CN112877418A - 一种用于检测血管通透性的产品及其制备方法 - Google Patents
一种用于检测血管通透性的产品及其制备方法 Download PDFInfo
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Abstract
本发明属于分子生物学领域,具体涉及一种用于检测血管通透性的产品及其制备方法。具体技术方案为:利用ARPC4和/或TRAF2或基因制备的检测用产品。本发明使用分子生物学技术,筛选出一种与血管通透性相关度高的靶基因。本领域技术人员可以利用业内常规技术制备出系列可用于检测血管通透性的试剂、试纸等相同产品。利用本发明提供的靶基因及制备的检测用产品,可及时、快速、准确判断血管通透性是否发生改变。
Description
技术领域
本发明属于分子生物学领域,具体涉及一种用于检测血管通透性的产品及其制备方法。
背景技术
血管是血液循环系统的重要组成部分。血液循环的完成离不开血管在组织中进行的血液与组织物质交换。血管通透性指物质穿过毛细血管壁的能力。
血管的通透性受到血管壁结构的影响。通常情况下,血管壁能够阻挡大分子通过血管壁。但当发生炎症、烧伤、休克、免疫反应、肿瘤转移等一系列病理过程时,血管壁通透性会发生改变。因此,及时、快速、准确检测血管通透性,对预先发现某些疾病、确诊某些疾病均具有重要意义。
现有检测血管通透性的方法往往依赖于炎症病理过程的发生,以及对尿蛋白的检测,即依赖于血管通透性已经改变甚至已经出现症状的“事后”指标,并不能够及时有效地对血管通透性的改变做出预先判断。
因此,如果能提供一种可对血管通透性改变做出快速、准确判断的产品,将具有重要的科研价值和现实意义。
发明内容
本发明的目的是提供一种用于检测血管通透性的产品及其制备方法。
为实现上述发明目的,本发明所采用的技术方案是:TRAF2和/或ARPC4基因在制备用于检测血管通透性产品中的应用。
优选的,所述产品为检测试剂、检测试纸、试剂盒中的一种。
相应的,一种用于检测血管通透性的产品,利用TRAF2和/或ARPC4基因制备而成。
优选的,含有TRAF2和/或ARPC4基因结合位点。
优选的,所述产品为检测试纸。
优选的,检测试纸的制备包括如下步骤:
进行ARPC4和/或TRAF2蛋白质的合成,得到蛋白质产物,将合成的蛋白质注射到小鼠体内,合成克隆抗体;
将得到的克隆抗体进行ELISA实验筛选,将一种抗体固定在ELISA底板上,作为反应1抗,加入患有与血管通透性改变相关疾病的人的血清样本;将另一种抗体结合免疫荧光酶,并作为二抗加入到反应中,检测荧光反应,挑选出荧光反应最大的抗体;
胶体金标记物的制备:取胶体金,加入碳酸钾溶液,混匀,加入筛选出的抗体,混匀,静置,再加入封闭液,混匀静置,静置后离心,弃上清,用胶体金复溶液将沉淀复溶,充分混匀,所得溶液即为胶体金标记物;
胶体金试纸制备:将玻璃纤维膜裁成所需大小,将样品垫置于胶体金标记物中,使其充分吸收。然后用吹风机将样品垫均匀吹干,制备成金标垫,依次贴硝酸纤维素膜、金标垫、样品垫、吸水纸,组装完成后剪成所需大小,放入容器内。
相应的,一种筛选用于检测毛细血管通透性的靶基因的方法,包括如下步骤:
(1)选择与血管通透性变化相关的疾病;
(2)分别对患有所述与血管通透性变化相关的疾病病人和正常人进行血小板转录组测序,获得疾病和正常样本的血小板基因表达量;
(3)以正常样本基因表达量作为对照,对与血管通透性变化相关的疾病的基因进行表达差异统计,筛选出基因表达倍数为|Log2FC|>2且P_value<0.05的差异基因;
(4)将筛选出的所述差异基因进行KEGG数据库比对,获得差异基因富集的通路;
(5)在疾病所述富集通路中,选出与膜转运调控相关的代谢途径,并从选出的代谢途径中挑选出差异基因;随后按如下(6-1)~(8-1)进行,或者按如下(6-2)~(8-2)进行;
