CN105037534B - 一种检测肺癌标志物myc抗原表位氨基酸序列及应用 - Google Patents
一种检测肺癌标志物myc抗原表位氨基酸序列及应用 Download PDFInfo
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Abstract
一种检测肺癌标志物MYC抗原表位的氨基酸序列及应用,属于免疫学技术领域,本发明提供了一种肺癌相关基因MYC的抗原氨基酸序列。应用MYC多肽抗原检测肺癌患者血中相应的特异性自身抗体,这种自身抗体可作为肺癌标志物评估肺癌发生的危险度及预后疗效。这种抗原多肽及其抗体可用于制备肺癌早期诊断、预后预测试剂和开发治疗肺癌的靶向药物。
Description
技术领域
本发明属于生物技术领域,具体涉及一种通过生物信息学模拟筛选的具有检测肺癌标志物MYC多肽片段。
背景技术
肺癌是胸部的主要肿瘤,其发病率和死亡率在我国恶性肿瘤中均居于首位。由于临床上缺乏特异性的诊断标志物,多数病人发现时已属晚期,失去根治性手术的机会。因此,早期发现、早期诊断、早期治疗对于肺癌来说仍是当务之急。大量研究表明,血清或血浆中的肿瘤相关抗原能诱导机体产生自身抗体,在肿瘤患者血清中既存在肿瘤抗原,也存在针对该肿瘤抗原的自身抗体。因此, 既可以利用抗体检测肿瘤抗原,也可以利用抗原检测肿瘤抗原的自身抗体,但利用肿瘤自身抗体检测肿瘤的特异性和敏感性均比利用肿瘤抗原检测肿瘤要高得多。很多肿瘤相关抗原不仅在肿瘤患者体内存在,在正常人体内也存在,因此检测肿瘤相关抗原作为诊断依据可信性差。而肿瘤自身抗体在正常人体内含量很低检测不到或根本不存在,若体内肿瘤自身抗体水平明显增高,则表明体内存在异常免疫情况,表明体内相关抗原水平发生波动,预示疾病的存在或原有疾病加重。
近年来的研究表明,在恶性肿瘤体积发展到可用现代影像学技术检出之前3-5年,患者血中可出现高浓度的肿瘤相关抗原自身抗体。因此,检测血中肿瘤相关抗原自身抗体具有预测肿瘤发病风险和早期诊断肿瘤的重要价值。是肿瘤临床诊断领域的重点发展方向之一。在国外已有诊断肺癌和乳腺癌的早期诊断试剂盒市售。然而,目前所报道的自身抗体检测方法敏感度低,特异性差,假阴性比率可高达50%以上。其主要原因是由于每一种肿瘤相关抗原自身抗体在癌症患者中的阳性检测率平均在10%左右。如何提高诊断试剂敏感度是当前需要亟待解决的关键问题。比较行之有效的方法是寻找新的可充当肿瘤标志物的自身抗体,然后与现有已知的肿瘤相关抗原自身抗体组合成具有敏感度高和特异性强的诊断试剂盒。
MYC是一种常见的原癌基因,属于myc家族的重要成员,它编码细胞核内磷酸化蛋白质。MYC基因参与细胞的增殖、分化及凋亡过程,在维持正常细胞功能活中起重要作用。MYC与人类肿瘤有密切关系,在许多肿瘤组织中均可以检测到原癌基因MYC及其蛋白表达产物的异常。而且体外实验证实MYC基因可与其他癌基因协同转化多种细胞。大量研究表明可在人类肿瘤中检测到原癌基因MYC的异常,如MYC基因扩增,高水平转录及蛋白产物的堆积。MYC基因既是一种可易位基因,又是一种受多种物质调节的基因,具有促进细胞分裂并获得永生化功能。当MYC基因被激活后蛋白过度表达而堆集在细胞内,使细胞获得永生化,降低他们生长因子的需求,改变细胞内的基因调控,使细胞易于转化为恶性表型,当与其他活性癌基因协同作用时可致细胞发生恶性转化。MYC基因首先在小细胞肺癌中发现了过表达的现象,有30%的病例出现MYC明显扩增。研究发现,MYC在肺癌中表达明显升高,在肺癌早期即发生,与肿瘤的分期无关,与对照组差异有统计学意义。与年龄、性别、分化程度、淋巴结转移及肿瘤直径等无相关性,可能对肺癌的早期诊断有帮助,有可能成为肺癌早期诊断标记物及治疗靶点。基于MYC在肿瘤组织和正常组织间表达的巨大差异和其在肿瘤发展中的重要作用,MYC作为一个预测、预后指标和抗癌治疗靶点吸引了越来越多的关注。同时提示我们其在肺癌早期诊断的应用价值较好,以及作为潜在生物标志物的可能。本发明通过自行设计的MYC抗原表位多肽,检测肿瘤患者血清及血浆中自身抗体水平并开发相应的试剂,预测肺癌发生的危险性及预后预测,并为肺癌新药研究提供可靠的数据。
