CN106680411A - 一种用于检测系统性红斑狼疮(sle)的试剂盒及其检测方法 - Google Patents
一种用于检测系统性红斑狼疮(sle)的试剂盒及其检测方法 Download PDFInfo
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Abstract
本发明公开了一种用于检测系统性红斑狼疮(SLE)的试剂盒及其检测方法,该试剂盒含有能特异性检测系统性红斑狼疮(SLE)的适配子,该适配子能特异性结合C4BPA。本发明的试剂盒能够用于定量检测C4BPA蛋白的表达量,从而能够用于精确检测系统性红斑狼疮疾病。
Description
技术领域
本发明涉及一种用于检测系统性红斑狼疮(SLE)的试剂盒及其检测方法。
背景技术
系统性红斑狼疮(SLE)是自体免疫病因学(auto immune etiology)的炎症性紊乱,主要发生在年轻女性中。SLE能影响许多的身体器官系统,包括肾、皮肤、关节、神经系统、浆膜、血细胞和脉管。虽然不清楚SLE的具体诱因,但许多因素与疾病的发展有关,包括基因、种族、激素和环境因素。
多种类型抗核抗体的存在是患SLE的重要血清学特点,所以目前SLE的快速诊断主要是依据血清自身抗体检查,再结合以临床表现观察。其中抗核抗体(ANA)对SLE的敏感性为95%,特异性大约为65%,是目前最佳的SLE病情筛选指标,如多次呈阴性,则患SLE的可能性不大;抗双链DNA抗体(dsDNA)对SLE的特异性高达95%,敏感性约为70%,对确诊SLE和判断狼疮活动具有较大的参考价值;抗Sm抗体对SLE的特异性高达99%,但敏感性仅为25%,在SLE不活动时也可呈阳性,故多作为回顾性诊断的重要根据;此外抗单链DNA抗体(ss-DNA)也可作为SLE诊断指标之一。目前临床血检中对以上所述四种指标的检测,主要是采用放射免疫测定法(RIA),虽然其具有较好的灵敏度、特异性和稳定性,但检测过程较为烦琐,操作不便,而且每次也只能分析一种指标,历时较长,所提供的诊断信息量有限而不全面,诸多的不足为临床诊断和治疗带来了不便。
CN 104655852 A中公开了一种特异性检测血清抗体中高甘露糖型N糖链水平的试剂在制备用于系统性红斑狼疮的诊断和/或预后评估的诊断工具中的应用。所述血清抗体中高甘露糖型N糖链水平的增高与系统性红斑狼疮的发生密切相关,可用于系统性红斑狼疮的早期诊断和/或预后评估,进而用于指导系统性红斑狼疮的治疗。但是该方法在小鼠层面上进行的实验,在人体中是否能够具有相应的鉴定效果无法预期。
CN 105229470 A公开了用于诊断、预后和治疗系统性红斑狼疮(SLE)的方法和试剂,其涉及根据红细胞C4d(EC4d)标志物、B细胞C4d(BC4d)标志物、抗核抗体(ANA)。该方法中涉及了众多的标志物检测种类较多,而且检测不便。
目前,临床治疗复杂的自身免疫性疾病(如SLE)的最艰巨的挑战之一是患者的疾病的准确和早期鉴别。此外,没有鉴别出使临床医师或其他人能够准确地确定SLE的病理生理表现、临床活动、对治疗的响应或预后的可靠性诊断标记。
发明内容
一种非诊断目的的系统性红斑狼疮阶段性蛋白表达差异图谱模型的构建方法,包括如下步骤:分别采集系统性红斑狼疮患者组、健康对照组的外周血单个核细胞标本;分别对各所述外周血单个核细胞标本进行细胞总蛋白抽提和蛋白浓度测定;将所述细胞总蛋白经蛋白酶消化后,用相对和绝对定量的等量异位标签标记,得到相对和绝对定量的等量异位标签多重标记的多肽;将所述多肽经强阳离子交换和反相液相色谱分离,再进行串联质谱鉴定和相对定量分析,得到所述系统性红斑狼疮阶段性蛋白表达差异图谱模型,通过表型鉴定,最终分析得到了SERPINA6和C4BPA这两个蛋白,以该蛋白作为生物标志物对其表达量进行检测可以用于指导SLE患者中的早期诊断。
本发明另外提供了SERPINA6和C4BPA这两个蛋白用于制备SLE患者诊断的标志物。
另外,本发明对C4BPA在系统性红斑狼疮病人血液中的表达进行了定量检测。
另外一方面,本发明提供了一种特异性的识别C4BPA的适配子,其序列如SEQ IDNO:1-13所示。
另外一方面,本发明提供一种试剂盒,用于特异性的诊断系统性红斑狼疮,其包含如SEQ ID NO:1-13所示的序列。
另外一方面,所述的适配子偶联有磁珠以及筛选标记。
有益效果:本发明通过差异蛋白筛选,实验结果表明C4BPA在系统性红斑狼疮出现明显上调表达,可作为系统性红斑狼疮疾病临床诊断标志物。并且本发明通过特异性的筛选获得了C4BPA蛋白特异性结合的适配子,能够用于定量检测C4BPA蛋白的表达量,从而能够用于精确检测系统性红斑狼疮疾病。
附图说明
图1是患者血液和健康对照组血液中C4BPA的浓度的统计分析图。
图2是ROC曲线显示蛋白诊断系统性红斑狼疮的特异性和灵敏度。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1
分别采集系统性红斑狼疮患者20例、健康对照组20例的外周血单个核细胞标本;分别对各所述外周血单个核细胞标本进行细胞总蛋白抽提和蛋白浓度测定;将所述细胞总蛋白经蛋白酶消化后,用相对和绝对定量的等量异位标签标记,得到相对和绝对定量的等量异位标签多重标记的多肽;将所述多肽经强阳离子交换和反相液相色谱分离,再进行串联质谱鉴定和相对定量分析,得到所述系统性红斑狼疮阶段性蛋白表达差异图谱模型,通过表型鉴定,最终分析得到了SERPINA6和C4BPA这两个蛋白,差异表达显著,可以作为生物标志物对其表达量进行检测可以用于指导SLE患者中的早期诊断。