(6-1)从步骤(5)的差异基因中,利用蛋白互作分析和受试者操作特征分析进一步筛选出具有预后意义的特征基因并作为预选靶基因;
(7-1)对预选靶基因进行扩增验证,并计算在疾病样本与正常样本中的拷贝倍数;
(8-1)从步骤(7-1)中进行的预选靶基因中,挑选出表达差异倍数最大的基因,即为所需靶基因;
(6-2)从步骤(5)的差异基因中,选择与血管通透性变化呈正相关的疾病和与血管通透性变化呈负相关的疾病共有的基因,作为预选靶基因;
(7-2)对预选靶基因分别在与血管通透性变化呈正相关的疾病中、在与血管通透性变化呈负相关的疾病中及正常样本中进行表达,并统计各预选靶基因的表达量;
(8-2)选出在与血管通透性变化呈正相关的疾病中表达量高于在正常样本中的表达量,且在与血管通透性变化呈负相关的疾病中表达量高于在正常样本中的表达量的预选靶基因,即为所需靶基因。
优选的,所述与血管通透性变化相关的疾病为动脉粥样硬化,和/或,易栓症,和/或,紫癜。
优选的,所述与血管通透性变化相关的疾病为易栓症和紫癜。
优选的,所述与血管通透性变化相关的疾病为动脉粥样硬化时,进行步骤(6-1)~(8-1);和/或,所述与血管通透性变化相关的疾病为易栓症和紫癜时,进行步骤(6-2)~(8-2)。
本发明具有以下有益效果:本发明使用分子生物学技术,筛选出两种与血管通透性相关度高的靶基因。本领域技术人员可以利用业内常规技术制备出系列可用于检测血管通透性的试剂、试纸等相关产品。利用本发明提供的靶基因及制备的检测用产品,可及时、快速、准确判断血管通透性是否发生改变。
附图说明
图1为动脉粥样硬化疾病差异基因富集通路示意图;
图2为ARPC4基因的qpcr基因拷贝数检测;
图3为易栓症差异基因富集通路示意图;
图4为紫癜差异基因富集通路示意图;
图5为PLCG1、TRAF2和LAD基因表达量结果示意图;
图6为胶体金试纸制备结构图;
图7为血管通透性检测试纸阴性测试结果;
图8为血管通透性检测试纸阳性测试结果。
具体实施方式
本发明提供了两者可用于检测血管通透性的靶基因:ARPC4、TRAF2。所述基因可进一步被制备为试剂、试纸、试剂盒、检测剂等,以及时、快速、便捷、准确地检测血管通透性的改变情况。
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述。显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例一:利用动脉粥样硬化筛选符合要求的靶基因
1、选择血管通透性改变密切相关的疾病:动脉粥样硬化。脂质代谢异常为动脉粥样硬化的发生基础,随着血管内壁脂质和复合糖类积聚,出血和血栓的形成,逐渐导致纤维组织增生及钙质沉着,从而造成动脉壁增厚变硬、血管腔狭窄,进而影响到血管壁的物质交换,改变血管的通透性。
基于上述理由,本实施例选择动脉粥样硬化作为毛细血管通透性改变的疾病表现形式。
2、对患有动脉粥样硬化的病人及正常人的血液样本进行血小板转录组测序。通过病例收集,共取得动脉粥样硬化样本3例,包括男性病例1例,女性病例2例,年龄段为45~77岁。同时收集正常人样本3例,男性样本2例,女性样本1例,年龄段为40~80岁。获得动脉粥样硬化和正常样本血小板基因表达量(共考察58303个基因)。
3、以正常样本基因表达量作为对照,对动脉粥样硬化的基因进行表达差异统计,筛选出基因表达倍数为|Log2FC|>2且P_value<0.05的差异基因(共获得3817个差异基因)。
4、将步骤3中筛选出的差异基因进行KEGG数据库比对,获得动脉粥样硬化疾病差异基因的富集通路。富集了53条差异显著(P<0.05)的代谢途径(如图1所示)。
5、在图1展示的53条代谢途径中,抗原处理和递呈(Antigen processing andpresentation)、NF-信号通路(NF-kappa B signaling pathway)、流体切应力和动脉粥样硬化通路(Fluid shear stress and atherosclerosis)参与和调控细胞的膜转运和物质交换、信息传递。挑选出上述3条代谢途径中富集的差异基因(共94个)。
