CN113481288A - Fxyd3蛋白和基因在炎症性肠道疾病的诊断和治疗中的应用 - Google Patents
Fxyd3蛋白和基因在炎症性肠道疾病的诊断和治疗中的应用 Download PDFInfo
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Abstract
本发明公开了FXYD3蛋白和基因在炎症性肠道疾病的诊断和治疗中的应用。本发明不仅发现了FXYD3基因主要表达在结肠杯状细胞,且其在炎症性肠病患者的炎症组织中表达显著下降,该发现为临床诊断炎症性肠病及判断易感性提供了重要的依据和生物标志物。进一步研究发现在肠道上皮细胞特异性敲除FXYD3的小鼠中,结肠杯状细胞数量和mucin 2的分泌明显受到抑制,且炎症性肠病的疾病程度更严重。细胞因子IL‑22,IL‑10可促进肠上皮细胞中FXYD3的表达,FXYD3通过正向调控STAT3信号活化从而促进杯状细胞分化和黏蛋白的分泌,增强肠道黏膜屏障,该发现预示着FXYD3可作为临床治疗炎症性肠病的新靶点。
Description
技术领域
本发明涉及疾病的临床诊断标志物及治疗靶点,具体地说,涉及离子通道调节蛋白FXYD3在炎症性肠病的诊断和治疗中的作用。
背景技术
肠道稳态发生紊乱时会导致炎性肠病,炎症性肠病(Inflammatory boweldiseases,IBD)是一类由免疫介导的、易复发的慢性非特异性肠道炎症性疾病,包括克罗恩病(Crohn’s Disease,CD)和溃疡性结肠炎(Ulcerative Colitis,UC)。IBD的发病高峰人群为20~40岁的青壮年,且具有容易复发、迁延不愈的特征,严重影响了患者的生活质量,由此,对IBD发病机制的深入研究具有重要意义,有望为IBD的临床治疗提供新的靶点。
肠道的黏膜屏障与炎症性肠病的发生密切相关,肠上皮中的杯状细胞所分泌的黏蛋白与水、无机盐等共同组成覆盖于上皮细胞表面的黏液,形成了黏膜上皮细胞与肠内容物之间的强大屏障,避免上皮细胞受到各种物理、化学及生物的损伤。黏液的缺失会导致炎症性肠病的发生。但目前对黏液的产生和黏膜屏障的完整性是如何被精细调控的机制尚不明确,需要开展更深入的研究来阐明其与炎症性肠病的关系,为临床治疗提供潜在的靶点。
FXYD(FXYD domain-containing ion transport regulator)家族分子是Na,K-ATP酶的特异性辅助亚基,其家族均由6个至9个小外显子的基因编码,蛋白大小在约60至160个氨基酸,共同结构为单个跨膜片段及其周围35个氨基酸残基组成的核心结构,主要负责维持细胞膜上的Na离子和K离子浓度的平衡(Lindzen M.,Gottschalk K.E.,Fuzesi M.,Garty H.&Karlish S.J.Structural interactions between FXYD proteins and Na+,K+-ATPase:alpha/beta/FXYD subunit stoichiometry and cross-linking.The Journalof biological chemistry 281,5947-5955(2006).)。FXYD3,又称Mat-8,与家族其它成员的I型膜蛋白结构不同,FXYD3具有两次跨膜样结构域(Crambert G.,Li C.,Claeys D.&Geering K.FXYD3(Mat-8),a new regulator of Na,K-ATPase.Molecular biology ofthe cell 16,2363-2371(2005).)。FXYD3主要表达于胃肠道、皮肤、肺部和膀胱等上皮组织中,有研究表明FXYD3在肿瘤的发生发展中有调控作用(Zhu Z.L.,Zhao Z.R.,Zhang Y.,Yang Y.H.,Wang Z.M.,Cui D.S.,Wang M.W.,Kleeff J.,Kayed H.,Yan B.Y.&SunX.F.Expression and significance of FXYD-3protein in gastricadenocarcinoma.Disease markers 28,63-69(2010).),但FXYD3在结肠稳态和炎症性肠病中的作用及其具体机制尚不清楚。
