctggactt ggtgaatgga gagaatgtgg agcagcatcc ttcaaccctg 180
agtgtccagg agggagacag cgctgttatc aagtgtactt attcagacag tgcctcaaac 240
tacttccctt ggtataagca agaacttgga aaaagacctc agcttattat agacattcgt 300
tcaaatgtgg gcgaaaagaa agaccaacga attgctgtta cattgaacaa gacagccaaa 360
catttctccc tgcacatcac agagacccaa cctgaagact cggctgtcta cttctgtgca 420
gcaagtgagt tcacgggagg aggaaacaaa ctcacctttg ggacaggcac tcagctaaaa 480
gtggaactc 489
<210> 21
<211> 479
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 21
ctcgggagtc agataatgtt cattcatttc ttatatgggg agaggacccc agtcagaggc 60
cccatctcag acccgaggct agcatgggct gcaggctgct ctgctgtgcg gttctctgtc 120
tcctgggagc ggtccccatg gaaacgggag ttacgcagac accaagacac ctggtcatgg 180
gaatgacaaa taagaagtct ttgaaatgtg aacaacatct gggtcataac gctatgtatt 240
ggtacaagca aagtgctaag aagccactgg agctcatgtt tgtctacagt cttgaagaac 300
gggttgaaaa caacagtgtg ccaagtcgct tctcacctga atgccccaac agctctcact 360
tattccttca cctacacacc ctgcagccag aagactcggc cctgtatctc tgcgccagca 420
gccaagattg ggggtcggac gagcagtact tcgggccggg caccaggctc acggtcaca 479
<210> 22
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 22
ctcgggagtc agataa 16
<210> 23
<211> 490
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 23
gctgggtgtt cgcgacagac gacagttttc ttatatggga gataagacaa tctgcatctt 60
cacaggaggg atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga 120
ctgggtaaac agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct 180
gagcgtccag gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga 240
ttatttccta tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag 300
ttccattaag gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa 360
gcacctctct ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc 420
agcaagcgtg ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca 480
agtcatccca 490
<210> 24
<211> 511
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 24
gctgggtgtt cgcgactttc aatggttttc ttatatgggg ctctctgctc ctgctcacag 60
tgatcctgat ctggtaaagc tcccatcctg ccctgaccct gccatgggca ccaggctcct 120
cttctgggtg gccttctgtc tcctgggggc atatcacaca ggagctggag tctcccagtc 180
ccccagtaac aaggtcacag agaagggaaa ggatgtagag ctcaggtgtg atccaatttc 240
aggtcatact gccctttact ggtaccgaca gaggctgggg cagggcctgg agtttttaat 300
ttacttccaa ggcaacagtg caccagacaa atcagggctg cccagtgatc gcttctctgc 360
agagaggact ggggaatccg tctccactct gacgatccag cgcacacagc aggaggactc 420
ggccgtgtat ctctgtgcca gcagaggggg cagggggctc tctggggcca acgtcctgac 480
tttcggggcc ggcagcaggc tgaccgtgct g 511
<210> 25
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 25
gctgggtgtt cgcgac 16
<210> 26
<211> 575
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 26
gtcttcgagc tgaacggttc tgatactttt cttatatggg gttcatatgt aaaatgaagg 60
gtctgtggaa ggacatgaat aaagcacagg aggttgaagt cagatttgca gctttctagg 120
caggagataa gacaatctgc atcttcacag gagggatggc catgctcctg ggggcatcag 180
tgctgattct gtggcttcag ccagactggg taaacagtca acagaagaat gatgaccagc 240
aagttaagca aaattcacca tccctgagcg tccaggaagg aagaatttct attctgaact 300
gtgactatac taacagcatg tttgattatt tcctatggta caaaaaatac cctgctgaag 360
gtcctacatt cctgatatct ataagttcca ttaaggataa aaatgaagat ggaagattca 420
ctgttttctt aaacaaaagt gccaagcacc tctctctgca cattgtgccc tcccagcctg 480
gagactctgc agtgtacttc tgtgcagcaa gcgtgggttc tgggggttac cagaaagtta 540
cctttggaac tggaacaaag ctccaagtca tccca 575
<210> 27
<211> 467
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 27
gtcttcgagc tgaacgcacg tctatctttc ttatatgggg atcctgccct gaccctgcca 60
tgggcaccag gctcctcttc tgggtggcct tctgtctcct gggggcatat cacacaggag 120
ctggagtctc ccagtccccc agtaacaagg tcacagagaa gggaaaggat gtagagctca 180
ggtgtgatcc aatttcaggt catactgccc tttactggta ccgacagagg ctggggcagg 240
gcctggagtt tttaatttac ttccaaggca acagtgcacc agacaaatca gggctgccca 300
gtgatcgctt ctctgcagag aggactgggg aatccgtctc cactctgacg atccagcgca 360
cacagcagga ggactcggcc gtgtatctct gtgccagcag agggggcagg gggctctctg 420
gggccaacgt cctgactttc ggggccggca gcaggctgac cgtgctg 467
<210> 28
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 28
gtcttcgagc tgaacg 16
<210> 29
<211> 513
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 29
gactaactcc tcctaggtca cgattgtttc ttatatggga gatttgcagc tttctaggca 60
ggagataaga caatctgcat cttcacagga gggatggcca tgctcctggg ggcatcagtg 120
ctgattctgt ggcttcagcc agactgggta aacagtcaac agaagaatga tgaccagcaa 180
gttaagcaaa attcaccatc cctgagcgtc caggaaggaa gaatttctat tctgaactgt 240
gactatacta acagcatgtt tgattatttc ctatggtaca aaaaataccc tgctgaaggt 300
cctacattcc tgatatctat aagttccatt aaggataaaa atgaagatgg aagattcact 360
gttttcttaa acaaaagtgc caagcacctc tctctgcaca ttgtgccctc ccagcctgga 420
gactctgcag tgtacttctg tgcagcaagc gtgggttctg ggggttacca gaaagttacc 480
tttggaactg gaacaaagct ccaagtcatc cca 513
<210> 30
<211> 495
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 30
gactaactcc tcctagcctc cgcagctttc ttatatgggg acagtgatcc tgatctggta 60
aagctcccat cctgccctga ccctgccatg ggcaccaggc tcctcttctg ggtggccttc 120
tgtctcctgg gggcatatca cacaggagct ggagtctccc agtcccccag taacaaggtc 180
acagagaagg gaaaggatgt agagctcagg tgtgatccaa tttcaggtca tactgccctt 240
tactggtacc gacagaggct ggggcagggc ctggagtttt taatttactt ccaaggcaac 300
agtgcaccag acaaatcagg gctgcccagt gatcgcttct ctgcagagag gactggggaa 360
tccgtctcca ctctgacgat ccagcgcaca cagcaggagg actcggccgt gtatctctgt 420
gccagcagag ggggcagggg gctctctggg gccaacgtcc tgactttcgg ggccggcagc 480
aggctgaccg tgctg 495
<210> 31
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 31
gactaactcc tcctag 16
<210> 32
<211> 578
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 32
tggccagagt acgatacgtt tgggattttc ttatatgggg aaggactgag cttgcctgtg 60
actggctagg gaggaacctg agactagggg acagaaagac tagggattca cccagtaaag 120
agagctcatc tgtgactgag gagccttgct ccatttcagg tcttctgtga tttcaataag 180
gaagaagaat ggaaactctc ctgggagtgt ctttggtgat tctatggctt caactggcta 240
gggtgaacag tcaacaggga gaagaggatc ctcaggcctt gagcatccag gagggtgaaa 300
atgccaccat gaactgcagt tacaaaacta gtataaacaa tttacagtgg tatagacaaa 360
attcaggtag aggccttgtc cacctaattt taatacgttc aaatgaaaga gagaaacaca 420
gtggaagatt aagagtcacg cttgacactt ccaagaaaag cagttccttg ttgatcacgg 480
cttcccgggc agcagacact gcttcttact tctgtgctac ggacaagccc ggtaaccagt 540
tctattttgg gacagggaca agtttgacgg tcattcca 578
<210> 33
<211> 453
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 33
tggccagagt acgatactct agcccgtttc ttatatggga gacccgaggc tagcatgggc 60
tgcaggctgc tctgctgtgc ggttctctgt ctcctgggag cagttcccat agacactgaa 120
gttacccaga caccaaaaca cctggtcatg ggaatgacaa ataagaagtc tttgaaatgt 180
gaacaacata tggggcacag ggctatgtat tggtacaagc agaaagctaa gaagccaccg 240
gagctcatgt ttgtctacag ctatgagaaa ctctctataa atgaaagtgt gccaagtcgc 300
ttctcacctg aatgccccaa cagctctctc ttaaaccttc acctacacgc cctgcagcca 360
gaagactcag ccctgtatct ctgcgccagc agcccgtaca ggggggagaa gaatgagcag 420
ttcttcgggc cagggacacg gctcaccgtg cta 453
<210> 34
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 34
tggccagagt acgata 16
<210> 35
<211> 553
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 35
cagcgaccag tataaggact ttatattttc ttatatgggg aggacagatt tcttttatga 60
ttcctacagc agaaaaatga gaaacgtttg ttattatttt tttttcgtgt ttaaggtttg 120
aatcctcagt gaaccagggc agaaaagaat gatgaaatcc ttgagagttt tactggtgat 180
cctgtggctt cagttaagct gggtttggag ccaacagaag gaggtggagc aggatcctgg 240
accactcagt gttccagagg gagccattgt ttctctcaac tgcacttaca gcaacagtgc 300
ttttcaatac ttcatgtggt acagacagta ttccagaaaa ggccctgagt tgctgatgta 360
cacatactcc agtggtaaca aagaagatgg aaggtttaca gcacaggtcg ataaatccag 420
caagtatatc tccttgttca tcagagactc acagcccagt gattcagcca cctacctctg 480
tgcaatgccc ggagatcaaa ctggggcaaa caacctcttc tttgggactg gaacgagact 540
caccgttatt ccc 553
<210> 36
<211> 473
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 36
cagcgaccag tataagccct tgcgcctttc ttatatgggg agcacagaca cagtgctgcc 60
tgcccctttg tgccatgggc tccaggctgc tctgttgggt gctgctttgt ctcctgggag 120
caggcccagt aaaggctgga gtcactcaaa ctccaagata tctgatcaaa acgagaggac 180