另外测量患者血清中C4BPA浓度明显高于对照组血清(P<0.05),参见图1。
另外,根据图2 ROC曲线设定了C4BPA的cut-off值为100.9ng/ml,AUC达到了0.9823,其敏感性和特异性均大于94%,阳性率为96.5%。统计分析结果显示,血液中C4BPA浓度与系统性红斑狼疮密切相关,综合上述实验研究,可以得出结论,C4BPA在系统性红斑狼疮患者中高分泌,可以作为C4BPA和正常人群区分的一个特别好的标志物。
实施例2适配子的获得
采用SELEX技术,筛选得到了特异性结合C4BPA蛋白的适配子,其序列如下文库为TACGTAGAATGACTCGTGAG(N)35CAGTACGATGGATGCAGTGA
C4BPA-1:
TACGTAGAATGACTCGTGAGCTCCACCAACCTCTTCTACTACATATATTCAAAACCAGTACGATGGATGCAGTGA
C4BPA-2:
TACGTAGAATGACTCGTGAGTCTCCTCCATTCATCCTCAAAATACTATTATCACTCAGTACGATGGATGCAGTGA
C4BPA-3:
TACGTAGAATGACTCGTGAGAATTTAATCTAAAACCCATATTTTCCACCACATTACAGTACGATGGATGCAGTGA
C4BPA-4:
TACGTAGAATGACTCGTGAGCCCATCTATCCTCCATTTCATTTACTAACCCCAAACAGTACGATGGATGCAGTGA
C4BPA-5:
TACGTAGAATGACTCGTGAGCACCTAAACATACTATACTTTAATTTAACTCCACTCAGTACGATGGATGCAGTGA
C4BPA-6:
TACGTAGAATGACTCGTGAGCTTTATCAAACAACACTTCATTTTCCCAATATTATCAGTACGATGGATGCAGTGA
C4BPA-7:
TACGTAGAATGACTCGTGAGATCATCACTCCACCCTACCCTTCCTCCCCAAACAACAGTACGATGGATGCAGTGA
C4BPA-8:
TACGTAGAATGACTCGTGAGTTCCTTTTTCTAACCATAACCTTCCCATCAATCTCCAGTACGATGGATGCAGTGA
C4BPA-9:
TACGTAGAATGACTCGTGAGTTCTAACTTACAATCTCTTTTTAATCTCATACCATCAGTACGATGGATGCAGTGA
C4BPA-10:
TACGTAGAATGACTCGTGAGAAAACTATTATATTCCTTTATACTATAATTTTACCCAGTACGATGGATGCAGTGA
C4BPA-11:
TACGTAGAATGACTCGTGAGATTATATCCACTAATCAAACCCATAAACTATTATCCAGTACGATGGATGCAGTGA
C4BPA-12:
TACGTAGAATGACTCGTGAGATCACTACATCATCACACTTTAACCACATCCCCTCCAGTACGATGGATGCAGTGA
C4BPA-13:
TACGTAGAATGACTCGTGAGCTCATTTCTCAATTCCCAACTTCAACCCCACCCCCCAGTACGATGGATGCAGTGA
实施例3蛋白结合适配子的性能测定
基于氧化石墨烯氧化具有吸附单链DNA的特性,构建了寡核苷酸适配子亲和性验证方法。将固定浓度的C4BPA蛋白靶标(1μΜ)分别与与其对应一系列不同浓度(10,25,50,75,100,150,200uΜ)的候选寡核苷酸适配子进行孵育,总体积为300uL,37℃避光孵育2h,并以BB缓冲液代替靶标作为阴性对照组。孵育结合后加入最佳用量比的GO吸附未与靶标结合的适配子,离心操作后采用F-7000荧光光度计测定上清液490nm激发下520nm发射的荧光强度,实验设置三次平行重复,实验采用避光处理。以实验组相对阴性对照组的荧光强度作为纵坐标,以适配子浓度作为横坐标,采用GraphPad Prism 5.0软件进行非线性回归拟合计算适配子的解离常数Kd值。结果如下:
另外,通过与血清中其它人体的蛋白相接触,本发明提供的适配子具有较好的结合特异性和稳定性,同时也没有任何溶血性,具有较好的生物学特性。
实施例4所述适配子疾病的诊断
将13个适配子分别与12个系统性红斑狼疮患者患者进行检测。
在酶标板孔中加入12个血清样品以及对照健康样本包被,并加入通过生物素标记的适配子13个,加入HRP标记的链酶亲和素,37℃孵育1.5小时;PBS洗三次后加入TMB进行显色5分钟;2M硫酸终止反应后酶标仪读数;进行检测,结果所示,与健康志愿者相比,系统性红斑狼疮患者血清中C4BPA蛋白的0D450均明显升高(p=0.0035)。通过标准曲线,计算得出,血清中C4BPA蛋白浓度均大于100.9ng/ml,而正常人群的C4BPA蛋白浓度均远远小于100.9ng/ml,结果证明13个适配子在系统性红斑狼疮诊断中具有应用前景。