发明内容
本发明针对现有技术中存在的不足,提供了FXYD3蛋白和基因在炎症性肠道疾病的诊断和治疗中的应用。
人FXYD3基因的序列如SEQ ID No.1所示,人FXYD3蛋白的氨基酸序列如SEQ IDNo.2所示。
本发明首先提供了人FXYD3基因mRNA作为靶点在制备用于诊断炎症性肠道疾病的试剂盒中的应用。
本发明又提供了一种用于诊断炎症性肠道疾病的试剂盒,包括:
(1)用于将mRNA反转录成cDNA的反转录试剂,
(2)用于PCR扩增人FXYD3基因cDNA序列的引物,
(3)已知浓度的、含有用(2)中引物PCR扩增人FXYD3基因cDNA所得扩增片段的阳性对照。
所述的试剂盒,还包括用于实时荧光定量PCR检测的荧光染料。
本发明还提供了人FXYD3蛋白作为靶点在制备用于诊断炎症性肠道疾病的试剂盒中的应用。
本发明还提供了一种用于诊断炎症性肠道疾病的试剂盒,包括:
(1)特异性结合人FXYD3蛋白的抗体,
(2)当作为一抗的(1)中抗体与人FXYD3蛋白结合时,能够结合于一抗的二抗,二抗为带有检测标记物的抗体。
优选的,所述的试剂盒,所述检测标记物为过氧化物酶、磷酸酯酶或荧光素酶,所述试剂盒还包括作为检测标记物的反应对象的化学发光化合物、显色底物或荧光染料。
本发明还提供了一种用于治疗炎症性肠道疾病的药物,有效成分为以下至少一种:
(1)能够外源表达FXYD3基因的重组表达载体;
(2)能够提高内源FXYD3基因表达的化合物。
所述的药物,(2)中化合物为细胞因子IL-22或IL-10。
本发明不仅发现了FXYD3基因主要表达在结肠杯状细胞,且其在炎症性肠病患者的炎症组织中表达显著下降,该发现为临床诊断炎症性肠病及判断易感性提供了重要的依据和生物标志物。进一步研究发现在肠道上皮细胞特异性敲除FXYD3的小鼠中,结肠杯状细胞数量和mucin 2的分泌明显受到抑制,且炎症性肠病的疾病程度更严重。细胞因子IL-22,IL-10可促进肠上皮细胞中FXYD3的表达,FXYD3通过正向调控STAT3信号活化从而促进杯状细胞分化和黏蛋白的分泌,增强肠道黏膜屏障,该发现预示着FXYD3可作为临床治疗炎症性肠病的新靶点。
附图说明
图1为发现FXYD3主要表达于结肠杯状细胞,A:人健康结肠组织FXYD3免疫组化染色(比例尺:50μm);B:人健康结肠组织mucin 2和FXYD3免疫荧光染色,mucin 2为绿色荧光,FXYD3为红色荧光,细胞核为蓝色荧光,可见绿色荧光和红色荧光有高度重合。
图2为发现炎性肠病中结肠上皮细胞FXYD3表达显著降低,A:对比健康组和克罗恩病人结肠组织中FXYD3免疫组化染色结果,比例尺:100μm;B:FXYD3免疫组化检测对照组和2%DSS小鼠造模后结肠组织,比例尺:50μm;C:qRT-PCR检测对照组和2%DSS小鼠造模组小鼠结肠上皮细胞中FXYD3表达情况;D:Western Blot实验检测对照组和2%DSS小鼠造模组小鼠结肠上皮细胞中FXYD3表达情况。
图3为发现结肠上皮特异性敲除FXYD3的转基因小鼠FXYD3IEC-KO的杯状细胞分泌功能受损,肠道黏液层减少。A:PAS-AB染色检测杯状细胞数量,比例尺:50μm,统计结肠中杯状细胞数和黏液层厚度;B:EUB338原位杂交检测肠腔中黏液层厚度,比例尺:50μm。C:透射电镜检测FXYD3结肠上皮细胞敲除后结肠组织中的变化。D:扫描电镜检测FXYD3结肠上皮细胞敲除后肠腔面变化。