6、在步骤5选出的94个差异基因中,挑选出平均表达量(base Mean)高的前20个基因作为预选靶基因。随后进行PPI蛋白互作筛选和受试者操作特征曲线分析,进行一步筛选得到10个预选靶基因(HLA-E、ARPC1B、CDS2、CALM1、ARPC4、HLA-B、HCLS1、HLA-C、DNM3、HLA-A)。
7、利用Primer Premier 6软件设计步骤6所选取的10个预选靶基因的qpcr引物,如表1所示。
表1各预选靶基因的qpcr引物
8、利用步骤7所设计的引物进行qpcr基因拷贝数检测,结果如表2所示。
表2qpcr基因拷贝数检测结果展示
基因 | 疾病1 | 疾病2 | 疾病3 | 正常1 | 正常2 | 正常3 |
HLA-E | 2.05 | 1.56 | 3.49 | 1.17 | 0.94 | 0.890892 |
ARPC1B | 1.42 | 0.70 | 1.75 | 1.02 | 1.10 | 0.876798 |
CDS2 | 7.14 | 3.55 | 6.17 | 0.42 | 1.24 | 1.342618 |
CALM1 | 1.47 | 1.17 | 2.27 | 0.65 | 1.28 | 1.070749 |
ARPC4 | 5.55 | 5.00 | 11.12 | 0.79 | 1.65 | 0.556974 |
HLA-B | 0.94 | 1.12 | 2.10 | 0.73 | 1.00 | 1.272211 |
HCLS1 | 7.72 | 2.82 | 6.00 | 0.93 | 0.88 | 1.188007 |
HLA-C | 1.24 | 0.76 | 1.75 | 0.49 | 0.71 | 1.802528 |
DNM3 | 1.01 | 0.69 | 1.50 | 1.30 | 0.742488 | 0.961776 |
HLA-A | 1.87 | 1.95 | 5.19 | 0.68 | 1.221628 | 1.095917 |
得到ARPC4基因拷贝数在疾病组和正常组中的差异倍数最大,即为所挑选的靶基因(如图2所示)。
实施例二:利用易栓症(YSZ)和血小板减少性紫癜(ITP)筛选符合要求的靶基因
1、选择与血管通透性改变密切相关的两种疾病:易栓症(YSZ)和血小板减少性紫癜(ITP)。其中,易栓症分为遗传性和获得性两种,获得性易栓症表现为血液成分(血小板、凝血因子、抗凝因子、纤溶和抗纤溶因子)的改变。血管通透性发生变化会引起血液成分的改变,从而可能引起获得性易栓症。血小板减少性紫癜表现为皮下出血,血液中血小板减少,血浆和血液中的其他成分向皮下组织中迁移等。因此,如果患有血小板减少性紫癜,其毛细血管的通透性往往会发生改变。
基于上述理由,本实施例选择获得性易栓症和血小板减少性紫癜作为毛细血管通透性改变的疾病表现形式。患有获得性易栓症,则毛细血管通透性变弱(即获得性易栓症与血管通透性变化呈负相关);患有血小板减少性紫癜,则毛细血管通透性变强(即血小板减少性紫癜与血管通透性变化呈正相关)。
2、分别对患有获得性易栓症的病人、患有血小板减少性紫癜的病人及正常人的血液样本进行血小板转录组测序。包括易栓症患者20名、血小板减少性紫癜患者51名、正常人36名;各患者和正常人中,各供血患者及正常人的年龄段在14到94周岁间,男女比例为1:2。分别获得疾病(YSZ、ITP)和正常样本(normal)血小板基因表达量。
3、以正常样本基因表达量作为对照,分别对易栓症(YSZ)和紫癜(ITP)的基因进行表达差异统计,筛选出基因表达倍数为|Log2FC|>2且P_value<0.05的差异基因。
