CN114113439A - 血清lrg1蛋白在制备肝内胆管癌***诊断试剂盒中的应用 - Google Patents
血清lrg1蛋白在制备肝内胆管癌***诊断试剂盒中的应用 Download PDFInfo
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- CN114113439A CN114113439A CN202110675135.8A CN202110675135A CN114113439A CN 114113439 A CN114113439 A CN 114113439A CN 202110675135 A CN202110675135 A CN 202110675135A CN 114113439 A CN114113439 A CN 114113439A
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Abstract
本发明涉及血清LRG1蛋白在制备肝内胆管癌***转移诊断试剂盒中的应用。本发明的有益效果主要体现在:本发明提供了血清LRG1蛋白作为肝内胆管癌病人血清***转移诊断标志物的新用途,可采用基于TMT标记的定量蛋白质组学技术,根据体内血清蛋白质组学数据对肝内胆管癌病人进行全面***的研究,该标志物经过ELISA验证,结果更为可靠,可为临床应用提供有价值的信息。
Description
(一)技术领域
本发明涉及血清LRG1蛋白在制备肝内胆管癌***转移诊断试剂 盒中的应用。
(二)背景技术
肝内胆管癌(intrahepatic cholangiocarcinoma,ICC)起源于肝内胆管 上皮细胞的恶性转化,是第二位常见的肝内恶性肿瘤,占原发性肝脏恶性 肿瘤的5-15%。近年来,ICC发病率在全世界范围内不断增加,其侵袭能 力强、恶性程度高、预后差。***转移是肝内胆管癌分期和预后的重要 因素,明确***转移状态对精准分期、预后判断及术后辅助治疗决策具 有重要意义。
富含亮氨酸的α-2-糖蛋白(leucine-richalpha-2-glycoprotein,LRG) 为富含亮氨酸重复序列家族中最早发现的成员,包含8个富亮氨酸重复 序列(leucine-richrepeat,LRR),每个LRR长度多为20~30个氨基 酸残基。LRG1可以与TGFβRⅡ/ALK1形成LRG1-ALK1-TGFβRⅡ -ENG复合物,该复合物会激活下游Smad1/5/8通路,发挥促进内皮细胞的迁移及血管生成的作用,因此LRG1表达水平的改变对肿瘤发生发展及 其生物学行为有重要影响。其在胃癌、结肠癌、胰腺癌等疾病进展中具有 重要意义,但血清LRG1在肝内胆管癌***转移诊断方面目前尚无报 道。
(三)发明内容
本发明是对LRG1能否作为肝内胆管癌***诊断标志物进行研究, 提供血清LRG1蛋白在肝内胆管癌***转移诊断中的应用。
本发明采用的技术方案是:
血清LRG1蛋白在制备肝内胆管癌***诊断试剂盒中的应用。本发 明利用TMT结合质谱技术检测肝内胆管癌病人术前血清,发现生物标志 物LRG1蛋白在肝内胆管癌***转移病人术前血清中浓度明显高于未 发生***转移病人的血清浓度,经ELISA验证表明LRG1蛋白在肝内 胆管癌***转移病人血清中浓度明显高于未发生***病人、且其血清 浓度与***转移阳性数目和阳性比呈正相关。
所述LRG1蛋白序列如下:
MSSWSRQRPKSPGGIQPHVSRTLFLLLLLAASAWGVTLSPKDCQ VFRSDHGSSISCQPPAEIPGYLPADTVHLAVEFFNLTHLPANLLQGASK LQELHLSSNGLESLSPEFLRPVPQLRVLDLTRNALTGLPPGLFQASATLDTLVLKENQLEVLEVSWLHGLKALGHLDLSGNRLRKLPPGLLANFTL LRTLDLGENQLETLPPDLLRGPLQLERLHLEGNKLQVLGKDLLLPQPD LRYLFLNGNKLARVAAGAFQGLRQLDMLDLSNNSLASVPEGLWASL GQPNWDMRDGFDISGNPWICDQNLSDLYRWLQAQKDKMFSQNDTR CAGPEAVKGQTLLAVAKSQ
优选的,所述试剂盒为诊断肝内胆管癌淋巴转移的试剂盒。
具体的,所述试剂盒可为检测LRG1蛋白浓度的TMT试剂盒或 ELISA试剂盒。
TMT技术是一种多肽体外标记技术,该技术采用10种同位素的标签, 特异性标记多肽的氨基基团,经过串联质谱LC-MS/MS分析,可同时比 较10组不同样品中蛋白质的相对含量。
本发明中用于检测LRG1蛋白的TMT试剂盒具体组成如下:
TMT10 126label reagent,2×0.8mg