图4为发现FXYD3IEC-KO小鼠对炎性肠病易感性增加。A:鼠类柠檬酸杆菌(Citrobacter rodentium,C.R.)感染后记录小鼠体重变化,七天后测量各组小鼠结肠长度,HE染色观察结肠组织破坏情况;B:喂食小鼠2%DSS建立肠炎模型,记录小鼠体重变化,九天后测量各组小鼠结肠长度,HE染色观察结肠组织破坏情况。
图5为发现FXYD3在肠道上皮细胞的缺失抑制细胞中STAT3信号的激活。A:WT和FXYD3IEC-KO小鼠结肠上皮细胞转录组测序,所得的基因表达变化火山图;B:分析KEGG信号通路变化差异;C:STAT3信号相关基因的表达差异;D:分离WT和FXYD3IEC-KO小鼠结肠上皮细胞,用IL-22、IL-10和IL-6处理后蛋白免疫印迹实验检测STAT3信号激活。
图6为发现FXYD3促进STAT3和JAK1结合。蛋白免疫共沉淀方法检测LS174T细胞中正常和沉默FXYD3表达后JAK1和FXYD3的结合变化情况。
图7为发现细胞因子IL-22,IL-10可促进LS174T细胞中FXYD3的表达。
具体实施方式
实施例1
取人的结肠组织样本(来源于浙江大学附属邵逸夫医院),10%福尔马林中固定,一周内石蜡包埋,切片。石蜡切片置于67℃烘箱中,烘片2小时,脱蜡至水,用pH7.4的PBS冲洗三次,每次3分钟(3×3’)。取一定量pH=6.0柠檬酸盐缓冲液,加入微波盒中,微波加热至沸腾,将脱蜡水化后的组织切片置于耐高温塑料切片架上,放入已沸腾的缓冲液中,中档微波处理10分钟,取出微波盒流水自然泠却,从缓冲液中取出玻片,先用蒸馏水冲洗两次,之后用PBS冲洗2×3’。每张切片加1滴3%H2O2,室温下孵育10分钟,以阻断内源性过氧化物酶的活性。PBS冲洗3×3’。除去PBS液,每张切片加1滴抗人FXYD3(abcom ab205534)(相应稀释倍数),室温下孵育2小时。PBS冲洗3×5’。除去PBS液,每张切片加1滴聚合物增强剂,室温下孵育20分钟。PBS冲洗3×3’。除去PBS液,每张切片加1滴酶标抗兔聚合物,室温下孵育30分钟。PBS冲洗3×5’。除去PBS液,每张切片加1滴新鲜配制的DAB液(二氨基联苯胺),显微镜下观察5分钟。
苏木素复染,0.1%HCl分化,自来水冲洗,蓝化,切片经梯度酒精脱水干燥,二甲苯透明,中性树胶封固,晾干后显微镜观察,图1A显示FXYD3主要表达在结肠杯状细胞。
取人的结肠组织样本冰冻后切片,厚度为20-30μm。挑选所需组织切片,置于PBS中,摇床上漂洗3次,5min/次。将组织切片置于封闭液(0.01M PBS,0.3%Triton X-100,3%山羊血清,原则上尽量选择与二抗种属同源的血清,切记不可使用与组织源相同的血清)中,室温、摇床慢摇、通透、封闭1h。FXYD3/MUC2一抗4℃孵育过夜。将组织切片置于PBS中,摇床上漂洗4次,10min/次,洗掉非特异结合的抗体。
加入二抗,室温、避光、摇床孵育2h。将组织切片置于PBS中,避光摇床上漂洗3次,10min/次。防淬灭封片剂封片,避光风干,共聚焦显微镜观察。图1B结果显示显绿色荧光的mucin 2与显红色荧光的FXYD3有高度重合,表明FXYD3主要定位于mucin2阳性细胞,即肠道杯状细胞。
实施例2
我们利用健康人和克罗恩病人及溃疡性结肠炎的结肠组织进行FXYD3免疫组化染色,免疫组化法同实施例1,图2A结果发现在FXYD3在病人样本中表达明显下降。同样地,在DSS诱导小鼠肠炎模型中,图2B结果显示结肠杯状细胞中FXYD3表达也显著下降。