4、将易栓症和紫癜中筛选出的差异基因进行KEGG数据库比对,获得两种疾病差异基因的富集通路。易栓症中差异基因共富集了44条差异显著(P<0.05)的代谢途径,(如图3所示),紫癜中差异基因共富集了33条差异显著的代谢途径(如图4所示)。
5、在图3展示了44条代谢途径,其中ABC转运途径、钙信号通路、局灶性粘连、MAPK信号通路和Rap1信号通路参与和调控细胞的膜转运和物质交换、信息传递。在图4展示的33条代谢途径中,NF-κB信号通路参与了膜转运调控。从上述代谢途径中分别挑选出差异基因。
从这些差异基因中选取三个共有基因(PLCG1、TRAF2和LAD),作为预选靶基因。
6、将三个基因在两种疾病中的表达量分别与正常样本中这三个基因的表达量进行T-test检验,结果如图5所示:在ITP中,TRAF2的表达量高于正常值;在YSZ中,TRAF2的表达量低于正常值。因此TRAF2为所需要的目的基因(靶基因)。
实施例三:ARPC4和TRAF2基因在制备检测毛细血管通透性试纸上的应用
在已筛选确定ARPC4、TRAF2为检测毛细血管通透性靶基因的情况下,本领域技术人员可以根据实际情况和本领域常规技术,单独使用ARPC4、TRAF2,或联合使用ARPC4和TRAF2,制备系列检测用产品,包括但不限于:检测剂、检测试纸、试剂盒等。本实施例提供一种可用于检测的试纸。
所述试纸的制备方法如图6所示,具体包括如下步骤:
1、通过NCBI网站得到ARPC4和TRAF2基因分别对应的产物蛋白质序列,分别如SEQID NO:1和SEQ ID NO:2所示。进行ARPC4和TRAF2蛋白质的合成,得到蛋白质产物。将合成的两种蛋白质作为多抗原注射到小鼠体内,合成多克隆抗体。
得到的多克隆抗体进行ELISA实验筛选。将一种抗体固定在ELISA底板上,作为反应1抗。加入动脉粥样硬化、多发性紫癜和易栓症的血清样本。将另一种抗体结合免疫荧光酶,并作为二抗加入到反应中。检测荧光反应,挑选出荧光反应最大的抗体(1抗和2抗)。
2、胶体金标记物的制备:取1mL胶体金,加入6μL碳酸钾溶液,迅速混匀,加入25μL筛选出的1抗,混匀,静置10min。加入10μl封闭液,混匀静置10分钟。静置后采用9000rpm离心7分钟,弃上清。用胶体金复溶液将沉淀复溶至1ml,用超声波清洗仪器将溶液充分混匀,所得溶液即为胶体金标记物。
3、胶体金试纸制备:将玻璃纤维膜裁成5×3cm2的小板,将样品垫置于胶体金标记物中,使其充分吸收。然后用吹风机将样品垫均匀吹干,制备成金标垫。如图6所示,依次贴硝酸纤维素膜、金标垫(两层)、样品垫、吸水纸,组装完成后用剪刀剪成宽度为4mm的试纸条,放入卡壳内。
分为两种方法进行检测。点样式:用毛细管吸取少量的所筛选的1抗,轻轻点在检测线处,点好样的膜室温下晾干。画线式:用移液器吸取30μL的1抗,然后用枪头从移液器上取下,用枪头配合直尺在NC膜检测线位置进行划线,同样用所筛选的2抗在质检线位置进行划线。
4、所述试纸的检测效果验证。利用制备的试纸分别对正常人、急性肾炎患者、动脉粥样硬化患者、多发性紫癜患者和易栓症患者的血液(各20名,男女各半)进行测试。血管通透性检测试纸呈阴性(通透性未改变)的结果如图7所示,血管通透性检测试纸呈阳性(通透性发生改变)的结果如图8所示。结果显示,试纸对正常人检测准确率和对血管通透性改变的患者检测准确性均为100%。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形、变型、修改、替换,均应落入本发明权利要求书确定的保护范围内。
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<110> 丰能医药科技(上海)有限责任公司
<120> 一种用于检测血管通透性的产品及其制备方法
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Claims (10)