TMT10 127N label reagent,2×0.8mg
TMT10 127C label reagent,2×0.8mg
TMT10 128N label reagent,2×0.8mg
TMT10 128C label reagent,2×0.8mg
TMT10 129N label reagent,2×0.8mg
TMT10 129C label reagent,2×0.8mg
TMT10 130N label reagent,2×0.8mg
TMT10 131label reagent,2×0.8mg
本发明中用于检测LRG1蛋白的ELISA试剂盒购自Invitrogen公司, 包括以下材料:
成分 | 数量 |
Human LRG1 Antibody Coated wells | 1板 |
Human LRG1 Biotin Conjugate | 2管 |
Human LRG1 Standard,recombinant | 2管 |
Wash Buffer Concentrate(20X) | 25mL |
Assay Diluent | 15ml |
Streptavidin-HRP(150X) | 0.125mL |
TMB Substrate | 12mL |
Stop Solution | 8mL |
Adhesive Plate Covers | 2个 |
本发明的有益效果主要体现在:本发明提供了血清LRG1蛋白作为 肝内胆管癌病人血清***诊断标志物的新用途,可采用基于TMT标记 的定量蛋白质组学技术,根据体内血清蛋白质组学数据对肝内胆管癌病人 进行全面***的研究,该标志物经过ELISA验证,结果更为可靠,可为 临床应用提供有价值的信息。
(四)附图说明
图1为血清蛋白质组技术检测的肝内胆管癌***转移病人和未发生淋 巴结转移病人血清中的LRG1浓度;*P<0.05,**P<0.01,***P<0.001;
图2为ELISA检测肝内胆管癌***转移病人和未发生***转移病人 血清中的LRG1浓度;*P<0.05,**P<0.01,***P<0.001;
图3为LRG1在肝内胆管癌***转移诊断中灵敏度和特异性的ROC曲 线评估。
图4为LRG1与***转移阳性数目及***阳性比的相关性分析。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围 并不仅限于此:
实施例1:
实施例中试剂来源:
主要仪器:
实施例1:
血清蛋白质组学技术检测肝内胆管癌***转移病人术前血清蛋白 LRG1浓度明显高于未发生***转移病人浓度。
1.检测样本:
对福建医科大学孟超肝胆医院诊断为肝内胆管癌的病人,术前空腹时 抽取静脉血5mL至生化管,冰盒取回后3000rpm,4℃离心10分钟,取 上层血清分装后保存于-80℃冰箱。***转移状态根据***清扫术后 病理诊断明确。分别收集8例肝内胆管癌***转移病人和8例未发生淋 巴结转移的肝内胆管癌术前血清。
2.检测方法:
(1)采用安捷伦多重亲和去除***MARS-Human 14液相色谱柱去除 血清中的高丰度蛋白;
(2)每组血清定量100μg,按照TMT试剂盒说明书进行胰蛋白酶酶解。 用2个TMT试剂盒,***转移病人低丰度蛋白肽段用一个TMT试剂 盒,未发生***转移病人低丰度蛋白肽段用一个TMT试剂盒。16个病 人及2个混合样品分别按以下顺序标记:
(3)将上述TMT标记样品混合干燥后用95%H2O+5%ACN,pH=10复 溶,通过C18反相色谱柱将混合样品分60个馏份;
(4)每个馏份分别进行低pH值纳升液相色谱串联质谱鉴定。
(5)质谱数据采用Thermo Scientific Proteome Discoverer version 1.4软 件搜索,与UniprotKB人类蛋白质组数据库进行匹配;
3.检测结果:
结果见图1。质谱结果显示,肝内胆管癌***转移病人术前血清蛋 白LRG1浓度明显高于未发生***转移病人浓度。
实施例2:ELISA检测肝内胆管癌***转移病人术前血清LRG1浓 度明显高于未发生***转移病人浓度
1.检测标本:
血清样本取自于2014年2月至2020年4月之间在福建医科大学孟超 肝胆医院行肝切除联合***清扫的肝内胆管癌术前血清。共收集了68 个肝内胆管癌临床样本,其中未发生***转移的肝内胆管癌病人32人, 发生***转移的肝内胆管癌病人36人。
2.检测方法:
(1)将LRG1蛋白的ELISA试剂盒试剂缓慢均衡至室温。
(2)按照试剂盒配制标准品:1管标准品加入标准品稀释液666.7uL, 轻轻混匀,配成75ng/mL标准品浓缩液。按说明书方法依次倍比稀释标 准品。标准品稀释液组成:AssayDiluent(ThermoFisher Scientific,Inc. Human LRG1 ELISA Kit,CatalogNO.EH305RB)。