qRT-PCR:分离肠上皮细胞,具体方法为:配制肠道上皮分离液:PBS缓冲液加MTT1mM、EDTA.2Na 5mM和5%FBS和青链霉素。小鼠处死后取结肠组织,预冷PBS冲出肠道内容物,纵切剪开结肠,切成5mm,放入肠道上皮分离液中,37℃,45分钟,摇床150转每分钟。之后涡旋细胞筛(200目)过滤,收集的细胞为结肠上皮细胞。
Trizol法按试剂说明书进行总RNA提取,按Toyobo逆转录试剂盒(Toyobo,日本)说明书步骤逆转录成cDNA。
逆转录体系:
cDNA用RNase-free water稀释10倍后进行real-time PCR,反应体系为:
引物序列为:
鼠FXYD3上游引物:TACAGCATGTCCTACTCGCAG,
鼠FXYD3下游引物:GAGGAAGAGGTAACCACAGGG;
人FXYD3上游引物:AGGTTGGCGGGCTCATC,
人FXYD3下游引物:CATTTGCATTTTGCACTCATGAC。
Real-time反应条件为:
50℃、2min;95℃、10min;95℃、15sec,60℃、35sec,40个循环。
加样完成后用封板膜封口并离心数秒,使用CFX-Touch荧光定量PCR仪进行扩增反应,图2C结果显示DSS诱导的炎性肠病小鼠肠道上皮细胞中FXYD3的表达较正常小鼠显著下降。
蛋白免疫印迹实验:分离小鼠结肠上皮细胞,300-500g离心5min,PBS洗1遍,加入Cell Lysis Buffer(Cell Signaling Technology#9803)裂解液冰上裂解细胞30min,12000g离心10min后,取上清加入5×loading buffer煮沸。SDS电泳后,转膜至PVDF膜,将PVDF膜浸入封闭液(5%脱脂奶粉)中,摇床上常温封闭1h。加入抗FXYD3(Santa Cruz67245)一抗抗体,4℃过夜孵育。洗去一抗后,加入二抗常温孵育1h。洗去多余二抗后,加入底物SupersignalTM West Pico PLUS Chemiluminescent(Thermo)曝光显色,图2D结果显示炎性肠病小鼠结肠上皮细胞中FXYD3的蛋白表达明显下降。
实施例3
通过Cre/loxp重组系统构建结肠上皮细胞特异性敲除FXYD3的转基因小鼠:即将FXYD3fl/fl小鼠(携带有loxp序列)与带有Pvillin-cre的转基因小鼠杂交繁殖,经鉴定后将Pvillin+FXYD3 f/f小鼠作为FXYD3IEC-KO实验小鼠。同时,将同窝生的Pvillin-FXYD3fl/fl小鼠作为WT对照组。其中,FXYD3fl/fl小鼠和Pvillin-cre品系小鼠均为C57BL/6背景,购买于南京大学-南京模式动物中心。
阿利新蓝糖原染色(AB-PAS):小鼠肠道组织石蜡包埋切片后,脱蜡至水:依次将切片放入二甲苯Ⅰ20min-二甲苯Ⅱ20min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-75%酒精5min,自来水洗。阿利新蓝染色:切片入阿利新蓝染液中染色5min,自来水洗2min;高碘酸染色:切片入高碘酸染液中15min,自来水洗,蒸馏水洗两遍;雪弗染色:切片入雪弗染液避光染色30min,,流水冲洗5min;苏木素染色:切片入苏木素染液染3-5min,自来水洗,分化液分化,自来水洗,返蓝液返蓝,流水冲洗。脱水封片:切片依次入无水乙醇I 5min-无水乙醇II5min-无水乙醇Ⅲ5min-二甲苯Ⅰ5min-二甲苯Ⅱ5min透明,中性树胶封片。显微镜镜检,图像采集分析。图3A结果发现,FXYD3IEC-KO小鼠结肠组织中杯状细胞数量和黏液层厚度明显减少。
