CN107245502B - Cd2结合蛋白(cd2ap)和其相互作用蛋白 - Google Patents

Cd2结合蛋白(cd2ap)和其相互作用蛋白 Download PDF

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CN107245502B
CN107245502B CN201710448431.8A CN201710448431A CN107245502B CN 107245502 B CN107245502 B CN 107245502B CN 201710448431 A CN201710448431 A CN 201710448431A CN 107245502 B CN107245502 B CN 107245502B
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李朝阳
张会侠
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Abstract

下调个体CD2AP表达的方法，该方法包括施用一个CD2AP下调配伍到个体，其中CD2AP下调配伍通过siRNA/shRNA,Crispr/cas9,crispr/cpf1,Talen或ZFNs来工作；因此，个体肝组织的CD2AP表达被下调。

Description

CD2结合蛋白(CD2AP)和其相互作用蛋白
技术领域
本发明涉及CD2相关蛋白(CD2AP)及其相互作用的蛋白，尤其是CD2AP与丙型肝炎病毒(HCV)非结构蛋白NS5A之间的相互作用，CD2AP和胰岛素受体底物1(IRS1)之间的相互作用，以及Cbl-b/Cbl和IRS1之间的相互作用，并进一步涉及下调CD2AP表达的试剂和方法，操纵CD2AP和HCV NS5A之间的相互作用而抑制HCV装配的试剂和方法，操纵CD2AP和IRS1之间的相互作用而治疗二型糖尿病病人的试剂和方法,和操纵Cbl-b/Cbl和IRS1之间的相互作用而治疗二型糖尿病病人的试剂和方法。此外，本发明也涉及到基于CD2AP的血清学和胆汁检测脂肪肝和二型糖尿病的试剂和方法。
背景技术
丙型肝炎病毒(HCV)，Flavivirus的一员，是一种单股正链RNA病毒，基因组9.6kb(1)。HCV感染全球约1亿8000万人，导致严重的慢性肝病(2)。HCV的正常靶点是肝细胞。进入宿主细胞后，HCV脱掉其基因组RNA的外壳，并翻译出多聚蛋白前体，然后在宿主和病毒蛋白酶的切割下产生三个结构蛋白和七个非结构蛋白，非结构蛋白在病毒RNA复制，装配和释放中起重要作用(3)。
慢性HCV感染通常会导致脂肪肝，主要病症是富含中性脂的囊泡的异常积累。HCV的脂滴主要组分也是中性脂，是病毒颗粒进行装配的场所。(4,5)。脂滴是细胞中广泛存在的一种独特的细胞器，具有一个磷脂单层。脂滴参与许多生物学过程，如能量储存、脂质代谢、免疫及信号转导。在HCV感染的细胞中，脂滴的表面布满HCV的核心蛋白和NS5A，NS5A分布在在外表面(5-7)，核心蛋白和NS5A双双附着到脂滴，对传染性HCV颗粒的组装和释放是必不可少的(6,8,9)。
NS5A可以分为三个结构域，D1，D2和D3。D1和D2为RNA复制所需，D3有助于病毒的组装和释放。另外，通常认为,NS5A D1负责靶向脂滴而D3负责结合核心蛋白(10,11)。
将含有NS5A的HCV复制复合体转运到脂滴，取决于核心蛋白/NS5A与细胞骨架之间的相互作用。微管和肌动蛋白丝抑制剂的处理会抑制HCV复制复合体的移动(12)。沉默微管动力蛋白dynein，显著降低NS5A阳性结构到脂滴的远程移动性(13,14)。然而，并没有证据显示dynein和NS5A存在直接作用。因此，转运过程中与NS5A相互作用的宿主蛋白了解的仍然不完全
肝脏是***代谢的主要器官，肝脏功能的失衡极大地促胰岛素抗性和2型糖尿病(T2DM)的发生(15)。在导致胰岛素抗性的分子中，IRS-1是一个支架蛋白，在胰岛素级联中起着重要作用。许多体内和体外研究表明，降低IRS的细胞水平可能是胰岛素抗性的一种机制(15-22)。胰岛素受体是一个酪氨酸激酶，其底物IRS1的稳定性，主要是通过蛋白酶体降解蛋白水平来调节。研究表明，蛋白酶体介导降解IRS-1可能参与了胰岛素和***(IGF-1)信号通路的下调，导致胰岛素抗性(23-27)。IRS1的泛素化显示为胰岛素诱导IRS1蛋白酶体降解的前提；IRS-1的N-端区域包括PH、PTB结构域，被证明是靶向IRS-1到泛素蛋白酶体降解途径所必须的(28)。
胰岛素抗性通常导致肝纤维化和脂肪变性，在HCV感染状况下尤其如此(29)。胰岛素通过诱导其受体顺式磷酸化，驱动营养储存和组织生长，其受体是一个二聚体跨膜受体酪氨酸激酶(RTK)。这导致胰岛素受体底物(IRS)1和2的磷酸化，激活一个广泛的信号网络包括但不限于phosphatidylinositol-2-kinase/AKT/mTOR和Ras/MEK/ERK通路。
IRS-1是一个信号适配器蛋白，在人类中由IRS-1基因编码。这是一个131kDa的蛋白质，氨基酸序列有1242个残基。它包含一个位于N-末端的pleckstrin同源(PH)结构域和位于40个残基的下游的PTB结构域，接着是不保守的C-末端尾巴。IRS-1在信号传输中起着关键的作用，信号从胰岛素和***-1(IGF-1)受体到胞内信号通路，如PI3K/Akt和Erk MAP激酶通路。胰岛素受体(IR)对IRS-1酪氨酸磷酸化引入了多个可以和带有SH2蛋白同源结构域的蛋白，如PI3K、Grb-2/Sos复合物和SHP2相互结合的位点。
发明内容
本发明提供下调个体CD2AP表达的方法。在一些实施例中，其特征在于，该方法包括：施用一个CD2AP下调配伍到个体，其中CD2AP下调配伍通过siRNA/shRNA,Crispr/cas9,crispr/cpf1,Talen或ZFNs来工作；因此，个体肝组织的CD2AP表达被下调。在一些实施例中，其特征在于，所述CD2AP下调配伍包括至少一个选自序列号3-20(针对人)或59-76(针对狗)的siRNA/shRNAi多聚核苷酸序列或至少一个Crispr/cas9,crispr/cpf1载体，其中所述Crispr/cas9,crispr/cpf1载体包含一个选自序列号21-56(针对人)或77-103(针对狗)的引导多聚核苷酸序列。
本发明提供一个下调CD2AP在个体肝组织表达的药物配伍，其特征在于，所述配伍包括至少一个选自序列号3-20(针对人)或59-76(针对狗)的siRNA/shRNAi多聚核苷酸序列或至少一个Crispr/cas9,crispr/cpf1载体，其中所述Crispr/cas9,crispr/cpf1载体包含一个选自序列号21-56(针对人)或77-103(针对狗)的引导多聚核苷酸序列。
本发明提供筛选能够下降CD2AP和HCV非结构蛋白NS5A相互作用的候选试剂的方法，其特征在于，所述方法包括：提供表达CD2AP和NS5A的细胞；让候选试剂作用于表达CD2AP和NS5A的细胞；检测候选试剂对CD2AP和NS5A相互作用的影响；其中，如果引起CD2AP和NS5A相互作用下降一个预定阈值，选定该候选试剂；其中，所述预定阈值被定义为CD2AP和NS5A相互作用下降至少70％，优选80％。
本发明提供降低CD2AP和NS5A相互作用的药物配伍，其特征在于，所述药物配伍包括至少一个多肽，含有5-40个氨基酸，优选10-30个氨基酸，更优选15-25个氨基酸；其中所述多肽衍生于序列号2的3-58氨基酸，序列号2的111-165氨基酸，序列号2的271-327氨基酸，和序列号105的353-466氨基酸。
本发明提供筛选能够下降CD2AP和IRS1相互作用的候选试剂的方法，其特征在于，所述方法包括：提供表达CD2AP和IRS1的细胞；让候选试剂作用于表达CD2AP和IRS1的细胞；检测候选试剂对CD2AP和IRS1相互作用的影响；其中，如果引起CD2AP和IRS1相互作用下降一个预定阈值，选定该候选试剂；其中，所述预定阈值被定义为CD2AP和IRS1相互作用下降至少70％，优选80％。
本发明提供降低CD2AP和IRS1相互作用的药物配伍，其特征在于，所述药物配伍包括至少一个多肽，含有5-40个氨基酸，优选10-30个氨基酸，更优选15-25个氨基酸；其中所述多肽衍生于序列号2或58的3-58氨基酸，序列号2或58的111-165氨基酸，和序列号2或58的271-327氨基酸。
本发明提供筛选能够下降Cbl-b/Cbl和IRS1相互作用的候选试剂的方法，其特征在于，所述方法包括：提供表达Cbl-b/Cbl和IRS1的细胞；让候选试剂作用于表达Cbl-b/Cbl和IRS1的细胞；检测候选试剂对Cbl-b/Cbl和IRS1相互作用的影响；其中，如果引起Cbl-b/Cbl和IRS1相互作用下降一个预定阈值，选定该候选试剂；其中，所述预定阈值被定义为Cbl-b/Cbl和IRS1相互作用下降至少70％，优选80％。
本发明提供下调个体肝组织中Cbl-b/Cbl表达的方法，其特征在于，该方法包括：施用一个Cbl-b/Cbl下调配伍到个体，其中Cbl-b/Cbl下调配伍通过siRNA/shRNA,Crispr/cas9,crispr/cpf1,Talen或ZFNs来工作；因此，个体肝组织的Cbl-b/Cbl表达被下调。在一些实施例中，所述Cbl-b/Cbl下调配伍包括至少一个选自序列号112-124和196-208(针对人)或161-170和246-255(针对狗)的siRNA/shRNAi多聚核苷酸序列或至少一个Crispr/cas9,crispr/cpf1载体，其中所述Crispr/cas9,crispr/cpf1载体包含一个选自序列号125-158和209-243(针对人)或171-192和256-280(针对狗)的引导多聚核苷酸序列。
本发明提供一个下调Cbl-b/Cbl在个体表达的药物配伍，其特征在于，所述Cbl-b/Cbl下调配伍包括至少一个选自序列号112-124和196-208(针对人)或161-170和246-255(针对狗)的siRNA/shRNAi多聚核苷酸序列或至少一个Crispr/cas9,crispr/cpf1载体，其中所述Crispr/cas9,crispr/cpf1载体包含一个选自序列号125-158和209-243(针对人)或171-192和256-280(针对狗)的引导多聚核苷酸序列。
本发明提供治疗个体的HCV感染，其特征在于，所述治疗包括：施用一个配伍，其包含至少一个选自序列号3-20的siRNA/shRNAi多聚核苷酸序列；施用一个配伍，其包含至少一个Crispr/cas9,crispr/cpf1载体，其中所述Crispr/cas9,crispr/cpf1载体包含一个选自序列号21-56的引导多聚核苷酸序列；或者施用一个配伍，其包含一个降低CD2AP和NS5A相互作用的试剂。
本发明提供治疗患有糖尿病的个体，其特征在于，所述治疗包括：施用一个配伍，其包含至少一个选自序列号3-20(针对人)或59-76(针对狗)的siRNA/shRNAi多聚核苷酸序列；施用一个配伍，其包含至少一个Crispr/cas9,crispr/cpf1载体，其中所述Crispr/cas9,crispr/cpf1载体包含一个选自序列号21-56(针对人)或77-103(针对狗)的引导多聚核苷酸序列；或者，施用一个配伍，其包含一个降低CD2AP和IRS1相互作用的试剂。
本发明提供治疗患有糖尿病的个体，其特征在于，所述治疗包括：施用一个配伍，其包含至少一个选自序列号112-124和195-208(针对人)或161-170和246-255(针对狗)的siRNA/shRNAi多聚核苷酸序列；施用一个配伍，其包含至少一个Crispr/cas9,crispr/cpf1载体，其中所述Crispr/cas9,crispr/cpf1载体包含一个选自序列号125-158和209-243(针对人)或171-192和256-280(针对狗)的引导多聚核苷酸序列；或者，施用一个配伍，其包含一个降低Cbl-b/Cbl和IRS1相互作用的试剂。
本发明提供检测个体肝脏异化的诊断方法。在实施例中，所述方法包括，提供来自个体的肝脏样品，让肝脏样品接触检测CD2AP表达的检测试剂；因此，当CD2AP表达被检测到时，提示该个体肝脏异化。异化包括HCV感染和糖尿病。
本发明提供检测个体肝脏异化的诊断试剂盒。在实施例中，所述试剂盒包括，CD2AP蛋白特异抗体或CD2AP mRNA特异的多聚核苷酸探针，和一个次级试剂，能够检测与CD2AP蛋白结合的抗体或来自CD2AP mRNA的信号。
通过如下结合附图对优选实施例的详细描述，本发明的目的和优点是显而易见的。
附图说明
本发明的优选实施方案现在将参考附图进行说明，其中类似的附图标记表示相同的元件。
图1显示BioID构件,NS5A-BirA*-HA的结构域示意图。
图2显示免疫印迹图片。Huh7细胞转导了表达NS5A BirA*-HA的慢病毒载体，在添加或不添加50μM生物素的完全培养基中孵育24h。全细胞裂解物进行10％SDS-PAGE分析；分离蛋白质用辣根过氧化物酶标记的链霉亲和素来检测。
图3所示SDS-PAGE凝胶与考马斯亮蓝染色的图片。细胞裂解物的制备与图2相同。细胞裂解物通过链霉亲和素琼脂糖珠进行亲和纯化。纯化的蛋白质进行10％SDS-PAGE分析和考马斯亮蓝染色。将箭头指示的生物素处理样品的特定条带进行质谱分析。
图4显示免疫印迹图片。免疫共沉淀分析293T细胞中CD2AP和NS5A蛋白之间的相互作用。293T细胞转染了单独的HA标记CD2AP(pRK-HA-CD2AP)或HA标记CD2AP(pRK-HA-CD2AP)连同Flag标记的NS5A；NS5A来自2a型HCV的JFH1菌株(pRK-Flag-NS5A)。转染36小时后，细胞裂解物进行免疫共沉淀(co-IP)，将细胞裂解液与鼠源的Flag抗体(标为F)或对照IgG1(标为IgG)进行co-IP。用兔源的HA抗体和Flag抗体进行WB检测。裂解液中HA-CD2AP的表达量相同。
图5显示免疫印迹照片。免疫共沉淀分析HCV感染期间CD2AP和NS5A蛋白之间的相互作用。Huh7.5.1细胞感染HCV JFH1或未感染为对照。感染后72h，收集细胞进行裂解。细胞裂解液与CD2AP抗体(左图)或抗NS5A抗体(中间图)进行co-IP，然后用NS5A抗体和CD2AP抗体进行WB检测。NS5A的表达证实HCV的感染(右上图)。
图6显示免疫印迹照片。HCV JFH1感染Huh7.5.1细胞72小时，细胞裂解液与NS5A抗体或对照小鼠IgG1进行co-IP，然后用NS5A抗体和CD2AP抗体进行WB检测IP复合物。只有NS5A抗体可以沉淀CD2AP，IgG1对照未拉下CD2AP。
图7显示免疫荧光染色照片。CD2AP和NS5A在HCV感染的Huh7.5.1细胞中共定位。Huh7.5.1细胞感染HCV J399EM(如图6所示HCV2a相同株，但NS5A是GFP标记的)(下面板)或未感染(上面板)72小时。然后用兔源的CD2AP抗体和Alex Fluor 555标记的二抗染色CD2AP(红色)，绿色代表NS5A-EGFP的定位。
图8所示全长和截短CD2AP示意图。CD2AP的N-端含有三个SH3结构域。从N端到C端分别是SH3-1，SH3-2和SH3-3。
图9显示免疫印迹照片。CD2AP截短体与NS5A的相互作用。将HA标记的NS5A和Flag标记的CD2AP截短体共转293T细胞。转染后36小时，将细胞裂解液与鼠源的Flag(F)抗体或者IgG1对照(IgG)进行co-IP。IP复合物用兔源的HA和Flag抗体进行检测。IP下来的CD2AP截短体用星号标记。分子量在20-35kDa之间的非特异性谱带未在此显示。
图10显示全长和截短NS5A的示意图。NS5A由三个结构域组成；如图所示，它们由两个低复杂度的序列连接(Lcs1和Lcs2)。
图11显示免疫印迹照片。NS5A截短体与CD2AP的相互作用。293T细胞转染了HA标记的CD2AP和其中一个flag标记的NS5A蛋白或截短突变体。转染后36小时，将细胞裂解液与鼠源的Flag(F)抗体或者IgG1对照(IgG)进行co-IP。IP复合物用兔源的HA和Flag抗体进行检测。发现NS5A的第三结构域与CD2AP特异性相互作用。抗flag抗体纯化的NS5A截短体用星号表示。
图12显示NS5A与全长及截短CD2AP的定位。在Huh7.5.1细胞中稳定表达带有mCherry标签的全长CD2AP或者SH3结构域缺失的截短CD2AP(CD2AP：331-639)，然后用HCV-J399EM感染该细胞。48h后，共聚焦显微镜观察CD2AP(红色)和NS5A(绿色)的运动并进行实时跟踪。NS5A与全长CD2AP共定位(左图)，但不与截短CD2AP共定位(右图)。
图13是一个曲线图显示一个NS5A/CD2AP复合物的共移动。如图12所示，感染后72h实时图像跟踪CD2AP(红色)和NS5A(绿色)显示了NS5A和CD2AP的共移动。使用Volocity软件分析图13方框1中所示的NS5A/CD2AP共定位复合物的速度。
图14所示照片显示NS5A/CD2AP复合物的共移动依赖于肌动蛋白聚合。稳定表达带有mCherry标签的全长CD2AP的Huh7.5.1细胞用HCV-J399EM感染48小时，然后用1μg/mL细胞松弛素B(上图)或10μM秋水仙素(下图)处理1小时。或者药物处理1小时后，更换不加药物的新鲜培养基培养4小时后(右图)对活细胞进行共聚焦显微镜观察。细胞松弛素B处理后NS5A和CD2AP没有共定位(左上图)。然而，秋水仙碱处理不影响NS5A和CD2AP的共定位(左下图)。更换培养基后培养四小时，CD2AP和NS5A的共定位重新出现(右上图面板)，而秋水仙素处理的细胞，在更换培养基后后并无区别(右下图)。
图15是一个曲线图显示秋水仙素处理后方框3所示的NS5A/CD2AP共定位点的速度。在微管聚合被抑制后，CD2AP/NS5A复合物没有任何共移动。
图16显示免疫印迹照片。将NC和CD2AP沉默细胞(c4#,c6#)进行脂滴分离，然后用特异性的抗体进行检测。左侧是NS5A在脂滴上的表达水平，右侧是细胞裂解液中各种蛋白的表达。ADRP和calnexin分别是脂滴和内质网的标记物。用Image J软件进行NS5A的定量分析。在HCV亚复制子细胞CON1中将CD2AP下调后，NS5A在脂滴上的表达水平下降。然而，细胞裂解液中NS5A的表达水平没有受到CD2AP下调的影响(右侧)。
图17是荧光照片，显示下调CD2AP减少脂滴的量。将对照NC和CD2AP沉默细胞6#用含有BSA或含有0.5mM油酸-BSA复合物的DMEM培养16小时。细胞用中性脂染料进行脂滴染色。下调CD2AP显著降低由OA刺激的脂滴量。通过Volocity软件分析油酸刺激后单个细胞中脂滴的区域面积，证明对照细胞和CD2AP下调细胞间存在显著性差异该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。(黑色柱状图，p＜0.01)。
图18是荧光照片，显示过表达CD2AP可以功能性拯救脂滴的生物合成。CD2AP下调细胞(6#)转染CD2AP拯救突变体(6#-res)或空载体(6#-NC)。然后，用单独BSA或0.5mM的OA-BSA复合物处理细胞16小时。然后细胞进行脂滴染。通过Volocity软件分析油酸刺激后单个细胞中脂滴的区域面积，证明脂滴的量在CD2AP拯救后基本恢复(黑色柱状图，p＜0.05)。
图19是免疫荧光照片，显示在CD2AP下调细胞(6#)中表达HCV核心蛋白没有拯救脂滴的积累。HA标签核心蛋白转染对照(NC)或CD2AP蛋白下调(6#)细胞，染色脂滴。HCV核心蛋白被抗HA抗体染色(绿色)。脂滴如上染色(红色)。通过Volocity软件分析油酸刺激后单个细胞中脂滴的区域面积，证明在CD2AP下调细胞中表达核心蛋白没有拯救脂滴脂滴的量(黑色柱状图，p＜0.05)。
图20是免疫荧光照片，显示在CD2AP拯救细胞(6#-res)和对照细胞(6#-NC)中表达HCV核心蛋白，CD2AP拯救细胞显示LD的积累比对照细胞显著增多。核心蛋白的表达并没有回复CD2AP下调细胞中脂滴的表达量。通过Volocity软件分析油酸刺激后单个细胞中脂滴的区域面积，证明脂滴的量在CD2AP拯救后基本恢复(黑色柱状图，p＜0.05)。
图21是一个柱状图，显示感染后72h，与对照细胞相比，下调CD2AP降低HCV mRNA水平。CD2AP下调(4#和6#)或对照(NC)Huh 7.5.1细胞感染HCV JFH1 72小时。提取细胞内总RNA并进行定量RT-PCR检测HCV mRNA。该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。**(P<0.01)，*(P<0.05)。
图22显示免疫印迹照片。全细胞裂解物免疫印迹CD2AP，HCV NS5A，核心蛋白和β肌动蛋白，表明CD2AP的下调后NS5A和核心蛋白的降低。
图23是一个柱状图，显示下调CD2AP显著降低上清中HCV RNA拷贝数。来自CD2AP下调(4#和6#)或对照(NC)细胞上清中HCV RNA拷贝数用RT-PCR定量。该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。**(P<0.01)，*(P<0.05)。
图24是一个柱状图，显示下调CD2AP显著抑制报告基因的荧光素酶活性。CD2AP下调(4#和6#)或对照(NC)细胞感染了含有renila荧光素酶基因的报告病毒J399EM+LM。72小时后检测荧光素酶活性。该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。**(P<0.01)，*(P<0.05)，n.s.(无显著差别)。
图25是一个柱状图，显示CD2AP拯救细胞部分恢复细胞内HCV mRNA。下调了CD2AP的Huh7.5.1细胞(6#)转染了CD2AP拯救突变体(6#-res)或对照质粒(6#-NC)然后在MOI为0.1感染JFH1。感染后72h，相对qRT-PCR分析检测到细胞内HCV RNA水平，CD2AP拯救细胞和对照细胞相比显著增加。该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。**(P<0.01)，*(P<0.05)，n.s.(无显著差别)。
图26显示免疫印迹照片。CD2AP拯救细胞部分恢复HCV蛋白。细胞裂解物来自感染HCV JFH1的CD2AP拯救细胞，对CD2AP,核心蛋白,NS5A进行免疫印迹实验，显示部分拯救NS5A和核心蛋白。
图27是一个柱状图，显示与对照细胞(NC)相比，CD2AP下调(4#&6#)不影响HCVpp进入。细胞转染了HCVpp，荧光素酶活性检测48小时后进行。CD2AP下调后对HCV进入无显著影响。该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。**(P<0.01)，*(P<0.05)，n.s.(无显著差别)。
图28是一个柱状图，显示比较对照细胞(NC)，在复制子细胞CON1中的CD2AP下调(4#&6#)没有减少HCV亚基因组的复制。与对照相比，下调CD2AP显著降低了CD2AP mRNA(空盒，P＜0.01)，下调CD2AP没有减少细胞内HCV RNA水平(黑盒)。该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。**(P<0.01)，*(P<0.05)，n.s.(无显著差别)。
图29是一个柱状图，显示与对照细胞(NC)相比，下调CD2AP(4#和6#)不影响依赖于HCV-IRES的翻译。细胞转染了pHCV-IRES。48h后，用双荧光素酶报告基因检测***(Promega)测定荧光素酶活性。翻译效率决定于萤火虫荧光素酶(F-Luc)活性与Renilla荧光素酶(R-Luc)活性的比例。该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。**(P<0.01)，*(P<0.05)，n.s.(无显著差别)。
图30是一个柱状图，显示下调CD2AP显著降低细胞内HCV滴度(p＜0.05)。CD2AP下调(4#&6#)或对照(NC)Huh7.5.1细胞感染了J399EM，MOI为1。收集细胞颗粒，定量测定细胞内病毒滴度。该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。**(P<0.01)，*(P<0.05)，n.s.(无显著差别)。
图31是一个柱状图，显示下调CD2AP还显著降低上清液HCV滴度(p＜0.01)。培养上清液72小时后收集，定量释放到胞外的病毒滴度。该实验重复3次，定量数据误差显示的是平均值±S.E.M，双尾的学生t检验用于统计分析。**(P<0.01)，*(P<0.05)，n.s.(无显著差别)。
图32所示(A)脂滴(LD)和HCV NS5A结合的照片，和(B)一个NS5A阳性LD的柱状图。对于(A)，稳定的CD2AP下调(4#和6#)和对照细胞(NC)感染了JFH-1，然后进行免疫荧光染色脂滴(红色)和HCV NS5A(绿色)。核用DAPI染色(蓝色)。下调CD2AP显著降低脂滴与HCV蛋白NS5A的共定位。对于(B)，NS5A阳性脂滴的定量显示，CD2AP的下调显著降低HCV感染的细胞中NS5A在脂滴上的定位。对照(NC)和CD2AP下调(4#&6#)细胞分别共有161，104，和87的细胞计数。统计分析不同组间差异为*(p＜0.05)，**(p＜0.01)。
图33所示(A)脂滴和HCV核心蛋白结合的照片，和(B)一个核心蛋白阳性LD的柱状图。对于(A)，稳定的CD2AP下调(4#和6#)和对照细胞(NC)感染了JFH-1，然后后免疫荧光染色脂滴(红色)和HCV核心蛋白(绿色)。核用DAPI染色(蓝色)。下调CD2AP显著降低脂滴与HCV核心蛋白的共定位。对于(B)，核心蛋白阳性脂脂滴的定量显示，CD2AP的下调显著降低HCV感染的细胞中核心蛋白在脂滴上的定位。对照(NC)和CD2AP下调(4#&6#)细胞分别共有161，104，和87的细胞计数。统计分析不同组间差异为*(P＜0.05)，**(P＜0.01)。
图34显示免疫印迹照片。胰岛素受体底物1(IRS1)的表达在CD2AP下调细胞中显著上调。CD2AP下调(4#&6#)和对照(NC)细胞感染了JFH-1。感染后72h，全细胞裂解物进行免疫印迹实验，针对IRS1或胰岛素受体(IR)及其磷酸化形式。比较对照细胞，显著增加的IRS1和p-IRS1在CD2AP下调细胞中检测到。比较对照细胞，在CD2AP下调细胞中IR和p-IR有小幅增加。
图35显示免疫印迹照片。降解IRS1是蛋白酶依赖的。MG132处理过的Huh7.5.1细胞进行免疫印迹实验，运用特异性抗体检测处理后不同时间IRS1的表达水平(0，0.25，0.5，1，2，4H 10μM MG132处理)。
图36显示免疫印迹照片。CD2AP下调细胞对蛋白酶体抑制剂处理更不敏感。CD2AP下调(4#&6#)和对照(NC)细胞经DMSO(-)或(+)μM MG132处理两小时。全细胞裂解物IRS1的特异性抗体进行免疫印迹实验。
图37显示免疫印迹照片。IRS1的泛素化在CD2AP下调细胞(4#&6#)中比对照(NC)细胞要少。细胞在完全培养基中培养48小时后收获。用抗IRS1抗体纯化细胞裂解物。纯化的蛋白为了polyubiquitin和IRS1进行免疫印迹实验。
图38显示免疫印迹照片。IRS1、CD2AP、cbl-b/cbl存在于同一蛋白复合物。Huh7.5.1细胞裂解物进行免疫共沉淀反应，抗IRS1抗体(左面板)。CD2AP和IRS1共纯化。Huh7.5.1细胞裂解物进行免疫共沉淀反应,抗cbl-b抗体(中面板)或抗cbl抗体(右面板)。IRS1与cbl-b/cbl共同纯化。
图39显示在Huh7.5.1细胞中IRS1、CD2AP、cbl-b/cbl的共定位照片。细胞进行染色，抗IRS1抗体(红色)和抗CD2AP抗体(绿色)。细胞质中观察到CD2AP和IRS1共定位(左面板)。细胞进行染色，抗IRS1抗体(红色)和抗cbl-b抗体(绿色)(中面板)或抗cbl抗体(绿色)(右面板)。细胞质中观察到IRS1和cbl-b或cbl共定位。
图40是免疫印迹照片。Huh7.5.1细胞中下调cbl-b或cbl的表达，运用siRNA，针对cbl-b或cbl；用相应抗体免疫印迹cbl-b或cbl蛋白。处理后的肌动蛋白印迹作为上样量对照。2#和3#是cbl-b特异的两个siRNA；1#和4#是cbl特异的两个siRNA。NC是阴性对照siRNA。数据显示，在cbl-b(左面板)或cbl(右面板)下调的Huh7.5.1细胞中IRS1的水平出现明显的上升。
图41显示免疫印迹照片。与对照(NC)细胞相比，Akt-AMPK-HSL轴线在CD2AP下调细胞(4#&6#)中被活化。总的细胞裂解物用针对AMPK信号通路的不同抗体进行了免疫印迹。P-Akt(S473)上升，但总Akt不上升；p-AMPK(T172)下降，但是总AMPK不减少；p-HSL(s554)下降，但总HSL不下降；Erk或p-Erk无变化。
图42显示免疫迹照片。P-Akt的表达水平，与对照细胞相比，在CD2AP拯救细胞中得以恢复。来自CD2AP拯救细胞和对照细胞的裂解物，用抗Akt(S473)和抗Akt抗体进行了免疫印迹。CD2AP下调细胞比对照细胞表达更多的p-Akt。当CD2AP下调细胞中CD2AP被拯救时，CD2AP拯救细胞中的p-Akt比对照细胞中更少。
图43显示免疫印迹照片。Dorsomophin(AMPK抑制剂)的处理，与对照细胞(NC)相比，CD2AP下调细胞(4#&6#)减少了p-AMPK和p-HSL水平。细胞在完全培养基中培养48小时，然后用DMSO或dorsomorphin(5μm)处理四小时。全细胞裂解物用特异抗体进行了免疫印迹。
图44是一个曲线图，显示HCV感染小鼠模型中肝组织HCV滴度的时间变化过程。感染后不同时间点用QPCR定量肝组织HCV滴度。前两周为HCV急性感染期而感染两周后是慢性感染期。
图45是一个曲线图，显示HCV感染小鼠模型中血清HCV滴度的时间过程。感染后不同时间点用QPCR定量血清HCV滴度。
图46显示感染HCV小鼠在感染后不同时间后经CD2AP染色的小鼠肝组织切片的照片。HCV感染诱导CD2AP表达。在感染后1个月，2个月和4个月的肝组织切片中出现CD2AP染色，和脂肪肝的出现相对应。
图47显示HCV感染和非HCV感染患者的肝组织经CCD2AP染色的照片。非HCV感染患者的肝组织没有显示CD2AP染色，但是HCV感染病人的肝组织中显示CD2AP染色。