agcaagtgac actgagctgc tcccctatct ctgggcatag gagtgtatcc tggtaccaac 240
agaccccagg acagggcctt cagttcctct ttgaatactt cagtgagaca cagagaaaca 300
aaggaaactt ccctggtcga ttctcagggc gccagttctc taactctcgc tctgagatga 360
atgtgagcac cttggagctg ggggactcgg ccctttatct ttgcgccagc agcttggtaa 420
gctctggggc caacgtcctg actttcgggg ccggcagcag gctgaccgtg ctg 473
<210> 37
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 37
tcagcgacca gtataa 16
<210> 38
<211> 514
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 38
aaccgcgagc tggaacattc taaaattttc ttatatgggg agatttgcag ctttctaggc 60
aggagacaag acaatctgca tcttcacagg agggatggcc atgctcctgg gggcatcagt 120
gctgattctg tggcttcagc cagactgggt aaacagtcaa cagaagaatg atgaccagca 180
agttaagcaa aattcaccat ccctgagcgt ccaggaagga agaatttcta ttctgaactg 240
tgactatact aacagcatgt ttgattattt cctatggtac aaaaaatacc ctgctgaagg 300
tcctacattc ctgatatcta taagttccat taaggataaa aatgaagatg gaagattcac 360
tgttttctta aacaaaagtg ccaagcacct ctctctgcac attgtgccct cccagcctgg 420
agactctgca gtgtacttct gtgcagcaag cgtgggttct gggggttacc agaaagttac 480
ctttggaact ggaacaaagc tccaagtcat ccca 514
<210> 39
<211> 468
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 39
aaccgcgagc tggaacagat acgtcctttc ttatatgggg agaggcccca tctcagaccc 60
gaggctagca tgggctgcag gctgctctgc tgtgcggttc tctgtctcct gggagcagtt 120
cccatagaca ctgaagttac ccagacacca aaacacctgg tcatgggaat gacaaataag 180
aagtctttga aatgtgaaca acatatgggg cacagggcta tgtattggta caagcagaaa 240
gctaagaagc caccggagct catgtttgtc tacagctatg agaaactctc tataaatgaa 300
agtgtgccaa gtcgcttctc acctgaatgc cccaacagct ctctcttaaa ccttcaccta 360
cacgccctgc agccagaaga ctcagccctg tatctctgcg ccagcagccc gtacaggggg 420
gagaagaatg agcagttctt cgggccaggg acacggctca ccgtgcta 468
<210> 40
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 40
aaccgcgagc tggaac 16
<210> 41
<211> 491
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 41
ttgtaggagg ctagcatata agtaggtttc ttatatgggg atctgtgact gaggagcctt 60
gctccatttc aggtcttctg tgatttcaat aaggaagaag aatggaaact ctcctgggag 120
tgtctttggt gattctatgg cttcaactgg ctagggtgaa cagtcaacag ggagaagagg 180
atcctcaggc cttgagcatc caggagggtg aaaatgccac catgaactgc agttacaaaa 240
ctagtataaa caatttacag tggtatagac aaaattcagg tagaggcctt gtccacctaa 300
ttttaatacg ttcaaatgaa agagagaaac acagtggaag attaagagtc acgcttgaca 360
cttccaagaa aagcagttcc ttgttgatca cggcttcccg ggcagcagac actgcttctt 420
acttctgtgc tacggacaag cccggtaacc agttctattt tgggacaggg acaagtttga 480
cggtcattcc a 491
<210> 42
<211> 468
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 42
ttgtaggagg ctagcatgta cagggttttc ttatatgggg agaggcccca tctcagaccc 60
gaggctagca tgggctgcag gctgctctgc tgtgcggttc tctgtctcct gggagcagtt 120
cccatagaca ctgaagttac ccagacacca aaacacctgg tcatgggaat gacaaataag 180
aagtctttga aatgtgaaca acatatgggg cacagggcta tgtattggta caagcagaaa 240
gctaagaagc caccggagct catgtttgtc tacagctatg agaaactctc tataaatgaa 300
agtgtgccaa gtcgcttctc acctgaatgc cccaacagct ctctcttaaa ccttcaccta 360
cacgccctgc agccagaaga ctcagccctg tatctctgcg ccagcagccc gtacaggggg 420
gagaagaatg agcagttctt cgggccaggg acacggctca ccgtgcta 468
<210> 43
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 43
ttgtaggagg ctagca 16
<210> 44
<211> 535
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 44
acaccggtcg gccgatatgt ttcgtctttc ttatatgggg aaagcacagg aggttgaagt 60
cagatttgca gctttctagg caggagataa gacaatctgc atcttcacag gagggatggc 120
catgctcctg ggggcatcag tgctgattct gtggcttcag ccagactggg taaacagtca 180
acagaagaat gatgaccagc aagttaagca aaattcacca tccctgagcg tccaggaagg 240
aagaatttct attctgaact gtgactatac taacagcatg tttgattatt tcctatggta 300
caaaaaatac cctgctgaag gtcctacatt cctgatatct ataagttcca ttaaggataa 360
aaatgaagat ggaagattca ctgttttctt aaacaaaagt gccaagcacc tctctctgca 420
cattgtgccc tcccagcctg gagactctgc agtgtacttc tgtgcagcaa gcgtgggttc 480
tgggggttac cagaaagtta cctttggaac tggaacaaag ctccaagtca tccca 535
<210> 45
<211> 467
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 45
acaccggtcg gccgatactc tcacagtttc ttatatgggg atcctgccct gaccctgcca 60
tgggcaccag gctcctcttc tgggtggcct tctgtctcct gggggcatat cacacaggag 120
ctggagtctc ccagtccccc agtaacaagg tcacagagaa gggaaaggat gtagagctca 180
ggtgtgatcc aatttcaggt catactgccc tttactggta ccgacagagg ctggggcagg 240
gcctggagtt tttaatttac ttccaaggca acagtgcacc agacaaatca gggctgccca 300
gtgatcgctt ctctgcagag aggactgggg aatccgtctc cactctgacg atccagcgca 360
cacagcagga ggactcggcc gtgtatctct gtgccagcag agggggcagg gggctctctg 420
gggccaacgt cctgactttc ggggccggca gcaggctgac cgtgctg 467
<210> 46
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 46
acaccggtcg gccgat 16
<210> 47
<211> 477
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 47
gacgtgccat cctagagccc cagaactttc ttatatgggg attgcagctc agacacagca 60
aaagagccta gaacctgggt cctagtttgc acctagaata tgaggcaagt ggcgagagtg 120
atcgtgttcc tgaccctgag tactttgagc cttgctaaga ccacccagcc catctccatg 180
gactcatatg aaggacaaga agtgaacata acctgtagcc acaacaacat tgctacaaat 240
gattatatca cgtggtacca acagtttccc agccaaggac cacgatttat tattcaagga 300
tacaagacaa aagttacaaa cgaagtggcc tccctgttta tccctgccga cagaaagtcc 360
agcactctga gcctgccccg ggtttccctg agcgacactg ctgtgtacta ctgcctcgtg 420
ggagatagca actatcagtt aatctggggc gctgggacca agctaattat aaagcca 477
<210> 48
<211> 498
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 48
gacgtgccat cctagacaca agtacatttc ttatatgggg tctttcccag gaggaccaag 60
ccctgagcac agacacagtg ctgcctgccc ctttgtgcca tgggctccag gctgctctgt 120
tgggtgctgc tttgtctcct gggagcaggc ccagtaaagg ctggagtcac tcaaactcca 180
agatatctga tcaaaacgag aggacagcaa gtgacactga gctgctcccc tatctctggg 240
cataggagtg tatcctggta ccaacagacc ccaggacagg gccttcagtt cctctttgaa 300
tacttcagtg agacacagag aaacaaagga aacttccctg gtcgattctc agggcgccag 360
ttctctaact ctcgctctga gatgaatgtg agcaccttgg agctggggga ctcggccctt 420
tatctttgcg ccagcagctt ggtaagctct ggggccaacg tcctgacttt cggggccggc 480
agcaggctga ccgtgctg 498
<210> 49
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 49
gacgtgccat cctaga 16
<210> 50
<211> 565
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 50
gcagttacat actcttgatc acgttgtttc ttatatgggg gttcatatgt aaaatgaagg 60
gtctgtggaa ggacatgaat aaagcacagg aggttgaagt cagatttgca gctttctagg 120
caggagataa gacaatctgc atcccacagg agggatggcc atgctcctgg gggcatcagt 180
gctgattctg tggcttcagc cagactgggt aaacagtcaa cagaagaatg atgaccagca 240
agttaagcaa aattcaccat ccctgagcgt ccaggaagga agaatttcta ttctgaactg 300
tgactatact aacagcatgt ttgattattt cctatggtac aaaaaatacc ctgctgaagg 360
tcctacattc ctgatatcta taagttccat taaggataaa aatgaagatg gaagattcac 420
tgttttctta aacaaaagtg ccaagcacct ctctctgcac attgtgccct cccagcctgg 480
agactctgca gtgtacttct gtgcagcaag ccctggaggc ttcaaaacta tctttggagc 540
aggaacaaga ctatttgtta aagca 565
<210> 51
<211> 485
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 51
gcagttacat actctttgcg ccggactttc ttatatgggg ggatctggta aagctcccat 60
cctgccctga ccctgccatg tgcaccaggc tcctcttctg ggtggccttc tgtctcctgg 120
gggcatatca cacaggagct ggagtctccc agtcccccag taacaaggtc acagagaagg 180
gaaaggatgt agagctcagg tgtgatccaa tttcaggtca tactgccctt tactggtacc 240
gacagaggct ggggcagggc ctggagtttt taatttactt ccaaggcaac agtgcaccag 300
acaaatcagg gctgcccagt gatcgcttct ctgcagagag gactggggaa tccgtctcca 360
ctctgacgat ccagcgcaca cagcaggagg actcggccgt gtatctctgt gccagcagag 420
ggggcagggg gctctctggg gccaacgtcc tgactttcgg ggccggcagc aggctgaccg 480
tgctg 485
<210> 52
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 52
gcagttacat actctt 16
<210> 53
<211> 553
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 53
gctgcgatca taaccgaatt ttcctatttc ttatatgggg aggacagatt tcttttatga 60
ttcctacagc agaaaaatga gaaacgtttg ttattatttt tttttcgtgt ttaaggtttg 120
aatcctcagt gaaccagggc agaaaagaat gatgaaatcc ttgagagttt tactggtgat 180
cctgtggctt cagttaagct gggtttggag ccaacagaag gaggtggagc aggatcctgg 240
accactcagt gttccagagg gagccattgt ttctctcaac tgcacttaca gcaacagtgc 300