EUB338原位杂交染色:小鼠肠道组织石蜡包埋切片后,将载玻片浸在二甲苯中,室温10min。重复两次,每次用新的二甲苯;将载玻片依次在100%乙醇中脱水,室温5min,85%乙醇3min,70%乙醇三分钟;将载玻片浸在含0.1%DEPC的1×PBS中,10min,重复一次。杂交:加200μl 3%BSA在载玻片上,37℃孵育2h;用杂交buffer稀释FISH探针到终浓度大概0.1~0.4μM,84℃变性5min,再加20-50μl到载玻片之前先37℃放置3min,盖上盖玻片用Rubber cement(橡胶黏合剂)封片;在预温为38-42℃的杂交室杂交,孵育过夜;杂交后洗脱:仔细除去封片胶,将载玻片在wash solution洗脱液中室温处理15min使盖玻片松落(直到盖玻片与载玻片分开),重复一次;在wash solution洗脱液中冲洗15min,重复一次;将载玻片浸在75%乙醇2min,100%乙醇2min,空气中晾干20min;在避光条件下加20μl DAPI-Antifade solution进行复染。盖上盖玻片,孵育10min,之后在荧光显微镜下观察染色情况。图3B结果显示FXYD3IEC-KO小鼠肠腔中黏液厚度明显薄于WT小鼠。
透射电镜:取结肠距肛门1cm处的2mm长的结肠组织用2.5%戊二醛溶液固定2个小时。样品四度过夜后用1ml 0.1M PBS缓冲液洗涤三次,每次15分钟。洗涤结束后用50-100μl1%锇酸固定1小时。固定之后用水漂洗,10分钟洗三次。洗涤后,用100μl 2%醋酸铀固定/染色30分钟。之后脱水:50%、70%、90%乙醇各15分钟,100%乙醇20分钟,100%丙酮20分钟两次。包埋剂+纯丙酮(1:1)室温2小时,包埋剂+纯丙酮(3:1)过夜。纯包埋剂换液,以正确方向包埋37℃聚合。聚合后超薄切片(Leica UC7),染色于透射电镜TECNAI仪器上机观察。图3C结果显示FXYD3IEC-KO小鼠结肠杯状细胞中黏液蛋白不能突破细胞膜分泌到肠腔中。
扫描电镜:取结肠距肛门1cm处的2mm长的结肠组织,纵切开,封口膜上展平2.5%戊二醛溶液固定2个小时,后四度过夜。固定结束后,用1ml 0.1M PBS缓冲液清洗三次,每次十五分钟。去除PBS,用1%锇酸溶液固定样品1.5小时。PBS缓冲液洗涤样品三次,每次十五分钟。之后50%、70%、90%乙醇各15分钟,100%乙醇20分钟两次。样品临近点干燥,样品镀膜,电镜观察。图3D结果显示,FXYD3IEC-KO小鼠肠腔面存在许多“泡泡”,呈现葡萄样分布在肠上皮组织,而WT小鼠杯状细胞的肠腔面则有许多已经分泌出的黏蛋白。这些结果表明将结肠杯状细胞的FXYD3敲除会导致杯状细胞的分泌功能受损。
实施例4
鼠柠檬酸杆菌感染:8周雄性WT和FXYD3IEC-KO小鼠柠檬酸杆菌感染前饲喂抗生素7天(链霉素1mg/ml,甲硝唑1mg/ml,万古霉素0.5mg/ml,新霉素1mg/ml),无菌水再饲喂一天后,2×109/200μl灌胃。统计各组小鼠体重变化。七天后处死小鼠,测量结肠长度,HE染色结肠组织。图4A结果显示,FXYD3IEC-KO小鼠体重下降程度明显高于WT。感染第七天处死小鼠,统计各组小鼠结肠长度发现FXYD3IEC-KO小鼠结肠长度明显短于WT组小鼠。结肠组织HE染色观察发现,FXYD3IEC-KO小鼠肠道组织破坏更严重。这些结果表明FXYD3在肠道上皮细胞的缺失加重了鼠柠檬酸杆菌感染引起的肠道炎症。
小鼠炎性肠病模型:8周雄性WT和FXYD3IEC-KO小鼠饲喂含2%DSS水,饲喂7天观察小鼠情况,统计各组小鼠体重变化。再饲喂两天无DSS的水之后处死小鼠,解剖出小鼠结肠,测量结肠长度,HE染色结肠组织。图4B结果发现FXYD3IEC-KO小鼠体重下降程度明显高于WT组。第九天处死小鼠,取出结肠,发现FXYD3IEC-KO小鼠结肠长度明显短于WT,结肠组织H&E染色显示FXYD3IEC-KO小鼠结肠破坏更加严重。这些结果表明FXYD3在肠道上皮细胞的缺失促进DSS诱导的炎性肠病。
实施例5