图48显示糖尿病患者的肝组织经CCD2AP染色的照片。全部7个病人在他们的肝组织中显示CD2AP染色。
具体实施方式
可以通过引用以下的本发明的某些实施例的详细描述而更容易地理解本发明。
在本申请中，为了更充分地描述本发明所涉及领域状态，当出版物被引用，这些出版物的公开内容的全部经引用而并入本申请。
除非另有说明，本发明的实践将采用分子生物学(包括重组技术)，微生物学，细胞生物学，生物化学，核酸化学和免疫学的现有技术，这些是在本领域的技能之内。这些技术在文献中已有完全解释，如分子克隆：实验室说明书，第三版(Sambrook和Russel，2001年)(30)；分子生物学当代程序(FM Ausubel等主编，1987年)(31)；蛋白分析与纯化-实验技术(Rosenberg，1996年)(32)；蛋白分析方法：实验室程序实用指南(Copeland，2013年)(33)；免疫学现有程序(Coligan等主编，1999年)(34)。
本发明发现HCV装配需要CD2相关蛋白(CD2AP)参与；CD2AP是一个支撑分子，调节肌动蛋白细胞骨架。CD2AP相互作用于HCV非结构蛋白NS5A，通过依赖肌动蛋白的方式转运NS5A到细胞机器，然后通过微管依赖的方式靶向到脂滴脂滴。NS5A和CD2AP之间的相互作用需要CD2AP的SH3结构域和NS5A的第三结构域。正常肝细胞不表达CD2AP。在CD2AP表达的细胞中，下调CD2AP表达显著降低HCV装配和增殖。
CD2AP是一个接头蛋白，具有三个SH3结构域；其单倍剂量不足是人类肾小球疾病易感性的一个决定因素(35)。CD2AP通过E3连接酶下调细胞表面受体酪氨酸激酶活性(36-39)。此外，CD2AP已被证明正刺激PI3K信号，参与脂质代谢的一个信号通路(40,41)。
本发明也发现CD2AP相互作用于IRS1。在CD2AP表达的肝细胞中，下调CD2AP增加IRS1蛋白的水平。糖尿病病人的肝组织中观察到CD2AP表达。
本发明也发现CD2AP，cbl-b/cbl和IRS1共定位于同一蛋白质复合物。Cbl-b/cbl是E3连接酶。已知cbl-b/cbl相互作用于CD2AP。本发明发现cbl-b/cbl相互作用于IRS1，显示为cbl-b/cbl与IRS1共纯化和共定位。当cbl-b/cbl水平用siRNA下调，IRS1水平上调。
在一些实施例中，本发明提供在一个主体中下调CD2AP表达的方法。主体是人或狗。在一些实施例中，优选下调主体肝组织的肝细胞中的CD2AP表达。该下调CD2AP表达的方法包括：给主体注射CD2AP下调配伍，从而下调主体肝组织的CD2AP表达。在一些实施例中，CD2AP下调配伍包括siRNA/shRNAi多核苷酸，特异针对CD2AP(序列号1(人)和57(狗))，编码由SEQ ID NO 2(人)和58(狗)分别代表的氨基酸序列。在一些实施例中，该CD2AP特异的siRNA/shRNAi多核苷酸与选自序列号3-20(针对人)(表1)或序列号59-76(针对狗)(表3)的核苷酸序列相互补。在一些实施例中，CD2AP下调配伍包括Crispr/cas9,crispr/cpf1载体，特异针对主体中的CD2AP。CD2AP特异的Crispr/cas9,crispr/cpf1载体包括先导多核苷酸，选自序列号21-56(针对人)(表2)或序列号77-103(针对狗)(表4)。此外，类转录活化因子核酸酶(Talen)和锌指核酸酶(ZFN)技术也能用于下调CD2AP表达。
表1.用于下调人CD2AP表达的siRNA/shRNAi序列
SEQ ID NO# 核苷酸序列
SEQ ID NO 3 GCTGGAAGGAGAACTAAATGG
SEQ ID NO 4 GGAGAACTAAATGGGAGAAGA
SEQ ID NO 5 GGACTTCCAGCTGGAGGAATT
SEQ ID NO 6 GGAGCTGAAAGTGGGAGATAT
SEQ ID NO 7 GCTGAAAGTGGGAGATATTAT
SEQ ID NO 8 GCTGAAAGTGGGAGATATTAT
SEQ ID NO 9 GCCCAGGACGATTCAGAAACT
SEQ ID NO 10 GCTGGGCCTACTTCACCTATA
SEQ ID NO 11 GCCAGTAATTTACTGAGATCT
SEQ ID NO 12 GCTTCATCTCACTGCAAATAG
SEQ ID NO 13 GGAAGTTTCCAGCAGATTTCA
SEQ ID NO 14 AGCCGAGGGTCTGGGCAAA
SEQ ID NO 15 AGCCGAGGGTCTGGGCAAA
SEQ ID NO 16 TGAAGAGACTGGTAGGAGA
SEQ ID NO 17 CTAAATGGGAGAAGAGGAA
SEQ ID NO 18 AGGATGAACTGGAGCTGAA
SEQ ID NO 19 GGTAACAGATGATGGTGAA
SEQ ID NO 20 GGAAACAGATGATGTGAAA
表2.用于下调人CD2AP表达的CRISPR/CAS9,CRISPR/CPF1靶标序列
Figure GDA0001389300800000141
Figure GDA0001389300800000151
表3.用于下调狗CD2AP表达的siRNA/shRNAi序列
Figure GDA0001389300800000152
Figure GDA0001389300800000161
表4.用于下调狗CD2AP表达的CRISPR/CAS9,CRISPR/CPF1靶标序列
SEQ ID NO# 核苷酸序列
SEQ ID NO 77 AAAGGCAGACACTCAACCGCCGG
SEQ ID NO 78 ATGTATTGAAGTGAGACACCTGG
SEQ ID NO 79 ATGATGTGGGACTCCATCCCAGG
SEQ ID NO 80 AGGGCGTGACCCCCAAGTCCTGG
SEQ ID NO 81 TGTATTGAAGTGAGACACCTGGG
SEQ ID NO 82 GGGCGTGACCCCCAAGTCCTGGG
SEQ ID NO 83 CCATGCAGGAAGCATGATGTGGG
SEQ ID NO 84 GGGGTCACGCCCTGAGCCAAAGG
SEQ ID NO 85 TCCATGCAGGAAGCATGATGTGG
SEQ ID NO 86 ATTGAAGTGAGACACCTGGGTGG
SEQ ID NO 87 GACTCCATCCCAGGACTTGGGGG
SEQ ID NO 88 GAGTGTCTGCCTTTGGCTCAGGG
SEQ ID NO 89 TGGGACTCCATCCCAGGACTTGG
SEQ ID NO 90 AGACACCTGGGTGGCTCCGGCGG
SEQ ID NO 91 TGAGTGTCTGCCTTTGGCTCAGG
SEQ ID NO 92 GGACTCCATCCCAGGACTTGGGG
SEQ ID NO 93 GTGACCCCCAAGTCCTGGGATGG
SEQ ID NO 94 GGCGGTTGAGTGTCTGCCTTTGG
SEQ ID NO 95 GTGAGACACCTGGGTGGCTCCGG
SEQ ID NO 96 CCCACATCATGCTTCCTGCATGG
SEQ ID NO 97 GGGACTCCATCCCAGGACTTGGG
SEQ ID NO 98 TAACGCAACTTTCTATTTTTTGG
SEQ ID NO 99 CTCACTTCAATACATTTTTAAGG
SEQ ID NO 100 CCAGTTAAAAAGAAAATCTAAGG
SEQ ID NO 101 CTCAACCGCCGGAGCCACCCAGG
SEQ ID NO 102 TAAAGCAACTTTCTATTTTTTGG
SEQ ID NO 103 CCTTAGATTTTCTTTTTAACTGG
在一些实施例中，本发明提供一个药物配伍，用于下调主体中CD2AP表达。主体是人或狗。在一些实施例中，优选下调主体肝组织的肝细胞中的CD2AP表达。该下调CD2AP表达的方法包括：给主体注射CD2AP下调配伍，从而下调主体肝组织的CD2AP表达。在一些实施例中，CD2AP下调配伍包括siRNA/shRNAi多核苷酸，特异针对CD2AP(序列号1(人)和57(狗))，编码由SEQ ID NO 2和58分别代表的氨基酸序列。在一些实施例中，该CD2AP特异的siRNA/shRNAi多核苷酸与选自序列号3-20(针对人)(表1)或序列号59-76(针对狗)(表3)的核苷酸序列相互补。在一些实施例中，CD2AP下调配伍包括Crispr/cas9,crispr/cpf1载体，特异针对主体中的CD2AP。CD2AP特异的Crispr/cas9,crispr/cpf1载体包括先导多核苷酸，选自SEQ是NOS 21-56(针对人)(表2)或序列号77-103(针对狗)(表4)。
在一些实施例中，本发明提供了一种方法筛选候选药物，能够降低CD2AP和HCV非结构蛋白NS5A蛋白之间的相互作用。CD2AP的氨基酸序列为序列号2或其变体，其中变体被定义为一种氨基酸序列，与序列号2有至少80％，更优选90％，或甚至更优选95％的同源性。序列号2由序列号1的核酸序列所编码，其中CD2AP变体的编码核酸序列，与序列号1具有至少80％，更优选90％，或甚至更优选95％的同源性。NS5A的氨基酸序列为序列号105或其变体，其中变体被定义为一种氨基酸序列，与序列号105有至少80％，更优选90％，或甚至更优选95％的同源性。序列号105是由序列号104的核酸序列所编码，其中NS5A变体的编码核酸序列，与序列号104具有至少80％，更优选90％，或甚至更优选95％的同源性。该方法包括提供表达CD2AP和NS5A细胞，用候选药物处理CD2AP和NS5A表达细胞，然后检测候选药物对CD2AP和NS5A相互作用的影响；如果候选药物减少了CD2AP和NS5A相互作用而达到一个预定的阈值，选定候选药物。
表达CD2AP和NS5A细胞可以是任何合适的原代细胞或细胞系。在一些实施例中，合适的细胞是这样的一些细胞系，自身表达CD2AP，而NS5A的表达可以由转染NS5A表达载体；细胞系优选是肝癌细胞系。在一些实施例中，合适的细胞是HCV感染的肝细胞。
测定CD2AP和NS5A的相互作用，是任何合适的一个可以测量或确定CD2AP和NS5A之间的相互作用的方法。在一些实施例中，该法是免疫共沉淀，共定位，CD2AP和NS5A联动的聚焦活细胞成像；如何进行这些实验是公知的；因此，细节不在此赘述。确定候选药物是否有效降低CD2AP和NS5A相互作用的预定阈值定义为降低至少70％，更优选80％，CD2AP和NS5A蛋白之间的相互作用。例如，在免疫共沉淀实验，预定阈值是，候选药物处理过的细胞中，CD2AP或NS5A的免疫共沉淀量，与没有经过候选药物处理的细胞相比，减少至少70％，更优选80％。
在一些实施例中，本发明提供了减少CD2AP和NS5A相互作用的药物配伍。在一些实施例中，该药物配伍包含一种多肽，具有5-40氨基酸，优选10-30个氨基酸，更优选15-25个氨基酸，其多肽是序列号2的氨基酸3-58，111-165和271-327，和序列号105的氨基酸353-466的衍生物。一个衍生物定义为一个多肽，与相应系列具有至少80％，优选90％，或更优选95％同源性。
在一些实施例中，本发明提供了一种方法筛选候选药物，能够降低CD2AP和IRS1之间的相互作用。CD2AP的氨基酸序列为序列号2或58或其变体，其中变体被定义为一种氨基酸序列，与序列号2或58有至少80％，更优选90％，或甚至更优选95％的同源性。序列号2或58由序列号1或57的核酸序列所编码，其中CD2AP变体的编码核酸序列，与序列号1或57具有至少80％，更优选90％，或甚至更优选95％的同源性。IRS1的氨基酸序列为序列号107(针对人)或109针对狗或其变体，其中变体被定义为一种氨基酸序列，与序列号107或109有至少80％，更优选90％，或甚至更优选95％的同源性。序列号107或109是分别由序列号107或108的核酸序列所编码，其中IRS1变体的编码核酸序列，与序列号106或108具有至少80％，更优选90％，或甚至更优选95％的同源性。该方法包括提供表达CD2AP和IRS1细胞，用候选药物处理CD2AP和IRS1表达细胞，然后检测候选药物对CD2AP和IRS1相互作用的影响；如果候选药物减少了CD2AP和IRS1相互作用而达到一个预定的阈值，选定候选药物。
表达CD2AP和IRS1细胞可以是任何合适的原代细胞或细胞系。在一些实施例中，合适的细胞是表达CD2AP和IRS1的细胞系。
测定CD2AP和IRS1的相互作用，是任何合适的一个可以测量或确定CD2AP和IRS1之间的相互作用的方法。在一些实施例中，该法是免疫共沉淀，共定位；如何进行这些实验是公知的；因此，细节不在此赘述。确定候选药物是否有效降低CD2AP和IRS1相互作用的预定阈值定义为降低至少70％，更优选80％，CD2AP和IRS1蛋白之间的相互作用。例如，在免疫共沉淀实验，预定阈值是，候选药物处理过的细胞中，CD2AP或IRS1的免疫共沉淀量，与没有经过候选药物处理的细胞相比，减少至少70％，更优选80％。
在一些实施例中，本发明提供了减少CD2AP和IRS1相互作用的药物配伍。在一些实施例中，该药物配伍包含一种多肽，具有5-40氨基酸，优选10-30个氨基酸，更优选15-25个氨基酸，其多肽是序列号2或58的氨基酸3-58，111-165和271-327的衍生物。一个衍生物定义为一个多肽，与相应系列具有至少80％，优选90％，或更优选95％同源性。
在一些实施例中，本发明提供了一种方法筛选候选药物，能够降低Cbl-b/Cbl和IRS1之间的相互作用。Cbl-b的氨基酸序列为序列号111或160，或其变体，其中变体被定义为一种氨基酸序列，与序列号111或160有至少80％，更优选90％，或甚至更优选95％的同源性。序列号111和160由序列号110和159的核酸序列所分别编码，其中Cbl-b变体的编码核酸序列，与序列号110或159具有至少80％，更优选90％，或甚至更优选95％的同源性。Cbl的氨基酸序列为序列号194和245或其变体，其中变体被定义为一种氨基酸序列，与序列号194或245有至少80％，更优选90％，或甚至更优选95％的同源性。序列号194和245由序列号193和244的核酸序列所编码，其中Cbl变体的编码核酸序列，与序列号193或244具有至少80％，更优选90％，或甚至更优选95％的同源性。IRS1的氨基酸序列为序列号107或109，或其变体，其中变体被定义为一种氨基酸序列，与序列号107或109有至少80％，更优选90％，或甚至更优选95％的同源性。序列号107或109是由序列号106或108的核酸序列所分别编码，其中IRS1变体的编码核酸序列，与序列号106或108具有至少80％，更优选90％，或甚至更优选95％的同源性。该方法包括提供表达Cbl-b/Cbl和IRS1细胞，用候选药物处理Cbl-b/Cbl和IRS1表达细胞，然后检测候选药物对Cbl-b/Cbl和IRS1相互作用的影响；如果候选药物减少了Cbl-b/Cbl和IRS1相互作用而达到一个预定的阈值，选定候选药物。
表达Cbl-b/Cbl和IRS1细胞可以是任何合适的原代细胞或细胞系。在一些实施例中，合适的细胞是表达Cbl-b/Cbl和IRS1的细胞系。
测定Cbl-b/Cbl和IRS1的相互作用，是任何合适的一个可以测量或确定Cbl-b/Cbl和IRS1之间的相互作用的方法。在一些实施例中，该法是免疫共沉淀，共定位；如何进行这些实验是公知的；因此，细节不在此赘述。确定候选药物是否有效降低Cbl-b/Cbl和IRS1相互作用的预定阈值定义为降低至少70％，更优选80％，Cbl-b/Cbl和IRS1蛋白之间的相互作用。例如，在免疫共沉淀实验，预定阈值是，候选药物处理过的细胞中，Cbl-b/Cbl或IRS1的免疫共沉淀量，与没有经过候选药物处理的细胞相比，减少至少70％，更优选80％。
在一些实施例中，本发明提供在一个主体中下调Cbl-b/Cbl表达的方法。主体是人或狗。在一些实施例中，优选下调主体肝组织的肝细胞中的Cbl-b/Cbl表达。该下调Cbl-b/Cbl表达的方法包括：给主体注射Cbl-b/Cbl下调配伍，从而下调主体肝组织的Cbl-b/Cbl表达。在一些实施例中，Cbl-b/Cbl下调配伍包括siRNA/shRNAi多核苷酸，特异针对Cbl-b/Cbl(序列号110或159或序列号193或244)，编码由SEQ ID NO 111或160或序列号110或245分别代表的氨基酸序列。在一些实施例中，该Cbl-b/Cbl特异的siRNA/shRNAi多核苷酸与选自序列号112-124(表5)或161-170(表7)和序列号195-208(表9)或246-255(表11)的核苷酸序列互补。在一些实施例中，Cbl-b/Cbl下调配伍包括Crispr/cas9,crispr/cpf1载体，特异针对主体中的Cbl-b/Cbl。Cbl-b/Cbl特异的Crispr/cas9,crispr/cpf1载体包括先导多核苷酸，选自序列号125-158(表6)或171-192(表8)和序列号209-243(表10)或256-280(表12)。此外，类转录活化因子核酸酶(Talen)和锌指核酸酶(ZFN)技术也能用于下调Cbl-b/Cbl表达。
表5.用于下调人Cbl-b表达的siRNA/shRNAi序列
SEQ ID NO# Nuclei acid sequence
SEQ ID NO 112 GCCTGATACATATCAGCAT
SEQ ID NO 113 GCGGAATTGGAATTTCTTA
SEQ ID NO 114 GCATGCCGATGCTAGACTT
SEQ ID NO 115 GCCTGATACATATCAGCAT
SEQ ID NO 116 GGAGAGAATGTATGAAGAACA
SEQ ID NO 117 GCGGAATTGGAATTTCTTAGC
SEQ ID NO 118 GCACGACTACAGAAATATAGC
SEQ ID NO 119 GGAATATCTTACAGACCATAC
SEQ ID NO 120 GCACCAAACCCGGAAGCTATA
SEQ ID NO 121 GCCTGGATCTAATTCAGAAAG
SEQ ID NO 122 GGAATCACAGCGAGTTCAAAT
SEQ ID NO 123 GGAACACATGGTCCATCTTCA
SEQ ID NO 124 GCATAGTCTCATTGAACATTC
表6.用于下调人Cbl-b表达的CRISPR/CAS9,CRISPR/CPF1靶标序列
Figure GDA0001389300800000201
Figure GDA0001389300800000211
表7.用于下调狗Cbl-b表达的siRNA/shRNAi序列
SEQ ID NO# Nuclei acid sequence
SEQ ID NO 161 CCCACCATATATACTTGAT
SEQ ID NO 162 CCTGATACATATCAGCATT
SEQ ID NO 163 GCGGGCAATAAGACTCTTT
SEQ ID NO 164 GCAGAAATACAGCACCAAA
SEQ ID NO 165 GCACCAAACCTGGAAGCTA
SEQ ID NO 166 GCAATATCTTACAGACCAT
SEQ ID NO 167 CCACACCACATGACCATAT
SEQ ID NO 168 GCCTCCTCCCTTAAGAGAT
SEQ ID NO 169 CCTTCATCCCATCCTGTTT
SEQ ID NO 170 CCTCTGATCCAGTGCCATT
表8.用于下调狗Cbl-b表达的CRISPR/CAS9,CRISPR/CPF1靶标序列
Figure GDA0001389300800000212
Figure GDA0001389300800000221
表9.用于下调人Cbl表达的siRNA/shRNAi序列
SEQ ID NO# Nuclei acid sequence
SEQ ID NO 195 CCAGACAATCCCTCACAAT
SEQ ID NO 196 GGACACCTCATGTGCACAT
SEQ ID NO 197 CCAGGCCTCTACGGCCTTT
SEQ ID NO 198 CCAGAAAGCTTTGGTCATT
SEQ ID NO 199 GCCTGATTGGGCTCATGAAGG
SEQ ID NO 200 GGGAACATTCTCCAGACAATC
SEQ ID NO 201 GCTTCAGGGAAGGCTTCTATT
SEQ ID NO 202 GGGAAGGCTTCTATTTGTTTC
SEQ ID NO 203 GGACACCTCATGTGCACATCC
SEQ ID NO 204 GCAGAATCCCGACCTCAAAGA
SEQ ID NO 205 GGAGCAATGTGAGGGTGAAGA
SEQ ID NO 206 GCCTCTACGGCCTTTGGATAC
SEQ ID NO 207 GCTGTACGTATGAAGCAATGT
SEQ ID NO 208 GGTACTCCTACCAGGACATCC
表10.用于下调人Cbl表达的CRISPR/CAS9,CRISPR/CPF1靶标序列
Figure GDA0001389300800000222
Figure GDA0001389300800000231
表11.用于下调狗Cbl表达的siRNA/shRNAi序列
SEQ ID NO# Nuclei acid sequence
SEQ ID NO 246 CCAGAAGTTCATTCACAAA
SEQ ID NO 247 GGAACATCCTCCAGACGAT
SEQ ID NO 248 CCAGACGATCCCTCACAAT
SEQ ID NO 249 GCTTCAGGGAAGGCTTCTA
SEQ ID NO 250 GCAGGAATCAGAAGGCCAA
SEQ ID NO 251 CCTTTCTGCCGATGTGAAA
SEQ ID NO 252 GCTGATGATTCTCTCTTTA
SEQ ID NO 253 GCTTCTGGCTCCCTTCATA
SEQ ID NO 254 GCATCTGCCAATGCCATTT
SEQ ID NO 255 GCTGCACATATGAAGCAAT
表12.用于下调狗Cbl表达的CRISPR/CAS9,CRISPR/CPF1靶标序列
SEQ ID NO# Nuclei acid sequence
SEQ ID NO 256 CCCGGAGCCGCCGCCGCCCCCGG
SEQ ID NO 257 TGCCGGGCGGGTGGGGGCTGAGG
SEQ ID NO 258 CGGCCTCATCGGGCTCATGAAGG
SEQ ID NO 259 GGAGCTCTTCTTCACGTTGCCGG
SEQ ID NO 260 CAACGTGAAGAAGAGCTCCGGGG
SEQ ID NO 261 GGGGCTCGGGCGGCCTCATCGGG
SEQ ID NO 262 GGCAACGTGAAGAAGAGCTCCGG
SEQ ID NO 263 GCAACGTGAAGAAGAGCTCCGGG
SEQ ID NO 264 GGGGGCTCGGGCGGCCTCATCGG
SEQ ID NO 265 GTGAAGAAGAGCTCCGGGGCCGG
SEQ ID NO 266 TGAAGAAGAGCTCCGGGGCCGGG
SEQ ID NO 267 CGTCCTTCATGAGCCCGATGAGG
SEQ ID NO 268 AAGAAGAGCTCCGGGGCCGGGGG
SEQ ID NO 269 GAAGAAGAGCTCCGGGGCCGGGG
SEQ ID NO 270 GATGAGGCCGCCCGAGCCCCCGG
SEQ ID NO 271 GTGGTGGTGGTGCGGCTGGAAGG
SEQ ID NO 272 AAGAGCTCCGGGGCCGGGGGCGG
SEQ ID NO 273 CACCTCAGCCCCCACCCGCCCGG
SEQ ID NO 274 CGGCGGCGGCTCCGGGGGCTCGG
SEQ ID NO 275 AGCTCCGGGGCCGGGGGCGGCGG
SEQ ID NO 276 GCGGGTGGGGGCTGAGGTGGTGG
SEQ ID NO 277 TCCGGGGCCGGGGGCGGCGGCGG
SEQ ID NO 278 GCCGCCGCCGCCCCCGGCCCCGG
SEQ ID NO 279 CGGGCGGGTGGGGGCTGAGGTGG
SEQ ID NO 280 GCCGGGGGCGGCGGCGGCTCCGG
在一些实施例中，本发明提供Cbl-b/Cbl下调配伍，用于下调主体中Cbl-b/Cbl表达。主体是人或狗。在一些实施例中，优选下调主体肝组织的肝细胞中的Cbl-b/Cbl表达。该下调Cbl-b/Cbl表达的方法包括：给主体注射Cbl-b/Cbl下调配伍，从而下调主体肝组织的Cbl-b/Cbl表达。在一些实施例中，Cbl-b/Cbl下调配伍包括siRNA/shRNAi多核苷酸，特异针对Cbl-b/Cbl(序列号110或159或序列号193或244)，编码由序列号111或169或序列号194或245分别代表的氨基酸序列。在一些实施例中，该Cbl-b/Cbl特异的siRNA/shRNAi多核苷酸与选自序列号112-124(表5)或161-170(表7)和序列号195-208(表9)或246-255(表11)的核苷酸序列互补。在一些实施例中，Cbl-b/Cbl下调配伍包括Crispr/cas9,crispr/cpf1载体，特异针对主体中的Cbl-b/Cbl。Cbl-b/Cbl特异的Crispr/cas9,crispr/cpf1载体包括先导多核苷酸，选自序列号125-158(表6)或171-192(表8)和序列号209-243(表10)或256-280(表12)。
在一些实施例中，本发明提供了主体HCV感染的治疗方法。在一些实施例中，主体是人。在一些实施例中，治疗是通过施用配伍特异性下调主体肝组织的肝细胞中CD2AP的表达，其配伍包含至少一个与选自由序列号3-20或59-76所代表序列互补的siRNA/shRNAi核苷酸序列。在一些实施例中，治疗是通过施用Crispr/cas9,crispr/cpf1载体特异性下调主体肝组织的肝细胞中CD2AP表达，其Crispr/cas9,crispr/cpf1载体包括引导序列，选自由序列号21-56或77-103。在一些实施例中，治疗是通过施用含有能降低CD2AP和NS5A之间相互作用的药物而特异性下降低在主体肝组织的肝细胞中的CD2AP和NS5A之间的相互作用。
在一些实施例中，本发明提供了主体糖尿病的治疗方法。在一些实施例中，主体是人和狗。在一些实施例中，治疗是通过施用配伍特异性下调主体肝组织的肝细胞中CD2AP的表达，其配伍包含至少一个与选自由序列号3-20或59-76所代表序列互补的siRNA/shRNAi核苷酸序列。在一些实施例中，治疗是通过施用Crispr/cas9,crispr/cpf1载体特异性下调主体肝组织的肝细胞中CD2AP表达，其Crispr/cas9,crispr/cpf1载体包括引导序列，其引导序列选自序列号21-56或77-103。在一些实施例中，治疗是通过施用含有此前所描述的能降低CD2AP和IRS1之间相互作用的药物而特异性下降低在主体肝组织的肝细胞中的CD2AP和IRS1之间的相互作用。
在一些实施例中，本发明提供了主体糖尿病的治疗方法。在一些实施例中，主体是人和狗。在一些实施例中，治疗是通过施用配伍特异性下调主体肝组织的肝细胞中Cbl-b/Cbl的表达，其配伍包含至少一个与选自由序列号112-124或161-170和序列号195-208或246-255所代表序列互补的siRNA/shRNAi核苷酸序列。在一些实施例中，治疗是通过施用Crispr/cas9,crispr/cpf1载体特异性下调主体肝组织的肝细胞中Cbl-b/Cbl表达，其Crispr/cas9,crispr/cpf1载体包括引导序列，其引导序列选自序列号125-158或171-192和序列号209-243或256-280。在一些实施例中，治疗是通过施用含有此前所描述的能降低Cbl-b/Cbl和IRS1之间相互作用的药物而特异性下降低在主体肝组织的肝细胞中的Cbl-b/Cbl和IRS1之间的相互作用。
在一些实施例中，本发明提供肝脏异化诊断方法。所述诊断方法包括提供来自个体的肝脏样品，让肝脏样品与检测CD2AP表达的检测试剂接触；因而，当在肝脏样品中检测到CD2AP表达时，提示肝脏异化。所述异化包括HCV感染和糖尿病。检测CD2AP表达的方法可以是任一合适方法，包括PCR和免疫染色。
在一些实施例中，本发明提供个体肝脏异化诊断试剂盒。所述试剂盒包括针对CD2AP蛋白的特异抗体或针对CD2AP mRNA的特异多聚核苷酸探针，和一个次级试剂，能够检测与CD2AP结合的抗体或来自CD2AP mRNA的信号。
提供下面实施例的唯一目的是说明本发明的原理；它们决不旨在限制或缩小本发明的范围。
实施例
1.材料与方法
1.1细胞系和病毒
人肝癌细胞Huh7，其衍生的Huh7-Lunet和Huh7.5.1细胞，和HEK293T细胞，在含有5％CO2的潮湿环境下，培养于Dulbecco改良的Eagle培养基(DMEM)(Gibco，#11965-092，美国)，补充3.17g/l碳酸氢钠、10％胎牛血清(Gibco，#10099-141)，3g/L HEPEs，100U/ml的青霉素和链霉素。携带HCV 1b亚基因组HCV复制子pFKI389neo/NS3-3’的CON1细胞，衍生自Huh7-Lunet，保持在与Huh7-Lunet细胞相同的培养基，添加0.5mg/ml G418(默克，345810)(42)。感染性HCV JFH1病毒含有HCV基因型2a株基因组全长cDNA序列(43)。HCV J399EM病毒衍生自JFH-1病毒，在NS5A的399个氨基酸后面***EGFP基因，引入五个适应突变到JFH1基因组，增强病毒生产能力(44)。JFH1Luc报告病毒由在武汉病毒研究所陈绪林教授提供(45)。为了产生病毒备份，原始HCV病毒用DMEM稀释，接种到Huh7.5.1细胞，感染复数(MOI)为0.1。被感染的细胞在72HPI传代一次。然后，在感染后7或8天收集上清液，分装并存储在80℃.
