CN109470855B - 肺癌早期诊断用蛋白芯片及试剂盒 - Google Patents
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Abstract
本发明涉及一种肺癌早期诊断用的蛋白芯片及试剂盒。该蛋白芯片包括如下十种抗原片段:MAGE A3抗原片段、ANXA1抗原片段、CTAG1B抗原片段、CAGE1抗原片段、AP3D1抗原片段、CCT8抗原片段、CHK抗原片段、PRKACA抗原片段、EIF4G1抗原片段及MAGE A1抗原片段。该肺癌早期诊断用蛋白芯片及试剂盒创造性的选择出在肺癌早期发生过程中显著表达且能够引起免疫***产生相关抗体的抗原,并筛选得到相应的抗原片段进行组合,利用蛋白芯片技术将抗原片段包被于固相支持物的表面,以检测人体血清中肿瘤相关的自身抗体,实现对肺癌的早期诊断,检测阳性率高,能大大提高早期肺癌诊断的敏感性和特异性。
Description
技术领域
本发明涉及癌症诊断和治疗领域,尤其是涉及一种肺癌早期诊断用蛋白芯片及试剂盒。
背景技术
肺癌是发病率和死亡率增长最快,对人群健康和生命威胁最大的恶性肿瘤之一。近50年来许多国家都报道肺癌的发病率和死亡率均明显增高。目前对肺癌的诊断主要依靠影像学和细胞学,但早期肺癌无明显体征,目前的检测方法检测阳性率很低,往往只有10~15%的病人能够检测并诊断出。肺癌的早期诊断及治疗是提高患者生存率的关键,而目前的诊断方法检测出肺癌大多数已是中晚期,错过了最佳治疗时期。并且传统的ELISA检测血清抗体,速度慢、效率低,需要的血清样本量大,具体检测起来非常不便。
发明内容
基于此,有必要提供一种使用方便的能够用于肺癌早期诊断用的蛋白芯片及试剂盒。
一种肺癌早期诊断用蛋白芯片,包括固相支持物及包被于所述固相支持物表面的抗原;所述抗原包括如下十种抗原片段:MAGE A3抗原片段、ANXA1抗原片段、CTAG1B抗原片段、CAGE1抗原片段、AP3D1抗原片段、CCT8抗原片段、CHK抗原片段、PRKACA抗原片段、EIF4G1抗原片段及MAGE A1抗原片段。
在其中一个实施例中,所述MAGE A3抗原片段的氨基酸序列包括SEQ ID No.1所示的抗原特征氨基酸序列;
所述ANXA1抗原片段的氨基酸序列包括SEQ ID No.2所示的抗原特征氨基酸序列;
所述CTAG1B抗原片段的氨基酸序列包括SEQ ID No.3所示的抗原特征氨基酸序列;
所述CAGE1抗原片段的氨基酸序列包括SEQ ID No.4所示的抗原特征氨基酸序列;
所述AP3D1抗原片段的氨基酸序列包括SEQ ID No.5所示的抗原特征氨基酸序列;
所述CCT8抗原片段的氨基酸序列包括SEQ ID No.6所示的抗原特征氨基酸序列;
所述CHK抗原片段的氨基酸序列包括SEQ ID No.7所示的抗原特征氨基酸序列;
所述PRKACA抗原片段的氨基酸序列包括SEQ ID No.8所示的抗原特征氨基酸序列;
所述EIF4G1抗原片段的氨基酸序列包括SEQ ID No.9所示的抗原特征氨基酸序列;
所述MAGE A1抗原片段的氨基酸序列包括SEQ ID No.10所示的抗原特征氨基酸序列。
在其中一个实施例中,所述抗原片段为重组融合蛋白,还包括位于相应抗原特征氨基酸序列的N端的Myc-Histag以及C端的链霉亲和素。
在其中一个实施例中,所述固相支持物为玻片、免疫印迹膜、微孔板或磁性微珠。
在其中一个实施例中,所述固相支持物为磁性微珠,所述十种抗原片段分别包被于十种不同颜色的磁性微珠表面。
在其中一个实施例中,所述抗原片段为亲和素化的抗原片段,所述固相支持物的表面通过肽键连接生物素化的牛血清白蛋白,所述抗原片段与所述固相支持物之间通过亲和素与生物素化的牛血清白蛋白间接连接。
一种肺癌早期诊断用试剂盒,包括上述任一实施例所述的肺癌早期诊断用蛋白芯片。
在其中一个实施例中,所述肺癌早期诊断用试剂盒还包括含有荧光物质标记的二抗试剂、标准品试剂和质控参比品试剂。
在其中一个实施例中,所述荧光物质标记的二抗试剂是抗人IgG Fc-PE抗体和抗人IgM Fc-PE抗体。
在其中一个实施例中,所述标准品试剂与所述质控参比品试剂均是Anti-Myc嵌合抗体。
上述肺癌早期诊断用蛋白芯片及试剂盒创造性的选择出在肺癌早期发生过程中显著表达且能够引起免疫***产生相关抗体的抗原,并筛选得到相应的抗原片段进行组合,利用蛋白芯片技术将抗原片段包被于固相支持物的表面,以检测人体血清中肿瘤相关的自身抗体,实现对肺癌的早期诊断。通过十种抗原的抗原谱,检测血清中的自身抗体含量,来判断被检者是否患肺癌,比传统方法(如影像学和细胞学等)诊断肺癌,特别是早期肺癌,阳性率高,敏感性强,而且可以同时检测多个(大于100种)血清自身抗体含量,对样品需求少,能大大提高早期肺癌诊断的敏感性和特异性。
附图说明
图1为本发明一实施方式的肺癌早期诊断用蛋白芯片检测原理示意图。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
请结合图1,一实施方式的肺癌早期诊断用蛋白芯片,包括固相支持物及包被于固相支持物表面的抗原。在本实施方式中,所述抗原包括至少如下十种抗原片段:MAGE A3抗原片段、ANXA1抗原片段、CTAG1B抗原片段、CAGE1抗原片段、AP3D1抗原片段、CCT8抗原片段、CHK抗原片段、PRKACA抗原片段、EIF4G1抗原片段及MAGE A1抗原片段。其中,MAGE A3抗原片段的氨基酸序列包括SEQ ID No.1所示的抗原特征氨基酸序列;ANXA1抗原片段的氨基酸序列包括SEQ ID No.2所示的抗原特征氨基酸序列;CTAG1B抗原片段的氨基酸序列包括SEQID No.3所示的抗原特征氨基酸序列;CAGE1抗原片段的氨基酸序列包括SEQ ID No.4所示的抗原特征氨基酸序列;AP3D1抗原片段的氨基酸序列包括SEQ ID No.5所示的抗原特征氨基酸序列;CCT8抗原片段的氨基酸序列包括SEQ ID No.6所示的抗原特征氨基酸序列;CHK抗原片段的氨基酸序列包括SEQ ID No.7所示的抗原特征氨基酸序列;PRKACA抗原片段的氨基酸序列包括SEQ ID No.8所示的抗原特征氨基酸序列;EIF4G1抗原片段的氨基酸序列包括SEQ ID No.9所示的抗原特征氨基酸序列;MAGE A1抗原片段的氨基酸序列包括SEQ IDNo.10所示的抗原特征氨基酸序列。
各抗原片段可以是含有相应抗原特征氨基酸序列的提纯的天然蛋白、或是重组表达的蛋白,或者重组表达的含有相应抗原特征氨基酸序列的重组融合蛋白,或者是直接合成的含相应抗原特征氨基酸序列的多肽等。优选的,在本实施方式中,抗原片段为含有相应抗原特征氨基酸序列的重组融合蛋白,包括在相应的氨基酸序列的N端连接的Myc-Histag片段和在C端连接的链霉亲和素(SA)片段,整个抗原片段的结构为Myc-Histag-肺癌特征抗原-SA。整个抗原片段的DNA片段的密码子为细菌的密码子,以适合于在细菌中表达。其中,Myc的氨基酸序列为EQKLISEEDL,具体如SEQ ID No.11所示;Myc-Histag的DNA序列为GAGCAGAAACTCATCTCTGAAGAGGATCTGCATCACCATCACCATCAC,具体如SEQ ID No.12所示,其中,Histag的DNA序列为CATCACCATCACCATCAC。
本实施方式所述的各抗原片段可采用但不限于下述方法制备得到:
步骤一:合成肺癌特征抗原的DNA片段,并在5’端连接Myc和Histag对应的DNA片段,得到Myc-Histag-肺癌特征抗原的重组融合DNA片段;
步骤二:合成链霉亲和素对应的DNA片段,并将所述链霉亲和素对应的DNA片段连接在Myc-Histag-肺癌特征抗原的重组融合DNA片段的3’端,得到Myc-Histag-肺癌特征抗原-SA的目的重组融合DNA片段;
步骤三:将所述目的重组融合DNA片段克隆入重组表达载体中,转化细菌;
步骤四:挑取阳性转化克隆进行目的重组融合DNA片段的诱导表达;
步骤五:破碎菌体,离心收集含重组融合蛋白的包涵体沉淀,纯化后得到Myc-Histag-肺癌特征抗原-SA的重组融合蛋白。
在本实施方式中,所述将链霉亲和素对应的DNA片段连接在Myc-Histag-肺癌特征抗原的融合DNA片段的3’端是但不限于通过overlap PCR(overlap polymerase chainreaction)方法将相应的DNA片段连接。进一步,在overlap PCR连接DNA片段时,还可以在外侧的引物两端加上限制性内切酶的酶切位点,如两端分别加上Nde I和Xho I酶切位点。
在本实施方式中,重组表达载体可以是但不限于pET30b(+)。细菌可以是但不限于BL21大肠杆菌。
在本实施方式中,所述挑取阳性转化克隆进行重组融合DNA片段的诱导表达包括如下步骤:挑取阳性转化克隆,在LB培养基中培养至OD 600至0.6,加入终浓度为2mM的IPTG诱导表达,4小时后离心收集菌体。
在本实施方式中,所述破碎菌体,离心收集含重组融合蛋白的包涵体沉淀,纯化后得到Myc-Histag-肺癌特征抗原-SA的重组融合蛋白包括如下步骤:
超声波震荡破碎菌体,离心收集沉淀,沉淀为含有目的重组融合蛋白的包涵体;
使用浓度为8M的尿素溶解沉淀,在4℃下放置12小时以上,离心去除不溶物,与Ni亲和层析柱混合结合1小时;
结合后装柱,采用pH6.5的8M尿素洗涤层析柱20倍柱体积,然后采用pH5.9的8M尿素洗脱层析柱10倍柱体积,分部收集,最后采用pH4.5的8M尿素洗脱层析柱10倍柱体积,分部收集;
洗脱各组分分别上样进行SDS-PAGE分析,选择目的重组融合蛋白所在的组分进行复性;