ttttcaatac ttcatgtggt acagacagta ttccagaaaa ggccctgagt tgctgatgta 360
cacatactcc agtggtaaca aagaagatgg aaggtttaca gcacaggtcg ataaatccag 420
caagtatatc tccttgttca tcagagactc acagcccagt gattcagcca cctacctctg 480
tgcaatgccc ggagatcaaa ctggggcaaa caacctcttc tttgggactg gaacgagact 540
caccgttatt ccc 553
<210> 54
<211> 470
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 54
gctgcgatca taaccgatta acacagtttc ttatatgggg acagacacag tgctgcctgc 60
ccctttgtgc catgggctcc aggctgctct gttgggtgct gctttgtctc ctgggagcag 120
gcccagtaaa ggctggagtc actcaaactc caagatatct gatcaaaacg agaggacagc 180
aagtgacact gagctgctcc cctatctctg ggcataggag tgtatcctgg taccaacaga 240
ccccaggaca gggccttcag ttcctctttg aatacttcag tgagacacag agaaacaaag 300
gaaacttccc tggtcgattc tcagggcgcc agttctctaa ctctcgctct gagatgaatg 360
tgagcacctt ggagctgggg gactcggccc tttatctttg cgccagcagc ttggtaagct 420
ctggggccaa cgtcctgact ttcggggccg gcagcaggct gaccgtgctg 470
<210> 55
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 55
gctgcgatca taaccg 16
<210> 56
<211> 514
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 56
tgggaagtct actcatcccc ctttagtttc ttatatgggg agatttgcag ctttctaggc 60
aggagataag acaatctgca tcttcacagg agggatggcc atgctcctgg gggcatcagt 120
gctgattctg tggcttcagc cagactgggt aaacagtcaa cagaagaatg atgaccagca 180
agttaagcaa aattcaccat ccctgagcgt ccaggaagga agaatttcta ttctgaactg 240
tgactatact aacagcatgt ttgattattt cctatggtac aaaaaatacc ctgctgaagg 300
tcctacattc ctgatatcta taagttccat taaggataaa aatgaagatg gaagattcac 360
tgttttctta aacaaaagtg ccaagcacct ctctctgcac attgtgccct cccagcctgg 420
agactctgca gtgtacttct gtgcagcaag cgtgggttct gggggttacc agaaagttac 480
ctttggaact ggaacaaagc tccaagtcat ccca 514
<210> 57
<211> 467
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 57
tgggaagtct actcatcaat tagcaatttc ttatatgggg atcctgccct gaccctgcca 60
tgggcaccag gctcctcttc tgggtggcct tctgtctcct gggggcatat cacacaggag 120
ctggagtctc ccagtccccc agtaacaagg tcacagagaa gggaaaggat gtagagctca 180
ggtgtgatcc aatttcaggt catactgccc tttactggta ccgacagagg ctggggcagg 240
gcctggagtt tttaatttac ttccaaggca acagtgcacc agacaaatca gggctgccca 300
gtgatcgctt ctctgcagag aggactgggg aatccgtctc cactctgacg atccagcgca 360
cacagcagga ggactcggcc gtgtatctct gtgccagcag agggggcagg gggctctctg 420
gggccaacgt cctgactttc ggggccggca gcaggctgac cgtgctg 467
<210> 58
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 58
tgggaagtct actcat 16
<210> 59
<211> 538
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 59
acgatgtcat cccactaaac aggctttttc ttatatggga tataaagcac aggaggttga 60
agtcagattt gcagctttct aggcaggaga taagacaatc tgcatcttca caggagggat 120
ggccatgctc ctgggggcat cagtgctgat tctgtggctt cagccagact gggtaaacag 180
tcaacagaag aatgatgacc agcaagttaa gcaaaattca ccatccctga gcgtccagga 240
aggaagaatc tctattctga actgtgacta tactaacagc atgtttgatt atttcctatg 300
gtacaaaaaa taccctgctg aaggtcctac attcctgata tctataagtt ccattaagga 360
taaaaatgaa gatggaagat tcactgtttt cttaaacaaa agtgccaagc acctctctct 420
gcacattgtg ccctcccagc ctggagactc tgcagtgtac ttctgtgcag caagcgtggg 480
ttctgggggt taccagaaag ttacctttgg aactggaaca aagctccaag tcatccca 538
<210> 60
<211> 493
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 60
acgatgtcat cccacttttt ccgttgtttc ttatatgggg agtgatcctg atctggtaaa 60
gctcccatcc tgccctgacc ctgccatggg caccaggctc ctcttctggg tggccttctg 120
tctcctgggg gcatatcaca caggagctgg agtctcccag tcccccagta acaaggtcac 180
agagaaggga aaggatgtag agctcaggtg tgatccaatt tcaggtcata ctgcccttta 240
ctggtaccga cagaggctgg ggcagggcct ggagttttta atttacttcc aaggcaacag 300
tgcaccagac aaatcagggc tgcccagtga tcgcttctct gcagagagga ctggggaatc 360
cgtctccact ctgacgatcc agcgcacaca gcaggaggac tcggccgtgt atctctgtgc 420
cagcagaggg ggcagggggc tctctggggc caacgtcctg actttcgggg ccggcagcag 480
gctgaccgtg ctg 493
<210> 61
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 61
acgatgtcat cccact 16
<210> 62
<211> 514
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 62
tactcgcaga tgttagtcct agtgggtttc ttatatggga gatttgcagc tttctaggca 60
ggagataaga caatctgcat cttcacagga gggatggcca tgctcctggg ggcatcagtg 120
ctgattctgt ggcttcagcc agactgggta aacagtcaac agaagaatga tgaccagcaa 180
gttaagcaaa attcaccatc cctgagcgtc caggaaggaa gaatttctat tctgaactgt 240
gactatacta acagcatgtt tgattatttc ctatggtaca aaaaataccc tgctgaaggt 300
cctacattcc tgatatctat aagttccatt aaggataaaa atgaagatgg aagattcact 360
gtcttcttaa acaaaagtgc caagcacctc tctctgcaca ttgtgccctc ccagcctgga 420
gactctgcag tgtacttctg tgcagcaagc gtgggttctg ggggttacca gaaagttacc 480
tttggaactg gaacaaagct ccaagtcatc ccaa 514
<210> 63
<211> 467
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 63
tactcgcaga tgttaggcag ataatttttc ttatatgggg atcctgccct gaccctgcca 60
tgggcaccag gctcctcttc tgggtggcct tctgtctcct gggggcatat cacacaggag 120
ctggagtctc ccagtccccc agtaacaagg tcacagagaa gggaaaggat gtagagctca 180
ggtgtgatcc aatttcaggt catactgccc tttactggta ccgacagagg ctggggcagg 240
gcctggagtt tttaatttac ttccaaggca acagtgcacc agacaaatca gggctgccca 300
gtgatcgctt ctctgcagag aggactgggg aatccgtctc cactctgacg atccagcgca 360
cacagcagga ggactcggcc gtgtatctct gtgccagcag agggggcagg gggctctctg 420
gggccaacgt cctgactttc ggggccggca gcaggctgac cgtgctg 467
<210> 64
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 64
tactcgcaga tgttag 16
<210> 65
<211> 574
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 65
taccttacat gttcccgcat catctttttc ttatatgggg ttcatatgta aaatgaaggg 60
tctgtggaag gacatgaata aagcacagga ggttgaagtc aaatttgcag ctttctaggc 120
aggagataag acaatctgca tcttcacagg agggatggcc atgctcctgg gggcatcagt 180
gctgattctg tggcttcagc cagactgggt aaacagtcaa cagaagaatg atgaccagca 240
agttaagcaa aattcaccat ccctgagcgt ccaggaagga agaatttcta ttctgaactg 300
tgactatact aacagcatgt ttgattattt cctatggtac aaaaaatacc ctgctgaagg 360
tcctacattc ctgatatcta taagttccat taaggataaa aatgaagatg gaagattcac 420
tgttttctta aacaaaagtg ccaagcacct ctctctgcac attgtgccct cccagcctgg 480
agactctgca gtgtacttct gtgcagcaag cgtgggttct gggggttacc agaaagttac 540
ctttggaact ggaacaaagc tccaagtcat ccca 574
<210> 66
<211> 495
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 66
taccttacat gttcccccta tatcactttc ttatatgggg acagtgatcc tgatctggta 60
aagctcccat cctgccctga ccctgccatg ggcaccaggc tcctcttctg ggtggccttc 120
tgtctcctgg gggcatatca cacaggagct ggagtctccc agtcccccag taacaaggtc 180
acagagaagg gaaaggatgt agagctcagg tgtgatccaa tttcaggtca tactgccctt 240
tactggtacc gacagaggct ggggcagggc ctggagtttt taatttactt ccaaggcaac 300
agtgcaccag acaaatcagg gctgcccagt gatcgcttct ctgcagagag gactggggaa 360
tccgtctcca ctctgacgat ccagcgcaca cagcaggagg actcggccgt gtatctctgt 420
gccagcagag ggggcagggg gctctctggg gccaacgtcc tgactttcgg ggtcggcagc 480
aggctgaccg tgctg 495
<210> 67
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 67
taccttacat gttccc 16
<210> 68
<211> 579
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 68
attggtgaga tcacgggtac tgtccttttc ttatatgggg gagcttttgt agttttaaat 60
aagcaaggag tttctccagc gaaacttcct cacacctctt ggtcttggtc tcttcagaca 120
ctttccttcc tgttctctgg agatcttgca gaaaagagcc tgcagtgttt cccttgttca 180
gccatgctcc tggagcttat cccactgctg gggatacatt ttgtcctgag aactgccaga 240
gcccagtcag tgacccagcc tgacatccac atcactgtct ctgaaggagc ctcactggag 300
ttgagatgta actattccta tggggcaaca ccttatctct tctggtatgt ccagtccccc 360
ggccaaggcc tccagctgct cctgaagtac ttttcaggag acactctggt tcaaggcatt 420
aaaggctttg aggctgaatt taagaggagt caatcttcct tcaatctgag gaaaccctct 480
gtgcattgga gtgatgctgc tgagtacttc tgtgctgtgg gtgcttctaa cgactacaag 540
ctcagctttg gagccggaac cacagtaact gtaagagca 579
<210> 69
<211> 497
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 69
attggtgaga tcacggtcgt taacagtttc ttatatgggg atgctcacag agggcctggt 60
ctagaatatt ccacatctgc tctcactctg ccatggactc ctggaccttc tgctgtgtgt 120
ccctttgcat cctggtagcg aagcatacag atgctggagt tatccagtca ccccgccatg 180
aggtgacaga gatgggacaa gaagtgactc tgagatgtaa accaatttca ggccacaact 240
cccttttctg gtacagacag accatgatgc ggggactgga gttgctcatt tactttaaca 300
acaacgttcc gatagatgat tcagggatgc ccgaggatcg attctcagct aagatgccta 360