1.2质粒构建与试剂
人源CD2AP(GenBank#NM_012120)(序列号1；序列号2为氨基酸序列)和来自HCV基因型2a(AB047639JFH1)的NS5A(序列号1；序列号2为氨基酸序列)分别用相应的引物克隆到真核表达载体质粒pRK-7HA和pRK-7Flag(Addgene)。取自感染了HCV JFH1的Huh7.5.1细胞和未感染的Huh7.5.1细胞的总RNA作为模板。用全长NS5A和CD2AP作为模板，通过聚合酶链反应(PCR)扩增截短NS5A和CD2AP。哺乳动物细胞表达质粒pcDNA3.1BirA(R118G)-HA(BirA*)购自Addgene。HCV NS5A亚克隆到BirA*的N端。整个NS5A-BirA*-HA序列被用限制性内切酶SalⅠ和NotⅠ从pcDNA3.1中切除并***到pHAGE。抗Flag,HA,或β-actin小鼠单克隆抗体(mAb)购自天津三江生物(天津，中国)；抗HCV核心蛋白小鼠多克隆抗体和抗CD2AP兔多克隆抗体(H-290)购自圣克鲁斯生物技术购买；抗HCV 2a NS5A单克隆抗体(7B5和2F6)购自BioFront；抗NS5A单克隆抗体9E10是由Charles Rice教授提供(洛克菲勒大学)(46)。抗Phospho(p)-Akt(Ser473)(4060),Akt(4691),p-Erk(9106S),Erk(4695P)and PI3K-Aktinhibitor LY294002(9901)的兔单克隆抗体购自细胞信号技术(马萨诸塞州、美国)；抗ADRP(ab52355)的兔多克隆抗体抗ADRP(ab52355)购自Abcam；兔抗calnexin(RLT0613)多克隆抗体购自Ruiyingbio(苏州，中国)；HCS LipidTox深红色中性脂质染色和Alexa Fluor标记的二抗购自Invitrogen公司(Carlsbad，美国)；辣根过氧化酶(HRP)标记的二抗购自antgene生物科技(武汉，中国)；小鼠IgG1同型对照和HRP-链霉亲和素蛋白购自BioLegend(圣地亚哥，美国)；4’,6-Diamidine-2’-phenylindole dihydro-chloride(DAPI)购自罗氏(曼海姆，德国)。所有其他试剂均购自AMRESCO(俄亥俄，美国)。
1.3细胞裂解物制备和免疫印迹(WB)
用冰冷的磷酸盐缓冲液(PBS)轻轻地冲洗细胞，然后溶于裂解液(20mM Tris-HCl(pH 7.5)，150mM NaCl,1mM EDTA,1mM EGTA、1％Triton X-100，2.5mM焦磷酸钠，1mMβ-磷酸甘油，1mM Na3VO4，1mM PMSF)(Li等，2009)。BCA法测定蛋白浓度。用10％SDS-PAGE电泳分开蛋白，然后转移到硝酸纤维素膜(#9004700、Billerica,美国)。用含有5％脱脂牛奶的TBST(Tris缓冲生理盐水(TBS)加上0.1％Tween-20))封闭，分开的蛋白用特定的初级抗体，辣根过氧化物酶标记的第二抗体来探测。
1.4免疫共沉淀(Co-IP)
对于外源表达质粒的co-IP，首先使用磷酸钙法将质粒转染到10cm培养皿中的HEK293T细胞中。36小时后，用1mL裂解液将细胞裂解。将等体积的裂解液与2μg特异性的抗体或同种型对照抗体和20μl Protein G琼脂糖珠(16-266，Merck Millipore)进行孵育，4℃旋转4小时。用细胞裂解液将珠子洗涤6次后，加20μl 2×SDS上样缓冲液中煮沸，用10％SDS-PAGE胶分离蛋白质。对于内源性的co-IP实验，将HCV JFH1感染72小时或者未感染的Huh7.5.1细胞直接裂解。利用特异性的抗体进行上面所述的co-IP。
1.5免疫荧光染色
在20mm玻璃共聚焦皿(NEST)上培养细胞。感染后72小时，然后将细胞固定在4％(w/v)多聚甲醛(PFA)，室温(RT)15分钟。在用含有10％山羊血清加1％的BSA的PBST封闭细胞后，细胞在含有指明的初级抗体的封闭缓冲液中孵育。结合的抗体用Alexa Fluor标记的二抗来检测。细胞核用DAPI复染。脂滴的染色用HCS LipidTOX深红中性脂肪染色剂。添加抗猝灭荧光介质后，用共聚焦显微镜拍摄照片(Perkin Elmer UltraView Vox共聚焦显微镜)。
1.6 RNA提取和实时定量RT-PCR(qPCR)
培养的细胞和培养的上清液中的总RNA的提取，按照制造商的说明分别采用RNA纯组织试剂盒和RNA纯病毒试剂盒(CWBiotech，北京)。使用PrimeScript RT试剂盒(TakaraBio，日本)从1微克总RNA合成cDNA第一条链。在Bio-Rad Connect TM QPCR仪器(CFX连接TM光学模块)上，使用SYBR Green Supermix(Bio-Rad，美国)定量RNA。胞内HCV RNA和细胞RNA的量相对于GAPDH RNA水平进行归化。由系列稀释的HCV JFH-1cDNA质粒制备标准曲线，参照标准曲线测定培养上清液中的HCV RNA水平。
1.7逆转录病毒的制备与转导
为了建立一个稳定的表达下调的细胞系，根据制造商的说明，将干扰靶标基因的短发夹RNA(shRNA)亚克隆到pSuper retro puro质粒(Oligoengine)。水疱性口炎病毒糖蛋白(VSV-G)-假型逆转录病毒颗粒，在293T细胞中采用磷酸钙法制备。简而言之，由pSuperretro puro构件和包装质粒pGag-pol和pVSV-G共转染HEK293T细胞。shRNA逆转录病毒的备份与7.5微克/毫升聚凝胺转导Huh7.5.1细胞。表达下调细胞用2毫克/毫升嘌呤霉素(Amersco)选择至少7天。存活克隆的干扰效果是由qPCR或免疫印迹(WB)分析证实。针对CD2AP mRNA的siRNA/shRNA序列列于表1。
1.8在CD2AP下调细胞中功能拯救CD2AP
为了功能性的拯救CD2AP干扰，CD2AP下调细胞Huh7.5.1(shCD2AP-6#)瞬时转染慢病毒载体pHAGE，表达外源摆动突变HA-CD2AP基因(sh-CD2AP 6#-HA-CD2AP)，其中靶标CD2AP序列GGAAACAGATGATGTGAAA(序列号1的2175-2193)变成GGAGACGGACGACGTAAAG(序列号281)。慢病毒制备参考杨等人的文章(48)。含有空载体的慢病毒颗粒转染shCD2AP-6#细胞作为对照。
1.9亲和捕获生物素化蛋白质
生物素化蛋白质在4℃进行分离，使用加以修改的以前程序(49)。简而言之，Huh7细胞稳定表达NS5A BirA*，在添加了50μM生物素的完全培养基中孵育24小时。五个10cm细胞培养板上长满细胞，用上述细胞裂解液裂解。生物素化蛋白与100μl链霉亲和素琼脂糖珠4℃震荡过夜后析出。然后深度洗净链霉亲和素琼脂糖珠(49)。NS5A的相互作用蛋白，经由质谱分析和免疫印迹法证实。
1.10 HCVpp进入与HCV IRES依赖性翻译实验
HCVpp的制备参照文献中的方法(50)。将质粒pNL4.3.lucRE和pcDNA3.1-E1E2按照3:1比例共转HEK 293T细胞。48小时后，收集培养上清，1000g离心后通过0.45μm过滤器(Merck Millipore)过滤，长期使用可分装储存在-80℃。对于HCVpp进入效率测定，将CD2AP沉默和对照Huh7.5.1细胞铺在24孔板中，细胞密度约30％，过夜培养后加入250μl HCVpp，6h后换成新鲜培养基。48h后，用试剂盒(E1500，Promega)检测萤火虫荧光素酶活性。对于HCV IRES介导的翻译实验：用Lipofectamine 2000将pHCV-IRES质粒转染到CD2AP沉默和对照Huh7.5.1细胞中。48小时后，用双荧光素酶试剂盒(E1910，Promega)检测F-Luc和R-Luc活性，二者的比值可以代表翻译效率(F-Luc/R-Luc)。
1.11分离脂滴
脂滴富集部分由密度梯度离心制备(51)。简而言之，约95％融合度的细胞，在PBS中收刮，通过离心分离沉淀，1000Xg，5分钟，然后溶解于1ml低渗缓冲液(50mM HEPES,1mMEDTA和2mM MgCl2at pH 7.4,1mM PMSF和混合蛋白酶抑制剂)。孵育悬浮混合物，4℃，20分钟，超声处理20下。细胞核通过离心去除，1000Xg，4℃，5分钟。收集1ml上清液，混合等体积的1.5M蔗糖等渗缓冲液(50mM HEPES,100mM KCl,2mM MgCl2)，放置在SW55Ti(Beckman)离心管的底部，然后在混合物上放置3ml含有1mM PMSF等渗缓冲液。离心样品，10000g，4℃，2小时。收集顶层LD组份，用10％的三氯乙酸沉淀(TCA)，用***：乙醇(1:1)洗一次，在2×SDS上样缓冲液中煮沸，SDS-PAGE分离。
1.12 OA刺激
为了确定OA刺激引起的脂滴脂滴积累，1.5×105cells接种到共聚焦皿，完全培养基中培养16小时。然后培养细胞12小时，其无血清DMEM培养液含0.5mM OA和2％BSA(w/v)；然后LD染色。
1.13丙型肝炎病毒滴定
用1个MOI的J399EM病毒感染目的细胞。72h后，收集培养基中的胞外病毒。胞内病毒的收集过程如下：用PBS清洗细胞以除去残留的细胞外病毒，将细胞在PBS中刮下来，1000g离心5min；将细胞沉淀重新悬浮于基础DMEM中，并在液氮中进行三轮反复冻融以释放细胞内的病毒颗粒；1000g离心5min除去细胞碎片得到细胞内的病毒。用Lindenbach等描述的终点稀释法测定病毒的滴度(52)。将胞外病毒和胞内病毒用DMEM培养基进行10倍系列稀释。用稀释的病毒样本感染96孔板中的Huh7.5.1细胞(每个稀释度6个复孔)。感染96h后，在荧光显微镜下观察每个稀释度中EGFP阳性的孔数，根据Reed and Muench公式计算病毒滴度(52)。
1.14细胞活力检测
通过MTS(G3581，Promega)测定CD2AP沉默细胞的活力。将待测细胞铺在96孔板中，每孔5×103个细胞。37℃培养箱中分别培养24h，48h，72h和96h。在相应的时间点每孔加入20μl MTS试剂，37℃孵育1小时。读板仪(Perkin Elmer，USA)测量490nm处的吸光值。
1.15统计分析
所有实验至少重复3次，使用GraphPad Prism软件作图。实验结果用双尾的学生t检验统计分析。定量数据误差显示的是平均值±标准误(Mean±Standard Error，SEM)。p<0.05视为有显著性差异，*(P<0.05)；p<0.01视为有极显著性差异，**(P<0.01)；ns表示统计学上无显著性差异。
IRS1泛素化实验
将CD2AP沉默和对照细胞在完全培养基中培养48小时，随后进行裂解。将细胞裂解液(1mL)分别与2μg兔源的IRS1抗体以及30μl Protein G琼脂糖珠在4℃孵育4小时。经过漂洗后，将珠子在30μl 2×SDS上样缓冲液中煮沸，得到蛋白样品。分别取分装的10μl样品进行跑胶，检测从CD2AP沉默和对照细胞中纯化的IRS1的量。将二者纯化的IRS1的总蛋白量调节一致，从而定量IRS1的泛素化水平。
胰岛素信号通路实验
胰岛素信号通路的分子用相应的的抗体来检测，检测细胞裂解物中这些分子，而这些裂解物来自对照和CD2AP下调细胞Huh7.5.1。
RNA干扰
Huh7.5.1细胞接种为50％融合度，然后用Cbl-b，Cbl特异的小干扰RNAs(siRNAs)或阴性对照siRNA转染细胞。siRNA的转染使用PepMute试剂，遵照制造商的说明(SignaGen，美国)。基因沉默在转染后48小时测量。Cbl-b，Cbl特异的小干扰RNAs系列列于表3和5。对IRS1的影响用IRS1,cbl-b,cbl和肌动蛋白的特异抗体来检测。
免疫组化(IHC)
为了对HCV感染小鼠的CD2AP染色，在指定的时间，收集HCV感染或模拟感染的小鼠的肝组织右页，进行切片，厚度为5微米。为了对来自于感染或未感染HCV的病人的肝活检标本的CD2AP染色，组织切片，厚度5微米。组织切片加热，65℃，1小时。经过除蜡，脱水和3％过氧化氢处理10分钟后，进行抗原回收。切片浸入10mM柠檬酸钠缓冲液(pH6.0)，95-100℃加热，30分钟；然后在缓冲液中冷却至室温。然后封闭切片，使用正常羊血清，稀释在0.02％PBST，室温，1小时。切片孵育于兔抗CD2AP抗体(GeneTex，美国)或同型对照兔IgG，室温，1小时。HRP耦合的羊抗兔二抗用于检测结合的初级抗体，室温，1小时。遵照产品说明，用DAB配伍显色(Maxim，中国)。切片用hematoxylin负染2分钟。经脱水和加盖玻片后，切片照相，使用Pannormic数字切片扫描仪(3DHISTECH，匈牙利)。肝切片的使用经过中国科学院武汉病毒研究所审查委员会批准。批准号：WIVH28201601。
2.结果
2.1用BioID方法在Huh7细胞中发现全新NS5A相关的宿主蛋白
Huh7细胞转染BioID构件NS5A-BirA*-HA，在50μM外源生物素存在下培养，标记与NS5A密切相关的蛋白(图1)。然后用链霉亲和素-HRP检测被外源生物素标记的细胞蛋白。与没有生物素的细胞相比，在生物素存在下，观察到生物素标记蛋白质的增加(图2)。对于图2，NS5A-BirA-HA构件转染Huh7细胞。在免疫检测NS5A或HA标签后，确认构件的表达。然后将细胞***成两份，一份经50μM生物素处理，另一份没有处理。然后细胞裂解物进行SDS-PAGE。用链霉亲和素-HRP进行免疫印迹，检测生物素化的细胞蛋白。我们发现的是，与没有生物素处理的细胞相比，生物素处理后，更多的蛋白质被生物素标记。
为确定生物素标记的宿主蛋白，链霉亲和素纯化的蛋白质进行分离与考马斯亮蓝亮蓝染色(图3)。对七个特定的条带进行质谱分析，并揭示了这些细胞蛋白的身份。有趣的是，这些蛋白质要么与运输胞器，如COPG2,CD2AP,GOLGA5和PACE1，或者与RNA生物学，如RPA34,EF2P和NP1L1，相关。我们首先集中在研究CD2AP，其是一个接头蛋白，含有SH3域，最早被发现与CD2胞内域结合(53)。CD2AP还结合肌动蛋白帽蛋白(CP)，具有高亲和力，降低肌动蛋白聚合的速率(54,55)，从而在肌动蛋白纤维组织中起到了重要的作用。
2.2 HCV NS5A蛋白与CD2AP相互作用
HCV非结构蛋白NS5A具有多个富含脯氨酸的序列，特异性地结合生长因子受体结合蛋白2(Grb2)接头蛋白，其含有SH3结构域(56,57)。由于CD2AP有三个SH3结构域(35)，我们直接测试CD2AP是否确实结合NS5A。当HA标记的CD2AP与FLAG标签的NS5A在HEK 293T细胞内过度表达，我们发现，由NS5A可以特异性地沉淀CD2AP(图4)。为确定在HCV感染期间NS5A是否结合CD2AP，我们对HCV JFH1感染的Huh7.5.1细胞或没有感染的Huh7.5.1细胞进行了免疫共沉淀(Co-IP)分析。我们发现，在受感染的细胞中，抗CD2AP抗体确实与NS5A免疫共沉淀。同时，CD2AP也可以被NS5A特异性抗体沉淀(图5)。为了进一步证明在受感染Huh7.5.1细胞中CD2AP与NS5A相互作用，我们采用鼠IgG亚型抗体作为NS5A抗体的对照，进行免疫共沉淀实验。我们发现，在HCV感染的细胞中，CD2AP确实结合NS5A(图6)。此外，通过共聚焦成像分析，我们观察到,在受HCV-J399EM感染的细胞中，CD2AP和NS5A共定位，HCV-J399EM是HCV2a株，其中NS5A带有GFP标记(图7)。另外，我们在受JFH1感染的Huh7.5.1细胞中，双染CD2AP和NS5A，发现CD2AP和NS5A共定位(结果未显示)。综上所述，这些结果表明，在HCV感染细胞中CD2AP与内源性NS5A相互作用。
2.3 NS5A的结构域III与CD2AP SH3结构域相互作用
由于CD2AP包含三个SH3结构域，我们进一步实验确定,在CD2AP中是一个特定的SH3结构域还是所有三个SH3结构域负责结合NS5A。我们制备了各种CD2AP的截短突变体，编码1-107aa,1-268aa,1-330aa,331-639aa,60-639aa,and 1-639aa(按照序列号2)，它分别包含第一，第二和第三，所有三个SH3结构域，没有SH3结构域但保留CD2AP的所有其他域，没有第一个SH3结构域但保留CD2AP的所有其他域，和全长CD2AP(图8)。我们然后在HEK 293T细胞中与HA标记的全长NS5A一起共表达这些CD2AP蛋白，进行免疫共沉淀实验。如图9所示，缺失SH3结构域的CD2AP突变体不与NS5A相互作用(图9，参见331-639)。相反地，全长CD2AP或包含SH3结构域的CD2AP蛋白结合NS5A；当CD2A的突变体保留更多的SH3结构域时，结合被增强(图9，比较1-107,1-268,和1-330)。第二和第三的SH3结构域参与NS5A结合，由下面的观察进一步证实，没有第一个SH3结构域的CD2AP仍然结合NS5A(图9，参见60-639)。
我们也鉴定了NS5A中参与和CD2AP结合的区域。NS5A包含N-末端的两亲性螺旋，锚定蛋白到胞质膜，和三个结构域(结构域I，结构域II和结构域III)，被两个低复杂度序列(LCS)分开(58,59)。我们制备全长NS5A和一系列分别缺乏结构域I，II，III的NS5A突变体(图10)，研究哪些结构域结合CD2AP。我们发现，删除NS5A的第三个结构域，CD2AP无法结合NS5A(图11)。然而，删除其他NS5A结构域，不影响NS5A结合CD2AP，从而提示，NS5A的第三结构域和CD2AP的SH3结构域相互作用。
2.4在靶向脂滴前CD2AP通过肌动蛋白依赖的方式运送NS5A
为了探讨CD2AP与NS5A相互作用的功能，Huh7.5.1细胞稳定表达mcherry标记的全长CD2AP或缺乏所有三个SH3结构域的CD2AP突变体，然后感染HCV-J399EM。通过实时图像跟踪，我们发现，只有全长CD2AP与GFP-NS5A共定位和与NS5A共移动，而没有SH3结构域的CD2AP突变体不与NS5A共定位(图12，左面板，框内的点)。对CD2AP和NS5A实况图像的量化，进一步证实全长CD2AP与NS5A共移动(图13)。观察到NS5A与缺失所有三个SH3结构域的CD2AP之间没有共定位，进一步支持了我们的结论，NS5A和CD2AP的SH3结构域相互作用。由于CD2AP点的移动依赖于肌动纤维聚合(60)，我们通过用秋水仙碱(一种微管蛋白聚合抑制剂)或细胞松弛素B(肌动蛋白聚合抑制剂)处理受感染细胞，来调查CD2AP与NS5A的共移动是否依赖于肌动纤维聚合。我们发现，细胞松弛素B而不是秋水仙素处理显著降低NS5A和CD2AP的共定位(图14，左面板)。然而，在培养基中用DMSO取代细胞松弛素B后4小时，恢复CD2AP和NS5A的共定位(图14，右上面板)。这些结果表明，CD2AP和NS5A的共定位依赖于肌动蛋白细胞骨架。NS5A必须通过微管***运往脂滴来组装(12)，我们发现，细胞用秋水仙素处理后，CD2AP与NS5A复合物没有移动(图15)。由于秋水仙碱处理不影响CD2AP和NS5A的共定位，但是细胞松弛素B处理阻止CD2AP和NS5A共定位，我们假设，依赖于肌动蛋白的CD2AP和NS5A共定位，是发生在HCV组装之前。如果这个假设是正确的，我们预见更少的NS5A与脂滴结合。为了检验这一假设，我们采用CON1复制***，其中脂滴在数量上大为减少。然后我们下调CD2AP表达，用生化手段检测NS5A与脂滴的结合是否会减轻。通过在CON1复制***中下调CD2AP(记为c4#和c6#)，我们发现在CON1中NS5A水平没有受到影响，然而，NS5A与脂滴组份的结合明显减少(图16)。其中calnexin和ADRP，分别为ER和LD标记物(图16)。由于总的NS5A的表达水平不受影响，但当CD2AP表达下调时脂滴结合NS5A的水平减少，我们得出的结论是，CD2AP通过肌动蛋白细胞骨架运输NS5A到脂滴。这些结果也表明，CD2AP的下调不影响HCV基因组复制而降低HCV装配。
2.5 CD2AP影响LD的生物合成
由于CD2AP可能在HCV的装配和释放发挥作用，同时我们已经表明CD2AP表达下调减少NS5A转运到脂滴，我们随后研究，在转运NS5A到脂滴之外，CD2AP是否在HCV组装中发挥其他作用。我们首先测试了下调CD2AP对脂滴生物合成的影响。下调CD2AP显著降低了脂滴的生物合成和积累(图17，BSA下左柱，NC和6#)。由于在非感染的条件下脂滴的生物合成是非常有限的，我们进一步探讨，在OA处理下，下调CD2AP对脂滴生物合成的影响。我们发现，CD2AP下调明显减轻脂滴的生物合成(图17，在OA右列)。在OA处理下，对超过200细胞中的脂滴计数，证实当CD2AP下调时，每个细胞有显著少的脂滴(图17，黑盒，比较NC和6#，P＜0.05)。为了证明CD2AP确实影响脂滴的生物合成，我们染色经过OA或BSA处理的CD2AP拯救细胞。我们发现，与对照细胞相比，CD2AP过表达细胞显示显著多的脂滴(图18)。在OA处理下，对超过200个细胞中的脂滴计数，证实当CD2AP上调时，每个细胞有显著上调的脂滴(图18，黑盒，比较NC和HA-CD2AP，P＜0.05)。
为了排除由于核心蛋白表达水平降低而导致减少脂滴的可能性，我们随后在CD2AP下调和对照细胞中过表达核心蛋白。我们发现，与对照细胞相比，在CD2AP下调细胞中上调核心蛋白不会显著增加脂滴的形成(图19，右边两个面板和黑框，显示在核心蛋白过表达后，脂滴明显减少，在P＜0.05)，从而进一步证明，当CD2AP表达下调，脂滴的生物合成与HCV核心蛋白定位于脂滴上受到阻碍。然而，在CD2AP下调细胞中增强CD2AP表达后，脂滴的水平显著提高，同样地核心蛋白在脂滴上的定位也显著增加(图20，右边两个面板和黑框，显示过表达CD2AP后位于脂滴的核心蛋白显著增加)。这些结果表明，CD2AP在细胞脂滴的生成和导向HCV组件到脂滴中起重要作用。
2.6下调CD2AP抑制HCV的繁殖
由于HCV基因组复制不受CD2AP和NS5A相互作用的影响，我们试图通过沉默CD2AP来检测这种相互作用对HCV繁殖的影响。我们制备了两个稳定CD2AP下调细胞系(Huh7.5.1-sh CD2AP-4，记为4#；Huh7.5.1-sh CD2AP-6，记为6#)；Huh7.5.1-sh CD2AP阴性对照，记为NC。下调CD2AP并不影响细胞的生长。然而，细胞感染HCV-JFH1 72小时后，与对照细胞相比，下调CD2AP显著降低HCV mRNA水平(图21)。免疫印迹分析证实，HCV的NS5A和核心蛋白表达显著减少(图22)。另外，释放到CD2AP下调细胞培养上清的病毒RNA拷贝数也明显减少(图23，P＜0.01)。通过使用一个带有renila荧光素酶报告基因的报告病毒J399EM+LM，进一步证实了CD2AP下调在HCV复制的影响(图24)。为了排除此影响是由于CD2AP下调的脱靶效应的可能性，我们进行了一项拯救实验。我们瞬时表达HA-CD2AP突变体，在CD2AP下调细胞shCD2AP-6#的靶点含有的摆动突变(记为6#-HA-CD2AP)。HCV JFH1感染后，HA-CD2AP突变体的表达(6#-HA-CD2AP+)，但不是空载体(6#-HA-CD2AP-)，拯救了细胞内HCV RNA水平(图25)。与RNA水平一致，相比转染空载体的细胞，在6#-HA-CD2AP中核心蛋白和NS5A蛋白水平也显示部分恢复(2道与3道，图26)。总之，这些结果表明，在Huh7.5.1细胞中，下调内源性CD2AP显著抑制HCV的繁殖。
2.7下调CD2AP不妨碍HCV侵入，基因组RNA复制和IRES依赖性翻译，但抑制HCV感染性颗粒的产生
由于CD2AP不影响HCV亚基因组复制但在HCV繁殖中起重要作用，我们进一步研究，CD2AP影响HCV感染的的根本机制。我们首先通过转导HCV假颗粒，探讨CD2AP是否影响HCV侵入。用HCVpps转导CD2AP稳定下调细胞。转导48小时后后，测定荧光素酶活性，作为HCV侵入效率的指标。如图27所示，CD2AP下调和对照细胞之间，HCVpp感染无显著差异，提示HCV侵入不受CD2AP下调的影响。我们随后探讨，下调CD2AP是否会影响HCV内部核糖体进入位点(IRES)导向的翻译。用双顺反子报告质粒pHCV-IRES来监测HCV IRES活性。在质粒pHCV-IRES中，上游Renilla荧光素酶基因(Rluc)的翻译是由5’帽子结构介导的，下游火萤荧光素酶基因(Fluc)是由HCV IRES元件控制的。HCV IRES依赖性翻译水平是针对Rluc活性利用Fluc活性进行归一化计算。与对照组相比，沉默CD2AP不显著影响HCV IRES依赖性翻译(图28，空盒子代表CD2AP相对翻译水平，而黑框测量归一化HCV IRES活性)。
我们进一步评估在亚基因组复制子Con1细胞中，CD2AP下调对HCV基因组RNA复制的影响。下调Con1细胞的CD2AP后，在CD2AP下调和对照Con1细胞之间，我们发现HCV RNA和蛋白质水平没有显着差异(图29)，从而证明CD2AP并不直接影响HCV亚基因组复制。然后我们测试CD2AP下调是否影响HCV的装配和释放。稳定表达sh-CD2AP-4#,6#或sh-NCHuh7.5.1细胞感染J399EM，MOI为1。在感染后72小时(HPI)，病毒滴度在细胞质和培养上清液均显著降低(图30和图31)，从而提示CD2AP参与HCV装配或/和释放。
2.8 CD2AP调控与脂滴相关联的多个HCV组分
由于在没有丙型肝炎病毒感染的情况下CD2AP的下调缓解脂滴生物合成，我们随后研究，当细胞在被丙型肝炎病毒感染的时候是否有同样的现象。我们用JFH1感染了CD2AP下调(4#和6#)和对照细胞(NC)，对脂滴，NS5A，或HCV核心蛋白染色。我们发现，HCV感染后，脂滴的形成在CD2AP下调的细胞中严重受损(图32(A)和图33(A)；LD面板下4#和6#)。此外，定位于脂滴的NS5A和核心蛋白明显下降(图32(A)和图33(A)；合并面板下4#和6#)。CD2AP下调细胞与对照细胞相比，其NS5A或核心蛋白的阳性脂滴百分比存在显著差异(图32B和33B、比较4#和6#)。前面我们证明了在Con1细胞中CD2AP下调不影响NS5A的表达水平，因此NS5A在脂滴上的定位减少是由于CD2AP下调后，脂滴生物合成减少从而造成转运缺陷而造成的。
在Huh7.5.1细胞中下调CD2AP增加IRS1和p-IRS1的总体水平(图34)。我们发现，IRS1可以被蛋白酶体途径降解。当用MG132处理2小时，IRS1水平显著性上调(图35)。为了证实CD2AP下调影响IRS1的蛋白酶依赖性的降解，我们比较了在MG132处理的条件下，对照细胞和CD2AP下调细胞的IRS1水平。我们发现，MG132显著增加对照细胞中IRS1水平，但是CD2AP下调细胞中没有增加(图36)。此外，通过从对照和CD2AP下调细胞纯化IRS1，我们发现，CD2AP下调显著降低IRS1的多位泛素化(图37)。为了弄清与CD2AP形成的蛋白复合物，我们用抗IRS1抗体进行了免疫共沉淀，发现CD2AP与IRS1可以共沉淀。我们用抗Cbl-b和抗Cbl抗体同样进行了免疫共沉淀实验，我们发现，IRS1与Cbl-b和Cbl同样可以共沉淀(图38)。为了进一步证明CD2AP，IRS1和Cbl-b/cbl存在于蛋白复合物，我们双染了CD2AP和IRS1与IRS1和Cbl-b/Cbl，发现IRS1和CD2AP，IRS1和Cbl-b/cbl确实共定位(图39)。为了证实Cbl-b/cbl是作用于IRS1的E3连接酶，我们下调Huh7.5.1细胞中的Cbl-b/cbl，发现IRS1的显著上调(图40)。因此，Cbl-b/cbl是作用于IRS1的E3连接酶。这些结果证明，CD2AP，IRS1和Cbl-b/cbl存在于同一蛋白复合物。
因为IRS1守卫胰岛素信号通路，我们检测在CD2AP下调后，胰岛素信号通路是否受到影响。我们发现，CD2AP下调增加p-Akt(s473)水平，但是下调p-AMPK(t172)和p-HSL(s554)的水平(图41)。预期地，当CD2AP下调的细胞Huh7.5.1中CD2AP被拯救时，p-Akt水平被下调(图42)。为了证明AMPK直接负责HSL的磷酸化，我们用AMPK的一个抑制剂dorsomophin处理细胞，发现dorsomophin确实降低p-AMPK水平，相应地降低HSL水平(图43)。
以上结果来自肿瘤细胞系。我们然后用HCV感染小鼠模型研究我们的结果是否有体内重要性。小鼠肝和血清中的HCV滴度在感染后不同时间点用QPCR进行监测(图44,45)。HCV滴度的趋势与已经发表的非常类似(61)。我们随后在来自HCV感染小鼠的肝组织中染色CD2AP，发现在感染后1,2和4个月时，CD2AP显著上调。由于在此时间范围之外的早或晚的时间没有明显CD2AP染色，提示CD2AP表达不与HCV滴度相关，而是HCV感染的结果(图46)。有趣的是，在这个小鼠模型中，CD2AP强染色的时间段与脂肪肝的发生有很好的相关性。
此外，我们研究在HCV感染的病人中CD2AP是否被上调。我们发现，HCV感染病人的肝活检样品，16份中有9份显示中到强CD2AP染色，而未感染HCV病人的肝活检样品，12份中只有1份显示强CD2AP染色(图47)。
最后，我们研究来自糖尿病病人的肝活检样品中，是否能检测到CD2AP免疫染色。我们发现，大多数糖尿病病人的肝组织显示强CD2AP染色。因此，在人肝活检样品中，CD2AP表达在糖尿病病人的肝中显著增加(图48)。
尽管本发明参照特异的实施方式来描述，但是将被理解的是，实施例是说明性的，本发明的范围并不局限于此。本发明的替代实施例对本发明涉及的领域的普通技术人员将变得显而易见。这样的替代实施例都被认为是包含在本发明的精神和范围之内。因此，本发明的范围由所附的权利要求被描述，由前面的描述所支持。
References
1.Choo QL,Kuo G,Weiner AJ,Overby LR,Bradley DW,HoughtonM.1989.Isolation of a cDNA clone derived from a blood-borne non-A,non-B viralhepatitis genome.Science 244:359-362.