本发明通过自行设计的MYC抗原表位多肽,检测肺癌患者血清及血浆中自身抗体表达水平并开发相应的试剂,预测肺癌发生的危险性,并为制备肺癌早期诊断及预后预测试剂盒奠定基础。
发明内容
本发明要解决的技术问题是公开一种检测肺癌标志物MYC自身抗体的抗原表位序列。
本发明同时公开了MYC抗原表位的用途。
本发明提供的一种检测肺癌标志物MYC自身抗体的抗原表位氨基酸序列为:
H- DHQHNYAAPPSTRKDYPSECIDPSVVFPYPLND-OH
其纯度>95%,pH>7.0。
本发明所述的MYC抗原表位多肽在制备肺癌早期诊断试剂盒中的应用。
本发明利用自行设计的MYC蛋白的线性多肽,采用ELISA法检测肺癌患者血清及血浆中抗MYC蛋白的特异性自身IgG抗体。自身IgG抗体水平升高表明肿瘤患者体内MYC蛋白的表达量增加,预示患者可能出现原发性或继发性肺癌,可以预测肺癌发生与复发的危险性,指导临床医生对肺癌的早期诊断及预后预测。
抗原抗体的结合实际上只发生在抗原决定簇和抗体的抗原结合位点之间,两者在空间结构和空间构型上完全互补。因此抗原决定簇就可以代表整个蛋白与抗体结合的状态与亲和特性。另外,以重组蛋白为抗原,要经过载体构建、转染、表达、筛选、纯化等繁琐的过程,蛋白空间结构复杂,抗原表位不易暴露,因此抗原抗体结合的特异性差。此外,ELISA法的高灵敏性对纯化技术的稳定性要求极高,成本昂贵。
发明人遵循以下原则设计线性多肽抗原:①选择细胞膜蛋白表面区域;②选择不形成a-helix的序列;③两端的肽段比中间的排列合理;④避免蛋白内部重复;⑤避免同源性强的肽段;⑥序列中尽量避免Cys和Glu,不可以有太多的Pro,但有1-2个Pro利于肽链结构稳定,对产生特异性抗体有益。此外,该多肽抗原必须含有人类白细胞二类抗原(HLA)系统的限制性表位,包括HLA-DR, HLA-DP and HLA-DQ的限制性表位。这些表位可被90%以上华人群体的HLA二类抗原系统所识别。基于以上抗原设计原则及MYC蛋白的生物学特性,本发明利用生物信息学和多个表位预测模拟软件,分析与抗原性相关的参数,设计了的线性氨基酸序列。MYC线性多肽抗原由33个氨基酸残基组成,共含9个重叠表位,可检测至少9种单克隆抗体,具有高度的特异性。
法检测抗原表位
我们采用ELISA方法,对收集的血液进行检测,并得到各样本OD值进行分析。
质控 各样本设双复孔,取平均OD值。OD值离散度判定:离散度=OD1-OD2/OD1+OD2,离散度≤0.1,为有效结果;离散度>0.1,为无效结果。取100份健康人血清等体积混合作为质控血(Quality control, QC),代表人群的普遍情况,每板均设2个QC血浆孔,以QC血浆孔的OD值变异水平判定结果的稳定性,批间变异CV=所有批次QC孔 SD/所有批次QC孔 OD均值<20%。 批内变异CV=每日各板QC孔SD/每日各板QC孔均值<10%。
数据分析 采用SPSS17.0 for windows进行统计学分析。采用特异结合指数(Specific binding index, SBI)来判定MYC抗原多肽与血浆自身抗体的结合程度,SBI=MYC OD值 – NC OD值/ QC OD值 – NC OD值,NC为各样本的阴性对照。利用t检验分别比较恶性肺癌组与健康对照之间SBI值之间的差异,a=0.05。