将重组融合蛋白所在的组分合并后调整蛋白浓度至0.25mg/ml,对含2M尿素的缓冲液透析,4℃透析12小时,再对含0.2M尿素的缓冲液透析,4℃透析12小时,再对含0.02M尿素的缓冲液透析,4℃透析12小时,再对不含尿素的缓冲液透析,4℃透析12小时;
采用PEG20000吸水浓缩的方法浓缩,离心去沉淀,获得目的重组融合蛋白。
进一步,在本实施方式中,还包括将得到的Myc-Histag-肺癌特征抗原的重组融合DNA片段克隆入pBluescript SK(+/-)原核克隆载体中以进行保存的步骤。
本实施方式的固相支持物可以为玻片、免疫印迹膜、微孔板或磁性微珠。当固相支持物为玻片、免疫印迹膜或微孔板时,抗原片段在固相支持物表面呈阵列分布,多种抗原片段可以是在同一固相支持物表面呈阵列分布或者分布于不同的固相支持物表面;当固相支持物为磁性微珠时,上述十种抗原片段分别包被于十种不同颜色的磁性微珠表面。抗原片段可采用吸附或共价交联等方式固定在固相支持物的表面。
在本实施方式中,抗原片段为亲和素化的抗原片段,如含链霉亲和素(streptavidin)标签的抗原片段;固相支持物表面通过肽键连接生物素化的牛血清白蛋白(biotin-BSA),如在固相支持物的表面修饰有-COOH,通过氨基偶联试剂将biotin-BSA连接在固相支持物表面。抗原片段与固相支持物之间通过亲和素与生物素化的牛血清白蛋白间接连接。抗原片段与固相支持物之间间接连接,较之吸附等直接连接,可以提高蛋白芯片的检测灵敏度。
以磁性微珠为例,磁性微珠表面包被抗原片段的过程可以但不限于如下:
步骤一:在避光震荡条件下,在羧基化的磁性微珠表面通过氨基偶联试剂连接上生物素化的牛血清白蛋白,得到磁性微珠-BSA-Biotin;
步骤二:在避光震荡条件下,将磁性微珠-BSA-Biotin与Myc-Histag-肺癌特征抗原-SA的重组融合蛋白混合,混合后链霉亲和素与生物素连接,即得包被有肺癌特征抗原的液相蛋白芯片。
通过将肺癌特征抗原通过重组融合蛋白的形式,带上链霉亲和素、Myc和Histag标签、并在细菌中大量表达并纯化,极大的方便了相关抗原的生产以及后续与磁性微珠的偶联过程,便于液相蛋白芯片的制作,从而为肺癌的早期诊断提供了新的技术支持。
在羧基化的磁性微珠表面通过氨基偶联试剂连接上生物素化的牛血清白蛋白可使用但不限于下述方法:
1.1磁性微珠氨基试剂及仪器:旋涡振荡仪、旋转混合仪、超声清洗机、磁性分离器、Luminex磁性微珠氨基偶联试剂盒及BSA-biotin;
1.2磁性微珠氨基偶联步骤:
1)将偶联试剂盒从冰箱取出,放置20~30min以恢复室温;
2)重悬磁性微珠:如果使用1mL小瓶保存的磁性微珠,涡旋小瓶10s,超声10s;如果使用4mL小瓶保存的磁性微珠,15~30rpm旋转混合小瓶15min;
3)根据期望偶联的磁性微珠数量,吸取适量的磁性微珠体积(原始浓度12.5×106个/mL)于偶联反应管,以5×106个磁性微珠为例,吸取400μl磁性微珠用于后续偶联反应;
注:一个反应管最大反应量为12.5×106个磁性微珠;
4)将反应管置于磁性分离器1~2min(或置于离心机,>8000g、1~2min),保持反应管置于磁性分离器,用滴管小心移除上清;
5)往反应管中加入500μl Activation buffer,涡旋反应管10s,超声10s;
6)重复步骤5~6一次;
7)将反应管置于磁性分离器1~2min,保持反应管置于磁性分离器,用滴管小心移除上清;
8)如果磁性微珠数量大于5×106个,往反应管内加入400μl Activation buffer;如果磁性微珠数量小于等于5×106个,加入480μl Activation buffer;
9)将反应管涡旋10s,超声10s;
10)将Sμlfo-NHS用最低转速涡旋10s;
11)如果磁性微珠数大于5×106个,往反应管中加入50μl的Sμlfo-NHS;如果磁性微珠数小于等于5×106个,往反应管加入10μl的Sμlfo-NHS;
12)用250μl Activation buffer溶解10mg EDC于一离心管,上下颠倒离心管数次,涡旋离心管10~12s以确保EDC完全溶解均匀;
注:EDC溶解后应尽快使用,一次性使用;
13)如果磁性微珠数大于5×106个,往反应管中加入50μl的EDC;如果磁性微珠数小于等于5×106个,往反应管加入10μl的EDC;
14)最低转速涡旋反应管10s,铝箔包住反应管避光,置于旋转混合仪,15~30rpm、20min;
15)将反应管置于磁性分离器1~2min,保持反应管置于磁性分离器,用滴管小心移除上清;
16)往管内加入500μl Activation buffer,涡旋10s,超声10s;
17)重复15~16步骤两次;
18)将反应管置于磁性分离器1~2min,保持反应管置于磁性分离器,用滴管小心移除上清;
19)加入待偶联的BSA-biotin:8ug/1×10*6个磁性微珠;计算反应总体积:如果磁性微珠数大于5×106个,往反应管中加入Activation buffer至总体积为1000μl;如果磁性微珠数不大于5×106个,往反应管加入Activation buffer至总体积为500μl;
20)最低转速涡旋反应管10s,铝箔包住反应管避光,置于旋转混合仪,15~30rpm、2h;
21)将反应管置于磁性分离器1~2min,保持反应管置于磁性分离器,用滴管小心移除上清;
22)往管内加入500μl Wash buffer,涡旋10s,超声10s;
23)重复21~22步骤两次;
24)将反应管置于磁性分离器1~2min,保持反应管置于磁性分离器,用滴管小心移除上清;
25)往反应管中加入1mL Wash buffer,涡旋10s,超声10s,2~8℃避光保存待用。
本实施方式还提供了一种肺癌早期诊断用试剂盒,其包括上述肺癌早期诊断用蛋白芯片。
进一步,在本实施方式中,该肺癌早期诊断用试剂盒还包括含有荧光物质标记的二抗试剂。所述荧光物质标记的二抗试剂可以但不限于是抗人IgG Fc-PE抗体和抗人IgMFc-PE抗体。
更进一步,在本实施方式中,该肺癌早期诊断用试剂盒还包括标准品试剂和质控参比品试剂。标准品试剂与质控参比品试剂均可以但不限于是Anti-Myc嵌合抗体,其中,标准品为系列梯度浓度的抗体试剂以用于绘制标准曲线,质控参比品试剂为特定浓度的抗体试剂。
各种稳定液(如PBS)、稀释液(如PBS)、洗涤液(如PBS或者PBST):在液体配制室和工作液配制室完成配制,在液体分装室除菌、分装,在包装、贴签室贴签,在试剂盒包装室包装入试剂盒的完整包装。其中,PBS及PBST的配方可采用但不限于如下:
Phosphate-buffered saline(PBS):NaCl 137mM;KCl 2.7mM;Na2HPO4 10mM;KH2PO41.8mM;
Phosphate-buffered saline Tween 20(PBST):NaCl 137mM;KCl 2.7mM;Na2HPO410mM;KH2PO4 1.8mM;0.1%(v/v)Tween 20。
以磁性微珠为例,在检测过程中,当磁性微珠包被完成后,加入被检血样品与磁性微珠混合孵育,使得被检血样品中的这十种抗原的相应抗体与磁性微珠表面的抗原结合,从而间接地吸附于磁性微珠表面。再经通常免疫学技术采用的各种洗涤剂洗去非特异吸附的杂蛋白,在磁性微珠表面留下相应的人抗体。最后,将磁性微珠分别与含有荧光物质标记的二抗(抗人IgG Fc-PE和抗人IgM Fc-PE)混合孵育,使得荧光物质标记的二抗与磁性微珠表面的抗体结合。可采用流式分选的方法探测与上述不同抗原反应的血清抗体,流式分选仪红色光源(如635nm光源)测量包被不同抗原的磁性微珠,绿色光源(如532nm光源)探测与抗原结合的血清抗体,结合抗原的血清抗体可通过二抗的荧光强度来定量。单个抗原结合的血清抗体是否为阳性,根据荧光定量的读数,血清抗体稀释倍数和标准品绘制的曲线加以判断。确定单个抗原是否为阳性后,联合分析10种抗原的11种自身抗体(CTT8对应结合两种血清抗体IgG和IgM)的结果,计算总的抗原阳性的个数,再与相应的判断标准进行比较,凡大于规定标准值者即可判断为肺癌患者。
上述肺癌早期诊断用蛋白芯片及试剂盒创造性的选择出在肺癌早期发生过程中显著表达且能够引起免疫***产生相关抗体的抗原,并筛选得到相应的抗原片段进行组合,利用蛋白芯片技术将抗原片段包被于固相支持物的表面,以检测人体血清中肿瘤相关的自身抗体,实现对肺癌的早期诊断。通过十种抗原的抗原谱,检测血清中的自身抗体含量,来判断被检者是否患肺癌,比传统方法(影像学和细胞学)诊断肺癌,特别是早期肺癌,阳性率高,敏感性强,而且可以同时检测多个(大于100种)血清自身抗体含量,对样品需求少,能大大提高早期肺癌的诊断的敏感性和特异性。
以下为具体实施例部分。
实施例1含肺癌早期诊断的蛋白芯片的试剂盒的制备(液态蛋白芯片诊断试剂盒)
抗原蛋白芯片的制备(液态芯片):购买Luminex公司商品化的磁性微珠,首先将biotin-BSA通过氨基偶联试剂偶联到磁性微珠-COOH上,采用上述制备的10种抗原的纯化重组蛋白(含有Streptavidin标签),通过磁性微珠表面的biotin与抗原含的Streptavidin标签的结合,将10种抗原分别结合到不同颜色的磁性微珠表面。然后在磁性微珠表面加上一层含20%胎牛血清的PBS,室温保湿放置1小时后4℃放置过夜,以充分封闭磁性微珠上的非特异蛋白交联或结合位点,最后分装,置于4℃下保存。以上过程制备成功液态蛋白芯片。
各种稳定、稀释、洗涤液、用于绘制标准曲线的各标准品溶液、质控参比品、荧光物质标记的二抗等均直接配制、分装。其中,标准品溶液采用特定稀释液,配制指定系列梯度浓度的Anti-Myc嵌合抗体,分装;质控参比品采用特定稀释液,配制指定浓度的Anti-Myc嵌合抗体,分装;荧光物质标记的二抗采用特定稳定液,配制指定浓度的抗人IgG Fc-PE和抗人IgM Fc-PE抗体,分装。