2.Rosen HR.2011.Clinical practice.Chronic hepatitis C infection.TheNew England journal of medicine 364:2429-2438.
3.Lohmann V,Korner F,Koch J,Herian U,Theilmann L,BartenschlagerR.1999.Replication of subgenomic hepatitis C virus RNAs in a hepatoma cellline.Science 285:110-113.
4.Romero-Brey I,Merz A,Chiramel A,Lee JY,Chlanda P,Haselman U,Santarella-Mellwig R,Habermann A,Hoppe S,Kallis S,Walther P,Antony C,Krijnse-Locker J,Bartenschlager R.2012.Three-dimensional architecture and biogenesisof membrane structures associated with hepatitis C virus replication.PLoSpathogens 8:e1003056.
5.Ferraris P,Beaumont E,Uzbekov R,Brand D,Gaillard J,Blanchard E,Roingeard P.2013.Sequential biogenesis of host cell membrane rearrangementsinduced by hepatitis C virus infection.Cellular and molecular life sciences:CMLS 70:1297-1306.
6.Appel N,Zayas M,Miller S,Krijnse-Locker J,Schaller T,Friebe P,Kallis S,Engel U,Bartenschlager R.2008.Essential role of domain III ofnonstructural protein 5A for hepatitis C virus infectious particleassembly.PLoS pathogens 4:e1000035.
7.Miyanari Y,Atsuzawa K,Usuda N,Watashi K,Hishiki T,Zayas M,Bartenschlager R,Wakita T,Hijikata M,Shimotohno K.2007.The lipid droplet isan important organelle for hepatitis C virus production.Nature cell biology9:1089-1097.
8.Shi ST,Polyak SJ,Tu H,Taylor DR,Gretch DR,Lai MM.2002.Hepatitis Cvirus NS5A colocalizes with the core protein on lipid droplets and interactswith apolipoproteins.Virology 292:198-210.
9.Abid K,Pazienza V,de Gottardi A,Rubbia-Brandt L,Conne B,Pugnale P,Rossi C,Mangia A,Negro F.2005.An in vitro model of hepatitis C virus genotype3a-associated triglycerides accumulation.Journal of hepatology 42:744-751.
10.Hinson ER,Cresswell P.2009.The antiviral protein,viperin,localizesto lipid droplets via its N-terminal amphipathic alpha-helix.Proceedings ofthe National Academy of Sciences of the United States of America 106:20452-20457.
11.Masaki T,Suzuki R,Murakami K,Aizaki H,Ishii K,Murayama A,Date T,Matsuura Y,Miyamura T,Wakita T.2008.Interaction of hepatitis C virusnonstructural protein 5A with core protein is critical for the production ofinfectious virus particles.Journal of virology 82:7964-7976.
12.Lai CK,Jeng KS,Machida K,Lai MM.2008.Association of hepatitis Cvirus replication complexes with microtubules and actin filaments isdependent on the interaction of NS3and NS5A.Journal of virology 82:8838-8848.
13.Eyre NS,Fiches GN,Aloia AL,Helbig KJ,McCartney EM,McErlean CS,LiK,Aggarwal A,Turville SG,Beard MR.2014.Dynamic imaging of the hepatitis Cvirus NS5A protein during a productive infection.Journal of virology 88:3636-3652.
14.Lai C-K,Saxena V,Tseng C-H,Jeng K-S,Kohara M,LaiMM.2014.Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubulesduring intracellular transport.PloS one 9:e99022.
15.Tilg H,Moschen AR,Roden M.2017.NAFLD and diabetes mellitus.NatureReviews Gastroenterology&Hepatology 14:32-42.
16.Anai M,Funaki M,Ogihara T,Terasaki J,Inukai K,Katagiri H,FukushimaY,Yazaki Y,Kikuchi M,Oka Y.1998.Altered expression levels and impaired stepsin the pathway to phosphatidylinositol 3-kinase activation via insulinreceptor substrates 1 and 2 in Zucker fatty rats.Diabetes 47:13-23.
17.Araki E,Llpes MA,Patti M-E.1994.signalling in mice with targeteddisruption.Nature 372.
18.Bruning JC,Winnay J,Bonner-Weir S,Taylor SI,Accili D,KahnCR.1997.Development of a novel polygenic model of NIDDM in mice heterozygousfor IR and IRS-1 null alleles.Cell 88:561-572.
19.Jiang ZY,Lin Y-W,Clemont A,Feener EP,Hein KD,Igarashi M,YamauchiT,White MF,King GL.1999.Characterization of selective resistance to insulinsignaling in the vasculature of obese Zucker(fa/fa)rats.The Journal ofclinical investigation 104:447-457.
20.Kerouz NJ,
Figure GDA0001389300800000391
D,Pons S,Kahn CR.1997.Differential regulation ofinsulin receptor substrates-1 and-2(IRS-1 and IRS-2)and phosphatidylinositol3-kinase isoforms in liver and muscle of the obese diabetic(ob/ob)mouse.Journal of Clinical Investigation 100:3164.
21.Tamemoto H,Kadowaki T,Tobe K,Yagi T,Sakura H,Hayakawa T,TerauchiY,Ueki K,Kaburagi Y,Satoh S.1994.Insulin resistance and growth retardation inmice lacking insulin receptor substrate-1.
22.Withers DJ,Gutierrez JS,Towery H,Burks DJ,Ren JM,Previs S,Zhang Y,Bernal D,Pons S,Shulman GI,Bonner-Weir S,White MF.1998.Disruption of IRS-2causes type 2 diabetes in mice.Nature 391:900-904.
23.Stephens JM,Lee J,Pilch PF.1997.Tumor necrosis factor-α-inducedinsulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulinreceptor substrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal transduction.Journal of Biological Chemistry 272:971-976.
24.Egawa K,Nakashima N,Sharma PM,Maegawa H,Nagai Y,Kashiwagi A,Kikkawa R,Olefsky JM.2000.Persistent Activation of Phosphatidylinositol 3-Kinase Causes Insulin Resistance Due to Accelerated Insulin-Induced InsulinReceptor Substrate-1 Degradation in 3T3-L1 Adipocytes 1.Endocrinology 141:1930-1935.
25.Sun XJ,Goldberg JL,Qiao L,Mitchell JJ.1999.Insulin-induced insulinreceptor substrate-1 degradation is mediated by the proteasome degradationpathway.Diabetes 48:1359-1364.
26.Haruta T,Uno T,Kawahara J,Takano A,Egawa K,Sharma PM,Olefsky JM,Kobayashi M.2000.A rapamycin-sensitive pathway down-regulates insulinsignaling via phosphorylation and proteasomal degradation of insulin receptorsubstrate-1.Molecular endocrinology 14:783-794.
27.Lee AV,Gooch JL,Oesterreich S,Guler RL,Yee D.2000.Insulin-likegrowth factor I-induced degradation of insulin receptor substrate 1 ismediated by the 26S proteasome and blocked by phosphatidylinositol 3′-kinaseinhibition.Molecular and cellular biology 20:1489-1496.
28.Zhande R,Mitchell JJ,Wu J,Sun XJ.2002.Molecular mechanism ofinsulin-induced degradation of insulin receptor substrate 1.Molecular andcellular biology 22:1016-1026.
29.Bose SK,Ray R.2014.Hepatitis C virus infection and insulinresistance.World J Diabetes 5:52-58.
30.John J.Sambrook DDWR.1989.Molecular Cloning:A Laboratory Mannual,second edition.CSHL Press.
31.Ausubel FM.1987.Current Protocols in Molecular Biology
32.Rosenberg IM.1996.Protein Analysis and Purification-BenchtopTechniques
33.Copeland RA.2013.Methods for Protein Analysis:a Practical Guidefor Laboratory Protocols.
34.John E.Coligan BB.1999 Current Protocols in Immunology.
35.Kim JM,Wu H,Green G,Winkler CA,Kopp JB,Miner JH,Unanue ER,ShawAS.2003.CD2-associated protein haploinsufficiency is linked to glomerulardisease susceptibility.Science 300:1298-1300.
36.Kobayashi S,Sawano A,Nojima Y,Shibuya M,Maru Y.2004.The c-Cbl/CD2AP complex regulates VEGF-induced endocytosis and degradation of Flt-1(VEGFR-1).The FASEB journal 18:929-931.
37.Bao M,Hanabuchi S,Facchinetti V,Du Q,Bover L,Plumas J,Chaperot L,Cao W,Qin J,Sun S-C.2012.CD2AP/SHIP1 complex positively regulatesplasmacytoid dendritic cell receptor signaling by inhibiting the E3 ubiquitinligase Cbl.The Journal of Immunology 189:786-792.
38.Calco GN,Stephens OR,Donahue LM,Tsui CC,Pierchala BA.2014.CD2-associated protein(CD2AP)enhances casitas B lineage lymphoma-3/c(Cbl-3/c)-mediated Ret isoform-specific ubiquitination and degradation via its amino-terminal Src homology 3 domains.Journal of Biological Chemistry 289:7307-7319.
39.Kowanetz K,Szymkiewicz I,Haglund K,Kowanetz M,Husnjak K,Taylor JD,Soubeyran P,Engstrom U,Ladbury JE,Dikic I.2003.Identification of a novelproline-arginine motif involved in CIN85-dependent clustering of Cbl anddown-regulation of epidermal growth factor receptors.Journal of BiologicalChemistry 278:39735-39746.
40.Gout I,Middleton G,Adu J,Ninkina NN,Drobot LB,Filonenko V,MatsukaG,Davies AM,Waterfield M,Buchman VL.2000.Negative regulation of PI 3-kinaseby Ruk,a novel adaptor protein.The EMBO journal 19:4015-4025.
41.Huber TB,Hartleben B,Kim J,Schmidts M,Schermer B,Keil A,Egger L,Lecha RL,Borner C,
Figure GDA0001389300800000401
H.2003.Nephrin and CD2AP associate withphosphoinositide 3-OH kinase and stimulate AKT-dependent signaling.Molecularand cellular biology 23:4917-4928.
42.Oprea C,Ianache I,Radoi R,Erscoiu S,Tardei G,Nicolaescu O,Nica M,Calistru P,Ruta S,Ceausu E.2014.Alarming increase in tuberculosis andhepatitis C virus(HCV)among HIV infected intravenous drug users.Journal ofthe International AIDS Society 17:19625.
43.Chamond N,Cosson A,Coatnoan N,Minoprio P.2009.Proline racemasesare conserved mitogens:characterization of a Trypanosoma vivax prolineracemase.Molecular and biochemical parasitology 165:170-179.
44.Zehmer JK,Bartz R,Liu P,Anderson RG.2008.Identification of a novelN-terminal hydrophobic sequence that targets proteins to lipiddroplets.Journal of cell science 121:1852-1860.
45.Wu Y,Liao Q,Yang R,Chen X,Chen X.2011.A novel luciferase and GFPdual reporter virus for rapid and convenient evaluation of hepatitis C virusreplication.Virus research 155:406-414.
46.Lindenbach BD,Evans MJ,Syder AJ,Wolk B,Tellinghuisen TL,Liu CC,Maruyama T,Hynes RO,Burton DR,McKeating JA,Rice CM.2005.Complete replicationof hepatitis C virus in cell culture.Science 309:623-626.
47.Li C,Yu S,Nakamura F,Yin S,Xu J,Petrolla AA,Singh N,Tartakoff A,Abbott DW,Xin W,Sy MS.2009.Binding of pro-prion to filamin A disruptscytoskeleton and correlates with poor prognosis in pancreatic cancer.TheJournal of clinical investigation 119:2725-2736.
48.Yang L,Gao Z,Hu L,Wu G,Yang X,Zhang L,Zhu Y,Wong B-S,Xin W,Sy M-S.2016.Glycosylphosphatidylinositol Anchor Modification Machinery DeficiencyIs Responsible for the Formation of Pro-Prion Protein(PrP)in BxPC-3 Proteinand Increases Cancer Cell Motility.Journal of Biological Chemistry 291:3905-3917.
49.Roux KJ,Kim DI,Raida M,Burke B.2012.A promiscuous biotin ligasefusion protein identifies proximal and interacting proteins in mammaliancells.The Journal of cell biology 196:801-810.
50.Xu S,Pei R,Guo M,Han Q,Lai J,Wang Y,Wu C,Zhou Y,Lu M,ChenX.2012.Cytosolic phospholipase A2 gamma is involved in hepatitis C virusreplication and assembly.Journal of virology 86:13025-13037.
51.Vogt DA,Camus G,Herker E,Webster BR,Tsou CL,Greene WC,Yen TS,OttM.2013.Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNAreplication through interaction with the viral NS5A protein.PLoS pathogens 9:e1003302.
52.Lindenbach BD.2009.Measuring HCV infectivity produced in cellculture and in vivo.Methods in molecular biology 510:329-336.
53.Dustin ML,Olszowy MW,Holdorf AD,Li J,Bromley S,Desai N,Widder P,Rosenberger F,van der Merwe PA,Allen PM.1998.A novel adaptor proteinorchestrates receptor patterning and cytoskeletal polarity in T-cellcontacts.Cell 94:667-677.
54.Tang VW,Brieher WM.2013.FSGS3/CD2AP is a barbed-end cappingprotein that stabilizes actin and strengthens adherens junctions.The Journalof cell biology 203:815-833.
55.Zhao J,Bruck S,Cemerski S,Zhang L,Butler B,Dani A,Cooper JA,ShawAS.2013.CD2AP links cortactin and capping protein at the cell periphery tofacilitate formation of lamellipodia.Molecular and cellular biology 33:38-47.
56.Macdonald A,Crowder K,Street A,McCormick C,Harris M.2004.Thehepatitis C virus NS5A protein binds to members of the Src family of tyrosinekinases and regulates kinase activity.The Journal of general virology 85:721-729.
57.Brasaemle DL,Dolios G,Shapiro L,Wang R.2004.Proteomic analysis ofproteins associated with lipid droplets of basal and lipolytically stimulated3T3-L1 adipocytes.The Journal of biological chemistry 279:46835-46842.
58.Brass V,Bieck E,Montserret R,
Figure GDA0001389300800000421
B,Hellings JA,Blum HE,Penin F,Moradpour D.2002.An amino-terminal amphipathicα-helix mediates membraneassociation of the hepatitis C virus nonstructural protein 5A.Journal ofBiological Chemistry 277:8130-8139.
59.Tellinghuisen TL,Marcotrigiano J,Gorbalenya AE,Rice CM.2004.TheNS5A protein of hepatitis C virus is a zinc metalloprotein.The Journal ofbiological chemistry 279:48576-48587.
60.Welsch T,Endlich N,
Figure GDA0001389300800000422
G,Doroshenko E,Simpson JC,Kriz W,Shaw AS,Endlich K.2005.Association of CD2AP with dynamic actin on vesicles inpodocytes.American Journal of Physiology-Renal Physiology 289:F1134-F1143.
61.Chen J,Zhao Y,Zhang C,Chen H,Feng J,Chi X,Pan Y,Du J,Guo M,Cao H,Chen H,Wang Z,Pei R,Wang Q,Pan L,Niu J,Chen X,Tang H.2014.Persistenthepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice.Cell research 24:1050-1066.
SEQUENCE LISTING
<110> 中国科学院武汉病毒研究所
<120> CD2结合蛋白（CD2AP）和其相互作用蛋白
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Gln Asp Asp Ser Glu Thr Val Leu Ala Gly Pro Thr Ser Pro Ile Pro
180 185 190
Ser Leu Gly Asn Val Ser Glu Thr Ala Ser Gly Ser Val Thr Gln Pro
195 200 205
Lys Lys Ile Arg Gly Ile Gly Phe Gly Asp Ile Phe Lys Glu Gly Ser
210 215 220
Val Lys Leu Arg Thr Arg Thr Ser Ser Ser Glu Thr Glu Glu Lys Lys
225 230 235 240
Pro Glu Lys Pro Leu Ile Leu Gln Ser Leu Gly Pro Lys Thr Gln Ser
245 250 255
Val Glu Ile Thr Lys Thr Asp Thr Glu Gly Lys Ile Lys Ala Lys Glu
260 265 270
Tyr Cys Arg Thr Leu Phe Ala Tyr Glu Gly Thr Asn Glu Asp Glu Leu
275 280 285
Thr Phe Lys Glu Gly Glu Ile Ile His Leu Ile Ser Lys Glu Thr Gly
290 295 300
Glu Ala Gly Trp Trp Arg Gly Glu Leu Asn Gly Lys Glu Gly Val Phe
305 310 315 320
Pro Asp Asn Phe Ala Val Gln Ile Asn Glu Leu Asp Lys Asp Phe Pro
325 330 335
Lys Pro Lys Lys Pro Pro Pro Pro Ala Lys Ala Pro Ala Pro Lys Pro
340 345 350
Glu Leu Ile Ala Ala Glu Lys Lys Tyr Phe Ser Leu Lys Pro Glu Glu
355 360 365
Lys Asp Glu Lys Ser Thr Leu Glu Gln Lys Pro Ser Lys Pro Ala Ala
370 375 380
Pro Gln Val Pro Pro Lys Lys Pro Thr Pro Pro Thr Lys Ala Ser Asn
385 390 395 400
Leu Leu Arg Ser Ser Gly Thr Val Tyr Pro Lys Arg Pro Glu Lys Pro
405 410 415
Val Pro Pro Pro Pro Pro Ile Ala Lys Ile Asn Gly Glu Val Ser Ser
420 425 430
Ile Ser Ser Lys Phe Glu Thr Glu Pro Val Ser Lys Leu Lys Leu Asp
435 440 445
Ser Glu Gln Leu Pro Leu Arg Pro Lys Ser Val Asp Phe Asp Ser Leu
450 455 460
Thr Val Arg Thr Ser Lys Glu Thr Asp Val Val Asn Phe Asp Asp Ile
465 470 475 480
Ala Ser Ser Glu Asn Leu Leu His Leu Thr Ala Asn Arg Pro Lys Met
485 490 495
Pro Gly Arg Arg Leu Pro Gly Arg Phe Asn Gly Gly His Ser Pro Thr
500 505 510
His Ser Pro Glu Lys Ile Leu Lys Leu Pro Lys Glu Glu Asp Ser Ala
515 520 525
Asn Leu Lys Pro Ser Glu Leu Lys Lys Asp Thr Cys Tyr Ser Pro Lys
530 535 540
Pro Ser Val Tyr Leu Ser Thr Pro Ser Ser Ala Ser Lys Ala Asn Thr
545 550 555 560
Thr Ala Phe Leu Thr Pro Leu Glu Ile Lys Ala Lys Val Glu Thr Asp
565 570 575
Asp Val Lys Lys Asn Ser Leu Asp Glu Leu Arg Ala Gln Ile Ile Glu
580 585 590
Leu Leu Cys Ile Val Glu Ala Leu Lys Lys Asp His Gly Lys Glu Leu
595 600 605
Glu Lys Leu Arg Lys Asp Leu Glu Glu Glu Lys Thr Met Arg Ser Asn
610 615 620
Leu Glu Met Glu Ile Glu Lys Leu Lys Lys Ala Val Leu Ser Ser
625 630 635
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA for CD2AP
<400> 3
gctggaagga gaactaaatg g 21
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 4
ggagaactaa atgggagaag a 21
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 5
ggacttccag ctggaggaat t 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 6
ggagctgaaa gtgggagata t 21
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 7
gctgaaagtg ggagatatta t 21
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 8
gctgaaagtg ggagatatta t 21
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 9
gcccaggacg attcagaaac t 21
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 10
gctgggccta cttcacctat a 21
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 11
gccagtaatt tactgagatc t 21
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 12
gcttcatctc actgcaaata g 21
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 13
ggaagtttcc agcagatttc a 21
<210> 14
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 14
agccgagggt ctgggcaaa 19
<210> 15
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 15
agccgagggt ctgggcaaa 19
<210> 16
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 16
tgaagagact ggtaggaga 19
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 17
ctaaatggga gaagaggaa 19
<210> 18
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 18
aggatgaact ggagctgaa 19
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 19
ggtaacagat gatggtgaa 19
<210> 20
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA for human CD2AP
<400> 20
ggaaacagat gatgtgaaa 19
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 21
aaaggcgaca ccgtagacta 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 22
cgacaccgta gactaaggtg 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 23
gtgggaaaac cgcggtcggg 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 24
ggcgacaccg tagactaagg 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 25
agggtgggaa aaccgcggtc 20
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 26
tgggaaaacc gcggtcgggc 20
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 27
gcgacaccgt agactaaggt 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 28
cagggtggga aaaccgcggt 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 29
cgaccgcggt tttcccaccc 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 30
aaaaccgcgg tcgggcgggc 20
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 31
cgaggctagg cgggcgctcg 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 32
gaaaaccgcg gtcgggcggg 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 33
gagggtctgg gcaaaccggt 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 34
tgggtcccca ccttagtcta 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 35
cgagggtctg ggcaaaccgg 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 36
gcgctcgggg ttggagccga 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 37
tccgaggcta ggcgggcgct 20
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 38
ttttctaact gcgagtgcta 20
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 39
ccgaggctag gcgggcgctc 20
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 40
aaaccgcggt cgggcgggcg 20
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 41
ttagcactcg cagttagaaa 20
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 42
gctaggcggg cgctcggggt 20
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 43
tccccactgc gggagcggcc 20
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 44
cccgagcgcc cgcctagcct 20
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 45
accctggccg ctcccgcagt 20
<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 46
cggccagggt gggaaaaccg 20
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 47
cgagtgctaa ggaagaggcg 20
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 48
aactgcgagt gctaaggaag 20
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 49
ggcgggctcc gaggctaggc 20
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 50
tccccaggag ccacggcggc 20
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 51
ctaccccgcc cgcccgaccg 20
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 52
gtagggccct cccgccgccg 20
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 53
caccggtttg cccagaccct 20
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 54
ccctggccgc tcccgcagtg 20
<210> 55
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 55
agccgagggt ctgggcaaac 20
<210> 56
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human CD2AP
<400> 56
tggccgctcc cgcagtgggg 20
<210> 57
<211> 2082
<212> DNA
<213> Canis lupus
<400> 57
atgcatttta aaagtttgct gaaaaacctg gaatggagac aaccaaccag gaggaaaaag 60
acacatagag aacatcagct gaaaaaggtc aaaagaactg gggatggcaa gctcagaaag 120
tgtctacaac ttctccggtg gagtcggatt tctggtcacg ggtcagttga ctatattgtg 180
gagtatgact acgatgctgt acatgatgat gaattaacta ttcgggttgg tgaaataatc 240
aggaatgtga aaaaactaca ggaggaagga tggctagaag gagagctaaa tgggagaaga 300
ggaatgtttc ctgataattt tgttaaggaa attaagagag agacagaacc caaggatgat 360
aatttgccca ttaaacggga aagacatggg aatgtagcaa gccttgtaca acgaataagc 420
acctatggac ttccagctgg aggaattcaa ccacatccac aaaccaaaaa cattaagaag 480
aagaccaaga agcgtcagtg taaagttctc tttgagtacc ttccacaaaa tgaggatgaa 540
ttggagctga aagtgggaga tattattgat attaatgatg aggtagaaga aggctggtgg 600
agtggaaccc tgaacaacaa gttgggactg tttccctcaa attttgtgaa agaattagag 660
gtaacagatg atggtgaaac tcatgaagcc caagaggatt cagaaacggt ttttactggg 720
cctacctcac ctttaccgtc tccggggaat gggaatgaaa ctgcacctgg atcagttaca 780
cagccaaaga aaattcgagg aattggattt ggagatattt ttaaagaagg ctctgtgaaa 840
cttagaacaa gaacatctgg tagtgaaata gaagagaaga aaacggaaaa gcccttaatt 900
atacagtcag taggatccaa aacacagagt ctggatgcaa caaaaacaga cacggaaaat 960
aaaagtaaag caaaggaata ttgtagaaca ttatttgcct atgaaggtac taatgaagac 1020
gagctttctt ttaaagaggg agagataatt cacttaataa gtaaggagac tggagaagct 1080
ggctggtgga agggtgaact taatggtaaa gaaggagtat ttccagataa ttttgctatt 1140
cagatacatg aactggataa agactttcca aaaccaaaga aaccaccacc tcctgctaaa 1200
ggtccagctc caaaacctga gctaatagct acagagaaga agtattttcc tataaagcca 1260
gaagaaaaag atgaaaaatc agtactggaa cagaaacctt ctaaaccagc agctccacaa 1320
gtcccaccta agaagcctac tccacccacc aaagccaata atttattgag atctcctggg 1380
acaatatacc caaagcgacc tgaaaaacca gtccctccac cacctcctat agccaagatt 1440
aatggggaag tatctaccat ttcatcaaaa tttgaaactg agccattatc aaaaccaaag 1500
ctagattctg aacaattacc acttagacca aaatcagtag acctagattc atttacagtt 1560
aggagctcta aagaaacaga tattgtaaat tttgatgaca tagcttcctc agaaaacttg 1620
ctacatctta ctgcaaacag accgaagatg cctggaagaa ggttgcctgg acgcttcaat 1680
ggtggacatt ctccaaccca aagcccagaa aaaaccttga agttaccaaa agaagaagat 1740