1.TRAF2和/或ARPC4基因在制备用于检测血管通透性产品中的应用。
2.根据权利要求1所述应用,其特征在于:所述产品为检测试剂、检测试纸、试剂盒中的一种。
3.一种用于检测血管通透性的产品,其特征在于:利用TRAF2和/或ARPC4基因制备而成。
4.根据权利要求3所述的用于检测血管通透性的产品,其特征在于:含有TRAF2和/或ARPC4基因结合位点。
5.根据权利要求3所述的用于检测血管通透性的产品,其特征在于:所述产品为检测试纸。
6.根据权利要求5所述的用于检测血管通透性的产品,其特征在于:检测试纸的制备包括如下步骤:
进行ARPC4和/或TRAF2蛋白质的合成,得到蛋白质产物,将合成的蛋白质注射到小鼠体内,合成克隆抗体;
将得到的克隆抗体进行ELISA实验筛选,将一种抗体固定在ELISA底板上,作为反应1抗,加入患有与血管通透性改变相关疾病的人的血清样本;将另一种抗体结合免疫荧光酶,并作为二抗加入到反应中,检测荧光反应,挑选出荧光反应最大的抗体;
胶体金标记物的制备:取胶体金,加入碳酸钾溶液,混匀,加入筛选出的抗体,混匀,静置,再加入封闭液,混匀静置,静置后离心,弃上清,用胶体金复溶液将沉淀复溶,充分混匀,所得溶液即为胶体金标记物;
胶体金试纸制备:将玻璃纤维膜裁成所需大小,将样品垫置于胶体金标记物中,使其充分吸收。然后用吹风机将样品垫均匀吹干,制备成金标垫,依次贴硝酸纤维素膜、金标垫、样品垫、吸水纸,组装完成后剪成所需大小,放入容器内。
7.一种筛选用于检测毛细血管通透性的靶基因的方法,其特征在于:包括如下步骤:
(1)选择与血管通透性变化相关的疾病;
(2)分别对患有所述与血管通透性变化相关的疾病病人和正常人进行血小板转录组测序,获得疾病和正常样本的血小板基因表达量;
(3)以正常样本基因表达量作为对照,对与血管通透性变化相关的疾病的基因进行表达差异统计,筛选出基因表达倍数为|Log2FC|>2且P_value<0.05的差异基因;
(4)将筛选出的所述差异基因进行KEGG数据库比对,获得差异基因富集的通路;
(5)在疾病所述富集通路中,选出与膜转运调控相关的代谢途径,并从选出的代谢途径中挑选出差异基因;随后按如下(6-1)~(8-1)进行,或者按如下(6-2)~(8-2)进行;
(6-1)从步骤(5)的差异基因中,利用蛋白互作分析和受试者操作特征分析进一步筛选出具有预后意义的特征基因并作为预选靶基因;
(7-1)对预选靶基因进行扩增验证,并计算在疾病样本与正常样本中的拷贝倍数;
(8-1)从步骤(7-1)中进行的预选靶基因中,挑选出表达差异倍数最大的基因,即为所需靶基因;
(6-2)从步骤(5)的差异基因中,选择与血管通透性变化呈正相关的疾病和与血管通透性变化呈负相关的疾病共有的基因,作为预选靶基因;
(7-2)对预选靶基因分别在与血管通透性变化呈正相关的疾病中、在与血管通透性变化呈负相关的疾病中及正常样本中进行表达,并统计各预选靶基因的表达量;
(8-2)选出在与血管通透性变化呈正相关的疾病中表达量高于在正常样本中的表达量,且在与血管通透性变化呈负相关的疾病中表达量高于在正常样本中的表达量的预选靶基因,即为所需靶基因。
8.根据权利要求7所述筛选用于检测毛细血管通透性的靶基因的方法,其特征在于:所述与血管通透性变化相关的疾病为动脉粥样硬化,和/或,易栓症,和/或,紫癜。
9.根据权利要求8所述筛选用于检测毛细血管通透性的靶基因的方法,其特征在于:所述与血管通透性变化相关的疾病为易栓症和紫癜。
10.根据权利要求9所述筛选用于检测毛细血管通透性的靶基因的方法,其特征在于:所述与血管通透性变化相关的疾病为动脉粥样硬化时,进行步骤(6-1)~(8-1);和/或,所述与血管通透性变化相关的疾病为易栓症和紫癜时,进行步骤(6-2)~(8-2)。
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