atgcatcatt ctccactctg aagatccagc cctcagaacc cagggactca gctgtgtact 420
tctgtgccag cagtttagga cacatctctg gggccaacgt cctgactttc ggggccggca 480
gcaggctgac cgtgctg 497
<210> 70
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 70
attggtgaga tcacgg 16
<210> 71
<211> 488
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 71
ccgttcagta caccgcaccc actttttttc ttatatggga gatataagat ttcagttctc 60
agtgagtcta agtgacagaa ggaatggaga ccctcttggg cctgcttatc ctttggctgc 120
agctgcaatg ggtgagcagc aaacaggagg tgacacagat tcctgcagct ctgagtgtcc 180
cagaaggaga aaacttggtt ctcaactgca gtttcactga tagcgctatt tacaacctcc 240
agtggtttag gcaggaccct gggaaaggtc tcacatctct gttgcttatt cagtcaagtc 300
agagagagca aacaagtgga agacttaatg cctcgctgga taaatcatca ggacgtagta 360
ctttatacat tgcagcttct cagcctggtg actcagccac ctacctctgt gctgtcaccc 420
ttaatgctgg tggtactagc tatggaaagc tgacatttgg acaagggacc atcttgactg 480
tccatcca 488
<210> 72
<211> 476
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<220>
<221> misc_feature
<222> (463)..(463)
<223> n is a, c, g, or t
<400> 72
ccgttcagta caccgcatca gactcgtttc ttatatgggg aaagctccca tcctgccctg 60
accctgccat gggcaccagg ctcctcttct gggtggcctt ctgtctcctg ggggcatatc 120
acacaagagc tggagtctcc cagtccccca gtaacaaggt cacagagaat ggaaaggatg 180
tagagctcag gtgtgatcca atttctggtc atactgccct ttactggtac cgacagaggc 240
tggggcaggg cctggatttt ttaatttact tccaaggcaa cagtgcacca gacaaatcag 300
ggctgcccag tgatcgcttc tctgcagaga ggactgggga atccgtctcc actctgacga 360
tccagcgcac acagcaggag gactcggccg tgtatctctg tgccagcaga gggggcaggg 420
ggctctctgg ggccaacgtc ctgactttcg gggccggcag cangctgacc gtgctg 476
<210> 73
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 73
ccgttcagta caccgc 16
<210> 74
<211> 552
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 74
tccacacaga aagtggacct acagtctttc ttatatggga ggacagattt cttttatgat 60
tcctacagca gaaaaatgag aaacgtttgt tattattttt ttttcgtgtt taaggtttga 120
atcctcagtg aaccagggca gaaaagaatg atgaaatcct tgagagtttt actggtgatc 180
ctgtggcttc agttaagctg ggtttggagc caacagaagg aggtggagca ggatcctgga 240
ccactcagtg ttccagaggg agccattgtt tctctcaact gcacttacag caacagtgct 300
tttcaatact tcatgtggta cagacagtat tccagaaaag gccctgagtt gctgatgtac 360
acatactcca gtggtaacaa agaagatgga aggtttacag cacaggtcga taaatccagc 420
aagtatatct ccttgttcat cagagactca cagcccagtg attcagccac ctacctctgt 480
gcaatgcccg gagatcaaac tggggcaaac aacctcttct ttgggactgg aacgagactc 540
accgttattc cc 552
<210> 75
<211> 467
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 75
tccacacaga aagtgggctg tcgtactttc ttatatgggg agacacagtg ctgcctgccc 60
ctttgtgcca tgggctccag gctgctctgt tgggtgctgc tttgtctcct gggagcaggc 120
ccagtaaagg ctggagtcac tcaaactcca agatatctga tcaaaacgag aggacagcaa 180
gtgacactga gctgctcccc tatctctggg cataggagtg tatcctggta ccaacagacc 240
ccaggacagg gccttcagtt cctctttgaa tacttcagtg agacacagag aaacaaagga 300
aacttccctg gtcgattctc agggcgccag ttctctaact ctcgctctga gatgaatgtg 360
agcaccttgg agctggggga ctcggccctt tatctttgcg ccagcagctt ggtaagctct 420
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgct 467
<210> 76
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 76
tccacacaga aagtgg 16
<210> 77
<211> 575
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 77
gcgcaaccat aggatataac atgtagtttc ttatatgggg gttcatatgt aaaatgaagg 60
gtctgtggaa ggacatgaat aaagcacagg aggttgaagt cagatttgca gctttctagg 120
caggagataa gacaatctgc atcttcacag gagggatggc catgctcctg ggggcatcag 180
tgctgattct gtggcttcag ccagactggg taaacagtca acagaagaat gatgaccagc 240
aagttaagca aaattcacca tccctgagcg tccaggaagg aagaatttct attctgaact 300
gtgactatac taacagcatg tttgattatt tcctatggta caaaaaatac cctgctgaag 360
gtcctacatt cctgatatct ataagttcca ttaaggataa aaatgaagat ggaagattca 420
ctgttttctt aaacaaaagt gccaagcacc tctctctgca cattgtgccc tcccagcctg 480
gagactctgc agtgtacttc tgtgcagcaa gcgtgggttc tgggggttac cagaaagtta 540
cctttggaac tggaacaaag ctccaagtca tccca 575
<210> 78
<211> 484
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 78
gcgcaaccat aggatatcca ataatatttc ttatatgggg gatctggtaa agctcccatc 60
ctgccctgac cctgccatgg gcaccaggct cctcttctgg gtggccttct gtctcctggg 120
ggcatatcac acaggagctg gagtctccca gtcccccagt aacaaggtca cagagaaggg 180
aaaggatgta gagctcaggt gtgatccaat ttcaggtcat actgcccttt actggtaccg 240
acagaggctg gggcagggcc tggagttttt aatttacttc caaggcaaca gtgcaccaga 300
caaatcaggg ctgcccagtg atcgcttctc tgcagagagg actggggaat ccgtctccac 360
tctgacgatc cagcgcacac agcaggagga ctcggccgtg tatctctgtg ccagcagagg 420
gggcaggggg ctctctgggg ccaacgtcct gactttcggg gccggcagca ggctgaccgt 480
gctg 484
<210> 79
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 79
gcgcaaccat aggata 16
<210> 80
<211> 411
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 80
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtc ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagccct 360
ggaggcttca aaactatctt tggagcagga acaagactat ttgttaaagc a 411
<210> 81
<211> 137
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 81
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Pro Gly Gly Phe Lys Thr Ile Phe Gly
115 120 125
Ala Gly Thr Arg Leu Phe Val Lys Ala
130 135
<210> 82
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 82
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 83
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 83
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 84
<211> 396
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 84
atgaactatt ctccaggctt agtatctctg atactcttac tgcttggaag aacccgtgga 60
gattcagtga cccagatgga agggccagtg actctctcag aagaggcctt cctgactata 120
aactgcacgt acacagccac aggataccct tcccttttct ggtatgtcca atatcctgga 180
gaaggtctac agctcctcct gaaagccacg aaggctgatg acaagggaag caacaaaggt 240
tttgaagcca cataccgtaa agaaaccact tctttccact tggagaaagg ctcagttcaa 300
gtgtcagact cagcggtgta cttctgtgct ctgagtgata aaactggagg cttcaaaact 360
atctttggag caggaacaag actatttgtt aaagca 396
<210> 85
<211> 132
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 85
Met Asn Tyr Ser Pro Gly Leu Val Ser Leu Ile Leu Leu Leu Leu Gly
1 5 10 15
Arg Thr Arg Gly Asp Ser Val Thr Gln Met Glu Gly Pro Val Thr Leu
20 25 30
Ser Glu Glu Ala Phe Leu Thr Ile Asn Cys Thr Tyr Thr Ala Thr Gly
35 40 45
Tyr Pro Ser Leu Phe Trp Tyr Val Gln Tyr Pro Gly Glu Gly Leu Gln
50 55 60
Leu Leu Leu Lys Ala Thr Lys Ala Asp Asp Lys Gly Ser Asn Lys Gly
65 70 75 80
Phe Glu Ala Thr Tyr Arg Lys Glu Thr Thr Ser Phe His Leu Glu Lys
85 90 95
Gly Ser Val Gln Val Ser Asp Ser Ala Val Tyr Phe Cys Ala Leu Ser
100 105 110
Asp Lys Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala Gly Thr Arg Leu
115 120 125
Phe Val Lys Ala
130
<210> 86
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 86
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 87
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 87
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 88
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 88
atgacatcca ttcgagctgt atttatattc ctgtggctgc agctggactt ggtgaatgga 60
gagaatgtgg agcagcatcc ttcaaccctg agtgtccagg agggagacag cgctgttatc 120
aagtgtactt attcagacag tgcctcaaac tacttccctt ggtataagca agaacttgga 180
aaaagacctc agcttattat agacattcgt tcaaatgtgg gcgaaaagaa agaccaacga 240
attgctgtta cattgaacaa gacagccaaa catttctccc tgcacatcac agagacccaa 300
cctgaagact cggctgtcta cttctgtgca gcaagtgagt tcacgggagg aggaaacaaa 360
ctcacctttg ggacaggcac tcagctaaaa gtggaactc 399
<210> 89
<211> 133
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 89
Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp
1 5 10 15
Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val
20 25 30
Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala
35 40 45
Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro Gln
50 55 60
Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg
65 70 75 80
Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile
85 90 95
Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser
100 105 110
