CN113804893A - 尿液I型胶原蛋白α1链及其多肽片段在烧伤中的应用 - Google Patents
尿液I型胶原蛋白α1链及其多肽片段在烧伤中的应用 Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
本发明提供一种尿液I型胶原蛋白α1链[Collagen alpha‑1(I) chain]及其多肽片段的应用,具体为尿液I型胶原蛋白α1链及其多肽片段在制备用于烧伤诊断、鉴别诊断、烧伤面积及程度评价、治疗效果评价、监测、预后评估及机理研究等制剂的应用。烧伤是日常生活中常见的重要创伤,每年每100万人中约有5000~100000人烧伤,据世界卫生组织统计,每年全球死于烧伤患者超过30万人,严重烧伤救治存活率仍处于较低水平。本发明通过研究证实,与健康人(正常对照组)相比,尿液I型胶原蛋白α1链及其多肽片段在烧伤患者中表达升高。可用于烧伤患者的各种目的应用检测。本发明发挥尿液标本获取无创、可大规模重复取样、保存方便的优势,利用尿液标本检测尿液I型胶原蛋白α1链及其多肽片段。
Description
技术领域
本发明涉及尿液I型胶原蛋白α1链及其多肽片段的新用途,具体涉及尿液I型胶原蛋白α1链及其多肽片段在于烧伤诊断、鉴别诊断、烧伤面积及程度评价、治疗效果评价、监测、预后评估及机理研究等的应用。
背景技术
烧伤是指由于火焰、高温气体、灼热的固体或液体、放射线、电能、强酸强碱等化学物质所引起的人体皮肤、组织甚至深层脏器的损伤,是一种全身性综合性疾病。烧伤后创面组织大量坏死、感染、休克及凝血功能障碍等反应可引起机体发生一系列的病理生理学变化。不同程度的烧伤对人体有着不同的影响,严重的烧伤可对人体内环境造成破坏,使烧伤患者出现各系统复杂性的病理生理变化,相关的检测指标也会随着烧伤严重程度的不同随之出现相应的改变。及时检测这些指标的变化可为临床医生对于疾病的诊断,病情的判断,治疗方法的选择以及患者预后的评估等多方面提供有价值的参考依据。
但是,严重烧伤的病人皮肤完整性较差,血液学检测作为皮肤有创性检查在此类患者的临床应用中存在着诸多困难,反复的抽血检测还会加剧患者痛苦。尿液作为血液的超滤液蕴含着丰富的生物学信息,其采集过程具有无创便捷等优势,这在烧伤患者的检测中尤为明显。在尿液中寻找有助于烧伤诊断及反映病情变化的生物标志物,可提高烧伤患者的生活质量和依从性,减轻多次采血的痛苦,更好的为临床医生提供有利于疾病诊断和治疗的参考依据。
胶原蛋白是人体结缔组织中的主要成分,是体内含量最多、分布最广的功能性蛋白,占蛋白质总量的25%~30%。其中I型胶原蛋白在目前已发现的29中胶原蛋白中含量最高,约占90%。它在组织的形态发生和细胞代谢中起着重要作用,其主要分布于皮肤、角膜、肌腱等部位。其具有多种生物学作用,例如维护细胞、组织的正常生理功能以及对其损伤进行修复。 I型胶原蛋白α1链[Collagen alpha-1(I) chain, COL1A1]由COL1A1基因编码。本研究烧伤患者尿液中I型胶原蛋白α1链较健康人组出现表达上调,烧伤患者尿液中该蛋白含量增加。
与常用的临床血液样本相比,尿液可以完全无创、连续、大量收集;没有稳态调节,可累积更多种类、更大幅度的变化,机体的很多病理生理变化可能体现在尿液中。一些激素和细胞因子等分子量相对较小的蛋白多肽入血后,会很快被排泄进入尿液,这些蛋白和多肽在尿液中被检测到的概率比在血中大很多;尿液收集之前,尿中可能的蛋白降解过程已经完成,所以尿蛋白可在较长时间内保持稳定。为减轻烧伤患者多次采血的痛苦,本实验在前期方法学摸索的基础上,期望通过尿液蛋白或多肽研究,实现用无痛、方便、快捷、易重复的尿液检测辅助烧伤患者的诊断及病情监测,也为进一步尿液多肽检测试剂盒的研究奠定基础。
发明内容
本发明的目的在于提供一种尿液I型胶原蛋白α1链及其多肽片段在制备用于烧伤诊断、鉴别诊断、烧伤面积及程度评价、治疗效果评价、监测、预后评估及机理研究等制剂的应用。
优选地,所述尿液I型胶原蛋白α1链的氨基酸序列如SEQ ID NO.1所示(MFSFVDLRLL LLLAATALLT HGQEEGQVEG QDEDIPPITC VQNGLRYHDR DVWKPEPCRI CVCDNGKVLCDDVICDETKN CPGAEVPEGE CCPVCPDGSE SPTDQETTGV EGPKGDTGPR GPRGPAGPPG RDGIPGQPGLPGPPGPPGPP GPPGLGGNFA PQLSYGYDEK STGGISVPGP MGPSGPRGLP GPPGAPGPQG FQGPPGEPGEPGASGPMGPR GPPGPPGKNG DDGEAGKPGR PGERGPPGPQ GARGLPGTAG LPGMKGHRGF SGLDGAKGDAGPAGPKGEPG SPGENGAPGQ MGPRGLPGER GRPGAPGPAG ARGNDGATGA AGPPGPTGPA GPPGFPGAVGAKGEAGPQGP RGSEGPQGVR GEPGPPGPAG AAGPAGNPGA DGQPGAKGAN GAPGIAGAPG FPGARGPSGPQGPGGPPGPK GNSGEPGAPG SKGDTGAKGE PGPVGVQGPP GPAGEEGKRG ARGEPGPTGL PGPPGERGGPGSRGFPGADG VAGPKGPAGE RGSPGPAGPK GSPGEAGRPG EAGLPGAKGL TGSPGSPGPD GKTGPPGPAGQDGRPGPPGP PGARGQAGVM GFPGPKGAAG EPGKAGERGV PGPPGAVGPA GKDGEAGAQG PPGPAGPAGERGEQGPAGSP GFQGLPGPAG PPGEAGKPGE QGVPGDLGAP GPSGARGERG FPGERGVQGP PGPAGPRGANGAPGNDGAKG DAGAPGAPGS QGAPGLQGMP GERGAAGLPG PKGDRGDAGP KGADGSPGKD GVRGLTGPIGPPGPAGAPGD KGESGPSGPA GPTGARGAPG DRGEPGPPGP AGFAGPPGAD GQPGAKGEPG DAGAKGDAGPPGPAGPAGPP GPIGNVGAPG AKGARGSAGP PGATGFPGAA GRVGPPGPSG NAGPPGPPGP AGKEGGKGPRGETGPAGRPG EVGPPGPPGP AGEKGSPGAD GPAGAPGTPG PQGIAGQRGV VGLPGQRGER GFPGLPGPSGEPGKQGPSGA SGERGPPGPM GPPGLAGPPG ESGREGAPGA EGSPGRDGSP GAKGDRGETG PAGPPGAPGAPGAPGPVGPA GKSGDRGETG PAGPTGPVGP VGARGPAGPQ GPRGDKGETG EQGDRGIKGH RGFSGLQGPPGPPGSPGEQG PSGASGPAGP RGPPGSAGAP GKDGLNGLPG PIGPPGPRGR TGDAGPVGPP GPPGPPGPPGPPSAGFDFSF LPQPPQEKAH DGGRYYRADD ANVVRDRDLE VDTTLKSLSQ QIENIRSPEG SRKNPARTCRDLKMCHSDWK SGEYWIDPNQ GCNLDAIKVF CNMETGETCV YPTQPSVAQK NWYISKNPKD KRHVWFGESMTDGFQFEYGG QGSDPADVAI QLTFLRLMST EASQNITYHC KNSVAYMDQQ TGNLKKALLL QGSNEIEIRAEGNSRFTYSV TVDGCTSHTG AWGKTVIEYK TTKTSRLPII DVAPLDVGAP DQEFGFDVGP VCFL);或由SEQ ID NO.1所示的氨基酸序列衍生的,且与SEQ ID NO.1所示的氨基酸序列具有相同功能的氨基酸序列。