(3)二抗工作液制备:1管二抗冻干粉中加入150μl二抗稀释液配成 100×浓缩液,临用前用二抗稀释液100倍稀释二抗浓缩液成二抗工作液。 二抗稀释液组成:AssayDiluent A(ThermoFisher Scientific,Inc.Human LRG1 ELISA Kit,CatalogNO.EH305RB)。
(4)Streptavidin HRP工作液制备:临用前用HRP稀释液100倍稀释 StreptavidinHRP浓缩液,混匀。HRP稀释液组成:Assay Diluent B (ThermoFisher Scientific,Inc.Human LRG1 ELISAKit,Catalog NO. EH305RB)。
(5)洗涤液配制:用475mL蒸馏水将25mL浓缩洗涤液稀释至500mL。
(6)用样品稀释液将血清稀释60倍,取100μl标准品及血清依次加入 96孔酶标板上,盖上封闭纸,37℃孵育2小时。浓缩洗涤液组成:Wash Buffer(30×)(ThermoFisherScientific,Inc.Human LRG1 ELISAKit, Catalog NO.EH305RB)。
(7)吸弃所有液体,洗涤3次。
(8)每孔加100μl二抗工作液,盖上封闭纸,37℃孵育1小时。
(9)吸弃所有液体,洗涤3次。
(10)每孔加入100μl HRP工作液,盖上封闭纸,37℃孵育30分钟。
(11)吸弃所有液体,洗涤3次。
(12)每孔加入100μl底物,液体慢慢变为蓝色,室温下孵育10分钟。
(13)每孔加入100μl终止液,液体由蓝色变为黄色。
(14)酶标仪用450nm波长测OD值。
(15)计算:根据标准品浓度和相应的OD值,利用CurveExpert软件 算出标准曲线的直线回归方程,利用样品OD值再在回归方程上计算出样 品的LRG1浓度。
3.检测结果:
结果显示***转移病人术前血清中LRG1浓度明显高于未发生淋 巴结转移病人,LRG1在未发生***转移病人术前血清中的浓度平均为 37.05±1.806ng/mL,而在***转移病人术前血清中则高达 42.85±1.926ng/mL,经统计二者具有显著差异(P<0.05),参见图2。
LRG1诊断肝内胆管癌***转移的ROC曲线参见图3(将*** 转移与未发生***转移病人血清中的LRG1含量进行ROC分析,结果 显示LRG1在预测肝内胆管癌***转移方面具有统计学意义(P<0.05), 其曲线下面积(AUC)为0.664),LRG1血清浓度与肝内胆管癌病人淋巴 结转移相关性关系参见图4(通过Spearman相关性分析术前LRG1血清 浓度与肝内胆管癌病人***转移阳性数及阳性比是否相关)。结果(图 4)显示LRG1浓度与LNR相关度为0.320,与NMLN相关度为0.344, 均具有统计学意义(P<0.05)。
序列表
<110> 福建医科大学孟超肝胆医院(福州市传染病医院)
<120> 血清LRG1蛋白在制备肝内胆管癌***诊断试剂盒中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 347
<212> PRT
<213> Human adenovirus type 1
<400> 1
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Pro His Val Ser Arg Thr Leu Phe Leu Leu Leu Leu Leu Ala Ala Ser
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Ala Trp Gly Val Thr Leu Ser Pro Lys Asp Cys Gln Val Phe Arg Ser
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Asp His Gly Ser Ser Ile Ser Cys Gln Pro Pro Ala Glu Ile Pro Gly
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Tyr Leu Pro Ala Asp Thr Val His Leu Ala Val Glu Phe Phe Asn Leu
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Thr His Leu Pro Ala Asn Leu Leu Gln Gly Ala Ser Lys Leu Gln Glu