分离未造模WT和FXYD3IEC-KO小鼠结肠上皮细胞,分离方法同实施例2,进行转录组测序,图5A分析差异基因的表达,图5B分析了差异基因相关的KEGG信号通路(pathway),图5C分析结果发现STAT3信号通路相关基因在FXYD3IEC-KO小鼠来源的肠上皮细胞中表达下降,表明FXYD3能够调控STAT3信号。
分离未造模WT和FXYD3IEC-KO小鼠结肠上皮细胞,分离方法同实施例2,IL-22(10ng/ml),IL-10(50ng/ml)和IL-6(50ng/ml)处理后蛋白免疫印迹实验检测信号激活情况。蛋白免疫印迹实验方法同实施例2。图5D结果显示FXYD3IEC-KO小鼠肠道上皮细胞,在IL-22,IL-6和IL-10刺激下,STAT3信号活化程度显著低于WT小鼠来源的肠道上皮细胞,表明FXYD3能够促进STAT3信号激活
实施例6
蛋白免疫共沉淀方法检测LS174T细胞中STAT3和FXYD3的相互作用,JAK1和FXYD3的相互作用。
蛋白免疫共沉淀方法:细胞经过刺激后使用NP40蛋白裂解液在冰上裂解20-30min后,12000g,4℃离心15min,蛋白上清使用BCA法测定细胞全裂解液的蛋白浓度,取200μg蛋白样品与Flag-M2磁珠或琼脂糖珠偶联好的目的抗体4℃摇匀结合过夜后,形成磁珠蛋白复合物的样品用IP洗涤液室温洗三次,每次15min,加入1x蛋白上样缓冲液,100℃煮沸15min后进行免疫印记实验。图6结果显示FXYD3能够与JAK1和STAT3结合。在LS174T细胞中敲减FXYD3则会造成JAK1和STAT3结合减弱,表明FXYD3能够促进JAK1-STAT3的结合从而促进信号的传导。
实施例7
培养LS174T细胞,IL-22(10ng/ml)或IL-10(50ng/ml)处理24、48h后,收集细胞,裂解。蛋白免疫印迹实验检测细胞中FXYD3的蛋白表达变化,蛋白免疫印迹实验方法同实施例5。图7结果显示细胞因子IL-22和IL-10能够促进肠道上皮细胞中FXYD3的表达。
序列表
<110> 浙江大学
<120> FXYD3蛋白和基因在炎症性肠道疾病的诊断和治疗中的应用
<160> 6
<170> SIPOSequenceListing 1.0
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<213> 智人(Homo sapiens)
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agaggcttaa gaggcccgag tttcacccag tccccactcc acggtgcagc tgcggcttat 120
ctctcagccc agcgagatgc cagccttcct gtcccgggcc agcgctctga catgcagaag 180
gtgaccctgg gcctgcttgt gttcctggca ggctttcctg tcctggacgc caatgaccta 240
gaagataaaa acagtccttt ctactatgac tggcacagcc tccaggttgg cgggctcatc 300
tgcgctgggg ttctgtgcgc catgggcatc atcatcgtca tgagtgcaaa atgcaaatgc 360
aagtttggcc agaagtccgg tcaccatcca ggggagactc cacctctcat caccccaggc 420
tcagcccaaa gctga 435
<210> 2
<211> 87
<212> PRT
<213> 智人(Homo sapiens)
<400> 2
Met Gln Lys Val Thr Leu Gly Leu Leu Val Phe Leu Ala Gly Phe Pro
1 5 10 15