agtgccaact taaagccgtc tgaatttaaa aaggattcaa gctactctcc aaagccatct 1800
ctgtaccttt caacaccttc aagtgcttcg aaaccaaata cagctgcttt tttaactcca 1860
ttagaaatca aagctaaagt agaatcagat gatgggaaaa aaaacccctt ggatgaactt 1920
agagctcaga ttattgaatt gctgtgcatt gtagaagcac tgaaaaagga tcatgggaaa 1980
gaactggaaa aactacgaaa ggatttggaa gaggagaagg caatgagaag taatctagag 2040
gtggaaatcg agaagctgaa aaaggcagtc ctgtcgtctt ga 2082
<210> 58
<211> 693
<212> PRT
<213> Canis lupus
<400> 58
Met His Phe Lys Ser Leu Leu Lys Asn Leu Glu Trp Arg Gln Pro Thr
1 5 10 15
Arg Arg Lys Lys Thr His Arg Glu His Gln Leu Lys Lys Val Lys Arg
20 25 30
Thr Gly Asp Gly Lys Leu Arg Lys Cys Leu Gln Leu Leu Arg Trp Ser
35 40 45
Arg Ile Ser Gly His Gly Ser Val Asp Tyr Ile Val Glu Tyr Asp Tyr
50 55 60
Asp Ala Val His Asp Asp Glu Leu Thr Ile Arg Val Gly Glu Ile Ile
65 70 75 80
Arg Asn Val Lys Lys Leu Gln Glu Glu Gly Trp Leu Glu Gly Glu Leu
85 90 95
Asn Gly Arg Arg Gly Met Phe Pro Asp Asn Phe Val Lys Glu Ile Lys
100 105 110
Arg Glu Thr Glu Pro Lys Asp Asp Asn Leu Pro Ile Lys Arg Glu Arg
115 120 125
His Gly Asn Val Ala Ser Leu Val Gln Arg Ile Ser Thr Tyr Gly Leu
130 135 140
Pro Ala Gly Gly Ile Gln Pro His Pro Gln Thr Lys Asn Ile Lys Lys
145 150 155 160
Lys Thr Lys Lys Arg Gln Cys Lys Val Leu Phe Glu Tyr Leu Pro Gln
165 170 175
Asn Glu Asp Glu Leu Glu Leu Lys Val Gly Asp Ile Ile Asp Ile Asn
180 185 190
Asp Glu Val Glu Glu Gly Trp Trp Ser Gly Thr Leu Asn Asn Lys Leu
195 200 205
Gly Leu Phe Pro Ser Asn Phe Val Lys Glu Leu Glu Val Thr Asp Asp
210 215 220
Gly Glu Thr His Glu Ala Gln Glu Asp Ser Glu Thr Val Phe Thr Gly
225 230 235 240
Pro Thr Ser Pro Leu Pro Ser Pro Gly Asn Gly Asn Glu Thr Ala Pro
245 250 255
Gly Ser Val Thr Gln Pro Lys Lys Ile Arg Gly Ile Gly Phe Gly Asp
260 265 270
Ile Phe Lys Glu Gly Ser Val Lys Leu Arg Thr Arg Thr Ser Gly Ser
275 280 285
Glu Ile Glu Glu Lys Lys Thr Glu Lys Pro Leu Ile Ile Gln Ser Val
290 295 300
Gly Ser Lys Thr Gln Ser Leu Asp Ala Thr Lys Thr Asp Thr Glu Asn
305 310 315 320
Lys Ser Lys Ala Lys Glu Tyr Cys Arg Thr Leu Phe Ala Tyr Glu Gly
325 330 335
Thr Asn Glu Asp Glu Leu Ser Phe Lys Glu Gly Glu Ile Ile His Leu
340 345 350
Ile Ser Lys Glu Thr Gly Glu Ala Gly Trp Trp Lys Gly Glu Leu Asn
355 360 365
Gly Lys Glu Gly Val Phe Pro Asp Asn Phe Ala Ile Gln Ile His Glu
370 375 380
Leu Asp Lys Asp Phe Pro Lys Pro Lys Lys Pro Pro Pro Pro Ala Lys
385 390 395 400
Gly Pro Ala Pro Lys Pro Glu Leu Ile Ala Thr Glu Lys Lys Tyr Phe
405 410 415
Pro Ile Lys Pro Glu Glu Lys Asp Glu Lys Ser Val Leu Glu Gln Lys
420 425 430
Pro Ser Lys Pro Ala Ala Pro Gln Val Pro Pro Lys Lys Pro Thr Pro
435 440 445
Pro Thr Lys Ala Asn Asn Leu Leu Arg Ser Pro Gly Thr Ile Tyr Pro
450 455 460
Lys Arg Pro Glu Lys Pro Val Pro Pro Pro Pro Pro Ile Ala Lys Ile
465 470 475 480
Asn Gly Glu Val Ser Thr Ile Ser Ser Lys Phe Glu Thr Glu Pro Leu
485 490 495
Ser Lys Pro Lys Leu Asp Ser Glu Gln Leu Pro Leu Arg Pro Lys Ser
500 505 510
Val Asp Leu Asp Ser Phe Thr Val Arg Ser Ser Lys Glu Thr Asp Ile
515 520 525
Val Asn Phe Asp Asp Ile Ala Ser Ser Glu Asn Leu Leu His Leu Thr
530 535 540
Ala Asn Arg Pro Lys Met Pro Gly Arg Arg Leu Pro Gly Arg Phe Asn
545 550 555 560
Gly Gly His Ser Pro Thr Gln Ser Pro Glu Lys Thr Leu Lys Leu Pro
565 570 575
Lys Glu Glu Asp Ser Ala Asn Leu Lys Pro Ser Glu Phe Lys Lys Asp
580 585 590
Ser Ser Tyr Ser Pro Lys Pro Ser Leu Tyr Leu Ser Thr Pro Ser Ser
595 600 605
Ala Ser Lys Pro Asn Thr Ala Ala Phe Leu Thr Pro Leu Glu Ile Lys
610 615 620
Ala Lys Val Glu Ser Asp Asp Gly Lys Lys Asn Pro Leu Asp Glu Leu
625 630 635 640
Arg Ala Gln Ile Ile Glu Leu Leu Cys Ile Val Glu Ala Leu Lys Lys
645 650 655
Asp His Gly Lys Glu Leu Glu Lys Leu Arg Lys Asp Leu Glu Glu Glu
660 665 670
Lys Ala Met Arg Ser Asn Leu Glu Val Glu Ile Glu Lys Leu Lys Lys
675 680 685
Ala Val Leu Ser Ser
690
<210> 59
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 59
gaggaatgtt tcctgataa 19
<210> 60
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 60
tcagtagacc tagattcat 19
<210> 61
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 61
gcgtcagtgt aaagttctc 19
<210> 62
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 62
tagctacaga gaagaagta 19
<210> 63
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 63
agagggagag ataattcac 19
<210> 64
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 64
atcagtagac ctagattca 19
<210> 65
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 65
ggtactaatg aagacgagc 19
<210> 66
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 66
agaagaagat agtgccaac 19
<210> 67
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 67
ctcatgaagc ccaagagga 19
<210> 68
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 68
cgaataagca cctatggac 19
<210> 69
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 69
ctggaatgga gacaaccaa 19
<210> 70
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 70
gcaagctcag aaagtgtct 19
<210> 71
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 71
gctcagaaag tgtctacaa 19
<210> 72
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 72
cagaaagtgt ctacaactt 19
<210> 73
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 73
gtctacaact tctccggtg 19
<210> 74
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 74
ggagtcggat ttctggtca 19
<210> 75
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 75
gtcacgggtc agttgacta 19
<210> 76
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine CD2AP
<400> 76
acgggtcagt tgactatat 19
<210> 77
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 77
aaaggcagac actcaaccgc cgg 23
<210> 78
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 78
atgtattgaa gtgagacacc tgg 23
<210> 79
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 79
atgatgtggg actccatccc agg 23
<210> 80
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 80
agggcgtgac ccccaagtcc tgg 23
<210> 81
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 81
tgtattgaag tgagacacct ggg 23
<210> 82
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 82
gggcgtgacc cccaagtcct ggg 23
<210> 83
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 83
ccatgcagga agcatgatgt ggg 23
<210> 84
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 84
ggggtcacgc cctgagccaa agg 23
<210> 85
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 85
tccatgcagg aagcatgatg tgg 23
<210> 86
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 86
attgaagtga gacacctggg tgg 23
<210> 87
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 87
gactccatcc caggacttgg ggg 23
<210> 88
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 88
gagtgtctgc ctttggctca ggg 23
<210> 89
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 89
tgggactcca tcccaggact tgg 23
<210> 90
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 90
agacacctgg gtggctccgg cgg 23
<210> 91
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 91
tgagtgtctg cctttggctc agg 23
<210> 92
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 92
ggactccatc ccaggacttg ggg 23
<210> 93
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 93
gtgaccccca agtcctggga tgg 23
<210> 94
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 94
ggcggttgag tgtctgcctt tgg 23
<210> 95
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 95
gtgagacacc tgggtggctc cgg 23
<210> 96
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 96
cccacatcat gcttcctgca tgg 23
<210> 97
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 97
gggactccat cccaggactt ggg 23
<210> 98
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 98
taacgcaact ttctattttt tgg 23
<210> 99
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 99
ctcacttcaa tacattttta agg 23
<210> 100
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 100
ccagttaaaa agaaaatcta agg 23
<210> 101
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 101
ctcaaccgcc ggagccaccc agg 23
<210> 102
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 102
taaagcaact ttctattttt tgg 23
<210> 103
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine CD2AP
<400> 103
ccttagattt tctttttaac tgg 23
<210> 104
<211> 1398
<212> DNA
<213> Hepatitis C virus
<400> 104
tccggatcct ggctccgcga cgtgtgggac tgggtttgca ccatcttgac agacttcaaa 60
aattggctga cctctaaatt gttccccaag ctgcccggcc tccccttcat ctcttgtcaa 120
aaggggtaca agggtgtgtg ggccggcact ggcatcatga ccacgcgctg cccttgcggc 180
gccaacatct ctggcaatgt ccgcctgggc tctatgagga tcacagggcc taaaacctgc 240
atgaacacct ggcaggggac ctttcctatc aattgctaca cggagggcca gtgcgcgccg 300
aaacccccca cgaactacaa gaccgccatc tggagggtgg cggcctcgga gtacgcggag 360
gtgacgcagc atgggtcgta ctcctatgta acaggactga ccactgacaa tctgaaaatt 420
ccttgccaac taccttctcc agagtttttc tcctgggtgg acggtgtgca gatccatagg 480
tttgcaccca caccaaagcc gtttttccgg gatgaggtct cgttctgcgt tgggcttaat 540
tcctatgctg tcgggtccca gcttccctgt gaacctgagc ccgacgcaga cgtattgagg 600
tccatgctaa cagatccgcc ccacatcacg gcggagactg cggcgcggcg cttggcacgg 660
ggatcacctc catctgaggc gagctcctca gtgagccagc tatcagcacc gtcgctgcgg 720
gccacctgca ccacccacag caacacctat gacgtggaca tggtcgatgc caacctgctc 780
atggagggcg gtgtggctca gacagagcct gagtccaggg tgcccgttct ggactttctc 840
gagccaatgg ccgaggaaga gagcgacctt gagccctcaa taccatcgga gtgcatgctc 900
cccaggagcg ggtttccacg ggccttaccg gcttgggcac ggcctgacta caacccgccg 960
ctcgtggaat cgtggaggag gccagattac caaccgccca ccgttgctgg ttgtgctctc 1020
ccccccccca agaaggcccc gacgcctccc ccaaggagac gccggacagt gggtctgagc 1080
gagagcacca tatcagaagc cctccagcaa ctggccatca agacctttgg ccagcccccc 1140
tcgagcggtg atgcaggctc gtccacgggg gcgggcgccg ccgaatccgg cggtccgacg 1200
tcccctggtg agccggcccc ctcagagaca ggttccgcct cctctatgcc ccccctcgag 1260
ggggagcctg gagatccgga cctggagtct gatcaggtag agcttcaacc tcccccccag 1320
gggggggggg tagctcccgg ttcgggctcg gggtcttggt ctacttgctc cgaggaggac 1380
gataccaccg tgtgctgc 1398
<210> 105
<211> 466
<212> PRT
<213> Hepatitis C virus
<400> 105
Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Val Cys Thr Ile Leu
1 5 10 15
Thr Asp Phe Lys Asn Trp Leu Thr Ser Lys Leu Phe Pro Lys Leu Pro
20 25 30
Gly Leu Pro Phe Ile Ser Cys Gln Lys Gly Tyr Lys Gly Val Trp Ala
35 40 45
Gly Thr Gly Ile Met Thr Thr Arg Cys Pro Cys Gly Ala Asn Ile Ser
50 55 60
Gly Asn Val Arg Leu Gly Ser Met Arg Ile Thr Gly Pro Lys Thr Cys
65 70 75 80
Met Asn Thr Trp Gln Gly Thr Phe Pro Ile Asn Cys Tyr Thr Glu Gly
85 90 95
Gln Cys Ala Pro Lys Pro Pro Thr Asn Tyr Lys Thr Ala Ile Trp Arg
100 105 110
Val Ala Ala Ser Glu Tyr Ala Glu Val Thr Gln His Gly Ser Tyr Ser
115 120 125
Tyr Val Thr Gly Leu Thr Thr Asp Asn Leu Lys Ile Pro Cys Gln Leu
130 135 140
Pro Ser Pro Glu Phe Phe Ser Trp Val Asp Gly Val Gln Ile His Arg
145 150 155 160
Phe Ala Pro Thr Pro Lys Pro Phe Phe Arg Asp Glu Val Ser Phe Cys
165 170 175
Val Gly Leu Asn Ser Tyr Ala Val Gly Ser Gln Leu Pro Cys Glu Pro
180 185 190
Glu Pro Asp Ala Asp Val Leu Arg Ser Met Leu Thr Asp Pro Pro His
195 200 205
Ile Thr Ala Glu Thr Ala Ala Arg Arg Leu Ala Arg Gly Ser Pro Pro
210 215 220
Ser Glu Ala Ser Ser Ser Val Ser Gln Leu Ser Ala Pro Ser Leu Arg
225 230 235 240
Ala Thr Cys Thr Thr His Ser Asn Thr Tyr Asp Val Asp Met Val Asp
245 250 255
Ala Asn Leu Leu Met Glu Gly Gly Val Ala Gln Thr Glu Pro Glu Ser
260 265 270
Arg Val Pro Val Leu Asp Phe Leu Glu Pro Met Ala Glu Glu Glu Ser
275 280 285
Asp Leu Glu Pro Ser Ile Pro Ser Glu Cys Met Leu Pro Arg Ser Gly
290 295 300
Phe Pro Arg Ala Leu Pro Ala Trp Ala Arg Pro Asp Tyr Asn Pro Pro
305 310 315 320
Leu Val Glu Ser Trp Arg Arg Pro Asp Tyr Gln Pro Pro Thr Val Ala
325 330 335
Gly Cys Ala Leu Pro Pro Pro Lys Lys Ala Pro Thr Pro Pro Pro Arg
340 345 350
Arg Arg Arg Thr Val Gly Leu Ser Glu Ser Thr Ile Ser Glu Ala Leu
355 360 365
Gln Gln Leu Ala Ile Lys Thr Phe Gly Gln Pro Pro Ser Ser Gly Asp
370 375 380
Ala Gly Ser Ser Thr Gly Ala Gly Ala Ala Glu Ser Gly Gly Pro Thr
385 390 395 400
Ser Pro Gly Glu Pro Ala Pro Ser Glu Thr Gly Ser Ala Ser Ser Met
405 410 415
Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Glu Ser Asp Gln
420 425 430
Val Glu Leu Gln Pro Pro Pro Gln Gly Gly Gly Val Ala Pro Gly Ser
435 440 445
Gly Ser Gly Ser Trp Ser Thr Cys Ser Glu Glu Asp Asp Thr Thr Val
450 455 460
Cys Cys
465
<210> 106
<211> 3729
<212> DNA
<213> Homo sapiens
<400> 106
atggcgagcc ctccggagag cgatggcttc tcggacgtgc gcaaggtggg ctacctgcgc 60
aaacccaaga gcatgcacaa acgcttcttc gtactgcgcg cggccagcga ggctgggggc 120
ccggcgcgcc tcgagtacta cgagaacgag aagaagtggc ggcacaagtc gagcgccccc 180
aaacgctcga tcccccttga gagctgcttc aacatcaaca agcgggctga ctccaagaac 240
aagcacctgg tggctctcta cacccgggac gagcactttg ccatcgcggc ggacagcgag 300
gccgagcaag acagctggta ccaggctctc ctacagctgc acaaccgtgc taagggccac 360
cacgacggag ctgcggccct cggggcggga ggtggtgggg gcagctgcag cggcagctcc 420
ggccttggtg aggctgggga ggacttgagc tacggtgacg tgcccccagg acccgcattc 480
aaagaggtct ggcaagtgat cctgaagccc aagggcctgg gtcagacaaa gaacctgatt 540
ggtatctacc gcctttgcct gaccagcaag accatcagct tcgtgaagct gaactcggag 600
gcagcggccg tggtgctgca gctgatgaac atcaggcgct gtggccactc ggaaaacttc 660
ttcttcatcg aggtgggccg ttctgccgtg acggggcccg gggagttctg gatgcaggtg 720
gatgactctg tggtggccca gaacatgcac gagaccatcc tggaggccat gcgggccatg 780
agtgatgagt tccgccctcg cagcaagagc cagtcctcgt ccaactgctc taaccccatc 840
agcgtccccc tgcgccggca ccatctcaac aatcccccgc ccagccaggt ggggctgacc 900
cgccgatcac gcactgagag catcaccgcc acctccccgg ccagcatggt gggcgggaag 960
ccaggctcct tccgtgtccg cgcctccagt gacggcgaag gcaccatgtc ccgcccagcc 1020
tcggtggacg gcagccctgt gagtcccagc accaacagaa cccacgccca ccggcatcgg 1080
ggcagcgccc ggctgcaccc cccgctcaac cacagccgct ccatccccat gccggcttcc 1140
cgctgctcgc cttcggccac cagcccggtc agtctgtcgt ccagtagcac cagtggccat 1200
ggctccacct cggattgtct cttcccacgg cgatctagtg cttcggtgtc tggttccccc 1260
agcgatggcg gtttcatctc ctcggatgag tatggctcca gtccctgcga tttccggagt 1320
tccttccgca gtgtcactcc ggattccctg ggccacaccc caccagcccg cggtgaggag 1380
gagctaagca actatatctg catgggtggc aaggggccct ccaccctgac cgcccccaac 1440
ggtcactaca ttttgtctcg gggtggcaat ggccaccgct gcaccccagg aacaggcttg 1500
ggcacgagtc cagccttggc tggggatgaa gcagccagtg ctgcagatct ggataatcgg 1560
ttccgaaaga gaactcactc ggcaggcaca tcccctacca ttacccacca gaagaccccg 1620
tcccagtcct cagtggcttc cattgaggag tacacagaga tgatgcctgc ctacccacca 1680
ggaggtggca gtggaggccg actgccggga cacaggcact ccgccttcgt gcccacccgc 1740
tcctacccag aggagggtct ggaaatgcac cccttggagc gtcggggggg gcaccaccgc 1800
ccagacagct ccaccctcca cacggatgat ggctacatgc ccatgtcccc aggggtggcc 1860
ccagtgccca gtggccgaaa gggcagtgga gactatatgc ccatgagccc caagagcgta 1920
tctgccccac agcagatcat caatcccatc agacgccatc cccagagagt ggaccccaat 1980
ggctacatga tgatgtcccc cagcggtggc tgctctcctg acattggagg tggccccagc 2040
agcagcagca gcagcagcaa cgccgtccct tccgggacca gctatggaaa gctgtggaca 2100
aacggggtag ggggccacca ctctcatgtc ttgcctcacc ccaaaccccc agtggagagc 2160
agcggtggta agctcttacc ttgcacaggt gactacatga acatgtcacc agtgggggac 2220
tccaacacca gcagcccctc cgactgctac tacggccctg aggaccccca gcacaagcca 2280
gtcctctcct actactcatt gccaagatcc tttaagcaca cccagcgccc cggggagccg 2340
gaggagggtg cccggcatca gcacctccgc ctttccacta gctctggtcg ccttctctat 2400
gctgcaacag cagatgattc ttcctcttcc accagcagcg acagcctggg tgggggatac 2460
tgcggggcta ggctggagcc cagccttcca catccccacc atcaggttct gcagccccat 2520
ctgcctcgaa aggtggacac agctgctcag accaatagcc gcctggcccg gcccacgagg 2580
ctgtccctgg gggatcccaa ggccagcacc ttacctcggg cccgagagca gcagcagcag 2640
cagcagccct tgctgcaccc tccagagccc aagagcccgg gggaatatgt caatattgaa 2700
tttgggagtg atcagtctgg ctacttgtct ggcccggtgg ctttccacag ctcaccttct 2760
gtcaggtgtc catcccagct ccagccagct cccagagagg aagagactgg cactgaggag 2820
tacatgaaga tggacctggg gccgggccgg agggcagcct ggcaggagag cactggggtc 2880
gagatgggca gactgggccc tgcacctccc ggggctgcta gcatttgcag gcctacccgg 2940
gcagtgccca gcagccgggg tgactacatg accatgcaga tgagttgtcc ccgtcagagc 3000
tacgtggaca cctcgccagc tgcccctgta agctatgctg acatgcgaac aggcattgct 3060
gcagaggagg tgagcctgcc cagggccacc atggctgctg cctcctcatc ctcagcagcc 3120
tctgcttccc cgactgggcc tcaaggggca gcagagctgg ctgcccactc gtccctgctg 3180
gggggcccac aaggacctgg gggcatgagc gccttcaccc gggtgaacct cagtcctaac 3240
cgcaaccaga gtgccaaagt gatccgtgca gacccacaag ggtgccggcg gaggcatagc 3300
tccgagactt tctcctcaac acccagtgcc acccgggtgg gcaacacagt gccctttgga 3360
gcgggggcag cagtaggggg cggtggcggt agcagcagca gcagcgagga tgtgaaacgc 3420
cacagctctg cttcctttga gaatgtgtgg ctgaggcctg gggagcttgg gggagccccc 3480
aaggagccag ccaaactgtg tggggctgct gggggtttgg agaatggtct taactacata 3540
gacctggatt tggtcaagga cttcaaacag tgccctcagg agtgcacccc tgaaccgcag 3600
cctcccccac ccccaccccc tcatcaaccc ctgggcagcg gtgagagcag ctccacccgc 3660
cgctcaagtg aggatttaag cgcctatgcc agcatcagtt tccagaagca gccagaggac 3720
cgtcagtag 3729
<210> 107
<211> 1242
<212> PRT
<213> Homo sapiens
<400> 107
Met Ala Ser Pro Pro Glu Ser Asp Gly Phe Ser Asp Val Arg Lys Val
1 5 10 15
Gly Tyr Leu Arg Lys Pro Lys Ser Met His Lys Arg Phe Phe Val Leu
20 25 30
Arg Ala Ala Ser Glu Ala Gly Gly Pro Ala Arg Leu Glu Tyr Tyr Glu
35 40 45
Asn Glu Lys Lys Trp Arg His Lys Ser Ser Ala Pro Lys Arg Ser Ile
50 55 60
Pro Leu Glu Ser Cys Phe Asn Ile Asn Lys Arg Ala Asp Ser Lys Asn
65 70 75 80
Lys His Leu Val Ala Leu Tyr Thr Arg Asp Glu His Phe Ala Ile Ala
85 90 95
Ala Asp Ser Glu Ala Glu Gln Asp Ser Trp Tyr Gln Ala Leu Leu Gln
100 105 110
Leu His Asn Arg Ala Lys Gly His His Asp Gly Ala Ala Ala Leu Gly
115 120 125
Ala Gly Gly Gly Gly Gly Ser Cys Ser Gly Ser Ser Gly Leu Gly Glu
130 135 140
Ala Gly Glu Asp Leu Ser Tyr Gly Asp Val Pro Pro Gly Pro Ala Phe
145 150 155 160
Lys Glu Val Trp Gln Val Ile Leu Lys Pro Lys Gly Leu Gly Gln Thr
165 170 175
Lys Asn Leu Ile Gly Ile Tyr Arg Leu Cys Leu Thr Ser Lys Thr Ile
180 185 190
Ser Phe Val Lys Leu Asn Ser Glu Ala Ala Ala Val Val Leu Gln Leu
195 200 205
Met Asn Ile Arg Arg Cys Gly His Ser Glu Asn Phe Phe Phe Ile Glu
210 215 220
Val Gly Arg Ser Ala Val Thr Gly Pro Gly Glu Phe Trp Met Gln Val
225 230 235 240
Asp Asp Ser Val Val Ala Gln Asn Met His Glu Thr Ile Leu Glu Ala
245 250 255
Met Arg Ala Met Ser Asp Glu Phe Arg Pro Arg Ser Lys Ser Gln Ser
260 265 270
Ser Ser Asn Cys Ser Asn Pro Ile Ser Val Pro Leu Arg Arg His His
275 280 285
Leu Asn Asn Pro Pro Pro Ser Gln Val Gly Leu Thr Arg Arg Ser Arg
290 295 300
Thr Glu Ser Ile Thr Ala Thr Ser Pro Ala Ser Met Val Gly Gly Lys
305 310 315 320
Pro Gly Ser Phe Arg Val Arg Ala Ser Ser Asp Gly Glu Gly Thr Met
325 330 335
Ser Arg Pro Ala Ser Val Asp Gly Ser Pro Val Ser Pro Ser Thr Asn
340 345 350
Arg Thr His Ala His Arg His Arg Gly Ser Ala Arg Leu His Pro Pro
355 360 365
Leu Asn His Ser Arg Ser Ile Pro Met Pro Ala Ser Arg Cys Ser Pro
370 375 380
Ser Ala Thr Ser Pro Val Ser Leu Ser Ser Ser Ser Thr Ser Gly His
385 390 395 400
Gly Ser Thr Ser Asp Cys Leu Phe Pro Arg Arg Ser Ser Ala Ser Val
405 410 415
Ser Gly Ser Pro Ser Asp Gly Gly Phe Ile Ser Ser Asp Glu Tyr Gly
420 425 430
Ser Ser Pro Cys Asp Phe Arg Ser Ser Phe Arg Ser Val Thr Pro Asp
435 440 445
Ser Leu Gly His Thr Pro Pro Ala Arg Gly Glu Glu Glu Leu Ser Asn
450 455 460
Tyr Ile Cys Met Gly Gly Lys Gly Pro Ser Thr Leu Thr Ala Pro Asn
465 470 475 480
Gly His Tyr Ile Leu Ser Arg Gly Gly Asn Gly His Arg Cys Thr Pro
485 490 495
Gly Thr Gly Leu Gly Thr Ser Pro Ala Leu Ala Gly Asp Glu Ala Ala
500 505 510
Ser Ala Ala Asp Leu Asp Asn Arg Phe Arg Lys Arg Thr His Ser Ala
515 520 525
Gly Thr Ser Pro Thr Ile Thr His Gln Lys Thr Pro Ser Gln Ser Ser
530 535 540
Val Ala Ser Ile Glu Glu Tyr Thr Glu Met Met Pro Ala Tyr Pro Pro
545 550 555 560
Gly Gly Gly Ser Gly Gly Arg Leu Pro Gly His Arg His Ser Ala Phe
565 570 575
Val Pro Thr Arg Ser Tyr Pro Glu Glu Gly Leu Glu Met His Pro Leu
580 585 590
Glu Arg Arg Gly Gly His His Arg Pro Asp Ser Ser Thr Leu His Thr
595 600 605
Asp Asp Gly Tyr Met Pro Met Ser Pro Gly Val Ala Pro Val Pro Ser
610 615 620
Gly Arg Lys Gly Ser Gly Asp Tyr Met Pro Met Ser Pro Lys Ser Val
625 630 635 640
Ser Ala Pro Gln Gln Ile Ile Asn Pro Ile Arg Arg His Pro Gln Arg
645 650 655
Val Asp Pro Asn Gly Tyr Met Met Met Ser Pro Ser Gly Gly Cys Ser
660 665 670
Pro Asp Ile Gly Gly Gly Pro Ser Ser Ser Ser Ser Ser Ser Asn Ala
675 680 685
Val Pro Ser Gly Thr Ser Tyr Gly Lys Leu Trp Thr Asn Gly Val Gly
690 695 700
Gly His His Ser His Val Leu Pro His Pro Lys Pro Pro Val Glu Ser
705 710 715 720
Ser Gly Gly Lys Leu Leu Pro Cys Thr Gly Asp Tyr Met Asn Met Ser
725 730 735
Pro Val Gly Asp Ser Asn Thr Ser Ser Pro Ser Asp Cys Tyr Tyr Gly
740 745 750
Pro Glu Asp Pro Gln His Lys Pro Val Leu Ser Tyr Tyr Ser Leu Pro
755 760 765
Arg Ser Phe Lys His Thr Gln Arg Pro Gly Glu Pro Glu Glu Gly Ala
770 775 780
Arg His Gln His Leu Arg Leu Ser Thr Ser Ser Gly Arg Leu Leu Tyr
785 790 795 800
Ala Ala Thr Ala Asp Asp Ser Ser Ser Ser Thr Ser Ser Asp Ser Leu
805 810 815
Gly Gly Gly Tyr Cys Gly Ala Arg Leu Glu Pro Ser Leu Pro His Pro
820 825 830
His His Gln Val Leu Gln Pro His Leu Pro Arg Lys Val Asp Thr Ala
835 840 845
Ala Gln Thr Asn Ser Arg Leu Ala Arg Pro Thr Arg Leu Ser Leu Gly
850 855 860
Asp Pro Lys Ala Ser Thr Leu Pro Arg Ala Arg Glu Gln Gln Gln Gln
865 870 875 880
Gln Gln Pro Leu Leu His Pro Pro Glu Pro Lys Ser Pro Gly Glu Tyr
885 890 895
Val Asn Ile Glu Phe Gly Ser Asp Gln Ser Gly Tyr Leu Ser Gly Pro
900 905 910
Val Ala Phe His Ser Ser Pro Ser Val Arg Cys Pro Ser Gln Leu Gln
915 920 925
Pro Ala Pro Arg Glu Glu Glu Thr Gly Thr Glu Glu Tyr Met Lys Met
930 935 940
Asp Leu Gly Pro Gly Arg Arg Ala Ala Trp Gln Glu Ser Thr Gly Val
945 950 955 960
Glu Met Gly Arg Leu Gly Pro Ala Pro Pro Gly Ala Ala Ser Ile Cys
965 970 975