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Leu His Leu Ser Ser Asn Gly Leu Glu Ser Leu Ser Pro Glu Phe Leu
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Arg Pro Val Pro Gln Leu Arg Val Leu Asp Leu Thr Arg Asn Ala Leu
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Thr Gly Leu Pro Pro Gly Leu Phe Gln Ala Ser Ala Thr Leu Asp Thr
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Leu Val Leu Lys Glu Asn Gln Leu Glu Val Leu Glu Val Ser Trp Leu
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His Gly Leu Lys Ala Leu Gly His Leu Asp Leu Ser Gly Asn Arg Leu
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Arg Lys Leu Pro Pro Gly Leu Leu Ala Asn Phe Thr Leu Leu Arg Thr
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Leu Asp Leu Gly Glu Asn Gln Leu Glu Thr Leu Pro Pro Asp Leu Leu
195 200 205
Arg Gly Pro Leu Gln Leu Glu Arg Leu His Leu Glu Gly Asn Lys Leu
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Gln Val Leu Gly Lys Asp Leu Leu Leu Pro Gln Pro Asp Leu Arg Tyr
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Leu Phe Leu Asn Gly Asn Lys Leu Ala Arg Val Ala Ala Gly Ala Phe
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Gln Gly Leu Arg Gln Leu Asp Met Leu Asp Leu Ser Asn Asn Ser Leu
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Ala Ser Val Pro Glu Gly Leu Trp Ala Ser Leu Gly Gln Pro Asn Trp
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Asp Met Arg Asp Gly Phe Asp Ile Ser Gly Asn Pro Trp Ile Cys Asp
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Gln Asn Leu Ser Asp Leu Tyr Arg Trp Leu Gln Ala Gln Lys Asp Lys
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Met Phe Ser Gln Asn Asp Thr Arg Cys Ala Gly Pro Glu Ala Val Lys
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Gly Gln Thr Leu Leu Ala Val Ala Lys Ser Gln
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Claims (4)
1.血清LRG1蛋白在制备肝内胆管癌***转移诊断试剂盒中的应用。
2.如权利要求1所述的应用,其特征在于所述试剂盒为诊断肝内胆管癌淋巴转移的试剂盒。
3.如权利要求2所述的应用,其特征在于所述试剂盒为检测LRG1蛋白浓度的TMT试剂盒。
4.如权利要求2所述的应用,其特征在于所述试剂盒为检测LRG1蛋白浓度的ELISA试剂盒。
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