实施例2采用实施例1的试剂盒诊断普查筛检肺癌疑似患者以及健康人群
采用实施例2所述的试剂盒,采用蛋白芯片加免疫荧光法检测肺癌患者200例、正常人对照200例、肺癌高危人群2000例、普通社区人群1000例。采用正常人对照200例划定肺癌诊断标准进行结果判断。结果如表1所示。
表1采用实施例1的试剂盒诊断普查筛检肺癌疑似患者以及健康人群
肺癌患者200例、正常人对照200例中,试剂盒检测出肺癌患者阳性的158例、正常人阳性的20例,因此该试剂盒诊断肺癌的敏感性为79.0%,特异性为90.0%,说明该试剂盒诊断肺癌的敏感性特异性均较高。
肺癌患者200例中,I期肺癌患者有50例,其中42例试剂盒检测结果为阳性,早期肺癌(I期)的敏感性为84.0%,说明该试剂盒早期诊断肺癌的敏感性特异性均较高。
肺癌高危人群2000例中282例被判断为肺癌,高危人群检测阳性率大约为14.1%。2000例高危人群经随访,其中46人在此后被临床确诊为肺癌,而这46人中28人试剂盒检测阳性,说明该试剂盒可以以较高的灵敏度将肺癌患者从被检人群中筛检出来,敏感性约60.87%,从而可以用于肺癌高危人群的筛查。
被检测的普通社区人群(健康人群)1000例中103例被判断为肺癌。1000例普通社区人群经随访,其中2人在此后被临床确诊为肺癌,而这2人中全部为试剂盒检测阳性,说明该试剂盒可以以较高的灵敏度将肺癌患者从健康人群中筛检出来,从而可以用于普通社区人群的筛查。
实施例3联合使用其他检测试剂盒,以提高肺癌早期诊断的特异性和敏感性。
申请号201610007369.4,名称为蛋白芯片、蛋白质芯片诊断试剂盒制备及使用方法的专利申请中的蛋白芯片及试剂盒通过检测6种肺癌相关的自身抗原对应的自身抗体(IgG和IgM)的相对含量,来判定被检人是否患有肺癌。这6种抗原分别是(1)P53抗原片段;(2)SOX2抗原片段;(3)COPB1抗原片段;(4)EFHD2抗原片段;(5)EIF4G3抗原片段;(6)PCNA抗原片段。该试剂盒诊断肺癌的敏感性较高,特别是对早期(I期)敏感性为82.0%,特异性为88.0%。
联合使用实施例1所述的试剂盒和上述6种肺癌抗原自身抗体检测试剂盒,采用蛋白芯片加免疫荧光法检测肺癌患者200例、正常人对照200例、用正常人对照200例划定肺癌诊断标准进行结果判断。结果如表2所示。
表2
肺癌患者200例、正常人对照200例中,联合使用两种试剂盒检测出肺癌患者阳性的186、正常人阳性的18,因此联合使用两种试剂盒诊断肺癌的敏感性为93.0%,特异性为91.0%,说明该试剂盒诊断肺癌的敏感性,特异性均较高。
肺癌患者200例中,I期肺癌患者有82例,其中76例联合使用试剂盒检测结果为阳性,早期肺癌(I期)的敏感性为92.6%,并未发生显著下降,说明该试剂盒早期诊断肺癌的敏感性特异性均较高。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 广州市丹蓝生物科技有限公司
<120> 肺癌早期诊断用蛋白芯片及试剂盒
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<400> 5
Met Ala Leu Lys Met Val Lys Gly Ser Ile Asp Arg Met Phe Asp Lys
1 5 10 15
Asn Leu Gln Asp Leu Val Arg Gly Ile Arg Asn His Lys Glu Asp Glu
20 25 30
Ala Lys Tyr Ile Ser Gln Cys Ile Asp Glu Ile Lys Gln Glu Leu Lys
35 40 45
Gln Asp Asn Ile Ala Val Lys Ala Asn Ala Val Cys Lys Leu Thr Tyr
50 55 60
Leu Gln Met Leu Gly Tyr Asp Ile Ser Trp Ala Ala Phe Asn Ile Ile
65 70 75 80
Glu Val Met Ser Ala Ser Lys Phe Thr Phe Lys Arg Ile Gly Tyr Leu
85 90 95
Ala Ala Ser Gln Ser Phe His Glu Gly Thr Asp Val Ile Met Leu Thr
100 105 110
Thr Asn Gln Ile Arg Lys Asp Leu Ser Ser Pro Ser Gln Tyr Asp Thr
115 120 125
Gly Val Ala Leu Thr Gly Leu Ser Cys Phe Val Thr Pro Asp Leu Ala
130 135 140
Arg Asp Leu Ala Asn Asp Ile Met Thr Leu Met Ser His Thr Lys Pro
145 150 155 160
Tyr Ile Arg Lys Lys Ala Val Leu Ile Met Tyr Lys Val Phe Leu Lys
165 170 175
Tyr Pro Glu Ser Leu Arg Pro Ala Phe Pro Arg Leu Lys Glu Lys Leu
180 185 190
Glu Asp Pro Asp Pro Gly Val Gln Ser Ala Ala Val Asn Val Ile Cys
195 200 205
Glu Leu Ala Arg Arg Asn Pro Lys Asn Tyr Leu Ser Leu Ala Pro Leu
210 215 220
Phe Phe Lys Leu Met Thr Ser Ser Thr Asn Asn Trp Val Leu Ile Lys
225 230 235 240
Ile Ile Lys Leu Phe Gly Ala Leu Thr Pro Leu Gly Pro Arg Leu Gly
245 250 255
Lys Lys Leu Ile Glu Pro Leu Thr Asn Leu Ile His Ser Thr Ser Ala
260 265 270
Met Ser Leu Leu Tyr Glu Cys Val Asn Thr Val Ile Ala Val Leu Ile
275 280 285
Ser Leu Ser Ser Gly Met Pro Asn His Ser Ala Ser Ile Gln Leu Cys
290 295 300
Val Gln Lys Leu Arg Ile Leu Ile Glu Asp Ser Asp Gln Asn Leu Lys
305 310 315 320
Tyr Leu Gly Leu Leu Ala Met Ser Lys Ile Leu Lys Thr His Pro Lys
325 330 335
Ser Val Gln Ser His Lys Asp Leu Ile Leu Gln Cys Leu Asp Asp Lys
340 345 350
Asp Glu Ser Ile Arg Leu Arg Ala Leu Asp Leu Leu Tyr Gly Met Val
355 360 365
Ser Lys Lys Asn Leu Met Glu Ile Val Lys Lys Leu Met Thr His Val
370 375 380
Asp Lys Ala Glu Gly Thr Thr Tyr Arg Asp Glu Leu Leu Thr Lys Ile
385 390 395 400
Ile Asp Ile Cys Ser Gln Ser Asn Tyr Gln Tyr Ile Thr Asn Phe Glu
405 410 415
Trp Tyr Ile Ser Ile Leu Val Glu Leu Thr Arg Leu Glu Gly Thr Arg
420 425 430
His Gly His Leu Ile Ala Ala Gln Met Leu Asp Val Ala Ile Arg Val
435 440 445
Lys Ala Ile Arg Lys Phe Ala Val Ser Gln Met Ser Ala Leu Leu Asp
450 455 460
Ser Ala His Leu Leu Ala Ser Ser Thr Gln Arg Asn Gly Ile Cys Glu
465 470 475 480
Val Leu Tyr Ala Ala Ala Trp Ile Cys Gly Glu Phe Ser Glu His Leu
485 490 495
Gln Glu Pro His His Thr Leu Glu Ala Met Leu Arg Pro Arg Val Thr
500 505 510
Thr Leu Pro Gly His Ile Gln Ala Val Tyr Val Gln Asn Val Val Lys
515 520 525
Leu Tyr Ala Ser Ile Leu Gln Gln Lys Glu Gln Ala Gly Glu Ala Glu
530 535 540
Gly Ala Gln Ala Val Thr Gln Leu Met Val Asp Arg Leu Pro Gln Phe
545 550 555 560
Val Gln Ser Ala Asp Leu Glu Val Gln Glu Arg Ala Ser Cys Ile Leu
565 570 575
Gln Leu Val Lys His Ile Gln Lys Leu Gln Ala Lys Asp Val Pro Val
580 585 590