以上仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,凡在本发明的精神和原则之内所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
〈110〉申冬昌
〈120〉一种用于检测系统性红斑狼疮(SLE)的试剂盒及其检测方法
〈160〉13
〈210〉1
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-1
TACGTAGAATGACTCGTGAGCTCCACCAACCTCTTCTACTACATATATTCAAAACCAGTACGATGGATGCAGTGA
〈210〉2
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-2
TACGTAGAATGACTCGTGAGTCTCCTCCATTCATCCTCAAAATACTATTATCACTCAGTACGATGGATGCAGTGA
〈210〉3
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-3
TACGTAGAATGACTCGTGAGAATTTAATCTAAAACCCATATTTTCCACCACATTACAGTACGATGGATGCAGTGA
〈210〉4
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-4
TACGTAGAATGACTCGTGAGCCCATCTATCCTCCATTTCATTTACTAACCCCAAACAGTACGATGGATGCAGTGA
〈210〉5
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-5
TACGTAGAATGACTCGTGAGCACCTAAACATACTATACTTTAATTTAACTCCACTCAGTACGATGGATGCAGTGA
〈210〉6
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-6
TACGTAGAATGACTCGTGAGCTTTATCAAACAACACTTCATTTTCCCAATATTATCAGTACGATGGATGCAGTGA
〈210〉7
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-7
TACGTAGAATGACTCGTGAGATCATCACTCCACCCTACCCTTCCTCCCCAAACAACAGTACGATGGATGCAGTGA
〈210〉8
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-8
TACGTAGAATGACTCGTGAGTTCCTTTTTCTAACCATAACCTTCCCATCAATCTCCAGTACGATGGATGCAGTGA
〈210〉9
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-9
TACGTAGAATGACTCGTGAGTTCTAACTTACAATCTCTTTTTAATCTCATACCATCAGTACGATGGATGCAGTGA
〈210〉10
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-10
TACGTAGAATGACTCGTGAGAAAACTATTATATTCCTTTATACTATAATTTTACCCAGTACGATGGATGCAGTGA
〈210〉11
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-11
TACGTAGAATGACTCGTGAGATTATATCCACTAATCAAACCCATAAACTATTATCCAGTACGATGGATGCAGTGA
〈210〉12
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-12
TACGTAGAATGACTCGTGAGATCACTACATCATCACACTTTAACCACATCCCCTCCAGTACGATGGATGCAGTGA
〈210〉13
〈211〉75
〈212〉DNA
〈213〉人工序列
〈400〉C4BPA-13
TACGTAGAATGACTCGTGAGCTCATTTCTCAATTCCCAACTTCAACCCCACCCCCCAGTACGATGGATGCAGTGA
Claims (5)
1.一种用于检测系统性红斑狼疮(SLE)的试剂盒,其特征在于:含有能特异性检测系统性红斑狼疮(SLE)的适配子。
2.如权利要求1所述的试剂盒,其特征在于:所述适配子能特异性结合C4BPA。
3. 如权利要求2所述的试剂盒,其特征在于:所述适配子的序列如SEQ ID NO:1-13任一所示。
4. 一种适配子,其特征在于:序列如SEQ ID NO:1-13任一所示。
5.权利要求4所述的适配子在制备用于检测系统性红斑狼疮(SLE)的试剂盒的应用。
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