Arg Pro Thr Arg Ala Val Pro Ser Ser Arg Gly Asp Tyr Met Thr Met
980 985 990
Gln Met Ser Cys Pro Arg Gln Ser Tyr Val Asp Thr Ser Pro Ala Ala
995 1000 1005
Pro Val Ser Tyr Ala Asp Met Arg Thr Gly Ile Ala Ala Glu Glu
1010 1015 1020
Val Ser Leu Pro Arg Ala Thr Met Ala Ala Ala Ser Ser Ser Ser
1025 1030 1035
Ala Ala Ser Ala Ser Pro Thr Gly Pro Gln Gly Ala Ala Glu Leu
1040 1045 1050
Ala Ala His Ser Ser Leu Leu Gly Gly Pro Gln Gly Pro Gly Gly
1055 1060 1065
Met Ser Ala Phe Thr Arg Val Asn Leu Ser Pro Asn Arg Asn Gln
1070 1075 1080
Ser Ala Lys Val Ile Arg Ala Asp Pro Gln Gly Cys Arg Arg Arg
1085 1090 1095
His Ser Ser Glu Thr Phe Ser Ser Thr Pro Ser Ala Thr Arg Val
1100 1105 1110
Gly Asn Thr Val Pro Phe Gly Ala Gly Ala Ala Val Gly Gly Gly
1115 1120 1125
Gly Gly Ser Ser Ser Ser Ser Glu Asp Val Lys Arg His Ser Ser
1130 1135 1140
Ala Ser Phe Glu Asn Val Trp Leu Arg Pro Gly Glu Leu Gly Gly
1145 1150 1155
Ala Pro Lys Glu Pro Ala Lys Leu Cys Gly Ala Ala Gly Gly Leu
1160 1165 1170
Glu Asn Gly Leu Asn Tyr Ile Asp Leu Asp Leu Val Lys Asp Phe
1175 1180 1185
Lys Gln Cys Pro Gln Glu Cys Thr Pro Glu Pro Gln Pro Pro Pro
1190 1195 1200
Pro Pro Pro Pro His Gln Pro Leu Gly Ser Gly Glu Ser Ser Ser
1205 1210 1215
Thr Arg Arg Ser Ser Glu Asp Leu Ser Ala Tyr Ala Ser Ile Ser
1220 1225 1230
Phe Gln Lys Gln Pro Glu Asp Arg Gln
1235 1240
<210> 108
<211> 3723
<212> DNA
<213> Canis lupus
<220>
<221> misc_feature
<222> (2356)..(2356)
<223> n is a, c, g, or t
<400> 108
atggcgagcc ctccggagac cgacggcttc tcggacgtgc gcaaggtggg ctacctgcgc 60
aaacccaaga gcatgcacaa gcgcttcttc gtgctgcggg cggccagcga ggcggggggc 120
ccggcgcgcc tcgagtacta cgagaacgag aagaagtggc ggcacaagtc gagcgccccc 180
aaacgctcga tccccctcga gagctgcttc aacatcaaca agcgggcgga ctccaagaac 240
aagcacctgg tggcccttta cacccgggac gagcactttg ccatcgcggc ggacagcgag 300
gccgagcagg acagctggta ccaggccctc ctgcagctgc acaaccgggc caagggccac 360
cacgacggcg cctcggcccc cggggcggga ggcggcgggg gcagctgcag cggcagctcg 420
ggcctcgggg aggccggcga ggacttgagc tacggggacg tgcccccggg acctgcgttc 480
aaggaggtct ggcaggtgat cctgaaaccc aagggcctgg ggcagacaaa gaacctgatt 540
ggcatctacc gcctctgcct gaccagcaag accatcagct tcgtgaagct gaactccgag 600
gcggcggccg tggtgctgca gctgatgaac atccgacgtt gcggccactc ggagaacttc 660
ttcttcatcg aagtgggccg ttccgcagtg acgggacccg gcgagttctg gatgcaggtg 720
gatgactccg tggtggccca gaacatgcac gagaccatcc tggaggccat gcgggccatg 780
agcgacgagt tccgccctcg gagtaagagc cagtcctcct ccaactgctc caaccccatc 840
agcgtccccc tgcgccggca ccacctcaac aacccccctc ccagccaggt ggggctgacg 900
cgccgctcgc gcaccgagag catcaccgcc acctctccgg ccagcatggt gggcgggaag 960
cagggctcct tccgtgtgcg cgcgtccagc gacggcgagg gcaccatgtc ccgcccggcc 1020
tcggtggacg gcagccccgt gagcccgagc accaccagga cccacgcgca ccggcatcgc 1080
ggcagctccc ggctgcaccc cccgctcaac cacagccgct ccatccccat gccttcctct 1140
cgctgctcgc cttccgccac cagcccggtc agcctgtcgt ccagcagcac cagtggccac 1200
ggctccacct cggactgcct cttcccccgg cgctctagtg cctctgtgtc gggttccccc 1260
agcgacggtg gtttcatctc ctctgacgag tacggctcga gtccctgcga tttccgaagt 1320
tccttccgca gtgtcacccc ggattccctg ggccacaccc ccccggcccg cggcgaggag 1380
gagctgagca actacatctg catgggaggc aaagggtcct ccaccctcac cgcccccaac 1440
ggtcactaca ttttgcctcg gggtggcaat ggccaccgct acatcccggg ggctggcttg 1500
ggcaccagcc cggccctggc tgcggatgaa gcggccgctg cggccgacct ggataaccgg 1560
ttccgaaagc ggactcactc cgcgggcaca tcccctacca tttcccacca gaagaccccg 1620
tcccagtctt ctgtggcttc cattgaggag tacacggaga tgatgcctgc ctacccgcca 1680
ggaggtggca gtggaggccg actgcctggc taccggcact ctgccttcgt gcccacccac 1740
tcctaccccg aggagggtct ggaaatgcac cctctggaca ggcgtggggg ccaccaccgg 1800
ccggacgccg ccgccctcca cacggatgat ggctacatgc ccatgtcccc gggagtggca 1860
ccggtgccca gcagccggaa gggcagtggg gactatatgc ccatgagccc caagagcgtg 1920
tccgcgccgc agcagatcat caaccccatt agacgccatc cccagagggt ggaccccaat 1980
ggctacatga tgatgtcccc aagcggcagc tgctctcctg acattggagg tgggcccggc 2040
agcagcagca gcggcagcgc cgccccttct gggagcagct atggcaagct gtggacaaac 2100
ggggtagggg gccaccaccc tcacgccctg ccgcacccca aactccccgt ggagagcggg 2160
agtggcaagc tcctgtcttg taccggcgac tacatgaaca tgtcgccggt gggggactcc 2220
aacaccagca gcccctccga cggctactac ggcccagagg acccccagca caagccagtt 2280
ctctcctact actcattgcc aaggtccttt aagcacaccc agcgccctgg ggagctggag 2340
gagagcgccc ggcacnagca cctccgcctc tcctccagct cgggtcgtct tctctacgcc 2400
gcgacggcgg aagattcctc ctcctccacc agcagcgaca gcctgggccc agggggatac 2460
tgtggggtca ggccggatcc cggcctcccg catatccacc atcaggtcct gcagcctcac 2520
ctgcctcgga aggtggacac ggccgcgcag accaacagcc gcctggctcg gcccacgagg 2580
ctgtccctgg gggaccccaa ggccagcacc ttacctcggg ttcgagagca gcagcacccg 2640
ccgcccctgc tgcaccctcc ggagcccaag agccccgggg aatatgtgaa tattgagttc 2700
gggagcgatc agccgggcta cttatcgggg ccggtggctg cccgcagctc gccttctgtc 2760
aggtgcccac cccagctcca gccagctccc cgcgaggaag agactggcac cgaggagtac 2820
atgaacatgg acctggggcc tggccggagg gcagcctggc aggagggtgc tggggtccag 2880
cccggcaggg tgggccccgc gccccccggg gccgctagcg tgtgcaggcc cacccgggca 2940
gtgcccagca gccggggcga ctacatgacc atgcaggtgg gctgtcccgg ccagggctac 3000
gtggacacct cgccagtggc ccccatcagc tacgctgaca tgcggacagg cattgtcgtg 3060
gaggaggcca gcctgccggg ggccacagcg gccgccccct cctcggcctc ggcagcctcg 3120
gcttccccca cggcgcctcc aaaagcgggg gagctggtgg cccgctcctc cctgctgggg 3180
ggcccgcagg gacccggggg catgagcgcc ttcacccggg tgaacctcag ccccaaccgc 3240
aaccagagtg ccaaagtgat ccgcgccgac ccgcaggggt gccggaggcg gcatagctct 3300
gagaccttct cctccacgcc cagtgccacc cgggcgggca acgcagtgcc cttcggcggg 3360
ggggcggccc tggggggcag cggtggcggc agcagcgcgg aggatatgaa acgccacagt 3420
tcggcttcct ttgagaacgt gtggctgagg cctggggagc tcgggggagc ccccaaggag 3480
ccggccccgc acgctggggc cgccgggggt ttggagaatg ggcttaacta catagacctg 3540
gatttggtca aggacttcaa acagtgctct caggagcgcc cccctcaacc gcagccgccc 3600
ccgcccccgg cccctcatca gcctctgggc agcagtgaga gcagttcaac cagccgctcc 3660
agcgaggatc taagcgccta tgccagcatc agtttccaga agcagccaga ggacctccag 3720
tag 3723
<210> 109
<211> 1240
<212> PRT
<213> Canis lupus
<220>
<221> misc_feature
<222> (786)..(786)
<223> Xaa can be any naturally occurring amino acid
<400> 109
Met Ala Ser Pro Pro Glu Thr Asp Gly Phe Ser Asp Val Arg Lys Val
1 5 10 15
Gly Tyr Leu Arg Lys Pro Lys Ser Met His Lys Arg Phe Phe Val Leu
20 25 30
Arg Ala Ala Ser Glu Ala Gly Gly Pro Ala Arg Leu Glu Tyr Tyr Glu
35 40 45
Asn Glu Lys Lys Trp Arg His Lys Ser Ser Ala Pro Lys Arg Ser Ile
50 55 60
Pro Leu Glu Ser Cys Phe Asn Ile Asn Lys Arg Ala Asp Ser Lys Asn
65 70 75 80
Lys His Leu Val Ala Leu Tyr Thr Arg Asp Glu His Phe Ala Ile Ala
85 90 95
Ala Asp Ser Glu Ala Glu Gln Asp Ser Trp Tyr Gln Ala Leu Leu Gln
100 105 110
Leu His Asn Arg Ala Lys Gly His His Asp Gly Ala Ser Ala Pro Gly
115 120 125
Ala Gly Gly Gly Gly Gly Ser Cys Ser Gly Ser Ser Gly Leu Gly Glu
130 135 140
Ala Gly Glu Asp Leu Ser Tyr Gly Asp Val Pro Pro Gly Pro Ala Phe
145 150 155 160
Lys Glu Val Trp Gln Val Ile Leu Lys Pro Lys Gly Leu Gly Gln Thr
165 170 175
Lys Asn Leu Ile Gly Ile Tyr Arg Leu Cys Leu Thr Ser Lys Thr Ile
180 185 190
Ser Phe Val Lys Leu Asn Ser Glu Ala Ala Ala Val Val Leu Gln Leu
195 200 205
Met Asn Ile Arg Arg Cys Gly His Ser Glu Asn Phe Phe Phe Ile Glu
210 215 220
Val Gly Arg Ser Ala Val Thr Gly Pro Gly Glu Phe Trp Met Gln Val
225 230 235 240
Asp Asp Ser Val Val Ala Gln Asn Met His Glu Thr Ile Leu Glu Ala
245 250 255
Met Arg Ala Met Ser Asp Glu Phe Arg Pro Arg Ser Lys Ser Gln Ser
260 265 270
Ser Ser Asn Cys Ser Asn Pro Ile Ser Val Pro Leu Arg Arg His His
275 280 285
Leu Asn Asn Pro Pro Pro Ser Gln Val Gly Leu Thr Arg Arg Ser Arg
290 295 300
Thr Glu Ser Ile Thr Ala Thr Ser Pro Ala Ser Met Val Gly Gly Lys
305 310 315 320
Gln Gly Ser Phe Arg Val Arg Ala Ser Ser Asp Gly Glu Gly Thr Met
325 330 335
Ser Arg Pro Ala Ser Val Asp Gly Ser Pro Val Ser Pro Ser Thr Thr
340 345 350
Arg Thr His Ala His Arg His Arg Gly Ser Ser Arg Leu His Pro Pro
355 360 365
Leu Asn His Ser Arg Ser Ile Pro Met Pro Ser Ser Arg Cys Ser Pro
370 375 380
Ser Ala Thr Ser Pro Val Ser Leu Ser Ser Ser Ser Thr Ser Gly His
385 390 395 400
Gly Ser Thr Ser Asp Cys Leu Phe Pro Arg Arg Ser Ser Ala Ser Val
405 410 415
Ser Gly Ser Pro Ser Asp Gly Gly Phe Ile Ser Ser Asp Glu Tyr Gly
420 425 430
Ser Ser Pro Cys Asp Phe Arg Ser Ser Phe Arg Ser Val Thr Pro Asp
435 440 445
Ser Leu Gly His Thr Pro Pro Ala Arg Gly Glu Glu Glu Leu Ser Asn
450 455 460
Tyr Ile Cys Met Gly Gly Lys Gly Ser Ser Thr Leu Thr Ala Pro Asn
465 470 475 480
Gly His Tyr Ile Leu Pro Arg Gly Gly Asn Gly His Arg Tyr Ile Pro
485 490 495
Gly Ala Gly Leu Gly Thr Ser Pro Ala Leu Ala Ala Asp Glu Ala Ala
500 505 510
Ala Ala Ala Asp Leu Asp Asn Arg Phe Arg Lys Arg Thr His Ser Ala
515 520 525
Gly Thr Ser Pro Thr Ile Ser His Gln Lys Thr Pro Ser Gln Ser Ser
530 535 540
Val Ala Ser Ile Glu Glu Tyr Thr Glu Met Met Pro Ala Tyr Pro Pro
545 550 555 560
Gly Gly Gly Ser Gly Gly Arg Leu Pro Gly Tyr Arg His Ser Ala Phe
565 570 575
Val Pro Thr His Ser Tyr Pro Glu Glu Gly Leu Glu Met His Pro Leu
580 585 590
Asp Arg Arg Gly Gly His His Arg Pro Asp Ala Ala Ala Leu His Thr
595 600 605
Asp Asp Gly Tyr Met Pro Met Ser Pro Gly Val Ala Pro Val Pro Ser
610 615 620
Ser Arg Lys Gly Ser Gly Asp Tyr Met Pro Met Ser Pro Lys Ser Val
625 630 635 640
Ser Ala Pro Gln Gln Ile Ile Asn Pro Ile Arg Arg His Pro Gln Arg
645 650 655
Val Asp Pro Asn Gly Tyr Met Met Met Ser Pro Ser Gly Ser Cys Ser
660 665 670
Pro Asp Ile Gly Gly Gly Pro Gly Ser Ser Ser Ser Gly Ser Ala Ala
675 680 685
Pro Ser Gly Ser Ser Tyr Gly Lys Leu Trp Thr Asn Gly Val Gly Gly
690 695 700
His His Pro His Ala Leu Pro His Pro Lys Leu Pro Val Glu Ser Gly
705 710 715 720
Ser Gly Lys Leu Leu Ser Cys Thr Gly Asp Tyr Met Asn Met Ser Pro
725 730 735
Val Gly Asp Ser Asn Thr Ser Ser Pro Ser Asp Gly Tyr Tyr Gly Pro
740 745 750
Glu Asp Pro Gln His Lys Pro Val Leu Ser Tyr Tyr Ser Leu Pro Arg
755 760 765
Ser Phe Lys His Thr Gln Arg Pro Gly Glu Leu Glu Glu Ser Ala Arg
770 775 780
His Xaa His Leu Arg Leu Ser Ser Ser Ser Gly Arg Leu Leu Tyr Ala
785 790 795 800
Ala Thr Ala Glu Asp Ser Ser Ser Ser Thr Ser Ser Asp Ser Leu Gly
805 810 815
Pro Gly Gly Tyr Cys Gly Val Arg Pro Asp Pro Gly Leu Pro His Ile
820 825 830
His His Gln Val Leu Gln Pro His Leu Pro Arg Lys Val Asp Thr Ala
835 840 845
Ala Gln Thr Asn Ser Arg Leu Ala Arg Pro Thr Arg Leu Ser Leu Gly
850 855 860
Asp Pro Lys Ala Ser Thr Leu Pro Arg Val Arg Glu Gln Gln His Pro
865 870 875 880
Pro Pro Leu Leu His Pro Pro Glu Pro Lys Ser Pro Gly Glu Tyr Val
885 890 895
Asn Ile Glu Phe Gly Ser Asp Gln Pro Gly Tyr Leu Ser Gly Pro Val
900 905 910
Ala Ala Arg Ser Ser Pro Ser Val Arg Cys Pro Pro Gln Leu Gln Pro
915 920 925
Ala Pro Arg Glu Glu Glu Thr Gly Thr Glu Glu Tyr Met Asn Met Asp
930 935 940
Leu Gly Pro Gly Arg Arg Ala Ala Trp Gln Glu Gly Ala Gly Val Gln
945 950 955 960
Pro Gly Arg Val Gly Pro Ala Pro Pro Gly Ala Ala Ser Val Cys Arg
965 970 975
Pro Thr Arg Ala Val Pro Ser Ser Arg Gly Asp Tyr Met Thr Met Gln
980 985 990
Val Gly Cys Pro Gly Gln Gly Tyr Val Asp Thr Ser Pro Val Ala Pro
995 1000 1005
Ile Ser Tyr Ala Asp Met Arg Thr Gly Ile Val Val Glu Glu Ala
1010 1015 1020
Ser Leu Pro Gly Ala Thr Ala Ala Ala Pro Ser Ser Ala Ser Ala
1025 1030 1035
Ala Ser Ala Ser Pro Thr Ala Pro Pro Lys Ala Gly Glu Leu Val
1040 1045 1050
Ala Arg Ser Ser Leu Leu Gly Gly Pro Gln Gly Pro Gly Gly Met
1055 1060 1065
Ser Ala Phe Thr Arg Val Asn Leu Ser Pro Asn Arg Asn Gln Ser
1070 1075 1080
Ala Lys Val Ile Arg Ala Asp Pro Gln Gly Cys Arg Arg Arg His
1085 1090 1095
Ser Ser Glu Thr Phe Ser Ser Thr Pro Ser Ala Thr Arg Ala Gly
1100 1105 1110
Asn Ala Val Pro Phe Gly Gly Gly Ala Ala Leu Gly Gly Ser Gly
1115 1120 1125
Gly Gly Ser Ser Ala Glu Asp Met Lys Arg His Ser Ser Ala Ser
1130 1135 1140
Phe Glu Asn Val Trp Leu Arg Pro Gly Glu Leu Gly Gly Ala Pro
1145 1150 1155
Lys Glu Pro Ala Pro His Ala Gly Ala Ala Gly Gly Leu Glu Asn
1160 1165 1170
Gly Leu Asn Tyr Ile Asp Leu Asp Leu Val Lys Asp Phe Lys Gln
1175 1180 1185
Cys Ser Gln Glu Arg Pro Pro Gln Pro Gln Pro Pro Pro Pro Pro
1190 1195 1200
Ala Pro His Gln Pro Leu Gly Ser Ser Glu Ser Ser Ser Thr Ser
1205 1210 1215
Arg Ser Ser Glu Asp Leu Ser Ala Tyr Ala Ser Ile Ser Phe Gln
1220 1225 1230
Lys Gln Pro Glu Asp Leu Gln
1235 1240
<210> 110
<211> 3033
<212> DNA
<213> Homo sapiens
<400> 110
atgggctatt tgtgtgttaa tttcatttgg ttcttgggaa taacgactca ccgcgttgat 60
ttaaagaaag aactaaaatt ccagatggca aactcaatga atggcagaaa ccctggtggt 120
cgaggaggaa atccccgaaa aggtcgaatt ttgggtatta ttgatgctat tcaggatgca 180
gttggacccc ctaagcaagc tgccgcagat cgcaggaccg tggagaagac ttggaagctc 240
atggacaaag tggtaagact gtgccaaaat cccaaacttc agttgaaaaa tagcccacca 300
tatatacttg atattttgcc tgatacatat cagcatttac gacttatatt gagtaaatat 360
gatgacaacc agaaacttgc ccaactcagt gagaatgagt actttaaaat ctacattgat 420
agccttatga aaaagtcaaa acgggcaata agactcttta aagaaggcaa ggagagaatg 480
tatgaagaac agtcacagga cagacgaaat ctcacaaaac tgtcccttat cttcagtcac 540
atgctggcag aaatcaaagc aatctttccc aatggtcaat tccagggaga taactttcgt 600
atcacaaaag cagatgctgc tgaattctgg agaaagtttt ttggagacaa aactatcgta 660
ccatggaaag tattcagaca gtgccttcat gaggtccacc agattagctc tggcctggaa 720
gcaatggctc taaaatcaac aattgattta acttgcaatg attacatttc agtttttgaa 780
tttgatattt ttaccaggct gtttcagcct tggggctcta ttttgcggaa ttggaatttc 840
ttagctgtga cacatccagg ttacatggca tttctcacat atgatgaagt taaagcacga 900
ctacagaaat atagcaccaa acccggaagc tatattttcc ggttaagttg cactcgattg 960
ggacagtggg ccattggcta tgtgactggg gatgggaata tcttacagac catacctcat 1020
aacaagccct tatttcaagc cctgattgat ggcagcaggg aaggatttta tctttatcct 1080
gatgggagga gttataatcc tgatttaact ggattatgtg aacctacacc tcatgaccat 1140
ataaaagtta cacaggaaca atatgaatta tattgtgaaa tgggctccac ttttcagctc 1200
tgtaagattt gtgcagagaa tgacaaagat gtcaagattg agccttgtgg gcatttgatg 1260
tgcacctctt gccttacggc atggcaggag tcggatggtc agggctgccc tttctgtcgt 1320
tgtgaaataa aaggaactga gcccataatc gtggacccct ttgatccaag agatgaaggc 1380
tccaggtgtt gcagcatcat tgaccccttt ggcatgccga tgctagactt ggacgacgat 1440
gatgatcgtg aggagtcctt gatgatgaat cggttggcaa acgtccgaaa gtgcactgac 1500
aggcagaact caccagtcac atcaccagga tcctctcccc ttgcccagag aagaaagcca 1560
cagcctgacc cactccagat cccacatcta agcctgccac ccgtgcctcc tcgcctggat 1620
ctaattcaga aaggcatagt tagatctccc tgtggcagcc caacgggttc accaaagtct 1680
tctccttgca tggtgagaaa acaagataaa ccactcccag caccacctcc tcccttaaga 1740
gatcctcctc caccgccacc tgaaagacct ccaccaatcc caccagacaa tagactgagt 1800
agacacatcc atcatgtgga aagcgtgcct tccagagacc cgccaatgcc tcttgaagca 1860
tggtgccctc gggatgtgtt tgggactaat cagcttgtgg gatgtcgact cctaggggag 1920
ggctctccaa aacctggaat cacagcgagt tcaaatgtca atggaaggca cagtagagtg 1980
ggctctgacc cagtgcttat gcggaaacac agacgccatg atttgccttt agaaggagct 2040
aaggtctttt ccaatggtca ccttggaagt gaagaatatg atgttcctcc ccggctttct 2100
cctcctcctc cagttaccac cctcctccct agcataaagt gtactggtcc gttagcaaat 2160
tctctttcag agaaaacaag agacccagta gaggaagatg atgatgaata caagattcct 2220
tcatcccacc ctgtttccct gaattcacaa ccatctcatt gtcataatgt aaaacctcct 2280
gttcggtctt gtgataatgg tcactgtatg ctgaatggaa cacatggtcc atcttcagag 2340
aagaaatcaa acatccctga cttaagcata tatttaaagg gagatgtttt tgattcagcc 2400
tctgatcccg tgccattacc acctgccagg cctccaactc gggacaatcc aaagcatggt 2460
tcttcactca acaggacgcc ctctgattat gatcttctca tccctccatt aggtgaagat 2520
gcttttgatg ccctccctcc atctctccca cctcccccac ctcctgcaag gcatagtctc 2580
attgaacatt caaaacctcc tggctccagt agccggccat cctcaggaca ggatcttttt 2640
cttcttcctt cagatccctt tgttgatcta gcaagtggcc aagttccttt gcctcctgct 2700
agaaggttac caggtgaaaa tgtcaaaact aacagaacat cacaggacta tgatcagctt 2760
ccttcatgtt cagatggttc acaggcacca gccagacccc ctaaaccacg accgcgcagg 2820
actgcaccag aaattcacca cagaaaaccc catgggcctg aggcggcatt ggaaaatgtc 2880
gatgcaaaaa ttgcaaaact catgggagag ggttatgcct ttgaagaggt gaagagagcc 2940
ttagagatag cccagaataa tgtcgaagtt gcccggagca tcctccgaga atttgccttc 3000
cctcctccag tatccccacg tctaaatcta tag 3033
<210> 111
<211> 1010
<212> PRT
<213> Homo sapiens
<400> 111
Met Gly Tyr Leu Cys Val Asn Phe Ile Trp Phe Leu Gly Ile Thr Thr
1 5 10 15
His Arg Val Asp Leu Lys Lys Glu Leu Lys Phe Gln Met Ala Asn Ser
20 25 30
Met Asn Gly Arg Asn Pro Gly Gly Arg Gly Gly Asn Pro Arg Lys Gly
35 40 45
Arg Ile Leu Gly Ile Ile Asp Ala Ile Gln Asp Ala Val Gly Pro Pro
50 55 60
Lys Gln Ala Ala Ala Asp Arg Arg Thr Val Glu Lys Thr Trp Lys Leu
65 70 75 80
Met Asp Lys Val Val Arg Leu Cys Gln Asn Pro Lys Leu Gln Leu Lys
85 90 95
Asn Ser Pro Pro Tyr Ile Leu Asp Ile Leu Pro Asp Thr Tyr Gln His
100 105 110
Leu Arg Leu Ile Leu Ser Lys Tyr Asp Asp Asn Gln Lys Leu Ala Gln
115 120 125
Leu Ser Glu Asn Glu Tyr Phe Lys Ile Tyr Ile Asp Ser Leu Met Lys
130 135 140
Lys Ser Lys Arg Ala Ile Arg Leu Phe Lys Glu Gly Lys Glu Arg Met
145 150 155 160
Tyr Glu Glu Gln Ser Gln Asp Arg Arg Asn Leu Thr Lys Leu Ser Leu
165 170 175
Ile Phe Ser His Met Leu Ala Glu Ile Lys Ala Ile Phe Pro Asn Gly
180 185 190
Gln Phe Gln Gly Asp Asn Phe Arg Ile Thr Lys Ala Asp Ala Ala Glu
195 200 205
Phe Trp Arg Lys Phe Phe Gly Asp Lys Thr Ile Val Pro Trp Lys Val
210 215 220
Phe Arg Gln Cys Leu His Glu Val His Gln Ile Ser Ser Gly Leu Glu
225 230 235 240
Ala Met Ala Leu Lys Ser Thr Ile Asp Leu Thr Cys Asn Asp Tyr Ile
245 250 255
Ser Val Phe Glu Phe Asp Ile Phe Thr Arg Leu Phe Gln Pro Trp Gly
260 265 270
Ser Ile Leu Arg Asn Trp Asn Phe Leu Ala Val Thr His Pro Gly Tyr
275 280 285
Met Ala Phe Leu Thr Tyr Asp Glu Val Lys Ala Arg Leu Gln Lys Tyr
290 295 300
Ser Thr Lys Pro Gly Ser Tyr Ile Phe Arg Leu Ser Cys Thr Arg Leu
305 310 315 320
Gly Gln Trp Ala Ile Gly Tyr Val Thr Gly Asp Gly Asn Ile Leu Gln
325 330 335
Thr Ile Pro His Asn Lys Pro Leu Phe Gln Ala Leu Ile Asp Gly Ser
340 345 350
Arg Glu Gly Phe Tyr Leu Tyr Pro Asp Gly Arg Ser Tyr Asn Pro Asp
355 360 365
Leu Thr Gly Leu Cys Glu Pro Thr Pro His Asp His Ile Lys Val Thr
370 375 380
Gln Glu Gln Tyr Glu Leu Tyr Cys Glu Met Gly Ser Thr Phe Gln Leu
385 390 395 400
Cys Lys Ile Cys Ala Glu Asn Asp Lys Asp Val Lys Ile Glu Pro Cys
405 410 415
Gly His Leu Met Cys Thr Ser Cys Leu Thr Ala Trp Gln Glu Ser Asp
420 425 430
Gly Gln Gly Cys Pro Phe Cys Arg Cys Glu Ile Lys Gly Thr Glu Pro
435 440 445
Ile Ile Val Asp Pro Phe Asp Pro Arg Asp Glu Gly Ser Arg Cys Cys
450 455 460
Ser Ile Ile Asp Pro Phe Gly Met Pro Met Leu Asp Leu Asp Asp Asp
465 470 475 480
Asp Asp Arg Glu Glu Ser Leu Met Met Asn Arg Leu Ala Asn Val Arg
485 490 495
Lys Cys Thr Asp Arg Gln Asn Ser Pro Val Thr Ser Pro Gly Ser Ser
500 505 510
Pro Leu Ala Gln Arg Arg Lys Pro Gln Pro Asp Pro Leu Gln Ile Pro
515 520 525
His Leu Ser Leu Pro Pro Val Pro Pro Arg Leu Asp Leu Ile Gln Lys
530 535 540
Gly Ile Val Arg Ser Pro Cys Gly Ser Pro Thr Gly Ser Pro Lys Ser
545 550 555 560
Ser Pro Cys Met Val Arg Lys Gln Asp Lys Pro Leu Pro Ala Pro Pro
565 570 575
Pro Pro Leu Arg Asp Pro Pro Pro Pro Pro Pro Glu Arg Pro Pro Pro
580 585 590
Ile Pro Pro Asp Asn Arg Leu Ser Arg His Ile His His Val Glu Ser
595 600 605
Val Pro Ser Arg Asp Pro Pro Met Pro Leu Glu Ala Trp Cys Pro Arg
610 615 620
Asp Val Phe Gly Thr Asn Gln Leu Val Gly Cys Arg Leu Leu Gly Glu
625 630 635 640
Gly Ser Pro Lys Pro Gly Ile Thr Ala Ser Ser Asn Val Asn Gly Arg
645 650 655
His Ser Arg Val Gly Ser Asp Pro Val Leu Met Arg Lys His Arg Arg
660 665 670
His Asp Leu Pro Leu Glu Gly Ala Lys Val Phe Ser Asn Gly His Leu
675 680 685
Gly Ser Glu Glu Tyr Asp Val Pro Pro Arg Leu Ser Pro Pro Pro Pro
690 695 700
Val Thr Thr Leu Leu Pro Ser Ile Lys Cys Thr Gly Pro Leu Ala Asn
705 710 715 720
Ser Leu Ser Glu Lys Thr Arg Asp Pro Val Glu Glu Asp Asp Asp Glu
725 730 735
Tyr Lys Ile Pro Ser Ser His Pro Val Ser Leu Asn Ser Gln Pro Ser
740 745 750
His Cys His Asn Val Lys Pro Pro Val Arg Ser Cys Asp Asn Gly His
755 760 765
Cys Met Leu Asn Gly Thr His Gly Pro Ser Ser Glu Lys Lys Ser Asn
770 775 780
Ile Pro Asp Leu Ser Ile Tyr Leu Lys Gly Asp Val Phe Asp Ser Ala
785 790 795 800
Ser Asp Pro Val Pro Leu Pro Pro Ala Arg Pro Pro Thr Arg Asp Asn
805 810 815
Pro Lys His Gly Ser Ser Leu Asn Arg Thr Pro Ser Asp Tyr Asp Leu
820 825 830
Leu Ile Pro Pro Leu Gly Glu Asp Ala Phe Asp Ala Leu Pro Pro Ser
835 840 845
Leu Pro Pro Pro Pro Pro Pro Ala Arg His Ser Leu Ile Glu His Ser
850 855 860
Lys Pro Pro Gly Ser Ser Ser Arg Pro Ser Ser Gly Gln Asp Leu Phe
865 870 875 880
Leu Leu Pro Ser Asp Pro Phe Val Asp Leu Ala Ser Gly Gln Val Pro
885 890 895
Leu Pro Pro Ala Arg Arg Leu Pro Gly Glu Asn Val Lys Thr Asn Arg
900 905 910
Thr Ser Gln Asp Tyr Asp Gln Leu Pro Ser Cys Ser Asp Gly Ser Gln
915 920 925
Ala Pro Ala Arg Pro Pro Lys Pro Arg Pro Arg Arg Thr Ala Pro Glu
930 935 940
Ile His His Arg Lys Pro His Gly Pro Glu Ala Ala Leu Glu Asn Val
945 950 955 960
Asp Ala Lys Ile Ala Lys Leu Met Gly Glu Gly Tyr Ala Phe Glu Glu
965 970 975
Val Lys Arg Ala Leu Glu Ile Ala Gln Asn Asn Val Glu Val Ala Arg
980 985 990
Ser Ile Leu Arg Glu Phe Ala Phe Pro Pro Pro Val Ser Pro Arg Leu
995 1000 1005
Asn Leu
1010
<210> 112
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 112
gcctgataca tatcagcat 19
<210> 113
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 113
gcggaattgg aatttctta 19
<210> 114
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 114
gcatgccgat gctagactt 19
<210> 115
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 115
gcctgataca tatcagcat 19
<210> 116
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 116
ggagagaatg tatgaagaac a 21
<210> 117
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 117
gcggaattgg aatttcttag c 21
<210> 118
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 118
gcacgactac agaaatatag c 21
<210> 119