Ala Glu Glu Val Ser Ala Leu Phe Ala Gly Glu Leu Asn Pro Val Ala
595 600 605
Pro Lys Ala Gln Lys Lys Val Pro Val Pro Glu Gly Leu Asp Leu Asp
610 615 620
Ala Trp Ile Asn Glu Pro Leu Ser Asp Ser Glu Ser Glu Asp Glu Arg
625 630 635 640
Pro Arg Ala Val Phe His Glu Glu Glu Gln Arg Arg Pro Lys His Arg
645 650 655
Pro Ser Glu Ala Asp Glu Glu Glu Leu Ala Arg Arg Arg Glu Ala Arg
660 665 670
Lys Gln Glu Gln Ala Asn Asn Pro Phe Tyr Ile Lys Ser Ser Pro Ser
675 680 685
Pro Gln Lys Arg Tyr Gln Asp Thr Pro Gly Val Glu His Ile Pro Val
690 695 700
Val Gln Ile Asp Leu Ser Val Pro Leu Lys Val Pro Gly Leu Pro Met
705 710 715 720
Ser Asp Gln Tyr Val Lys Leu Glu Glu Glu Arg Arg His Arg Gln Lys
725 730 735
Leu Glu Lys Asp Lys Arg
740
<210> 6
<211> 497
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Asn Lys Met Val Ile Asn His Leu Glu Lys Leu Phe Val Thr Asn
1 5 10 15
Asp Ala Ala Thr Ile Leu Arg Glu Leu Glu Val Gln His Pro Ala Ala
20 25 30
Lys Met Ile Val Met Ala Ser His Met Gln Glu Gln Glu Val Gly Asp
35 40 45
Gly Thr Asn Phe Val Leu Val Phe Ala Gly Ala Leu Leu Glu Leu Ala
50 55 60
Glu Glu Leu Leu Arg Ile Gly Leu Ser Val Ser Glu Val Ile Glu Gly
65 70 75 80
Tyr Glu Ile Ala Cys Arg Lys Ala His Glu Ile Leu Pro Asn Leu Val
85 90 95
Cys Cys Ser Ala Lys Asn Leu Arg Asp Ile Asp Glu Val Ser Ser Leu
100 105 110
Leu Arg Thr Ser Ile Met Ser Lys Gln Tyr Gly Asn Glu Val Phe Leu
115 120 125
Ala Lys Leu Ile Ala Gln Ala Cys Val Ser Ile Phe Pro Asp Ser Gly
130 135 140
His Phe Asn Val Asp Asn Ile Arg Val Cys Lys Ile Leu Gly Ser Gly
145 150 155 160
Ile Ser Ser Ser Ser Val Leu His Gly Met Val Phe Lys Lys Glu Thr
165 170 175
Glu Gly Asp Val Thr Ser Val Lys Asp Ala Lys Ile Ala Val Tyr Ser
180 185 190
Cys Pro Phe Asp Gly Met Ile Thr Glu Thr Lys Gly Thr Val Leu Ile
195 200 205
Lys Thr Ala Glu Glu Leu Met Asn Phe Ser Lys Gly Glu Glu Asn Leu
210 215 220
Met Asp Ala Gln Val Lys Ala Ile Ala Asp Thr Gly Ala Asn Val Val
225 230 235 240
Val Thr Gly Gly Lys Val Ala Asp Met Ala Leu His Tyr Ala Asn Lys
245 250 255
Tyr Asn Ile Met Leu Val Arg Leu Asn Ser Lys Trp Asp Leu Arg Arg
260 265 270
Leu Cys Lys Thr Val Gly Ala Thr Ala Leu Pro Arg Leu Thr Pro Pro
275 280 285
Val Leu Glu Glu Met Gly His Cys Asp Ser Val Tyr Leu Ser Glu Val
290 295 300
Gly Asp Thr Gln Val Val Val Phe Lys His Glu Lys Glu Asp Gly Ala
305 310 315 320
Ile Ser Thr Ile Val Leu Arg Gly Ser Thr Asp Asn Leu Met Asp Asp
325 330 335
Ile Glu Arg Ala Val Asp Asp Gly Val Asn Thr Phe Lys Val Leu Thr
340 345 350
Arg Asp Lys Arg Leu Val Pro Gly Gly Gly Ala Thr Glu Ile Glu Leu
355 360 365
Ala Lys Gln Ile Thr Ser Tyr Gly Glu Thr Cys Pro Gly Leu Glu Gln
370 375 380
Tyr Ala Ile Lys Lys Phe Ala Glu Ala Phe Glu Ala Ile Pro Arg Ala
385 390 395 400
Leu Ala Glu Asn Ser Gly Val Lys Ala Asn Glu Val Ile Ser Lys Leu
405 410 415
Tyr Ala Val His Gln Glu Gly Asn Lys Asn Val Gly Leu Asp Ile Glu
420 425 430
Ala Glu Val Pro Ala Val Lys Asp Met Leu Glu Ala Gly Ile Leu Asp
435 440 445
Thr Tyr Leu Gly Lys Tyr Trp Ala Ile Lys Leu Ala Thr Asn Ala Ala
450 455 460
Val Thr Val Leu Arg Val Asp Gln Ile Ile Met Ala Lys Pro Ala Gly
465 470 475 480
Gly Pro Lys Pro Pro Ser Gly Lys Lys Asp Trp Asp Asp Asp Gln Asn
485 490 495
Asp
<210> 7
<211> 457
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Lys Thr Lys Phe Cys Thr Gly Gly Glu Ala Glu Pro Ser Pro Leu
1 5 10 15
Gly Leu Leu Leu Ser Cys Gly Ser Gly Ser Ala Ala Pro Ala Pro Gly
20 25 30
Val Gly Gln Gln Arg Asp Ala Ala Ser Asp Leu Glu Ser Lys Gln Leu
35 40 45
Gly Gly Gln Gln Pro Pro Leu Ala Leu Pro Pro Pro Pro Pro Leu Pro
50 55 60
Leu Pro Leu Pro Leu Pro Gln Pro Pro Pro Pro Gln Pro Pro Ala Asp
65 70 75 80
Glu Gln Pro Glu Pro Arg Thr Arg Arg Arg Ala Tyr Leu Trp Cys Lys
85 90 95
Glu Phe Leu Pro Gly Ala Trp Arg Gly Leu Arg Glu Asp Glu Phe His
100 105 110
Ile Ser Val Ile Arg Gly Gly Leu Ser Asn Met Leu Phe Gln Cys Ser
115 120 125
Leu Pro Asp Thr Thr Ala Thr Leu Gly Asp Glu Pro Arg Lys Val Leu
130 135 140
Leu Arg Leu Tyr Gly Ala Ile Leu Gln Met Arg Ser Cys Asn Lys Glu
145 150 155 160
Gly Ser Glu Gln Ala Gln Lys Glu Asn Glu Phe Gln Gly Ala Glu Ala
165 170 175
Met Val Leu Glu Ser Val Met Phe Ala Ile Leu Ala Glu Arg Ser Leu
180 185 190
Gly Pro Lys Leu Tyr Gly Ile Phe Pro Gln Gly Arg Leu Glu Gln Phe
195 200 205