Glu Phe Thr Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr Gln
115 120 125
Leu Lys Val Glu Leu
130
<210> 90
<211> 421
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 90
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
a 421
<210> 91
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 91
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 92
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 92
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 93
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 93
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 94
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 94
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 95
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 95
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 96
<211> 390
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 96
atggaaactc tcctgggagt gtctttggtg attctatggc ttcaactggc tagggtgaac 60
agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 120
atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 180
agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 240
ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 300
gcagcagaca ctgcttctta cttctgtgct acggacaagc ccggtaacca gttctatttt 360
gggacaggga caagtttgac ggtcattcca 390
<210> 97
<211> 130
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 97
Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu
1 5 10 15
Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser
20 25 30
Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser
35 40 45
Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val
50 55 60
His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg
65 70 75 80
Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile
85 90 95
Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp
100 105 110
Lys Pro Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr Val
115 120 125
Ile Pro
130
<210> 98
<211> 405
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 98
atgatgaaat ccttgagagt tttactggtg atcctgtggc ttcagttaag ctgggtttgg 60
agccaacaga aggaggtgga gcaggatcct ggaccactca gtgttccaga gggagccatt 120
gtttctctca actgcactta cagcaacagt gcttttcaat acttcatgtg gtacagacag 180
tattccagaa aaggccctga gttgctgatg tacacatact ccagtggtaa caaagaagat 240
ggaaggttta cagcacaggt cgataaatcc agcaagtata tctccttgtt catcagagac 300
tcacagccca gtgattcagc cacctacctc tgtgcaatgc ccggagatca aactggggca 360
aacaacctct tctttgggac tggaacgaga ctcaccgtta ttccc 405
<210> 99
<211> 135
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 99
Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu
1 5 10 15
Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro
20 25 30
Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser
35 40 45
Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys
50 55 60
Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp
65 70 75 80
Gly Arg Phe Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu
85 90 95
Phe Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala
100 105 110
Met Pro Gly Asp Gln Thr Gly Ala Asn Asn Leu Phe Phe Gly Thr Gly
115 120 125
Thr Arg Leu Thr Val Ile Pro
130 135
<210> 100
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 100
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 101
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 101
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 102
<211> 391
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 102
atggaaactc tcctgggagt gtctttggtg attctatggc ttcaactggc tagggtgaac 60
agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 120
atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 180
agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 240
ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 300
gcagcagaca ctgcttctta cttctgtgct acggacaagc ccggtaacca gttctatttt 360
gggacaggga caagtttgac ggtcattcca a 391
<210> 103
<211> 130
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 103
Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu
1 5 10 15
Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser
20 25 30
Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser
35 40 45
Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val
50 55 60
His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg
65 70 75 80
Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile
85 90 95
Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp
100 105 110
Lys Pro Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr Val
115 120 125
Ile Pro
130
<210> 104
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 104
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 105
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 105
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 106
<211> 378
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 106
atgaggcaag tggcgagagt gatcgtgttc ctgaccctga gtactttgag ccttgctaag 60
accacccagc ccatctccat ggactcatat gaaggacaag aagtgaacat aacctgtagc 120
cacaacaaca ttgctacaaa tgattatatc acgtggtacc aacagtttcc cagccaagga 180
ccacgattta ttattcaagg atacaagaca aaagttacaa acgaagtggc ctccctgttt 240
atccctgccg acagaaagtc cagcactctg agcctgcccc gggtttccct gagcgacact 300
gctgtgtact actgcctcgt gggagatagc aactatcagt taatctgggg cgctgggacc 360
aagctaatta taaagcca 378
<210> 107
<211> 126
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 107
Met Arg Gln Val Ala Arg Val Ile Val Phe Leu Thr Leu Ser Thr Leu
1 5 10 15
Ser Leu Ala Lys Thr Thr Gln Pro Ile Ser Met Asp Ser Tyr Glu Gly
20 25 30
Gln Glu Val Asn Ile Thr Cys Ser His Asn Asn Ile Ala Thr Asn Asp
35 40 45
Tyr Ile Thr Trp Tyr Gln Gln Phe Pro Ser Gln Gly Pro Arg Phe Ile
50 55 60
Ile Gln Gly Tyr Lys Thr Lys Val Thr Asn Glu Val Ala Ser Leu Phe
65 70 75 80
Ile Pro Ala Asp Arg Lys Ser Ser Thr Leu Ser Leu Pro Arg Val Ser
85 90 95
Leu Ser Asp Thr Ala Val Tyr Tyr Cys Leu Val Gly Asp Ser Asn Tyr
100 105 110
Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ile Ile Lys Pro
115 120 125
<210> 108
<211> 411
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 108
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagccct 360
ggaggcttca aaactatctt tggagcagga acaagactat ttgttaaagc a 411
<210> 109
<211> 137
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 109
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Pro Gly Gly Phe Lys Thr Ile Phe Gly
115 120 125
Ala Gly Thr Arg Leu Phe Val Lys Ala
130 135
<210> 110
<211> 404
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 110
atgatgaaat ccttgagagt tttactggtg atcctgtggc ttcagttaag ctgggtttgg 60
agccaacaga aggaggtgga gcaggatcct ggaccactca gtgttccaga gggagccatt 120
gtttctctca actgcactta cagcaacagt gcttttcaat acttcatgtg gtacagacag 180
tattccagaa aaggccctga gttgctgatg tacacatact ccagtggtaa caaagaagat 240
ggaaggttta cagcacaggt cgataaatcc agcaagtata tctccttgtt catcagagac 300
tcacagccca gtgattcagc cacctacctc tgtgcaatgc ccggagatca aactggggca 360
aacaacctct tctttgggac tggaacgaga ctcaccgtta ttcc 404
<210> 111
<211> 134
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 111
Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu
1 5 10 15
Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro
20 25 30
Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser
35 40 45
Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys
50 55 60
Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp
65 70 75 80
Gly Arg Phe Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu
85 90 95
Phe Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala
100 105 110
Met Pro Gly Asp Gln Thr Gly Ala Asn Asn Leu Phe Phe Gly Thr Gly
115 120 125
Thr Arg Leu Thr Val Ile
130
<210> 112
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 112
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 113
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 113
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 114
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 114