优选地,所述制剂为烧伤患者尿液I型胶原蛋白α1链及其多肽片段检测试剂盒。
优选地,所述试剂盒包括抗原抗体反应的免疫方法及其试剂盒如能够特异性结合I型胶原蛋白α1链及其多肽片段的适配体抗体或抗体片段中的一种或多种。
优选地,所述检测方法包括直接检测I型胶原蛋白α1链及其多肽片段的质谱等方法及其相关试剂盒。
优选地,所述检测方法包括直接检测I型胶原蛋白α1链及其多肽片段的相关核酸检测等方法及其相关试剂盒。
优选地,所述试剂盒还包括选自下组的成分:固相载体,稀释液,对照品,标准品,质控品,检测抗体,第二抗体、第二抗体稀释液,发光试剂,洗涤液、显色液、终止液中的任意一种或几种的组合。
优选地,所述标准品包括I型胶原蛋白α1链标准品、人源化标签抗体标准品;较佳地,所述质控品包括:I型胶原蛋白α1链质控品、人源化标签抗体质控品;较佳地,所述固相载体包括:微粒、微球、玻片、试纸条、塑料珠、液相芯片、微孔板或亲和膜等以及同等功能的其他载体。
优选地,所述固相载体的材质为聚氯乙烯、聚苯乙烯、聚丙酰胺、纤维素中的任意一种及具有类似功能的载体。
发明人首先收集了健康人、烧伤患者的尿液标本, 4000r/min离心5min后,吸取上清,采用Bradford法测定提取的蛋白浓度,进行SDS-PAGE酶解。尿液样本的Label-free质谱分析由OrbitrapFusion型质谱完成。将烧伤组和正常对照组在质谱中得到的数据进行定量计算。以蛋白表达量差异在1.5倍以上且经统计检验P<0.05作为参考标准筛选差异性多肽。然后发明人对具有统计学意义的差异性多肽进行鉴定,利用数据库检索得到差异蛋白I型胶原蛋白α1链。
本发明通过研究证实与健康人相比,I型胶原蛋白α1链及其多肽片段在烧伤患者的尿液中呈高表达,与临床诊断有较好的一致性。从而提出检测尿液I型胶原蛋白α1链及其多肽片段可用于烧伤的辅助诊断或病情监测。
本发明发挥尿液标本获取无创、可大规模重复取样、保存方便的优势,利用尿液标本检测尿液I型胶原蛋白α1链及其多肽片段。
为让本发明的上述和其它目的、特征和优点能更明显易懂,下文特举较佳实施例,并配合附图,作详细说明如下。
附图说明
图1是尿液I型胶原蛋白α1链及其多肽片段在烧伤组及健康对照组中含量图。
图2是I型胶原蛋白α1链参与主要生物过程示意图。
具体实施方式
实施例1 尿液标本的收集与处理
选取烧伤患者作为烧伤组,选取同期健康体检者作为正常对照组。收集各组研究对象入院后的新鲜晨尿样本30ml,不能正常排尿者收集其早晨导尿管中尿液,置于干燥、洁净的容器内。将收集的尿液标本4000r/min离心5min后,吸取上清,每管2ml分装,-80℃冰箱保存。
实施例2 质谱分析和尿液多肽的筛选
对尿液样品蛋白提取,并对提取的蛋白浓度进行测定。尿液样本的质谱分析由OrbitrapFusion型质谱完成。将实验组和正常对照组在质谱中得到的数据进行定量计算。组间比较采用t-test进行差异分析,以蛋白表达量差异在1.5倍以上且经统计检验P<0.05作为参考标准筛选差异表达蛋白。
实施例3 差异多肽的鉴定及分析
使用的数据库为Uniprot_Homo数据库,产生的质谱原始文件采用MaxQuant软件处理,检索参数设置见表1。
与健康人相比,I型胶原蛋白α1链在烧伤患者的尿液中高表达,如图1所示,其参与的主要生物过程如图2所示,I型胶原蛋白α1链在正常对照组和烧伤组尿液中的表达有显著性差异。
虽然本发明已以较佳实施例披露如上,然其并非用以限定本发明,任何所属技术领域的技术人员,在不脱离本发明的精神和范围内,当可作些许的更动与改进,因此本发明的保护范围当视权利要求所界定者为准。
SEQUENCE LISTING
<110> 首都医科大学附属北京世纪坛医院
<120> 尿液I型胶原蛋白α1链及其多肽片段在烧伤中的应用
<130> 1
<140> 20PCOL1A1
<141> 2020-05-10
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1464
<212> PRT
<213> Human Urine
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Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His
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Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp
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Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn
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Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly
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Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro
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Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro
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Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala
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Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser
165 170 175
Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro
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Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro
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Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly
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Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg
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Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro
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Gly Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly
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Leu Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu
275 280 285
Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg
290 295 300
Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly
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Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro
325 330 335
Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys
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Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly
355 360 365
Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro
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Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro
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Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly
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Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro
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Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly
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Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln
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Ala Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro
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Gly Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly
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Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro
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Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro
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Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly
645 650 655
Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro
660 665 670
Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln
675 680 685
Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly
690 695 700
Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser
705 710 715 720
Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala
725 730 735
Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly
740 745 750
Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro
755 760 765
Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser
770 775 780
Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly
785 790 795 800
Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro
805 810 815
Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu Pro Gly Asp Ala
820 825 830
Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly
835 840 845
Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala
850 855 860
Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala
865 870 875 880
Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly
885 890 895
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Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly
930 935 940
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945 950 955 960
Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro
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Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly
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Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu
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Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala
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Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu
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Thr Gly Pro Ala Gly Pro Thr Gly Pro Val Gly Pro Val Gly Ala
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Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu
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Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe
1100 1105 1110
Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu
1115 1120 1125
Gln Gly Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro
1130 1135 1140
Pro Gly Ser Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu
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Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp
1160 1165 1170
Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro
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Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln
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Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala
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Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro
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Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys
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Met Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro
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Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met
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Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala
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Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His
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Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr
1325 1330 1335
Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr
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Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr
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His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn
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Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile
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Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp
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Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu
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Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val Ala
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Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val
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Gly Pro Val Cys Phe Leu
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Claims (9)
1.尿液I型胶原蛋白α1链及其多肽片段在制备用于烧伤诊断、鉴别诊断、烧伤面积及程度评价、治疗效果评价、监测、预后评估及机理研究等制剂的应用。
2.根据权利要求1所述的应用,其特征在于,所述尿液I型胶原蛋白α1链的氨基酸序列如SEQ ID NO.1所示;或由SEQ ID NO.1所示的氨基酸序列衍生的,且与SEQ ID NO.1所示的氨基酸序列具有相同功能的氨基酸序列。
3.根据权利要求1所述的应用,其特征在于,所述制剂为烧伤患者尿液I型胶原蛋白α1链及其多肽片段检测试剂盒。
4.根据权利要求3所述的应用,其特征在于,所述试剂盒包括抗原抗体反应的免疫方法及其试剂盒如能够特异性结合I型胶原蛋白α1链及其多肽片段的适配体抗体或抗体片段中的一种或多种。
5.根据权利要求3所述的应用,其特征在于,所述检测方法包括直接检测I型胶原蛋白α1链及其多肽片段的质谱等方法及其相关试剂盒。
6.根据权利要求3所述的应用,其特征在于,所述检测方法包括直接检测I型胶原蛋白α1链及其多肽片段或其相关核酸检测等方法及其相关试剂盒。
7.根据权利要求3所述的应用,其特征在于,所述试剂盒还包括选自下组的成分:固相载体,稀释液,对照品,标准品,质控品,检测抗体,第二抗体、第二抗体稀释液,发光试剂,洗涤液、显色液、终止液中的任意一种或几种的组合。
8.根据权利要求7所述的应用,其特征在于,所述标准品包括I型胶原蛋白α1链标准品、人源化标签抗体标准品;较佳地,所述质控品包括:I型胶原蛋白α1链控品、人源化标签抗体质控品;较佳地,所述固相载体包括:微粒、微球、玻片、试纸条、塑料珠、液相芯片、微孔板或亲和膜等以及同等功能的其他载体。
9.根据权利要求8所述的应用,其特征在于,所述固相载体的材质为聚氯乙烯、聚苯乙烯、聚丙酰胺、纤维素中的任意一种及具有类似功能的载体。
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