Ile Pro Ser Arg Arg Leu Asp Thr Glu Glu Leu Ser Leu Pro Asp Ile
210 215 220
Ser Ala Glu Ile Ala Glu Lys Met Ala Thr Phe His Gly Met Lys Met
225 230 235 240
Pro Phe Asn Lys Glu Pro Lys Trp Leu Phe Gly Thr Met Glu Lys Tyr
245 250 255
Leu Lys Glu Val Leu Arg Ile Lys Phe Thr Glu Glu Ser Arg Ile Lys
260 265 270
Lys Leu His Lys Leu Leu Ser Tyr Asn Leu Pro Leu Glu Leu Glu Asn
275 280 285
Leu Arg Ser Leu Leu Glu Ser Thr Pro Ser Pro Val Val Phe Cys His
290 295 300
Asn Asp Cys Gln Glu Gly Asn Ile Leu Leu Leu Glu Gly Arg Glu Asn
305 310 315 320
Ser Glu Lys Gln Lys Leu Met Leu Ile Asp Phe Glu Tyr Ser Ser Tyr
325 330 335
Asn Tyr Arg Gly Phe Asp Ile Gly Asn His Phe Cys Glu Trp Met Tyr
340 345 350
Asp Tyr Ser Tyr Glu Lys Tyr Pro Phe Phe Arg Ala Asn Ile Arg Lys
355 360 365
Tyr Pro Thr Lys Lys Gln Gln Leu His Phe Ile Ser Ser Tyr Leu Pro
370 375 380
Ala Phe Gln Asn Asp Phe Glu Asn Leu Ser Thr Glu Glu Lys Ser Ile
385 390 395 400
Ile Lys Glu Glu Met Leu Leu Glu Val Asn Arg Phe Ala Leu Ala Ser
405 410 415
His Phe Leu Trp Gly Leu Trp Ser Ile Val Gln Ala Lys Ile Ser Ser
420 425 430
Ile Glu Phe Gly Tyr Met Asp Tyr Ala Gln Ala Arg Phe Asp Ala Tyr
435 440 445
Phe His Gln Lys Arg Lys Leu Gly Val
450 455
<210> 8
<211> 672
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ala Asp Val Phe Pro Gly Asn Asp Ser Thr Ala Ser Gln Asp Val
1 5 10 15
Ala Asn Arg Phe Ala Arg Lys Gly Ala Leu Arg Gln Lys Asn Val His
20 25 30
Glu Val Lys Asp His Lys Phe Ile Ala Arg Phe Phe Lys Gln Pro Thr
35 40 45
Phe Cys Ser His Cys Thr Asp Phe Ile Trp Gly Phe Gly Lys Gln Gly
50 55 60
Phe Gln Cys Gln Val Cys Cys Phe Val Val His Lys Arg Cys His Glu
65 70 75 80
Phe Val Thr Phe Ser Cys Pro Gly Ala Asp Lys Gly Pro Asp Thr Asp
85 90 95
Asp Pro Arg Ser Lys His Lys Phe Lys Ile His Thr Tyr Gly Ser Pro
100 105 110
Thr Phe Cys Asp His Cys Gly Ser Leu Leu Tyr Gly Leu Ile His Gln
115 120 125
Gly Met Lys Cys Asp Thr Cys Asp Met Asn Val His Lys Gln Cys Val
130 135 140
Ile Asn Val Pro Ser Leu Cys Gly Met Asp His Thr Glu Lys Arg Gly
145 150 155 160
Arg Ile Tyr Leu Lys Ala Glu Val Ala Asp Glu Lys Leu His Val Thr
165 170 175
Val Arg Asp Ala Lys Asn Leu Ile Pro Met Asp Pro Asn Gly Leu Ser
180 185 190
Asp Pro Tyr Val Lys Leu Lys Leu Ile Pro Asp Pro Lys Asn Glu Ser
195 200 205
Lys Gln Lys Thr Lys Thr Ile Arg Ser Thr Leu Asn Pro Gln Trp Asn
210 215 220
Glu Ser Phe Thr Phe Lys Leu Lys Pro Ser Asp Lys Asp Arg Arg Leu
225 230 235 240
Ser Val Glu Ile Trp Asp Trp Asp Arg Thr Thr Arg Asn Asp Phe Met
245 250 255
Gly Ser Leu Ser Phe Gly Val Ser Glu Leu Met Lys Met Pro Ala Ser
260 265 270
Gly Trp Tyr Lys Leu Leu Asn Gln Glu Glu Gly Glu Tyr Tyr Asn Val
275 280 285
Pro Ile Pro Glu Gly Asp Glu Glu Gly Asn Met Glu Leu Arg Gln Lys
290 295 300
Phe Glu Lys Ala Lys Leu Gly Pro Ala Gly Asn Lys Val Ile Ser Pro
305 310 315 320
Ser Glu Asp Arg Lys Gln Pro Ser Asn Asn Leu Asp Arg Val Lys Leu
325 330 335
Thr Asp Phe Asn Phe Leu Met Val Leu Gly Lys Gly Ser Phe Gly Lys
340 345 350
Val Met Leu Ala Asp Arg Lys Gly Thr Glu Glu Leu Tyr Ala Ile Lys
355 360 365
Ile Leu Lys Lys Asp Val Val Ile Gln Asp Asp Asp Val Glu Cys Thr
370 375 380
Met Val Glu Lys Arg Val Leu Ala Leu Leu Asp Lys Pro Pro Phe Leu
385 390 395 400
Thr Gln Leu His Ser Cys Phe Gln Thr Val Asp Arg Leu Tyr Phe Val
405 410 415
Met Glu Tyr Val Asn Gly Gly Asp Leu Met Tyr His Ile Gln Gln Val
420 425 430
Gly Lys Phe Lys Glu Pro Gln Ala Val Phe Tyr Ala Ala Glu Ile Ser
435 440 445
Ile Gly Leu Phe Phe Leu His Lys Arg Gly Ile Ile Tyr Arg Asp Leu
450 455 460
Lys Leu Asp Asn Val Met Leu Asp Ser Glu Gly His Ile Lys Ile Ala
465 470 475 480
Asp Phe Gly Met Cys Lys Glu His Met Met Asp Gly Val Thr Thr Arg
485 490 495
Thr Phe Cys Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile Ile Ala Tyr
500 505 510
Gln Pro Tyr Gly Lys Ser Val Asp Trp Trp Ala Tyr Gly Val Leu Leu
515 520 525
Tyr Glu Met Leu Ala Gly Gln Pro Pro Phe Asp Gly Glu Asp Glu Asp
530 535 540
Glu Leu Phe Gln Ser Ile Met Glu His Asn Val Ser Tyr Pro Lys Ser
545 550 555 560
Leu Ser Lys Glu Ala Val Ser Ile Cys Lys Gly Leu Met Thr Lys His
565 570 575
Pro Ala Lys Arg Leu Gly Cys Gly Pro Glu Gly Glu Arg Asp Val Arg
580 585 590
Glu His Ala Phe Phe Arg Arg Ile Asp Trp Glu Lys Leu Glu Asn Arg
595 600 605