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 119
ggaatatctt acagaccata c 21
<210> 120
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 120
gcaccaaacc cggaagctat a 21
<210> 121
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 121
gcctggatct aattcagaaa g 21
<210> 122
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 122
ggaatcacag cgagttcaaa t 21
<210> 123
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 123
ggaacacatg gtccatcttc a 21
<210> 124
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl-b
<400> 124
gcatagtctc attgaacatt c 21
<210> 125
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 125
gttgcgtttc cacgtctcgg 20
<210> 126
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 126
gaacagctcg ctcccgaaga 20
<210> 127
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 127
attgttgcgt ttccacgtct 20
<210> 128
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 128
agtgctgctg cggcgtcccg 20
<210> 129
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 129
aggaggagga gaccgctcgc 20
<210> 130
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 130
gaaggagcaa cccagcgcgc 20
<210> 131
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 131
gcgcgcaggc ctccgagacg 20
<210> 132
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 132
cgtctcggag gcctgcgcgc 20
<210> 133
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 133
gtcccgcggc ctccccgagt 20
<210> 134
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 134
ctcccctccc gcccgactcg 20
<210> 135
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 135
gacgccgcag cagcactagc 20
<210> 136
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 136
gtctcggagg cctgcgcgct 20
<210> 137
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 137
gcggcctccc cgagtcgggc 20
<210> 138
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 138
ccctcccgcc cgactcgggg 20
<210> 139
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 139
cgcggcctcc ccgagtcggg 20
<210> 140
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 140
ctccccgagt cgggcgggag 20
<210> 141
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 141
cgggtgtgga tttgtcttga 20
<210> 142
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 142
gcctccccga gtcgggcggg 20
<210> 143
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 143
tcccgcggcc tccccgagtc 20
<210> 144
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 144
cgcccgactc ggggaggccg 20
<210> 145
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 145
ctctcccctc ccgcccgact 20
<210> 146
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 146
tctcccctcc cgcccgactc 20
<210> 147
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 147
agcgatccca ctcccagccg 20
<210> 148
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 148
tcagcgatcc cactcccagc 20
<210> 149
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 149
cgctgggttg ctccttcttc 20
<210> 150
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 150
gcccgactcg gggaggccgc 20
<210> 151
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 151
gcgctgggtt gctccttctt 20
<210> 152
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 152
cctccccgag tcgggcggga 20
<210> 153
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 153
tgtgtgtggg gagccccggc 20
<210> 154
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 154
gtgtgtgggg agccccggct 20
<210> 155
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 155
cgctggacac cccacccctg 20
<210> 156
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 156
gccgcagcag cactagcagg 20
<210> 157
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 157
cggggctccc cacacacact 20
<210> 158
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl-b
<400> 158
ctgggtcctg tgtgtgccac 20
<210> 159
<211> 2952
<212> DNA
<213> Canis lupus
<400> 159
atggcaaatt ctatgaatgg cagaaaccct ggtggtcgag gaggaaaccc ccgaaaagga 60
cggattttgg gtatcattga tgctattcaa gatgcagttg gacctccgaa gcaagcagca 120
gcagatcgca ggacggtgga gaaaacttgg aaactcatgg acaaagtggt cagactgtgt 180
caaaatccca agcttcagtt gaaaaatagc ccaccatata tacttgatat cttacctgat 240
acatatcagc atttacgact tatactgagt aaatatgatg acaaccagaa acttgcccaa 300
ctcagtgaga atgagtattt taaaatctac atcgatagtc taatgaaaaa gtcaaagcgg 360
gcaataagac tctttaaaga aggcaaggag aggatgtatg aagagcagtc acaggacaga 420
cgaaatctca caaaactgtc ccttatcttc agtcacatgc tggcagaaat caaagcaatc 480
tttcccaatg ggcagttcca gggagataac tttcgtatca cgaaagcaga tgctgctgaa 540
ttctggagaa agttttttgg agacaaaact attgtaccat ggaaagtatt cagacagtgc 600
cttcatgagg ttcatcaaat tagctctggc ctggaagcaa tggctctgaa atcaacaatt 660
gatttaactt gtaatgatta catttcagtt tttgaatttg atatttttac caggctcttt 720
cagccttggg gctctatttt acggaattgg aatttcttag ctgtaacaca tccaggttac 780
atggcatttc tcacatacga tgaagttaaa gcacgactgc agaaatacag caccaaacct 840
ggaagctaca ttttccggtt aagctgcacc agattgggac agtgggccat tggctatgtg 900
acaggggatg gcaatatctt acagaccata ccacataaca agcccttgtt tcaagccctg 960
attgatggca gcagggaagg attctatctt tatcctgatg ggaggagtta taatcctgat 1020
ttaactggat tatgtgaacc cacaccacat gaccatataa aagttacgca ggaacaatat 1080
gaattatatt gtgaaatggg ctccactttt cagctctgta aaatttgtgc tgagaacgac 1140
aaagatgtca agattgagcc ctgtgggcat ttgatgtgca cctcttgcct tacagcgtgg 1200
caggagtcgg acggccaagg ctgccccttt tgccgctgtg aaataaaagg aacagagccc 1260
ataatcgtgg acccctttga tccaagagat gaaggttcca ggtgctgtag catcattgac 1320
ccctttggaa tgccaatgct ggacctggat gatgacgatg accgagaaga gtccttgatg 1380
atgaatcggt tggcaaatgt tcgaaagtgc actgataggc aaaattcacc agtcacatca 1440
ccaggatcct ctccccttgc acagagaaga aagccacatc cagatcctct ccagatccca 1500
catctgagcc tgccaccagt acctcctcgc ctggatctaa ttcagaaagg catagttcgg 1560
tctccctgtg gcagtcccac tggttcacca aagtcttctc cttgcatggt gagaaaacaa 1620
gataaaccac tcccagcacc gcctcctccc ttaagagatc ctcctccacc tccccctgag 1680
agacctcccc cgatcccacc tgacaacaga ctgagtcgac acttccatca cgtggaaagt 1740
gtgccttcta gagaccagcc aatgcctctt gaagcctggt gccctcggga tgtgtttgga 1800
actaatcagt cagtgggttg tcgacaatta ggggatggct ctccaaagcc tggaatcaca 1860
gcaagttcaa atgtaaatgg aaggcacagt agaatgggct ctgaccctgt gcttctgcga 1920
aaacacagac gccacgattt gcctttagaa ggagccaagg tcttttccaa tggtcacctg 1980
ggaagcgaag agtacgatgt tcctccccgg ctttcacctc ctcctccagc tgccaccctt 2040
gtccctagca tcaagtgtac tggcccgtta gcaaatcccc tttcagagaa aaccagagac 2100
ccagtcgagg aagatgatga tgaatacaag attccttcat cccatcctgt ttccctgaat 2160
tcacaaccat ctcattgcca taacgtaaaa cctcctctta ggtcttgtga taatggtcat 2220
tgtgtattga atggaacaca tggtacatct tcagaggtga agaaatcaaa catccctgaa 2280
ttaggcattt atttaaaggg agatgttttt gattcagcct ctgatccagt gccattacca 2340
cctgccaggc ctccaactcg ggacaatcca aagcatggtt cttcactcaa caggacgccc 2400
tctgattatg atcttctcat ccctccatta ggtgaagatg cttttgatgc cctcccccca 2460
tccctcccgc ctcccccacc tcccgcaagg cacagcctca tcgaacactc taaacctccc 2520
ggctccaata gccgaccatc ctcaggacag gaccttttcc ttcttccttc agaccccttc 2580
tttgatccag taagtggtca agtccctctg cctcctgcta ggagattacc aggggaaaat 2640
gtcaaatcca acagaacatc acaggactat gatcagcttc cttcagcttc agatggttcg 2700
caggcaccag cccggcctcc caagccgcgc ccgcgcagga ccgcccccga ggtccagcac 2760
cggaagcccc acgggcccga ggcagcgtcg gaaaacgtgg acgcgaagat cgccaaactc 2820
atgggggagg gctacgcctt cgaggaagtg aagagggcgc tggagatcgc ccagaacaac 2880
gtcgaggtgg cccggagcat cctgcgcgag ttcgcctacc cgccgcccgt ctccccgcgg 2940
ctgcacctct ag 2952
<210> 160
<211> 983
<212> PRT
<213> Canis lupus
<400> 160
Met Ala Asn Ser Met Asn Gly Arg Asn Pro Gly Gly Arg Gly Gly Asn
1 5 10 15
Pro Arg Lys Gly Arg Ile Leu Gly Ile Ile Asp Ala Ile Gln Asp Ala
20 25 30
Val Gly Pro Pro Lys Gln Ala Ala Ala Asp Arg Arg Thr Val Glu Lys
35 40 45
Thr Trp Lys Leu Met Asp Lys Val Val Arg Leu Cys Gln Asn Pro Lys
50 55 60
Leu Gln Leu Lys Asn Ser Pro Pro Tyr Ile Leu Asp Ile Leu Pro Asp
65 70 75 80
Thr Tyr Gln His Leu Arg Leu Ile Leu Ser Lys Tyr Asp Asp Asn Gln
85 90 95
Lys Leu Ala Gln Leu Ser Glu Asn Glu Tyr Phe Lys Ile Tyr Ile Asp
100 105 110
Ser Leu Met Lys Lys Ser Lys Arg Ala Ile Arg Leu Phe Lys Glu Gly
115 120 125
Lys Glu Arg Met Tyr Glu Glu Gln Ser Gln Asp Arg Arg Asn Leu Thr
130 135 140
Lys Leu Ser Leu Ile Phe Ser His Met Leu Ala Glu Ile Lys Ala Ile
145 150 155 160
Phe Pro Asn Gly Gln Phe Gln Gly Asp Asn Phe Arg Ile Thr Lys Ala
165 170 175
Asp Ala Ala Glu Phe Trp Arg Lys Phe Phe Gly Asp Lys Thr Ile Val
180 185 190
Pro Trp Lys Val Phe Arg Gln Cys Leu His Glu Val His Gln Ile Ser
195 200 205
Ser Gly Leu Glu Ala Met Ala Leu Lys Ser Thr Ile Asp Leu Thr Cys
210 215 220
Asn Asp Tyr Ile Ser Val Phe Glu Phe Asp Ile Phe Thr Arg Leu Phe
225 230 235 240
Gln Pro Trp Gly Ser Ile Leu Arg Asn Trp Asn Phe Leu Ala Val Thr
245 250 255
His Pro Gly Tyr Met Ala Phe Leu Thr Tyr Asp Glu Val Lys Ala Arg
260 265 270
Leu Gln Lys Tyr Ser Thr Lys Pro Gly Ser Tyr Ile Phe Arg Leu Ser
275 280 285
Cys Thr Arg Leu Gly Gln Trp Ala Ile Gly Tyr Val Thr Gly Asp Gly
290 295 300
Asn Ile Leu Gln Thr Ile Pro His Asn Lys Pro Leu Phe Gln Ala Leu
305 310 315 320
Ile Asp Gly Ser Arg Glu Gly Phe Tyr Leu Tyr Pro Asp Gly Arg Ser
325 330 335
Tyr Asn Pro Asp Leu Thr Gly Leu Cys Glu Pro Thr Pro His Asp His
340 345 350
Ile Lys Val Thr Gln Glu Gln Tyr Glu Leu Tyr Cys Glu Met Gly Ser
355 360 365
Thr Phe Gln Leu Cys Lys Ile Cys Ala Glu Asn Asp Lys Asp Val Lys
370 375 380
Ile Glu Pro Cys Gly His Leu Met Cys Thr Ser Cys Leu Thr Ala Trp
385 390 395 400
Gln Glu Ser Asp Gly Gln Gly Cys Pro Phe Cys Arg Cys Glu Ile Lys
405 410 415
Gly Thr Glu Pro Ile Ile Val Asp Pro Phe Asp Pro Arg Asp Glu Gly
420 425 430
Ser Arg Cys Cys Ser Ile Ile Asp Pro Phe Gly Met Pro Met Leu Asp
435 440 445
Leu Asp Asp Asp Asp Asp Arg Glu Glu Ser Leu Met Met Asn Arg Leu
450 455 460
Ala Asn Val Arg Lys Cys Thr Asp Arg Gln Asn Ser Pro Val Thr Ser
465 470 475 480
Pro Gly Ser Ser Pro Leu Ala Gln Arg Arg Lys Pro His Pro Asp Pro
485 490 495
Leu Gln Ile Pro His Leu Ser Leu Pro Pro Val Pro Pro Arg Leu Asp
500 505 510
Leu Ile Gln Lys Gly Ile Val Arg Ser Pro Cys Gly Ser Pro Thr Gly
515 520 525
Ser Pro Lys Ser Ser Pro Cys Met Val Arg Lys Gln Asp Lys Pro Leu
530 535 540
Pro Ala Pro Pro Pro Pro Leu Arg Asp Pro Pro Pro Pro Pro Pro Glu
545 550 555 560
Arg Pro Pro Pro Ile Pro Pro Asp Asn Arg Leu Ser Arg His Phe His
565 570 575
His Val Glu Ser Val Pro Ser Arg Asp Gln Pro Met Pro Leu Glu Ala
580 585 590
Trp Cys Pro Arg Asp Val Phe Gly Thr Asn Gln Ser Val Gly Cys Arg
595 600 605
Gln Leu Gly Asp Gly Ser Pro Lys Pro Gly Ile Thr Ala Ser Ser Asn
610 615 620
Val Asn Gly Arg His Ser Arg Met Gly Ser Asp Pro Val Leu Leu Arg
625 630 635 640
Lys His Arg Arg His Asp Leu Pro Leu Glu Gly Ala Lys Val Phe Ser
645 650 655
Asn Gly His Leu Gly Ser Glu Glu Tyr Asp Val Pro Pro Arg Leu Ser
660 665 670
Pro Pro Pro Pro Ala Ala Thr Leu Val Pro Ser Ile Lys Cys Thr Gly
675 680 685
Pro Leu Ala Asn Pro Leu Ser Glu Lys Thr Arg Asp Pro Val Glu Glu
690 695 700
Asp Asp Asp Glu Tyr Lys Ile Pro Ser Ser His Pro Val Ser Leu Asn
705 710 715 720
Ser Gln Pro Ser His Cys His Asn Val Lys Pro Pro Leu Arg Ser Cys
725 730 735
Asp Asn Gly His Cys Val Leu Asn Gly Thr His Gly Thr Ser Ser Glu
740 745 750
Val Lys Lys Ser Asn Ile Pro Glu Leu Gly Ile Tyr Leu Lys Gly Asp
755 760 765
Val Phe Asp Ser Ala Ser Asp Pro Val Pro Leu Pro Pro Ala Arg Pro
770 775 780
Pro Thr Arg Asp Asn Pro Lys His Gly Ser Ser Leu Asn Arg Thr Pro
785 790 795 800
Ser Asp Tyr Asp Leu Leu Ile Pro Pro Leu Gly Glu Asp Ala Phe Asp
805 810 815
Ala Leu Pro Pro Ser Leu Pro Pro Pro Pro Pro Pro Ala Arg His Ser
820 825 830
Leu Ile Glu His Ser Lys Pro Pro Gly Ser Asn Ser Arg Pro Ser Ser
835 840 845
Gly Gln Asp Leu Phe Leu Leu Pro Ser Asp Pro Phe Phe Asp Pro Val
850 855 860
Ser Gly Gln Val Pro Leu Pro Pro Ala Arg Arg Leu Pro Gly Glu Asn
865 870 875 880
Val Lys Ser Asn Arg Thr Ser Gln Asp Tyr Asp Gln Leu Pro Ser Ala
885 890 895
Ser Asp Gly Ser Gln Ala Pro Ala Arg Pro Pro Lys Pro Arg Pro Arg
900 905 910
Arg Thr Ala Pro Glu Val Gln His Arg Lys Pro His Gly Pro Glu Ala
915 920 925
Ala Ser Glu Asn Val Asp Ala Lys Ile Ala Lys Leu Met Gly Glu Gly
930 935 940
Tyr Ala Phe Glu Glu Val Lys Arg Ala Leu Glu Ile Ala Gln Asn Asn
945 950 955 960
Val Glu Val Ala Arg Ser Ile Leu Arg Glu Phe Ala Tyr Pro Pro Pro
965 970 975
Val Ser Pro Arg Leu His Leu
980
<210> 161
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 161
cccaccatat atacttgat 19
<210> 162
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 162
cctgatacat atcagcatt 19
<210> 163
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 163
gcgggcaata agactcttt 19
<210> 164
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 164
gcagaaatac agcaccaaa 19
<210> 165
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 165
gcaccaaacc tggaagcta 19
<210> 166
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 166
gcaatatctt acagaccat 19
<210> 167
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 167
ccacaccaca tgaccatat 19
<210> 168
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 168
gcctcctccc ttaagagat 19
<210> 169
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 169
ccttcatccc atcctgttt 19
<210> 170
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNA sequences for canine Cbl-b
<400> 170
cctctgatcc agtgccatt 19
<210> 171
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 171
cccccgaaaa ggacggattt tgg 23
<210> 172
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 172
ccccgaaaag gacggatttt ggg 23
<210> 173
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 173
ccaaaatccg tccttttcgg ggg 23
<210> 174
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 174
cccaaaatcc gtccttttcg ggg 23
<210> 175
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 175
cgaggaggaa acccccgaaa agg 23
<210> 176
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 176
gggtttcctc ctcgaccacc agg 23
<210> 177
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 177
tacccaaaat ccgtcctttt cgg 23
<210> 178
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 178
agcaagcagc agcagatcgc agg 23
<210> 179
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 179
acccaaaatc cgtccttttc ggg 23
<210> 180
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 180
ggtttcctcc tcgaccacca ggg 23
<210> 181
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 181
tctgctgctg cttgcttcgg agg 23
<210> 182
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 182
agaaaccctg gtggtcgagg agg 23
<210> 183
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 183
ggcagaaacc ctggtggtcg agg 23
<210> 184
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 184
agcagcagca gatcgcagga cgg 23
<210> 185
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 185
agcagcagat cgcaggacgg tgg 23
<210> 186
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 186
gaggaaaccc ccgaaaagga cgg 23
<210> 187
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 187
gatgctattc aagatgcagt tgg 23
<210> 188
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 188
tctatgaatg gcagaaaccc tgg 23
<210> 189
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 189
cgatctgctg ctgcttgctt cgg 23
<210> 190
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 190
gcaggacggt ggagaaaact tgg 23
<210> 191
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 191
atgaatggca gaaaccctgg tgg 23
<210> 192
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl-b
<400> 192
ggagaaaact tggaaactca tgg 23
<210> 193
<211> 2721
<212> DNA
<213> Homo sapiens
<400> 193
atggccggca acgtgaagaa gagctctggg gccgggggcg gcagcggctc cgggggctcg 60
ggttcgggtg gcctgattgg gctcatgaag gacgccttcc agccgcacca ccaccaccac 120
caccacctca gcccccaccc gccggggacg gtggacaaga agatggtgga gaagtgctgg 180
aagctcatgg acaaggtggt gcggttgtgt cagaacccaa agctggcgct aaagaatagc 240
ccaccttata tcttagacct gctaccagat acctaccagc atctccgtac tatcttgtca 300
agatatgagg ggaagatgga gacacttgga gaaaatgagt attttagggt gtttatggag 360
aatttgatga agaaaactaa gcaaaccata agcctcttca aggagggaaa agaaagaatg 420
tatgaggaga attctcagcc taggcgaaac ctaaccaaac tgtccctcat cttcagccac 480
atgctggcag aactaaaagg aatctttcca agtggactct ttcagggaga cacatttcgg 540
attactaaag cagatgctgc ggaattttgg agaaaagctt ttggggaaaa gacaatagtc 600
ccttggaaga gctttcgaca ggctctacat gaagtgcatc ccatcagttc tgggctggag 660
gccatggctc tgaaatccac tattgatctg acctgcaatg attatatttc ggtttttgaa 720
tttgacatct ttacccgact ctttcagccc tggtcctctt tgctcaggaa ttggaacagc 780
cttgctgtaa ctcatcctgg ctacatggct tttttgacgt atgacgaagt gaaagctcgg 840
ctccagaaat tcattcacaa acctggcagt tatatcttcc ggctgagctg tactcgtctg 900
ggtcagtggg ctattgggta tgttactgct gatgggaaca ttctccagac aatccctcac 960
aataaacctc tcttccaagc actgattgat ggcttcaggg aaggcttcta tttgtttcct 1020
gatggacgaa atcagaatcc tgatctgact ggcttatgtg aaccaactcc ccaagaccat 1080
atcaaagtga cccaggaaca atatgaatta tactgtgaga tgggctccac attccaacta 1140
tgtaaaatat gtgctgaaaa tgataaggat gtaaagattg agccctgtgg acacctcatg 1200
tgcacatcct gtcttacatc ctggcaggaa tcagaaggtc agggctgtcc tttctgccga 1260
tgtgaaatta aaggtactga acccatcgtg gtagatccgt ttgatcctag agggagtggc 1320
agcctgttga ggcaaggagc agagggagct ccctccccaa attatgatga tgatgatgat 1380
gaacgagctg atgatactct cttcatgatg aaggaattgg ctggtgccaa ggtggaacgg 1440
ccgccttctc cattctccat ggccccacaa gcttcccttc ccccggtgcc accacgactt 1500
gaccttctgc cgcagcgagt atgtgttccc tcaagtgctt ctgctcttgg aactgcttct 1560
aaggctgctt ctggctccct tcataaagac aaaccattgc cagtacctcc cacacttcga 1620
gatcttccac caccaccgcc tccagaccgg ccatattctg ttggagcaga atcccgacct 1680
caaagacgcc ccttgccttg tacaccaggc gactgtccct ccagagacaa actgccccct 1740
gtcccctcta gccgccttgg agactcatgg ctgccccggc caatccccaa agtaccagta 1800
tctgccccaa gttccagtga tccctggaca ggaagagaat taaccaaccg gcactcactt 1860
ccattttcat tgccctcaca aatggagccc agaccagatg tgcctaggct cggaagcacg 1920
ttcagtctgg atacctccat gagtatgaat agcagcccat tagtaggtcc agagtgtgac 1980
caccccaaaa tcaaaccttc ctcatctgcc aatgccattt attctctggc tgccagacct 2040
cttcctgtgc caaaactgcc acctggggag caatgtgagg gtgaagagga cacagagtac 2100
atgactccct cttccaggcc tctacggcct ttggatacat cccagagttc acgagcatgt 2160
gattgcgacc agcagattga tagctgtacg tatgaagcaa tgtataatat tcagtcccag 2220
gcgccatcta tcaccgagag cagcaccttt ggtgaaggga atttggccgc agcccatgcc 2280
aacactggtc ccgaggagtc agaaaatgag gatgatgggt atgatgtccc aaagccacct 2340
gtgccggccg tgctggcccg ccgaactctc tcagatatct ctaatgccag ctcctccttt 2400
ggctggttgt ctctggatgg tgatcctaca acaaatgtca ctgaaggttc ccaagttccc 2460
gagaggcctc caaaaccatt cccgcggaga atcaactctg aacggaaagc tggcagctgt 2520
cagcaaggta gtggtcctgc cgcctctgct gccaccgcct cacctcagct ctccagtgag 2580
atcgagaacc tcatgagtca ggggtactcc taccaggaca tccagaaagc tttggtcatt 2640
gcccagaaca acatcgagat ggccaaaaac atcctccggg aatttgtttc catttcttct 2700
cctgcccatg tagctaccta g 2721
<210> 194
<211> 906
<212> PRT
<213> Homo sapiens
<400> 194
Met Ala Gly Asn Val Lys Lys Ser Ser Gly Ala Gly Gly Gly Ser Gly
1 5 10 15
Ser Gly Gly Ser Gly Ser Gly Gly Leu Ile Gly Leu Met Lys Asp Ala
20 25 30
Phe Gln Pro His His His His His His His Leu Ser Pro His Pro Pro
35 40 45
Gly Thr Val Asp Lys Lys Met Val Glu Lys Cys Trp Lys Leu Met Asp
50 55 60
Lys Val Val Arg Leu Cys Gln Asn Pro Lys Leu Ala Leu Lys Asn Ser
65 70 75 80
Pro Pro Tyr Ile Leu Asp Leu Leu Pro Asp Thr Tyr Gln His Leu Arg
85 90 95
Thr Ile Leu Ser Arg Tyr Glu Gly Lys Met Glu Thr Leu Gly Glu Asn
100 105 110
Glu Tyr Phe Arg Val Phe Met Glu Asn Leu Met Lys Lys Thr Lys Gln
115 120 125
Thr Ile Ser Leu Phe Lys Glu Gly Lys Glu Arg Met Tyr Glu Glu Asn
130 135 140
Ser Gln Pro Arg Arg Asn Leu Thr Lys Leu Ser Leu Ile Phe Ser His
145 150 155 160
Met Leu Ala Glu Leu Lys Gly Ile Phe Pro Ser Gly Leu Phe Gln Gly
165 170 175
Asp Thr Phe Arg Ile Thr Lys Ala Asp Ala Ala Glu Phe Trp Arg Lys
180 185 190
Ala Phe Gly Glu Lys Thr Ile Val Pro Trp Lys Ser Phe Arg Gln Ala
195 200 205
Leu His Glu Val His Pro Ile Ser Ser Gly Leu Glu Ala Met Ala Leu
210 215 220
Lys Ser Thr Ile Asp Leu Thr Cys Asn Asp Tyr Ile Ser Val Phe Glu
225 230 235 240
Phe Asp Ile Phe Thr Arg Leu Phe Gln Pro Trp Ser Ser Leu Leu Arg
245 250 255
Asn Trp Asn Ser Leu Ala Val Thr His Pro Gly Tyr Met Ala Phe Leu
260 265 270
Thr Tyr Asp Glu Val Lys Ala Arg Leu Gln Lys Phe Ile His Lys Pro
275 280 285
Gly Ser Tyr Ile Phe Arg Leu Ser Cys Thr Arg Leu Gly Gln Trp Ala
290 295 300
Ile Gly Tyr Val Thr Ala Asp Gly Asn Ile Leu Gln Thr Ile Pro His
305 310 315 320
Asn Lys Pro Leu Phe Gln Ala Leu Ile Asp Gly Phe Arg Glu Gly Phe
325 330 335
Tyr Leu Phe Pro Asp Gly Arg Asn Gln Asn Pro Asp Leu Thr Gly Leu
340 345 350
Cys Glu Pro Thr Pro Gln Asp His Ile Lys Val Thr Gln Glu Gln Tyr
355 360 365
Glu Leu Tyr Cys Glu Met Gly Ser Thr Phe Gln Leu Cys Lys Ile Cys
370 375 380
Ala Glu Asn Asp Lys Asp Val Lys Ile Glu Pro Cys Gly His Leu Met
385 390 395 400
Cys Thr Ser Cys Leu Thr Ser Trp Gln Glu Ser Glu Gly Gln Gly Cys
405 410 415
Pro Phe Cys Arg Cys Glu Ile Lys Gly Thr Glu Pro Ile Val Val Asp
420 425 430
Pro Phe Asp Pro Arg Gly Ser Gly Ser Leu Leu Arg Gln Gly Ala Glu
435 440 445
Gly Ala Pro Ser Pro Asn Tyr Asp Asp Asp Asp Asp Glu Arg Ala Asp
450 455 460
Asp Thr Leu Phe Met Met Lys Glu Leu Ala Gly Ala Lys Val Glu Arg
465 470 475 480
Pro Pro Ser Pro Phe Ser Met Ala Pro Gln Ala Ser Leu Pro Pro Val
485 490 495
Pro Pro Arg Leu Asp Leu Leu Pro Gln Arg Val Cys Val Pro Ser Ser
500 505 510
Ala Ser Ala Leu Gly Thr Ala Ser Lys Ala Ala Ser Gly Ser Leu His
515 520 525
Lys Asp Lys Pro Leu Pro Val Pro Pro Thr Leu Arg Asp Leu Pro Pro
530 535 540
Pro Pro Pro Pro Asp Arg Pro Tyr Ser Val Gly Ala Glu Ser Arg Pro
545 550 555 560
Gln Arg Arg Pro Leu Pro Cys Thr Pro Gly Asp Cys Pro Ser Arg Asp
565 570 575