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tctctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 115
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 115
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 116
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 116
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtc ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 117
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 117
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 118
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 118
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 119
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 119
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 120
<211> 396
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 120
atgctcctgg agcttatccc actgctgggg atacattttg tcctgagaac tgccagagcc 60
cagtcagtga cccagcctga catccacatc actgtctctg aaggagcctc actggagttg 120
agatgtaact attcctatgg ggcaacacct tatctcttct ggtatgtcca gtcccccggc 180
caaggcctcc agctgctcct gaagtacttt tcaggagaca ctctggttca aggcattaaa 240
ggctttgagg ctgaatttaa gaggagtcaa tcttccttca atctgaggaa accctctgtg 300
cattggagtg atgctgctga gtacttctgt gctgtgggtg cttctaacga ctacaagctc 360
agctttggag ccggaaccac agtaactgta agagca 396
<210> 121
<211> 132
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 121
Met Leu Leu Glu Leu Ile Pro Leu Leu Gly Ile His Phe Val Leu Arg
1 5 10 15
Thr Ala Arg Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val
20 25 30
Ser Glu Gly Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala
35 40 45
Thr Pro Tyr Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln
50 55 60
Leu Leu Leu Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys
65 70 75 80
Gly Phe Glu Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg
85 90 95
Lys Pro Ser Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val
100 105 110
Gly Ala Ser Asn Asp Tyr Lys Leu Ser Phe Gly Ala Gly Thr Thr Val
115 120 125
Thr Val Arg Ala
130
<210> 122
<211> 405
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 122
atggagaccc tcttgggcct gcttatcctt tggctgcagc tgcaatgggt gagcagcaaa 60
caggaggtga cacagattcc tgcagctctg agtgtcccag aaggagaaaa cttggttctc 120
aactgcagtt tcactgatag cgctatttac aacctccagt ggtttaggca ggaccctggg 180
aaaggtctca catctctgtt gcttattcag tcaagtcaga gagagcaaac aagtggaaga 240
cttaatgcct cgctggataa atcatcagga cgtagtactt tatacattgc agcttctcag 300
cctggtgact cagccaccta cctctgtgct gtcaccctta atgctggtgg tactagctat 360
ggaaagctga catttggaca agggaccatc ttgactgtcc atcca 405
<210> 123
<211> 135
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 123
Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp
1 5 10 15
Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val
20 25 30
Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala
35 40 45
Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr
50 55 60
Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg
65 70 75 80
Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile
85 90 95
Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr
100 105 110
Leu Asn Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr Phe Gly Gln Gly
115 120 125
Thr Ile Leu Thr Val His Pro
130 135
<210> 124
<211> 405
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 124
atgatgaaat ccttgagagt tttactggtg atcctgtggc ttcagttaag ctgggtttgg 60
agccaacaga aggaggtgga gcaggatcct ggaccactca gtgttccaga gggagccatt 120
gtttctctca actgcactta cagcaacagt gcttttcaat acttcatgtg gtacagacag 180
tattccagaa aaggccctga gttgctgatg tacacatact ccagtggtaa caaagaagat 240
ggaaggttta cagcacaggt cgataaatcc agcaagtata tctccttgtt catcagagac 300
tcacagccca gtgattcagc cacctacctc tgtgcaatgc ccggagatca aactggggca 360
aacaacctct tctttgggac tggaacgaga ctcaccgtta ttccc 405
<210> 125
<211> 135
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 125
Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu
1 5 10 15
Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro
20 25 30
Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser
35 40 45
Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys
50 55 60
Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp
65 70 75 80
Gly Arg Phe Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu
85 90 95
Phe Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala
100 105 110
Met Pro Gly Asp Gln Thr Gly Ala Asn Asn Leu Phe Phe Gly Thr Gly
115 120 125
Thr Arg Leu Thr Val Ile Pro
130 135
<210> 126
<211> 420
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 126
atggccatgc tcctgggggc atcagtgctg attctgtggc ttcagccaga ctgggtaaac 60
agtcaacaga agaatgatga ccagcaagtt aagcaaaatt caccatccct gagcgtccag 120
gaaggaagaa tttctattct gaactgtgac tatactaaca gcatgtttga ttatttccta 180
tggtacaaaa aataccctgc tgaaggtcct acattcctga tatctataag ttccattaag 240
gataaaaatg aagatggaag attcactgtt ttcttaaaca aaagtgccaa gcacctctct 300
ctgcacattg tgccctccca gcctggagac tctgcagtgt acttctgtgc agcaagcgtg 360
ggttctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 420
<210> 127
<211> 140
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 127
Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro
1 5 10 15
Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln
20 25 30
Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn
35 40 45
Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys
50 55 60
Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys
65 70 75 80
Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala
85 90 95
Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Ala Ser Val Gly Ser Gly Gly Tyr Gln Lys Val
115 120 125
Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro
130 135 140
<210> 128
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 128
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 129
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 129
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 130
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 130
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 131
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 131
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 132
<211> 405
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 132
atggactcct ggaccttctg ctgtgtgtcc ctttgcatcc tggtagcgaa gcatacagat 60
gctggagtta tccagtcacc ccgccatgag gtgacagaga tgggacaaga agtgactctg 120
agatgtaaac caatttcagg ccacaactcc cttttctggt acagacagac catgatgcgg 180
ggactggagt tgctcattta ctttaacaac aacgttccga tagatgattc agggatgccc 240
gaggatcgat tctcagctaa gatgcctaat gcatcattct ccactctgaa gatccagccc 300
tcagaaccca gggactcagc tgtgtacttc tgtgccagca gtttaggaca catctctggg 360
gccaacgtcc tgactttcgg ggccggcagc aggctgaccg tgctg 405
<210> 133
<211> 135
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 133
Met Asp Ser Trp Thr Phe Cys Cys Val Ser Leu Cys Ile Leu Val Ala
1 5 10 15
Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr
20 25 30
Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His
35 40 45
Asn Ser Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu
50 55 60
Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro
65 70 75 80
Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu
85 90 95
Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala
100 105 110
Ser Ser Leu Gly His Ile Ser Gly Ala Asn Val Leu Thr Phe Gly Ala
115 120 125
Gly Ser Arg Leu Thr Val Leu
130 135
<210> 134
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 134
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 135
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 135
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 136
<211> 395
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 136
atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcggt ccccatggaa 60
acgggagtta cgcagacacc aagacacctg gtcatgggaa tgacaaataa gaagtctttg 120
aaatgtgaac aacatctggg tcataacgct atgtattggt acaagcaaag tgctaagaag 180
ccactggagc tcatgtttgt ctacagtctt gaagaacggg ttgaaaacaa cagtgtgcca 240