Glu Ile Gln Pro Pro Phe Lys Pro Lys Val Cys Gly Lys Gly Ala Glu
610 615 620
Asn Phe Asp Lys Phe Phe Thr Arg Gly Gln Pro Val Leu Thr Pro Pro
625 630 635 640
Asp Gln Leu Val Ile Ala Asn Ile Asp Gln Ser Asp Phe Glu Gly Phe
645 650 655
Ser Tyr Val Asn Pro Gln Phe Val His Pro Ile Leu Gln Ser Ala Val
660 665 670
<210> 9
<211> 1606
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Asn Lys Ala Pro Gln Ser Thr Gly Pro Pro Pro Ala Pro Ser Pro
1 5 10 15
Gly Leu Pro Gln Pro Ala Phe Pro Pro Gly Gln Thr Ala Pro Val Val
20 25 30
Phe Ser Thr Pro Gln Ala Thr Gln Met Asn Thr Pro Ser Gln Pro Arg
35 40 45
Gln Gly Gly Phe Arg Ser Leu Gln His Phe Tyr Pro Ser Arg Ala Gln
50 55 60
Pro Pro Ser Ser Ala Ala Ser Arg Val Gln Ser Ala Ala Pro Ala Arg
65 70 75 80
Pro Gly Pro Ala Ala His Val Tyr Pro Ala Gly Ser Gln Val Met Met
85 90 95
Ile Pro Ser Gln Ile Ser Tyr Pro Ala Ser Gln Gly Ala Tyr Tyr Ile
100 105 110
Pro Gly Gln Gly Arg Ser Thr Tyr Val Val Pro Thr Gln Gln Tyr Pro
115 120 125
Val Gln Pro Gly Ala Pro Gly Phe Tyr Pro Gly Ala Ser Pro Thr Glu
130 135 140
Phe Gly Thr Tyr Ala Gly Ala Tyr Tyr Pro Ala Gln Gly Val Gln Gln
145 150 155 160
Phe Pro Thr Gly Val Ala Pro Ala Pro Val Leu Met Asn Gln Pro Pro
165 170 175
Gln Ile Ala Pro Lys Arg Glu Arg Lys Thr Ile Arg Ile Arg Asp Pro
180 185 190
Asn Gln Gly Gly Lys Asp Ile Thr Glu Glu Ile Met Ser Gly Ala Arg
195 200 205
Thr Ala Ser Thr Pro Thr Pro Pro Gln Thr Gly Gly Gly Leu Glu Pro
210 215 220
Gln Ala Asn Gly Glu Thr Pro Gln Val Ala Val Ile Val Arg Pro Asp
225 230 235 240
Asp Arg Ser Gln Gly Ala Ile Ile Ala Asp Arg Pro Gly Leu Pro Gly
245 250 255
Pro Glu His Ser Pro Ser Glu Ser Gln Pro Ser Ser Pro Ser Pro Thr
260 265 270
Pro Ser Pro Ser Pro Val Leu Glu Pro Gly Ser Glu Pro Asn Leu Ala
275 280 285
Val Leu Ser Ile Pro Gly Asp Thr Met Thr Thr Ile Gln Met Ser Val
290 295 300
Glu Glu Ser Thr Pro Ile Ser Arg Glu Thr Gly Glu Pro Tyr Arg Leu
305 310 315 320
Ser Pro Glu Pro Thr Pro Leu Ala Glu Pro Ile Leu Glu Val Glu Val
325 330 335
Thr Leu Ser Lys Pro Val Pro Glu Ser Glu Phe Ser Ser Ser Pro Leu
340 345 350
Gln Ala Pro Thr Pro Leu Ala Ser His Thr Val Glu Ile His Glu Pro
355 360 365
Asn Gly Met Val Pro Ser Glu Asp Leu Glu Pro Glu Val Glu Ser Ser
370 375 380
Pro Glu Leu Ala Pro Pro Pro Ala Cys Pro Ser Glu Ser Pro Val Pro
385 390 395 400
Ile Ala Pro Thr Ala Gln Pro Glu Glu Leu Leu Asn Gly Ala Pro Ser
405 410 415
Pro Pro Ala Val Asp Leu Ser Pro Val Ser Glu Pro Glu Glu Gln Ala
420 425 430
Lys Glu Val Thr Ala Ser Met Ala Pro Pro Thr Ile Pro Ser Ala Thr
435 440 445
Pro Ala Thr Ala Pro Ser Ala Thr Ser Pro Ala Gln Glu Glu Glu Met
450 455 460
Glu Glu Glu Glu Glu Glu Glu Glu Gly Glu Ala Gly Glu Ala Gly Glu
465 470 475 480
Ala Glu Ser Glu Lys Gly Gly Glu Glu Leu Leu Pro Pro Glu Ser Thr
485 490 495
Pro Ile Pro Ala Asn Leu Ser Gln Asn Leu Glu Ala Ala Ala Ala Thr
500 505 510
Gln Val Ala Val Ser Val Pro Lys Arg Arg Arg Lys Ile Lys Glu Leu
515 520 525
Asn Lys Lys Glu Ala Val Gly Asp Leu Leu Asp Ala Phe Lys Glu Ala
530 535 540
Asn Pro Ala Val Pro Glu Val Glu Asn Gln Pro Pro Ala Gly Ser Asn
545 550 555 560
Pro Gly Pro Glu Ser Glu Gly Ser Gly Val Pro Pro Arg Pro Glu Glu
565 570 575
Ala Asp Glu Thr Trp Asp Ser Lys Glu Asp Lys Ile His Asn Ala Glu
580 585 590
Asn Ile Gln Pro Gly Glu Gln Lys Tyr Glu Tyr Lys Ser Asp Gln Trp
595 600 605
Lys Pro Leu Asn Leu Glu Glu Lys Lys Arg Tyr Asp Arg Glu Phe Leu
610 615 620
Leu Gly Phe Gln Phe Ile Phe Ala Ser Met Gln Lys Pro Glu Gly Leu
625 630 635 640
Pro His Ile Ser Asp Val Val Leu Asp Lys Ala Asn Lys Thr Pro Leu
645 650 655
Arg Pro Leu Asp Pro Thr Arg Leu Gln Gly Ile Asn Cys Gly Pro Asp
660 665 670
Phe Thr Pro Ser Phe Ala Asn Leu Gly Arg Thr Thr Leu Ser Thr Arg
675 680 685
Gly Pro Pro Arg Gly Gly Pro Gly Gly Glu Leu Pro Arg Gly Pro Ala
690 695 700
Gly Leu Gly Pro Arg Arg Ser Gln Gln Gly Pro Arg Lys Glu Pro Arg
705 710 715 720
Lys Ile Ile Ala Thr Val Leu Met Thr Glu Asp Ile Lys Leu Asn Lys
725 730 735
Ala Glu Lys Ala Trp Lys Pro Ser Ser Lys Arg Thr Ala Ala Asp Lys
740 745 750
Asp Arg Gly Glu Glu Asp Ala Asp Gly Ser Lys Thr Gln Asp Leu Phe
755 760 765
Arg Arg Val Arg Ser Ile Leu Asn Lys Leu Thr Pro Gln Met Phe Gln
770 775 780
Gln Leu Met Lys Gln Val Thr Gln Leu Ala Ile Asp Thr Glu Glu Arg
785 790 795 800