Lys Leu Pro Pro Val Pro Ser Ser Arg Leu Gly Asp Ser Trp Leu Pro
580 585 590
Arg Pro Ile Pro Lys Val Pro Val Ser Ala Pro Ser Ser Ser Asp Pro
595 600 605
Trp Thr Gly Arg Glu Leu Thr Asn Arg His Ser Leu Pro Phe Ser Leu
610 615 620
Pro Ser Gln Met Glu Pro Arg Pro Asp Val Pro Arg Leu Gly Ser Thr
625 630 635 640
Phe Ser Leu Asp Thr Ser Met Ser Met Asn Ser Ser Pro Leu Val Gly
645 650 655
Pro Glu Cys Asp His Pro Lys Ile Lys Pro Ser Ser Ser Ala Asn Ala
660 665 670
Ile Tyr Ser Leu Ala Ala Arg Pro Leu Pro Val Pro Lys Leu Pro Pro
675 680 685
Gly Glu Gln Cys Glu Gly Glu Glu Asp Thr Glu Tyr Met Thr Pro Ser
690 695 700
Ser Arg Pro Leu Arg Pro Leu Asp Thr Ser Gln Ser Ser Arg Ala Cys
705 710 715 720
Asp Cys Asp Gln Gln Ile Asp Ser Cys Thr Tyr Glu Ala Met Tyr Asn
725 730 735
Ile Gln Ser Gln Ala Pro Ser Ile Thr Glu Ser Ser Thr Phe Gly Glu
740 745 750
Gly Asn Leu Ala Ala Ala His Ala Asn Thr Gly Pro Glu Glu Ser Glu
755 760 765
Asn Glu Asp Asp Gly Tyr Asp Val Pro Lys Pro Pro Val Pro Ala Val
770 775 780
Leu Ala Arg Arg Thr Leu Ser Asp Ile Ser Asn Ala Ser Ser Ser Phe
785 790 795 800
Gly Trp Leu Ser Leu Asp Gly Asp Pro Thr Thr Asn Val Thr Glu Gly
805 810 815
Ser Gln Val Pro Glu Arg Pro Pro Lys Pro Phe Pro Arg Arg Ile Asn
820 825 830
Ser Glu Arg Lys Ala Gly Ser Cys Gln Gln Gly Ser Gly Pro Ala Ala
835 840 845
Ser Ala Ala Thr Ala Ser Pro Gln Leu Ser Ser Glu Ile Glu Asn Leu
850 855 860
Met Ser Gln Gly Tyr Ser Tyr Gln Asp Ile Gln Lys Ala Leu Val Ile
865 870 875 880
Ala Gln Asn Asn Ile Glu Met Ala Lys Asn Ile Leu Arg Glu Phe Val
885 890 895
Ser Ile Ser Ser Pro Ala His Val Ala Thr
900 905
<210> 195
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 195
ccagacaatc cctcacaat 19
<210> 196
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 196
ggacacctca tgtgcacat 19
<210> 197
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 197
ccaggcctct acggccttt 19
<210> 198
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 198
ccagaaagct ttggtcatt 19
<210> 199
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 199
gcctgattgg gctcatgaag g 21
<210> 200
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 200
gggaacattc tccagacaat c 21
<210> 201
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 201
gcttcaggga aggcttctat t 21
<210> 202
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 202
gggaaggctt ctatttgttt c 21
<210> 203
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 203
ggacacctca tgtgcacatc c 21
<210> 204
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 204
gcagaatccc gacctcaaag a 21
<210> 205
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 205
ggagcaatgt gagggtgaag a 21
<210> 206
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 206
gcctctacgg cctttggata c 21
<210> 207
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 207
gctgtacgta tgaagcaatg t 21
<210> 208
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA/shRNAi sequences for human Cbl
<400> 208
ggtactccta ccaggacatc c 21
<210> 209
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 209
ctcggctcga ctgcgagcga 20
<210> 210
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 210
gccgccgccg gctatccggg 20
<210> 211
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 211
tccgcccgga tagccggcgg 20
<210> 212
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 212
gctcggctcg actgcgagcg 20
<210> 213
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 213
tcgcagtcga gccgagccgg 20
<210> 214
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 214
cttcttcacg ttgccggcca 20
<210> 215
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 215
cgggttcggg tggcctgatt 20
<210> 216
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 216
cgctcgcagt cgagccgagc 20
<210> 217
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 217
ccgagccggc ggacccgcct 20
<210> 218
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 218
tcgggttcgg gtggcctgat 20
<210> 219
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 219
gccgagccgg cggacccgcc 20
<210> 220
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 220
agagctcttc ttcacgttgc 20
<210> 221
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 221
gccgccgccg ccggctatcc 20
<210> 222
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 222
cccaggcggg tccgccggct 20
<210> 223
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 223
cgtccttcat gagcccaatc 20
<210> 224
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 224
cggagcccag gcgggtccgc 20
<210> 225
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 225
tggcctgatt gggctcatga 20
<210> 226
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 226
tcacgttgcc ggccatggcc 20
<210> 227
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 227
cgccgccgcc gccggctatc 20
<210> 228
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 228
ggcaacgtga agaagagctc 20
<210> 229
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 229
cggctccggg ggctcgggtt 20
<210> 230
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 230
tccgggggct cgggttcggg 20
<210> 231
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 231
ggctccgggg gctcgggttc 20
<210> 232
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 232
gcaacgtgaa gaagagctct 20
<210> 233
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 233
gcaacgtgaa gaagagctct 20
<210> 234
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 234
gccacccgaa cccgagcccc 20
<210> 235
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 235
cacgttgccg gccatggcct 20
<210> 236
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 236
gcccggatag ccggcggcgg 20
<210> 237
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 237
gaagaagagc tctggggccg 20
<210> 238
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 238
caacgtgaag aagagctctg 20
<210> 239
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 239
aagaagagct ctggggccgg 20
<210> 240
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 240
gggagagaag cagggcgtga 20
<210> 241
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 241
cggcagcggc tccgggggct 20
<210> 242
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 242
cctgggcagg gtcggagccc 20
<210> 243
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for human Cbl
<400> 243
agagaagcag ggcgtgaagg 20
<210> 244
<211> 2727
<212> DNA
<213> Canis lupus
<220>
<221> misc_feature
<222> (142)..(142)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1876)..(1876)
<223> n is a, c, g, or t
<400> 244
atggccggca acgtgaagaa gagctccggg gccgggggcg gcggcggctc cgggggctcg 60
ggcggcctca tcgggctcat gaaggacgcc ttccagccgc accaccacca ccaccacctc 120
agcccccacc cgcccggcac cngtgacaag aagatggtgg agaagtgctg gaagctcatg 180
gacaaggtgg tgcggttgtg tcagaaccca aagctggcgc taaagaatag cccaccttat 240
atcttagacc tgctgccaga tacctaccag catctccgca ctatcttgtc aagatatgag 300
gggaagatgg agacacttgg agaaaatgag tattttaggg tgttcatgga gaatttgatg 360
aagaaaacta agcagaccat aagcctcttc aaggagggga aagaaagaat gtatgaggag 420
aattctcagc ctaggcgaaa cctaaccaaa ttgtccctga tcttcagcca catgctggca 480
gaactaaaag gaatctttcc aagtggactc tttcaaggag acacatttcg gattactaaa 540
gcagatgctg cagaattttg gaggaaagct tttggggaaa agacaatcgt cccttggaag 600
agtttccgcc aggcccttca tgaagtgcat cccatcagtt ctgggctcga ggccatggct 660
ctgaaatcca ctattgatct gacctgcaat gattatattt ctgtttttga atttgacatc 720
ttcacacgac tctttcagcc ctggtcctct ttgctcagga actggaacag tcttgctgta 780
actcatcctg gttacatggc tttcctgacg tatgatgaag tgaaagctcg gctccagaag 840
ttcattcaca aacctggcag ttacattttc cggttgagct gtactcgttt gggacagtgg 900
gctattgggt atgtcactgc tgatgggaac atcctccaga cgatccctca caataaacct 960
ctcttccaag ccctgattga cggcttcagg gaaggcttct atttgtttcc agatggacgg 1020
aatcagaatc ctgacctgac aggcctatgt gaaccaactc cccaagacca catcaaagtg 1080
acccaggaac aatatgaatt atactgtgag atgggctcca ccttccaact gtgtaaaata 1140
tgtgctgaga acgataagga tgtgaaaatt gagccctgtg gacacctcat gtgcacatcc 1200
tgtcttacat cctggcagga atcagaaggc caaggctgcc ctttctgccg atgtgaaatt 1260
aaaggtactg agcccattgt ggtagatccg tttgaccctc gaggaagtgg cagcctactg 1320
aggcaaggag ctgagggagc tccctcccca aattatgaag atgatgacga tgaacgagct 1380
gatgattctc tctttatgat gaaggaactg gctggtgcca aggtggaacg gcctccttct 1440
ccgttctcga tggccccaca ggctcccctg cccccagtac caccacgtct tgacctccta 1500
caacagcgag tgtctgttcc ttctagtgct tctggtcttg gaactgcttc taaggtagct 1560
tctggctccc ttcataagga caaaccatta ccaatacccc ccacacttcg agatcttcca 1620
ccaccacccc ctccagaccg accatattct gttggaacag acacccggcc tcagagacgt 1680
cccttgcctt gtacaccggg cgactgtcca tccagggaca aactgccgcc tgttccctct 1740
agccgtctcg gggaatcatg gctgcctcgg ccaatcccca aagtaccagt ggttgctcca 1800
aactcgagtg acccctggac ctctggtaga gaattaacca acaggcactc acttccattt 1860
tcattgccct cacaanatga acccagaaca gatgtgccta ggcttggagg cacattcaat 1920
gtggatactt ccatgaatgt gaataacagc ccactagcaa gttctgagtg tgagcacccc 1980
aaaatcaaac cttccgcatc tgccaatgcc atttattctc tggctgccag gcctcttcct 2040
gtgccaaagc tgccccctgg ggagcagtgt gaaggtgagg aggacacaga gtatatgacc 2100
ccctcctcta gacctctagg gcttccaaag ccagatggga aacggccttt ggagacaacc 2160
cagagttcac gagcatgtga ttgtgaccag cagatcgata gctgcacata tgaagcaatg 2220
tataatattc agtcccaagc gacaccatct gtcacagaga gcagcacctt tggtgaaggg 2280
agtctggctg cagcccacat cagcaccggc cccgaggaat cagaaaatga ggaggacggg 2340
tatgatgtcc ctaagccgcc catgccagca gtgctggccc gccggactct ctcagacatc 2400
tccaatgcca gttcctcctt tggctggttg tctctggaag gcgatcccac cacaaacttc 2460
actgagggtt cccaagttcc tgaaaggcct cccaaaccgt tccctcggag aatcaactct 2520
gaacgaaaag caggcagctg tcagcagggt ggtgccgctg ctgcctcacc acagctctcc 2580
agtgagattg agaacctcct gagccaggga tactcctacc aggacattca gaaagctctg 2640
gtcattgccc acaacaacat tgaaatggcc aagaacatcc tccgggaatt tgtttctatc 2700
tcttctcccg cccacgtagc cacctag 2727
<210> 245
<211> 908
<212> PRT
<213> Canis lupus
<220>
<221> misc_feature
<222> (48)..(48)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (626)..(626)
<223> Xaa can be any naturally occurring amino acid
<400> 245
Met Ala Gly Asn Val Lys Lys Ser Ser Gly Ala Gly Gly Gly Gly Gly
1 5 10 15
Ser Gly Gly Ser Gly Gly Leu Ile Gly Leu Met Lys Asp Ala Phe Gln
20 25 30
Pro His His His His His His Leu Ser Pro His Pro Pro Gly Thr Xaa
35 40 45
Asp Lys Lys Met Val Glu Lys Cys Trp Lys Leu Met Asp Lys Val Val
50 55 60
Arg Leu Cys Gln Asn Pro Lys Leu Ala Leu Lys Asn Ser Pro Pro Tyr
65 70 75 80
Ile Leu Asp Leu Leu Pro Asp Thr Tyr Gln His Leu Arg Thr Ile Leu
85 90 95
Ser Arg Tyr Glu Gly Lys Met Glu Thr Leu Gly Glu Asn Glu Tyr Phe
100 105 110
Arg Val Phe Met Glu Asn Leu Met Lys Lys Thr Lys Gln Thr Ile Ser
115 120 125
Leu Phe Lys Glu Gly Lys Glu Arg Met Tyr Glu Glu Asn Ser Gln Pro
130 135 140
Arg Arg Asn Leu Thr Lys Leu Ser Leu Ile Phe Ser His Met Leu Ala
145 150 155 160
Glu Leu Lys Gly Ile Phe Pro Ser Gly Leu Phe Gln Gly Asp Thr Phe
165 170 175
Arg Ile Thr Lys Ala Asp Ala Ala Glu Phe Trp Arg Lys Ala Phe Gly
180 185 190
Glu Lys Thr Ile Val Pro Trp Lys Ser Phe Arg Gln Ala Leu His Glu
195 200 205
Val His Pro Ile Ser Ser Gly Leu Glu Ala Met Ala Leu Lys Ser Thr
210 215 220
Ile Asp Leu Thr Cys Asn Asp Tyr Ile Ser Val Phe Glu Phe Asp Ile
225 230 235 240
Phe Thr Arg Leu Phe Gln Pro Trp Ser Ser Leu Leu Arg Asn Trp Asn
245 250 255
Ser Leu Ala Val Thr His Pro Gly Tyr Met Ala Phe Leu Thr Tyr Asp
260 265 270
Glu Val Lys Ala Arg Leu Gln Lys Phe Ile His Lys Pro Gly Ser Tyr
275 280 285
Ile Phe Arg Leu Ser Cys Thr Arg Leu Gly Gln Trp Ala Ile Gly Tyr
290 295 300
Val Thr Ala Asp Gly Asn Ile Leu Gln Thr Ile Pro His Asn Lys Pro
305 310 315 320
Leu Phe Gln Ala Leu Ile Asp Gly Phe Arg Glu Gly Phe Tyr Leu Phe
325 330 335
Pro Asp Gly Arg Asn Gln Asn Pro Asp Leu Thr Gly Leu Cys Glu Pro
340 345 350
Thr Pro Gln Asp His Ile Lys Val Thr Gln Glu Gln Tyr Glu Leu Tyr
355 360 365
Cys Glu Met Gly Ser Thr Phe Gln Leu Cys Lys Ile Cys Ala Glu Asn
370 375 380
Asp Lys Asp Val Lys Ile Glu Pro Cys Gly His Leu Met Cys Thr Ser
385 390 395 400
Cys Leu Thr Ser Trp Gln Glu Ser Glu Gly Gln Gly Cys Pro Phe Cys
405 410 415
Arg Cys Glu Ile Lys Gly Thr Glu Pro Ile Val Val Asp Pro Phe Asp
420 425 430
Pro Arg Gly Ser Gly Ser Leu Leu Arg Gln Gly Ala Glu Gly Ala Pro
435 440 445
Ser Pro Asn Tyr Glu Asp Asp Asp Asp Glu Arg Ala Asp Asp Ser Leu
450 455 460
Phe Met Met Lys Glu Leu Ala Gly Ala Lys Val Glu Arg Pro Pro Ser
465 470 475 480
Pro Phe Ser Met Ala Pro Gln Ala Pro Leu Pro Pro Val Pro Pro Arg
485 490 495
Leu Asp Leu Leu Gln Gln Arg Val Ser Val Pro Ser Ser Ala Ser Gly
500 505 510
Leu Gly Thr Ala Ser Lys Val Ala Ser Gly Ser Leu His Lys Asp Lys
515 520 525
Pro Leu Pro Ile Pro Pro Thr Leu Arg Asp Leu Pro Pro Pro Pro Pro
530 535 540
Pro Asp Arg Pro Tyr Ser Val Gly Thr Asp Thr Arg Pro Gln Arg Arg
545 550 555 560
Pro Leu Pro Cys Thr Pro Gly Asp Cys Pro Ser Arg Asp Lys Leu Pro
565 570 575
Pro Val Pro Ser Ser Arg Leu Gly Glu Ser Trp Leu Pro Arg Pro Ile
580 585 590
Pro Lys Val Pro Val Val Ala Pro Asn Ser Ser Asp Pro Trp Thr Ser
595 600 605
Gly Arg Glu Leu Thr Asn Arg His Ser Leu Pro Phe Ser Leu Pro Ser
610 615 620
Gln Xaa Glu Pro Arg Thr Asp Val Pro Arg Leu Gly Gly Thr Phe Asn
625 630 635 640
Val Asp Thr Ser Met Asn Val Asn Asn Ser Pro Leu Ala Ser Ser Glu
645 650 655
Cys Glu His Pro Lys Ile Lys Pro Ser Ala Ser Ala Asn Ala Ile Tyr
660 665 670
Ser Leu Ala Ala Arg Pro Leu Pro Val Pro Lys Leu Pro Pro Gly Glu
675 680 685
Gln Cys Glu Gly Glu Glu Asp Thr Glu Tyr Met Thr Pro Ser Ser Arg
690 695 700
Pro Leu Gly Leu Pro Lys Pro Asp Gly Lys Arg Pro Leu Glu Thr Thr
705 710 715 720
Gln Ser Ser Arg Ala Cys Asp Cys Asp Gln Gln Ile Asp Ser Cys Thr
725 730 735
Tyr Glu Ala Met Tyr Asn Ile Gln Ser Gln Ala Thr Pro Ser Val Thr
740 745 750
Glu Ser Ser Thr Phe Gly Glu Gly Ser Leu Ala Ala Ala His Ile Ser
755 760 765
Thr Gly Pro Glu Glu Ser Glu Asn Glu Glu Asp Gly Tyr Asp Val Pro
770 775 780
Lys Pro Pro Met Pro Ala Val Leu Ala Arg Arg Thr Leu Ser Asp Ile
785 790 795 800
Ser Asn Ala Ser Ser Ser Phe Gly Trp Leu Ser Leu Glu Gly Asp Pro
805 810 815
Thr Thr Asn Phe Thr Glu Gly Ser Gln Val Pro Glu Arg Pro Pro Lys
820 825 830
Pro Phe Pro Arg Arg Ile Asn Ser Glu Arg Lys Ala Gly Ser Cys Gln
835 840 845
Gln Gly Gly Ala Ala Ala Ala Ser Pro Gln Leu Ser Ser Glu Ile Glu
850 855 860
Asn Leu Leu Ser Gln Gly Tyr Ser Tyr Gln Asp Ile Gln Lys Ala Leu
865 870 875 880
Val Ile Ala His Asn Asn Ile Glu Met Ala Lys Asn Ile Leu Arg Glu
885 890 895
Phe Val Ser Ile Ser Ser Pro Ala His Val Ala Thr
900 905
<210> 246
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 246
ccagaagttc attcacaaa 19
<210> 247
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 247
ggaacatcct ccagacgat 19
<210> 248
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 248
ccagacgatc cctcacaat 19
<210> 249
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 249
gcttcaggga aggcttcta 19
<210> 250
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 250
gcaggaatca gaaggccaa 19
<210> 251
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 251
cctttctgcc gatgtgaaa 19
<210> 252
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 252
gctgatgatt ctctcttta 19
<210> 253
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 253
gcttctggct cccttcata 19
<210> 254
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 254
gcatctgcca atgccattt 19
<210> 255
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> siRNA sequences for canine Cbl
<400> 255
gctgcacata tgaagcaat 19
<210> 256
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 256
cccggagccg ccgccgcccc cgg 23
<210> 257
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 257
tgccgggcgg gtgggggctg agg 23
<210> 258
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 258
cggcctcatc gggctcatga agg 23
<210> 259
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 259
ggagctcttc ttcacgttgc cgg 23
<210> 260
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 260
caacgtgaag aagagctccg ggg 23
<210> 261
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 261
ggggctcggg cggcctcatc ggg 23
<210> 262
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 262
ggcaacgtga agaagagctc cgg 23
<210> 263
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 263
gcaacgtgaa gaagagctcc ggg 23
<210> 264
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 264
gggggctcgg gcggcctcat cgg 23
<210> 265
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 265
gtgaagaaga gctccggggc cgg 23
<210> 266
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 266
tgaagaagag ctccggggcc ggg 23
<210> 267
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 267
cgtccttcat gagcccgatg agg 23
<210> 268
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 268
aagaagagct ccggggccgg ggg 23
<210> 269
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 269
gaagaagagc tccggggccg ggg 23
<210> 270
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 270
gatgaggccg cccgagcccc cgg 23
<210> 271
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 271
gtggtggtgg tgcggctgga agg 23
<210> 272
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 272
aagagctccg gggccggggg cgg 23
<210> 273
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 273
cacctcagcc cccacccgcc cgg 23
<210> 274
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 274
cggcggcggc tccgggggct cgg 23
<210> 275
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 275
agctccgggg ccgggggcgg cgg 23
<210> 276
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 276
gcgggtgggg gctgaggtgg tgg 23
<210> 277
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 277
tccggggccg ggggcggcgg cgg 23
<210> 278
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 278
gccgccgccg cccccggccc cgg 23
<210> 279
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 279
cgggcgggtg ggggctgagg tgg 23
<210> 280
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CRISPR/CAS9 target sequences for canine Cbl
<400> 280
gccgggggcg gcggcggctc cgg 23
<210> 281
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> human CD2AP wobble mutant sequence
<400> 281
ggagacggac gacgtaaag 19

Claims (4)

1.下调个体CD2AP表达的配伍在制备预防由慢性丙型肝炎病毒感染导致的脂肪肝相关症状的药物中的应用，其特征在于，所述配伍通过siRNA/shRNA,Crispr/cas9,crispr/cpf1,Talen或ZFNs来工作；所述配伍包括至少一个选自针对人的序列号3-20或针对狗的序列号59-76的siRNA/shRNAi多聚核苷酸序列或至少一个Crispr/cas9,crispr/cpf1载体，其中所述Crispr/cas9,crispr/cpf1载体包含一个选自针对人的序列号21-56或针对狗的序列号77-103的多聚核苷酸序列。
2.一个下调CD2AP在个体肝组织表达的药物配伍在制备治疗丙型肝炎病毒感染的药物中的应用，其特征在于，所述配伍包括至少一个选自针对人的序列号3-20或针对狗的序列号59-76的siRNA/shRNAi多聚核苷酸序列或至少一个Crispr/cas9,crispr/cpf1载体，其中所述Crispr/cas9,crispr/cpf1载体包含一个选自针对人的序列号21-56或针对狗的序列号77-103的引导多聚核苷酸序列。
3.CD2AP表达的检测试剂在制备个体丙型肝炎病毒感染的检测试剂盒中的应用。
4.根据权利要求3所述应用，其特征在于，所述CD2AP表达的检测试剂包括，CD2AP蛋白特异抗体或CD2AP mRNA特异的多聚核苷酸探针，和一个次级试剂，能够检测与CD2AP蛋白结合的抗体或来自CD2AP mRNA的信号。
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Publication number Priority date Publication date Assignee Title
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US9228207B2 (en) 2013-09-06 2016-01-05 President And Fellows Of Harvard College Switchable gRNAs comprising aptamers
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US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
WO2018227432A1 (en) * 2017-06-14 2018-12-20 Wuhan Institute Of Virology, Chinese Academy Of Sciences Cd2-associated protein (cd2ap) and its interactive proteins
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CN111757937A (zh) 2017-10-16 2020-10-09 布罗德研究所股份有限公司 腺苷碱基编辑器的用途
CN107988255A (zh) * 2017-10-20 2018-05-04 深圳市龙岗区妇幼保健院 一种确定atr与erk结合区域的方法
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CN110438160B (zh) * 2019-07-18 2021-06-29 浙江大学 一种Cd2ap基因敲除动物的构建方法及应用
EP4146804A1 (en) 2020-05-08 2023-03-15 The Broad Institute Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003068808A1 (en) * 2002-02-13 2003-08-21 Garvan Institute Of Medical Research Novel protein complexes and uses therefor
WO2006108225A1 (en) * 2005-04-11 2006-10-19 Garvan Institute Of Medical Research Method of screening for compounds that modulate cell proliferation
WO2010054167A2 (en) * 2008-11-06 2010-05-14 University Of Miami Limited proteolysis of cd2ap and progression of renal disease
WO2012068344A2 (en) * 2010-11-17 2012-05-24 University Of Miami Regulation of cathepsin l by its transcription factor dendrin
CN103045729A (zh) * 2012-12-12 2013-04-17 芮屈生物技术（上海）有限公司 老年性痴呆病变前期CD2AP基因mRNA水平原位杂交筛查试剂盒及筛查方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040029192A1 (en) * 2002-08-08 2004-02-12 Shaw Andrey S. Compositions and methods for the diagnosis and treatment of kidney disease

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003068808A1 (en) * 2002-02-13 2003-08-21 Garvan Institute Of Medical Research Novel protein complexes and uses therefor
WO2006108225A1 (en) * 2005-04-11 2006-10-19 Garvan Institute Of Medical Research Method of screening for compounds that modulate cell proliferation
WO2010054167A2 (en) * 2008-11-06 2010-05-14 University Of Miami Limited proteolysis of cd2ap and progression of renal disease
WO2012068344A2 (en) * 2010-11-17 2012-05-24 University Of Miami Regulation of cathepsin l by its transcription factor dendrin
CN103045729A (zh) * 2012-12-12 2013-04-17 芮屈生物技术（上海）有限公司 老年性痴呆病变前期CD2AP基因mRNA水平原位杂交筛查试剂盒及筛查方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CD2-Associated Protein Contributes to Hepatitis C, Virus Propagation and Steatosis by Disrupting Insulin Signaling;Huixia Zhang等;《Hepatology》;20181231;第68卷(第5期);第1710-1725页 *
敲低CD2AP基因表达对足细胞增殖和***的影响;张春等;《中华肾脏病杂志》;20071231;第23卷(第12期);第778-783页 *

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