Val Leu Asp Ala Asn Asp Leu Glu Asp Lys Asn Ser Pro Phe Tyr Tyr
20 25 30
Asp Trp His Ser Leu Gln Val Gly Gly Leu Ile Cys Ala Gly Val Leu
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Cys Ala Met Gly Ile Ile Ile Val Met Ser Ala Lys Cys Lys Cys Lys
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Phe Gly Gln Lys Ser Gly His His Pro Gly Glu Thr Pro Pro Leu Ile
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Thr Pro Gly Ser Ala Gln Ser
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<210> 3
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<213> 人工序列(Artificial Sequence)
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tacagcatgt cctactcgca g 21
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<213> 人工序列(Artificial Sequence)
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gaggaagagg taaccacagg g 21
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aggttggcgg gctcatc 17
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catttgcatt ttgcactcat gac 23
Claims (8)
1.人FXYD3基因mRNA作为靶点在制备用于诊断炎症性肠道疾病的试剂盒中的应用。
2.一种用于诊断炎症性肠道疾病的试剂盒,其特征在于,包括:
(1)用于将mRNA反转录成cDNA的反转录试剂,
(2)用于PCR扩增人FXYD3基因cDNA序列的引物,
(3)已知浓度的、含有用(2)中引物PCR扩增人FXYD3基因cDNA所得扩增片段的阳性对照。
3.如权利要求2所述的试剂盒,其特征在于,还包括用于实时荧光定量PCR检测的荧光染料。
4.人FXYD3蛋白作为靶点在制备用于诊断炎症性肠道疾病的试剂盒中的应用。
5.一种用于诊断炎症性肠道疾病的试剂盒,其特征在于,包括:
(1)特异性结合人FXYD3蛋白的抗体,
(2)当作为一抗的(1)中抗体与人FXYD3蛋白结合时,能够结合于一抗的二抗,二抗为带有检测标记物的抗体。
6.如权利要求5所述的试剂盒,其特征在于,所述检测标记物为过氧化物酶、磷酸酯酶或荧光素酶,所述试剂盒还包括作为检测标记物的反应对象的化学发光化合物、显色底物或荧光染料。
7.一种用于治疗炎症性肠道疾病的药物,其特征在于,有效成分为以下至少一种:
(1)能够外源表达FXYD3基因的重组表达载体;
(2)能够提高内源FXYD3基因表达的化合物。
8.如权利要求7所述的药物,其特征在于,(2)中化合物为细胞因子IL-22或IL-10。
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