Leu Lys Gly Val Ile Asp Leu Ile Phe Glu Lys Ala Ile Ser Glu Pro
805 810 815
Asn Phe Ser Val Ala Tyr Ala Asn Met Cys Arg Cys Leu Met Ala Leu
820 825 830
Lys Val Pro Thr Thr Glu Lys Pro Thr Val Thr Val Asn Phe Arg Lys
835 840 845
Leu Leu Leu Asn Arg Cys Gln Lys Glu Phe Glu Lys Asp Lys Asp Asp
850 855 860
Asp Glu Val Phe Glu Lys Lys Gln Lys Glu Met Asp Glu Ala Ala Thr
865 870 875 880
Ala Glu Glu Arg Gly Arg Leu Lys Glu Glu Leu Glu Glu Ala Arg Asp
885 890 895
Ile Ala Arg Arg Arg Ser Leu Gly Asn Ile Lys Phe Ile Gly Glu Leu
900 905 910
Phe Lys Leu Lys Met Leu Thr Glu Ala Ile Met His Asp Cys Val Val
915 920 925
Lys Leu Leu Lys Asn His Asp Glu Glu Ser Leu Glu Cys Leu Cys Arg
930 935 940
Leu Leu Thr Thr Ile Gly Lys Asp Leu Asp Phe Glu Lys Ala Lys Pro
945 950 955 960
Arg Met Asp Gln Tyr Phe Asn Gln Met Glu Lys Ile Ile Lys Glu Lys
965 970 975
Lys Thr Ser Ser Arg Ile Arg Phe Met Leu Gln Asp Val Leu Asp Leu
980 985 990
Arg Gly Ser Asn Trp Val Pro Arg Arg Gly Asp Gln Gly Pro Lys Thr
995 1000 1005
Ile Asp Gln Ile His Lys Glu Ala Glu Met Glu Glu His Arg Glu His
1010 1015 1020
Ile Lys Val Gln Gln Leu Met Ala Lys Gly Ser Asp Lys Arg Arg Gly
1025 1030 1035 1040
Gly Pro Pro Gly Pro Pro Ile Ser Arg Gly Leu Pro Leu Val Asp Asp
1045 1050 1055
Gly Gly Trp Asn Thr Val Pro Ile Ser Lys Gly Ser Arg Pro Ile Asp
1060 1065 1070
Thr Ser Arg Leu Thr Lys Ile Thr Lys Pro Gly Ser Ile Asp Ser Asn
1075 1080 1085
Asn Gln Leu Phe Ala Pro Gly Gly Arg Leu Ser Trp Gly Lys Gly Ser
1090 1095 1100
Ser Gly Gly Ser Gly Ala Lys Pro Ser Asp Ala Ala Ser Glu Ala Ala
1105 1110 1115 1120
Arg Pro Ala Thr Ser Thr Leu Asn Arg Phe Ser Ala Leu Gln Gln Ala
1125 1130 1135
Val Pro Thr Glu Ser Thr Asp Asn Arg Arg Val Val Gln Arg Ser Ser
1140 1145 1150
Leu Ser Arg Glu Arg Gly Glu Lys Ala Gly Asp Arg Gly Asp Arg Leu
1155 1160 1165
Glu Arg Ser Glu Arg Gly Gly Asp Arg Gly Asp Arg Leu Asp Arg Ala
1170 1175 1180
Arg Thr Pro Ala Thr Lys Arg Ser Phe Ser Lys Glu Val Glu Glu Arg
1185 1190 1195 1200
Ser Arg Glu Arg Pro Ser Gln Pro Glu Gly Leu Arg Lys Ala Ala Ser
1205 1210 1215
Leu Thr Glu Asp Arg Asp Arg Gly Arg Asp Ala Val Lys Arg Glu Ala
1220 1225 1230
Ala Leu Pro Pro Val Ser Pro Leu Lys Ala Ala Leu Ser Glu Glu Glu
1235 1240 1245
Leu Glu Lys Lys Ser Lys Ala Ile Ile Glu Glu Tyr Leu His Leu Asn
1250 1255 1260
Asp Met Lys Glu Ala Val Gln Cys Val Gln Glu Leu Ala Ser Pro Ser
1265 1270 1275 1280
Leu Leu Phe Ile Phe Val Arg His Gly Val Glu Ser Thr Leu Glu Arg
1285 1290 1295
Ser Ala Ile Ala Arg Glu His Met Gly Gln Leu Leu His Gln Leu Leu
1300 1305 1310
Cys Ala Gly His Leu Ser Thr Ala Gln Tyr Tyr Gln Gly Leu Tyr Glu
1315 1320 1325
Ile Leu Glu Leu Ala Glu Asp Met Glu Ile Asp Ile Pro His Val Trp
1330 1335 1340
Leu Tyr Leu Ala Glu Leu Val Thr Pro Ile Leu Gln Glu Gly Gly Val
1345 1350 1355 1360
Pro Met Gly Glu Leu Phe Arg Glu Ile Thr Lys Pro Leu Arg Pro Leu
1365 1370 1375
Gly Lys Ala Ala Ser Leu Leu Leu Glu Ile Leu Gly Leu Leu Cys Lys
1380 1385 1390
Ser Met Gly Pro Lys Lys Val Gly Thr Leu Trp Arg Glu Ala Gly Leu
1395 1400 1405
Ser Trp Lys Glu Phe Leu Pro Glu Gly Gln Asp Ile Gly Ala Phe Val
1410 1415 1420
Ala Glu Gln Lys Val Glu Tyr Thr Leu Gly Glu Glu Ser Glu Ala Pro
1425 1430 1435 1440
Gly Gln Arg Ala Leu Pro Ser Glu Glu Leu Asn Arg Gln Leu Glu Lys
1445 1450 1455
Leu Leu Lys Glu Gly Ser Ser Asn Gln Arg Val Phe Asp Trp Ile Glu
1460 1465 1470
Ala Asn Leu Ser Glu Gln Gln Ile Val Ser Asn Thr Leu Val Arg Ala
1475 1480 1485
Leu Met Thr Ala Val Cys Tyr Ser Ala Ile Ile Phe Glu Thr Pro Leu
1490 1495 1500
Arg Val Asp Val Ala Val Leu Lys Ala Arg Ala Lys Leu Leu Gln Lys
1505 1510 1515 1520
Tyr Leu Cys Asp Glu Gln Lys Glu Leu Gln Ala Leu Tyr Ala Leu Gln
1525 1530 1535
Ala Leu Val Val Thr Leu Glu Gln Pro Pro Asn Leu Leu Arg Met Phe
1540 1545 1550
Phe Asp Ala Leu Tyr Asp Glu Asp Val Val Lys Glu Asp Ala Phe Tyr
1555 1560 1565
Ser Trp Glu Ser Ser Lys Asp Pro Ala Glu Gln Gln Gly Lys Gly Val
1570 1575 1580
Ala Leu Lys Ser Val Thr Ala Phe Phe Lys Trp Leu Arg Glu Ala Glu
1585 1590 1595 1600
Glu Glu Ser Asp His Asn
1605
<210> 10
<211> 309
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu
1 5 10 15
Glu Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Ala Ala
20 25 30
Ser Ser Ser Ser Pro Leu Val Leu Gly Thr Leu Glu Glu Val Pro Thr
