CN106093393A - 一种基于XBP1和XBP1s表达水平的检测胃癌组织的试剂盒 - Google Patents

一种基于XBP1和XBP1s表达水平的检测胃癌组织的试剂盒 Download PDF

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CN106093393A
CN106093393A CN201610387223.7A CN201610387223A CN106093393A CN 106093393 A CN106093393 A CN 106093393A CN 201610387223 A CN201610387223 A CN 201610387223A CN 106093393 A CN106093393 A CN 106093393A
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xbp1
detection
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protein
xbp1s
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田梗
王滨
姜文国
米佳
李旭日
杨春华
刘芳
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Binzhou Medical College
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

本发明涉及一种基于XBP1和XBP1s表达水平的检测胃癌组织的试剂盒，包含以下试剂：用于检测XBP1和XBP1s的蛋白水平的蛋白质检测试剂组和用于检测XBP1和XBP1s的mRNA水平的核酸检测试剂组；所述蛋白质检测试剂组包含抗XBP1抗体；所述核酸检测试剂组包含检测引物组；所述XBP1的蛋白质序列如SEQ ID NO:1所示，所述XBP1的cDNA编码序列如SEQ ID NO:2所示；所述XBP1s的蛋白质序列如SEQ ID NO:3所示，所述XBP1s的cDNA编码序列如SEQ ID NO:4所示。使用本发明，可有效地甄别正常组织和胃癌组织，既可用于科研工作中，也有助于胃癌早期诊断。

Description

一种基于XBP1和XBP1s表达水平的检测胃癌组织的试剂盒
技术领域
本发明涉及肿瘤检测领域，更特别地，涉及一种检测胃癌组织的试剂盒。
背景技术
我国是胃癌高发国家，其发病率和死亡率分别高居全部恶性肿瘤的第3位和第2位。由于胃癌起病隐匿，早期多无明显症状，不易觉察，使得胃癌的早期发现非常困难。特别是在我国，超过80％的胃癌在确诊时就已处于进展期，预后很差，严重威胁着人们的生命健康。
目前，除了影像学检查和内镜检查之外，生物大分子肿瘤标志物的检测也可用于胃癌的诊断。临床上常用的生物大分子肿瘤标志物如癌胚抗原(CEA)、糖蛋白类抗原(CA19-9、CA125)能提高体内恶性肿瘤特别是胃肠道恶性肿瘤的检出率，但这些标志物并不能做到早期高灵敏度、高特异性地诊断胃癌。由于体内各器官分化来源、肿瘤的成瘤原因以及肿瘤分泌的肿瘤因子均不相同，因此要特异性地、高灵敏地早期检测出某一种特定肿瘤，仍是一项很庞大的课题。
内质网是一种重要的真核细胞器，主要负责可溶性蛋白和膜蛋白的生物合成、折叠以及修饰，同时也是细胞内的动态Ca2+储存库。由于病毒感染、药物损伤等原因引起的内质网中出现错误折叠、或未折叠蛋白在内质网腔内聚集以及Ca2+平衡紊乱的状态称为内质网应激(endoplasmic reticulum stress，ERS)。为了确保蛋白折叠的正确性以及预防非折叠或者错误折叠蛋白在细胞内的聚集，抵抗ERS的不利作用，细胞初期呈现出保护性的未折叠蛋白反应(Unfold Protein Response，UPR)，其改变细胞的转录和翻译程序以缓解ERS，因此一定程度的ERS能激活细胞保护性适应机制。而严重的应激损伤内质网功能，则引发细胞的自发性死亡程序，最终诱导细胞凋亡。内质网应激发生后可通过IRE1、PERK和ATF6三个重要蛋白诱导三种不同的信号途径来介导UPR。而正确的蛋白折叠和转录后修饰需要大量的内质网分子伴侣蛋白的参与，比如GRP78、钙连接蛋白(calnexin)、钙网蛋白(calreticulin，CALR)和蛋白质二硫键异构酶(protein disulfide isomerases,PDI)等。
X盒连接蛋白1(X-box binding protein-1,XBP1)，以活化形式XBP1s来激活目的基因的表达，其功能非常庞杂。它一方面促进细胞适应环境在缺氧条件下生存和增殖，另一方面诱导蛋白折叠、ER相关蛋白降解(ERAD)、蛋白易位和蛋白分泌来减轻ERS。在一些研究中也发现，ERS激活有助于对抗肿瘤。特别是对抗肠道肿瘤的发生方面，XBP1具有保护作用。
发明内容
发明人在研究胃癌的过程中，发现胃癌患者的胃癌组织样本中XBP1的表达受到显著抑制。在该发现的基础上，本发明提供了一种基于XBP1和XBP1s表达水平的检测胃癌组织的试剂盒，包含以下试剂：用于检测XBP1和XBP1s的蛋白水平的蛋白质检测试剂组和用于检测XBP1和XBP1s的mRNA水平的核酸检测试剂组；
所述蛋白质检测试剂组包含抗XBP1抗体；所述核酸检测试剂组包含检测引物组；
所述XBP1的蛋白质序列如SEQ ID NO:1所示，所述XBP1的cDNA编码序列如SEQ IDNO:2所示；
所述XBP1s的蛋白质序列如SEQ ID NO:3所示，所述XBP1s的cDNA编码序列如SEQID NO:4所示。
进一步地，所述蛋白质检测试剂组中还包含针对所述抗XBP1抗体的具有显示标志物的二抗、与所述显示标志物对应的显示系统。所述显示标志物可为辣根过氧化物酶，所述显示系统可为DAB显示系统或ECL显示系统。
进一步地，所述蛋白质检测试剂组中还包含上样量标度，例如β-actin，以保证上样量相同。
进一步地，所述检测引物对由如SEQ ID NO:5所示序列的引物1、如SEQ ID NO:6所示序列的引物2、如SEQ ID NO:7所示序列的引物3和如SEQ ID NO:8所示序列的引物4组成。其中，，所述引物1与所述引物2用于检测所述XBP1的mRNA水平，所述引物3和所述引物4用于检测所述XBP1s的mRNA水平。
进一步地，所述核酸检测试剂组还包含逆转录酶及其缓冲液、荧光探针、dNTP、RNA酶抑制剂。
使用本发明，可有效地甄别正常组织和胃癌组织，既可用于科研工作中，也有助于胃癌早期诊断。
附图说明
图1为使用本发明的试剂盒检测正常组织和胃癌组织中的XBP1以及XBP1s蛋白含量的Westren blot图；
图2为使用本发明的试剂盒检测正常组织和胃癌组织中的XBP1 mRNA水平的统计图。
图3为使用本发明的试剂盒检测正常组织和胃癌组织中的XBP1s mRNA水平的统计图。
具体实施方式
以下结合实施例和附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
发明人在研究胃癌的过程中对ESR蛋白例如CALR、PDIA3、PDIA6和GRP94进行了检测，发现在胃癌组织中以上蛋白质的表达均呈不同程度的升高，通过进而研究ERS相关蛋白的信号通路上的因子，发明人意为地发现，XPB1和XPB1s的mRNA水平和蛋白质水平在胃癌组织中都显示出显著降低，这提示，XPB1和XPB1s的表达水平可用于鉴别胃癌组织和正常胃部组织。
实施例1基于XBP1和XPB1s表达水平的检测胃癌组织的试剂盒
本实施例的试剂盒包含以下试剂：
1)用于检测XBP1和XPB1s的蛋白水平的蛋白质检测试剂组，包含抗XBP1抗体(abcam3715，Abcam)、抗XPB1s抗体()、辣根过氧化物酶标记的二抗、DAB显示系统、抗β-actin抗体；
2)用于检测XBP1和XPB1s mRNA水平的核酸检测试剂组，包含如SEQ ID NO:5所示序列的引物1、如SEQ ID NO:6所示序列的引物2、如SEQ ID NO:7所示序列的引物3和如SEQID NO:8所示序列的引物4，以及逆转录酶M-MLV及其缓冲液、荧光探针SYBR-Green I、dNTP、RNA酶抑制剂。
实施例2Western blot测定XBP1和XPB1s的表达
从正常组织和胃癌组织中提取总蛋白，进行SDS-PAGE电泳。使用蛋白质检测试剂组，按照常规的Western blot法，对总蛋白中的XBP1和XPB1s分别用抗XBP1抗体和抗XPB1s抗体进行染色，结果如图1所示，胃癌组织(T泳道)中XBP1和XPB1s的蛋白含量显著低于正常组织(N泳道)。
实施例3
从正常组织和胃癌组织中提取总RNA，使用核酸检测试剂组，按照常规的一步式RT-PCR法，用引物1和引物2检测总RNA中的XBP1 mRNA的含量，用引物3和引物4检测总RNA中的XBP1s mRNA的含量。结果如图2和图3所示，胃癌组织中XBP1和XPB1s的mRNA水平远低于正常组织。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (8)