ROC曲线是根据一系列不同的二分类方式(分界值或决定阈),以真阳性率(灵敏度)为纵坐标,假阳性率(1-特异度)为横坐标绘制的曲线。ROC曲线下的面积值在1.0和0.5之间。在AU>0.5的情况下,AU越接近于1,说明诊断准确度越好。ROC曲线将灵敏度与特异性以图示方法结合在一起,可准确反映某分析方法特异性和敏感性的关系,是试验准确性的综合代表。该发明采用Analyse-it for Microsoft Excel软件绘制ROC曲线,计算曲线下面积(AU),判定灵敏度和特异度。
本发明应用MYC抗原表位多肽检测到肺癌患者血清及血浆中的MYC特异性自身IgG抗体,并且该反应具有高特异性和高灵敏性。
MYC抗原表位多肽可用于制备肺癌早期诊断及预后预测试剂盒。
具体实施方式
下面结合具体实施例子,进一步阐述本发明。应理解,这些实施例子仅用于说明本发明而不用于限制本发明的范围。
实施例1
试剂盒制备
1 试剂 试剂配制见Tab.2~7。
2 操作
(1)包被:酶标板应用洗涤缓冲液清洗3遍,工作抗原用包被液稀释至工作浓度,包被于酶标板,4℃过夜。
(2)加谷氨酸:洗涤缓冲液清洗3遍,用包被液稀释谷氨酸至浓度100µg/ml,每孔200µl,37℃或室温孵育1h;
(3)加血浆和质控对照(一抗):酶标板应用洗涤缓冲液清洗3遍,利用包被液将血浆稀释至合适浓度,一般为1:100~1:500,每孔100µl,37℃或室温孵育1h;
(4)二抗孵育:洗涤缓冲液清洗3遍,利用包被液稀释二抗标准液IgG,工作浓度1:20000,每孔加100µl,37℃或室温孵育1h;
(5)显色:洗涤缓冲液清洗3遍,每孔加100µl底物显色液,室温避光30~45min。
(6)检测:每孔加50µl终止液,10min内检测,检测波长为450nm,参考波长为630nm。
实施例2
肺癌患者的MYC自身IgG抗体检测
1样本收集:本研究从吉林大学第三医院、省肿瘤医院选取经放射学检查和组织学检查确诊肺癌样本225例,其中肺腺癌124例,肺鳞癌101例。所有血清样本采集前未经过任何抗癌治疗,并具有全面的临床资料和信息。同时招募了健康对照组样本198例。临床访谈及影像学检查均排除肺癌患病可能。健康组与肺癌组在性别、年龄匹配,具有可比性(P>0.05)
2检测结果:MYC的自身抗体表达水平 (Tab.8):肺癌组与健康对照组相比存在统计学差异(z =-2.269,P <0.05)。
ROC曲线分析:肺癌患者MYC自身IgG抗体检测在ROC曲线下面积为0.723(SE=0.027, 95%CI:0.509~0.615)。
以上数据充分表明,利用本发明所设计的抗原表位多肽检测得到的肺癌患者自身抗体IgG水平与正常健康组比较有显著性统计学差异。
Tab.8抗MYC自身IgG抗体在肺癌患者和健康对照样本中的表达水平
Tab.9 肺癌中抗MYC自身IgG抗体的ROC曲线分析
H- DHQHNYAAPPSTRKDYPSECIDPSVVFPYPLND -OH
其纯度>95%,pH>7.0。
Claims (2)
1.一种检测肺癌标志物MYC抗原表位多肽,其特征在于:氨基酸序列为
H-DHQHNYAAPPSTRKDYPSECIDPSVVFPYPLND-OH。
2.根据权利要求1所述的检测肺癌标志物MYC抗原表位多肽在制备肺癌早期诊断及预后预测试剂盒的应用。
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