35 40 45
Ala Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser Ala Phe
50 55 60
Pro Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser
65 70 75 80
Ser Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys Ile Leu Glu Ser
85 90 95
Leu Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp Leu Val Gly Phe
100 105 110
Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met
115 120 125
Leu Glu Ser Val Ile Lys Asn Tyr Lys His Cys Phe Pro Glu Ile Phe
130 135 140
Gly Lys Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys
145 150 155 160
Glu Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly
165 170 175
Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Thr
180 185 190
Gly Phe Leu Ile Ile Val Leu Val Met Ile Ala Met Glu Gly Gly His
195 200 205
Ala Pro Glu Glu Glu Ile Trp Glu Glu Leu Ser Val Met Glu Val Tyr
210 215 220
Asp Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr
225 230 235 240
Gln Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp
245 250 255
Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala
260 265 270
Glu Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala
275 280 285
Arg Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg Glu
290 295 300
Glu Glu Glu Gly Val
305
<210> 11
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 12
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gagcagaaac tcatctctga agaggatctg catcaccatc accatcac 48
Claims (9)
1.一种蛋白芯片在制备用于非小细胞肺癌的诊断产品中的应用,其特征在于,所述蛋白芯片包括固相支持物及包被于所述固相支持物表面的抗原;所述抗原包括如下十种抗原片段:MAGE A3抗原片段、ANXA1抗原片段、CTAG1B抗原片段、CAGE1抗原片段、AP3D1抗原片段、CCT8抗原片段、CHK抗原片段、PRKACA抗原片段、EIF4G1抗原片段及MAGE A1抗原片段;
所述MAGE A3抗原片段的氨基酸序列包括SEQ ID No.1所示的抗原特征氨基酸序列;
所述ANXA1抗原片段的氨基酸序列包括SEQ ID No.2所示的抗原特征氨基酸序列;
所述CTAG1B抗原片段的氨基酸序列包括SEQ ID No.3所示的抗原特征氨基酸序列;
所述CAGE1抗原片段的氨基酸序列包括SEQ ID No.4所示的抗原特征氨基酸序列;
所述AP3D1抗原片段的氨基酸序列包括SEQ ID No.5所示的抗原特征氨基酸序列;
所述CCT8抗原片段的氨基酸序列包括SEQ ID No.6所示的抗原特征氨基酸序列;
所述CHK抗原片段的氨基酸序列包括SEQ ID No.7所示的抗原特征氨基酸序列;
所述PRKACA抗原片段的氨基酸序列包括SEQ ID No.8所示的抗原特征氨基酸序列;
所述EIF4G1抗原片段的氨基酸序列包括SEQ ID No.9所示的抗原特征氨基酸序列;
所述MAGE A1抗原片段的氨基酸序列包括SEQ ID No.10所示的抗原特征氨基酸序列。
2.如权利要求1所述的应用,其特征在于,所述抗原片段为重组融合蛋白,还包括位于相应抗原特征氨基酸序列的N端的Myc-Histag以及C端的链霉亲和素。
3.如权利要求1或2所述的应用,其特征在于,所述固相支持物为玻片、免疫印迹膜、微孔板或磁性微珠。
4.如权利要求3所述的应用,其特征在于,所述固相支持物为磁性微珠,所述十种抗原片段分别包被于十种不同颜色的磁性微珠表面。
5.如权利要求3所述的应用,其特征在于,所述抗原片段为亲和素化的抗原片段,所述固相支持物的表面通过肽键连接生物素化的牛血清白蛋白,所述抗原片段与所述固相支持物之间通过亲和素与生物素化的牛血清白蛋白间接连接。
6.一种试剂盒在制备用于非小细胞肺癌的诊断产品中的应用,其特征在于,包括权利要求1~5中任一项所述的蛋白芯片在制备用于非小细胞肺癌的诊断产品中的应用中所使用的所述蛋白芯片。
7.如权利要求6所述的应用,其特征在于,所述试剂盒还包括含有荧光物质标记的二抗试剂、标准品试剂和质控参比品试剂。
8.如权利要求7所述的应用,其特征在于,所述荧光物质标记的二抗试剂是抗人IgGFc-PE抗体和抗人IgM Fc-PE抗体。
9.如权利要求7所述的应用,其特征在于,所述标准品试剂与所述质控参比品试剂均是Anti-Myc嵌合抗体。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1852974A (zh) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | 用于治疗和诊断癌症的组合物和方法 |
CN1873417A (zh) * | 2005-06-02 | 2006-12-06 | 中国医学科学院肿瘤医院肿瘤研究所 | 一种用于诊断肺癌的蛋白质芯片 |
WO2012154203A3 (en) * | 2010-10-28 | 2013-03-21 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Filovirus fusion proteins and their uses |
EP2806274A1 (en) * | 2013-05-24 | 2014-11-26 | AIT Austrian Institute of Technology GmbH | Lung cancer diagnostic method and means |
CN104777313A (zh) * | 2010-07-09 | 2015-07-15 | 私募蛋白质体公司 | 肺癌生物标记及其用途 |
CN106662586A (zh) * | 2014-06-20 | 2017-05-10 | 昂西免疫有限公司 | 改进的免疫测定方法 |
CN107075557A (zh) * | 2014-05-30 | 2017-08-18 | 香港大学 | 使用嗜中性粒细胞弹性蛋白酶和蛋白酶3作为诊断生物标志物的方法和组合物 |
-
2017
- 2017-09-08 CN CN201710807813.5A patent/CN109470855B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1852974A (zh) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | 用于治疗和诊断癌症的组合物和方法 |
CN1873417A (zh) * | 2005-06-02 | 2006-12-06 | 中国医学科学院肿瘤医院肿瘤研究所 | 一种用于诊断肺癌的蛋白质芯片 |
CN104777313A (zh) * | 2010-07-09 | 2015-07-15 | 私募蛋白质体公司 | 肺癌生物标记及其用途 |
WO2012154203A3 (en) * | 2010-10-28 | 2013-03-21 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Filovirus fusion proteins and their uses |
EP2806274A1 (en) * | 2013-05-24 | 2014-11-26 | AIT Austrian Institute of Technology GmbH | Lung cancer diagnostic method and means |
CN107075557A (zh) * | 2014-05-30 | 2017-08-18 | 香港大学 | 使用嗜中性粒细胞弹性蛋白酶和蛋白酶3作为诊断生物标志物的方法和组合物 |
CN106662586A (zh) * | 2014-06-20 | 2017-05-10 | 昂西免疫有限公司 | 改进的免疫测定方法 |
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