1.一种基于XBP1和XBP1s表达水平的检测胃癌组织的试剂盒，其特征在于，包含以下试剂：用于检测XBP1和XBP1s的蛋白水平的蛋白质检测试剂组和用于检测XBP1和XBP1s的mRNA水平的核酸检测试剂组；
所述蛋白质检测试剂组包含抗XBP1抗体；所述核酸检测试剂组包含检测引物组；
所述XBP1的蛋白质序列如SEQ ID NO:1所示，所述XBP1的cDNA编码序列如SEQ ID NO:2所示；
所述XBP1s的蛋白质序列如SEQ ID NO:3所示，所述XBP1s的cDNA编码序列如SEQ IDNO:4所示。
2.根据权利要求1所述的检测胃癌组织的试剂盒，其特征在于，所述蛋白质检测试剂组中还包含针对所述抗XBP1抗体的具有显示标志物的二抗、与所述显示标志物对应的显示系统。
3.根据权利要求2所述的检测胃癌组织的试剂盒，其特征在于，所述显示标志物为辣根过氧化物酶。
4.根据权利要求2所述的检测胃癌组织的试剂盒，其特征在于，所述显示系统为DAB显示系统或ECL显示系统。
5.根据权利要求1所述的检测胃癌组织的试剂盒，其特征在于，所述蛋白质检测试剂组中还包含上样量标度。
6.根据权利要求5所述的检测胃癌组织的试剂盒，其特征在于，所述上样量标度为抗β-actin抗体。
7.根据权利要求1所述的检测胃癌组织的试剂盒，其特征在于，所述检测引物组由如SEQ ID NO:5所示序列的引物1、如SEQ ID NO:6所示序列的引物2、如SEQ ID NO:7所示序列的引物3和如SEQ ID NO:8所示序列的引物4组成。
8.根据权利要求1-7中任一项所述的检测胃癌组织的试剂盒，其特征在于，所述核酸检测试剂组还包含逆转录酶及其缓冲液、荧光探针、dNTP、RNA酶抑制剂。
CN201610387223.7A 2016-06-02 2016-06-02 一种基于XBP1和XBP1s表达水平的检测胃癌组织的试剂盒 Pending CN106093393A (zh)

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