agtcgcttct cacctgaatg ccccaacagc tctcacttat tccttcacct acacaccctg 300
cagccagaag actcggccct gtatctctgc gccagcagcc aagattgggg gtcggacgag 360
cagtacttcg ggccgggcac caggctcacg gtcac 395
<210> 137
<211> 132
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 137
Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala
1 5 10 15
Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His Leu Val Met
20 25 30
Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Leu Gly His
35 40 45
Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro Leu Glu Leu
50 55 60
Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn Ser Val Pro
65 70 75 80
Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu Phe Leu His
85 90 95
Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Gln Asp Trp Gly Ser Asp Glu Gln Tyr Phe Gly Pro Gly Thr Arg
115 120 125
Leu Thr Val Thr
130
<210> 138
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 138
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 139
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 139
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 140
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 140
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 141
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 141
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 142
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 142
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 143
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 143
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 144
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 144
atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcagt tcccatagac 60
actgaagtta cccagacacc aaaacacctg gtcatgggaa tgacaaataa gaagtctttg 120
aaatgtgaac aacatatggg gcacagggct atgtattggt acaagcagaa agctaagaag 180
ccaccggagc tcatgtttgt ctacagctat gagaaactct ctataaatga aagtgtgcca 240
agtcgcttct cacctgaatg ccccaacagc tctctcttaa accttcacct acacgccctg 300
cagccagaag actcagccct gtatctctgc gccagcagcc cgtacagggg ggagaagaat 360
gagcagttct tcgggccagg gacacggctc accgtgcta 399
<210> 145
<211> 133
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 145
Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala
1 5 10 15
Val Pro Ile Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met
20 25 30
Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His
35 40 45
Arg Ala Met Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu
50 55 60
Met Phe Val Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro
65 70 75 80
Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His
85 90 95
Leu His Ala Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Pro Tyr Arg Gly Glu Lys Asn Glu Gln Phe Phe Gly Pro Gly Thr
115 120 125
Arg Leu Thr Val Leu
130
<210> 146
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 146
atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60
gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120
agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180
ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240
ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300
gagctggggg actcggccct ttatctttgc gccagcagct tggtaagctc tggggccaac 360
gtcctgactt tcggggccgg cagcaggctg accgtgctg 399
<210> 147
<211> 133
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 147
Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala
1 5 10 15
Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys
20 25 30
Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His
35 40 45
Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe
50 55 60
Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro
65 70 75 80
Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn
85 90 95
Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Leu Val Ser Ser Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser
115 120 125
Arg Leu Thr Val Leu
130
<210> 148
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 148
atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcagt tcccatagac 60
actgaagtta cccagacacc aaaacacctg gtcatgggaa tgacaaataa gaagtctttg 120
aaatgtgaac aacatatggg gcacagggct atgtattggt acaagcagaa agctaagaag 180
ccaccggagc tcatgtttgt ctacagctat gagaaactct ctataaatga aagtgtgcca 240
agtcgcttct cacctgaatg ccccaacagc tctctcttaa accttcacct acacgccctg 300
cagccagaag actcagccct gtatctctgc gccagcagcc cgtacagggg ggagaagaat 360
gagcagttct tcgggccagg gacacggctc accgtgcta 399
<210> 149
<211> 133
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 149
Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala
1 5 10 15
Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys
20 25 30
Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His
35 40 45
Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe
50 55 60
Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro
65 70 75 80
Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn
85 90 95
Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Leu Val Ser Ser Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser
115 120 125
Arg Leu Thr Val Leu
130
<210> 150
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 150
atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcagt tcccatagac 60
actgaagtta cccagacacc aaaacacctg gtcatgggaa tgacaaataa gaagtctttg 120
aaatgtgaac aacatatggg gcacagggct atgtattggt acaagcagaa agctaagaag 180
ccaccggagc tcatgtttgt ctacagctat gagaaactct ctataaatga aagtgtgcca 240
agtcgcttct cacctgaatg ccccaacagc tctctcttaa accttcacct acacgccctg 300
cagccagaag actcagccct gtatctctgc gccagcagcc cgtacagggg ggagaagaat 360
gagcagttct tcgggccagg gacacggctc accgtgcta 399
<210> 151
<211> 133
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 151
Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala
1 5 10 15
Val Pro Ile Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met
20 25 30
Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His
35 40 45
Arg Ala Met Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu
50 55 60
Met Phe Val Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro
65 70 75 80
Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His
85 90 95
Leu His Ala Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Pro Tyr Arg Gly Glu Lys Asn Glu Gln Phe Phe Gly Pro Gly Thr
115 120 125
Arg Leu Thr Val Leu
130
<210> 152
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 152
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 153
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 153
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 154
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 154
atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60
gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120
agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180
ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240
ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300
gagctggggg actcggccct ttatctttgc gccagcagct tggtaagctc tggggccaac 360
gtcctgactt tcggggccgg cagcaggctg accgtgctg 399
<210> 155
<211> 133
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 155
Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala
1 5 10 15
Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys
20 25 30
Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His
35 40 45
Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe
50 55 60
Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro
65 70 75 80
Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn
85 90 95
Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Leu Val Ser Ser Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser
115 120 125
Arg Leu Thr Val Leu
130
<210> 156
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 156
atgtgcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 157
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 157
Met Cys Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 158
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 158
atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60
gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120
agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180
ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240
ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300
gagctggggg actcggccct ttatctttgc gccagcagct tggtaagctc tggggccaac 360
gtcctgactt tcggggccgg cagcaggctg accgtgctg 399
<210> 159
<211> 133
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 159
Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala
1 5 10 15
Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys
20 25 30
Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His
35 40 45
Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe
50 55 60
Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro
65 70 75 80
Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn
85 90 95
Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Leu Val Ser Ser Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser
115 120 125
Arg Leu Thr Val Leu
130
<210> 160
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 160
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 161
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 161
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 162
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 162
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 163
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 163
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 164
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 164
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 165
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 165
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
<210> 166
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 166
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggtcggc agcaggctga ccgtgctg 408
<210> 167
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 167
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Val Gly Ser Arg Leu Thr Val Leu
130 135
<210> 168
<211> 405
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 168
atggactcct ggaccttctg ctgtgtgtcc ctttgcatcc tggtagcgaa gcatacagat 60
gctggagtta tccagtcacc ccgccatgag gtgacagaga tgggacaaga agtgactctg 120
agatgtaaac caatttcagg ccacaactcc cttttctggt acagacagac catgatgcgg 180
ggactggagt tgctcattta ctttaacaac aacgttccga tagatgattc agggatgccc 240
gaggatcgat tctcagctaa gatgcctaat gcatcattct ccactctgaa gatccagccc 300
tcagaaccca gggactcagc tgtgtacttc tgtgccagca gtttaggaca catctctggg 360
gccaacgtcc tgactttcgg ggccggcagc aggctgaccg tgctg 405
<210> 169
<211> 135
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 169
Met Asp Ser Trp Thr Phe Cys Cys Val Ser Leu Cys Ile Leu Val Ala
1 5 10 15
Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr
20 25 30
Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His
35 40 45
Asn Ser Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu
50 55 60
Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro
65 70 75 80
Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu
85 90 95
Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala
100 105 110
Ser Ser Leu Gly His Ile Ser Gly Ala Asn Val Leu Thr Phe Gly Ala
115 120 125
Gly Ser Arg Leu Thr Val Leu
130 135
<210> 170
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<220>
<221> misc_feature
<222> (395)..(395)
<223> n is a, c, g, or t
<400> 170
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacaaga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga atggaaagga tgtagagctc 120
aggtgtgatc caatttctgg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggatt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcangctga ccgtgctg 408
<210> 171
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<220>
<221> misc_feature
<222> (132)..(132)
<223> Xaa can be any naturally occurring amino acid
<400> 171
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Arg Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Asn Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Asp Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Xaa Leu Thr Val Leu
130 135
<210> 172
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 172
atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60
gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120
agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180
ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240
ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300
gagctggggg actcggccct ttatctttgc gccagcagct tggtaagctc tggggccaac 360
gtcctgactt tcggggccgg cagcaggctg accgtgctg 399
<210> 173
<211> 133
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 173
Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala
1 5 10 15
Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys
20 25 30
Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His
35 40 45
Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe
50 55 60
Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro
65 70 75 80
Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn
85 90 95
Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Leu Val Ser Ser Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser
115 120 125
Arg Leu Thr Val Leu
130
<210> 174
<211> 408
<212> DNA
<213> 人工序列
<220>
<223> 多核苷酸
<400> 174
atgggcacca ggctcctctt ctgggtggcc ttctgtctcc tgggggcata tcacacagga 60
gctggagtct cccagtcccc cagtaacaag gtcacagaga agggaaagga tgtagagctc 120
aggtgtgatc caatttcagg tcatactgcc ctttactggt accgacagag gctggggcag 180
ggcctggagt ttttaattta cttccaaggc aacagtgcac cagacaaatc agggctgccc 240
agtgatcgct tctctgcaga gaggactggg gaatccgtct ccactctgac gatccagcgc 300
acacagcagg aggactcggc cgtgtatctc tgtgccagca gagggggcag ggggctctct 360
ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctg 408
<210> 175
<211> 136
<212> PRT
<213> 人工序列
<220>
<223> 多肽
<400> 175
Met Gly Thr Arg Leu Leu Phe Trp Val Ala Phe Cys Leu Leu Gly Ala
1 5 10 15
Tyr His Thr Gly Ala Gly Val Ser Gln Ser Pro Ser Asn Lys Val Thr
20 25 30
Glu Lys Gly Lys Asp Val Glu Leu Arg Cys Asp Pro Ile Ser Gly His
35 40 45
Thr Ala Leu Tyr Trp Tyr Arg Gln Arg Leu Gly Gln Gly Leu Glu Phe
50 55 60
Leu Ile Tyr Phe Gln Gly Asn Ser Ala Pro Asp Lys Ser Gly Leu Pro
65 70 75 80
Ser Asp Arg Phe Ser Ala Glu Arg Thr Gly Glu Ser Val Ser Thr Leu
85 90 95
Thr Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala
100 105 110
Ser Arg Gly Gly Arg Gly Leu Ser Gly Ala Asn Val Leu Thr Phe Gly
115 120 125
Ala Gly Ser Arg Leu Thr Val Leu
130 135
Claims (13)
1.一种获得正确配对的TCR的方法,所述方法包括以下步骤:
1)获得肿瘤细胞激活的T细胞;
2)从步骤1)获得的激活的T细胞获得带有Barcode的单个T细胞的TCR alpha可变区和TCR beta可变区的cDNA;
3)将步骤2)获得的带有Barcode的TCR alpha可变区和TCR beta可变区的cDNA分别重组到携带TCR alpha恒定区和TCR beta恒定区的载体中,从而扩增TCR alpha可变区序列和TCR beta可变区序列;
4)从步骤3)获得的TCR alpha可变区序列和TCR beta可变区序列中挑选具有相同细胞Barcode的TCR alpha可变区序列和TCR beta可变区序列,从而获得正确配对的TCR。
2.如权利要求1所述的方法,其特征在于,所述步骤1)包括:
1-1.从肿瘤患者样品中分离单个核细胞;
1-2.从步骤1-1得到的单个核细胞中分选出T细胞和肿瘤细胞;
1-3.利用步骤1-2得到的肿瘤细胞刺激T细胞;
1-4.任选检测激活标志物的表达。
3.如权利要求1所述的方法,其特征在于,所述步骤2)包括以下步骤:
2-1.分选细胞活率在80%以上,优选在85%以上的激活的T细胞,并获得单细胞悬液;
2-2.获得细胞的mRNA;
2-3.利用Barcode引物序列与逆转录酶对获得的mRNA进行逆转录,从而得到带有Barcode的单个细胞的TCR alpha可变区和TCR beta可变区的cDNA;
2-4.任选对获得的cDNA进行纯化和PCR扩增;
2-5.对PCR产物进行环化和线性化。
4.如权利要求3所述的方法其特征在于,所述步骤2-2包括:获得100-2000个,优选100-800个,最优选200-500个激活的T细胞的mRNA。
5.如权利要求1所述的方法,其特征在于,所述步骤3)中,携带TCR alpha恒定区的载体具有启动子、TCR alpha恒定区序列和PolyA位点;携带TCR beta恒定区的载体具有启动子、TCR beta恒定区序列和PolyA位点。
6.如权利要求1所述的方法,其特征在于,所述步骤4)中,对步骤3)获得的TCR alpha可变区序列和TCR beta可变区序列进行测序并挑选具有相同细胞Barcode的TCR alpha可变区序列和TCR beta可变区序列,从而获得正确配对的TCR。
7.如权利要求1所述的方法,其特征在于,所述方法还包括以下步骤:
5)任选对获得的TCR进行功能评价。
8.一种TCR,所述TCR采用权利要求1-7中任一项所述方法制备得到。
9.如权利要求8所述的TCR,其特征在于,所述TCR的alpha可变区氨基酸序列选自表2,和/或所述TCR的beta可变区氨基酸序列选自表3。
10.权利要求8或9所述的TCR在制备遗传改造的T细胞中的用途。
11.一种遗传改造的T细胞,所述T细胞表达采用权利要求1-7中任一项所述方法制备得到的TCR或权利要求8或9所述的TCR。
12.权利要求11所述的遗传改造的T细胞在制备***的药物组合物中的用途。
13.一种药物组合物,所述药物组合物包含权利要求11所述的遗传改造的T细胞以及药学上可接受的赋形剂。
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