CN113234172B - 一种梅毒特异抗体的检测探针及其制备方法 - Google Patents
一种梅毒特异抗体的检测探针及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种梅毒特异性抗体的检测探针及其制备方法。本发明探针基于金葡菌的检测载体，三种梅毒抗原和噬菌体裂解酶结合域融合，并标记在金葡菌检测载体上。本发明利用噬菌体裂解酶结合域和金葡肽聚糖的结合能力，将三种梅毒抗原同时标记在金葡菌载体的表面，获得一种梅毒特异抗体检测的新型生物探针。可以利用这种新型探针对梅毒血清特异抗体进行快速的协同凝集检测。
Description
技术领域
本发明涉及性传播疾病病原体梅毒螺旋体的特异性抗体检测领域，具体来说，涉及一种梅毒特异性抗体的新型检测探针及其制备方法。
背景技术
梅毒(Syphilis)由细菌梅毒螺旋体(Treponema pallidum, TP)亚种引起，可通过直接性接触、输血、母婴等方式传播。梅毒是报告病例数最多的性病，严重危害人类健康。梅毒的检测为梅毒的感染诊断、分期指导、治疗方案和治疗效果提供评价依据。早期梅毒仍可治愈，因此早期检测与术前筛查对于患者准确诊断、及时治疗、有效预后、尽早切断传染源以及有效管理都意义重大，可有效降低梅毒长期并发症的风险，降低死胎、先天性梅毒以及传播风险，能极大减轻梅毒带来的家庭、社会和国家负担。
梅毒检测主要分为病原体与血清学检测。通常，患者出现明显症状时，可进行病原体检测；患者无明显症状，进行血清学检测。病原体检测包括暗视野显微镜法、镀银染色法和核酸检测法。由于大多数梅毒感染者早期无明显症状，血清学检测是主要的实验室梅毒检测方法。
梅毒螺旋体是一种非常复杂的微生物，含有很多抗原物质。电镜下梅毒螺旋体的最外层为外膜，外膜内是胞浆膜，两者之间是鞭毛。人体感染梅毒后，会产生两类抗体，一类是抗梅毒螺旋体的特异抗体，另一类是抗类脂质的梅毒非特异抗体，主要是心磷脂(Cardiolipin)抗体。
发明内容
本发明所要解决的技术问题是提供一种梅毒特异性抗体的检测探针及其制备方法。可以利用这种新型探针对梅毒血清特异抗体进行快速的协同凝集检测。
本发明基于发明人之前参与发明的新型多色多功能金黄色葡萄球菌（Staphylococcus aureus，SA）检测载体（中国专利ZL 2014 1 0462482.2）、金黄色葡萄球菌噬菌体裂解酶（Lysin）结合域（Cell binding domain, CBD）等研究成果，利用CBD和梅毒抗原融合蛋白标记金葡菌载体，用于梅毒特异抗体的快速检测。该方法不依赖仪器，通过裸眼观察即可在5-10分钟内实现对特异抗体的检测。
目前，血清学诊断常用梅毒抗原有Tpp15, Tpp17, Tpp47，这三种抗原在各期梅毒中都有较强的表达，可用于梅毒筛查。多种抗原融合可提高检测的敏感性和特异性，常用的嵌合基因有Tpp15-47, Tpp15-17, Tpp17-47, Tpp15-47-17。
在本发明中，不通过抗原基因间的融合，而是Tpp15, Tpp17, Tpp47通过(G4S)3柔性linker同CBD分别融合，得到的蛋白Tpp15-(G4S)3-CBD，Tpp17-(G4S)3-CBD, Tpp47-(G4S)3-CBD同新型金葡菌载体共同孵育，得到结合有这三种蛋白的金葡探针，不仅可以充分的暴露抗原位点，而且(G4S)3linker可以减少空间位阻，会有更好的灵敏度和特异性。
本发明所述的梅毒特异性抗体的检测探针，是将梅毒螺旋体三种抗原Tpp15,Tpp17, Tpp47通过(G4S)3柔性linker同CBD分别融合，得到的蛋白Tpp15-(G4S)3-CBD、Tpp17-(G4S)3-CBD、Tpp47-(G4S)3-CBD同时标记在红色灭活的金葡菌载体的表面。
所述金葡菌载体制备按照中国专利ZL 2014 1 0462482.2制备。即所述金葡菌载体被四唑氯化物染料染至红色；所述四唑氯化物染料具体为5-氰基-2，3-二(4-甲基苯基)四唑氯化物。
本发明上述检测探针的制备方法，是将梅毒螺旋体抗原和噬菌体裂解酶CBD通过基因重组，构建表达质粒，在大肠杆菌表达系统进行蛋白表达，可溶蛋白纯化后同新型金葡菌载体共同孵育，可得到检测梅毒特异抗体的协同凝集探针。
具体地，本发明的梅毒特异抗体检测探针制备按照以下步骤：
S1，将梅毒螺旋体抗原Tpp15, Tpp17, Tpp47通过(G4S)3柔性linker同噬菌体裂解酶（lysin） PlyV12 CBD通过基因重组并在大肠杆菌中表达融合蛋白，获得蛋白Tpp15-(G4S)3-CBD、Tpp17-(G4S)3-CBD、Tpp47-(G4S)3-CBD，冷冻保存备用；
S2，Tpp15-(G4S)3-CBD、Tpp17-(G4S)3-CBD、 Tpp47-(G4S)3-CBD标记在红色灭活的金黄色葡萄球菌载体上。
S1中，蛋白生产和纯化的过程为常规基因重组和蛋白表达纯化过程。
S2中，更具体步骤如下：
(1) 取红色灭活的1*1010 Particle/ml金黄色葡萄球菌0.1M Tris-Cl（pH8.0）悬液，CBD-(G4S)3-TpN15，CBD-(G4S)3-TpN17，CBD-(G4S)3-TpN47按1:1:1的摩尔比加入悬液，37℃,180rpm孵育30min；
(2) 4℃，4000rpm，5min收集沉淀，上清可回收再利用，相同体积0.1M Tris-Cl（pH8.0）缓冲液洗三遍后，相同体积0.1M Tris-Cl（pH8.0）重悬；
(3) 加入质量体积比1%的脱脂奶粉后，轻微颠倒溶解，37℃,180rpm孵育30min。
(4) 4℃，4000rpm，5min收集沉淀，相同体积0.1M Tris-Cl（pH8.0）缓冲液洗三遍后，1/10体积0.1M Tris-Cl（pH8.0）重悬备用。
梅毒特异抗体检测探针的制备方法利用常规基因重组和蛋白原核表达纯化两项技术以及金葡表面具有与噬菌体裂解酶CBD结合位点的天然性质。探针制备方法设计科学，有理有据，可以成功制备梅毒特异抗体检测探针。
本发明基于新型梅毒特异抗体检测探针使用时，采用的方法是协同凝集，只需要在玻片上进行凝集试验，且5min种读取结果，方法不需要仪器、简单、快速，且和临床使用的TPPA技术操作类似，易被接受。
附图说明
图1梅毒特异抗体检测新型探针制备流程图。
图2梅毒特异抗体检测新型探针对抗体检测的原理。
图3梅毒特异抗体新型检测探针，制备方法及应用技术路线图。
图4 梅毒病原体三种基因TPP15, TPP17和TPP47的核酸扩增结果。TPP15基因大小为423bp, Tpp17基因大小为468bp, TPP47基因大小为1302bp。
图5 Tpp15-(G4S)3-CBD，Tpp17-(G4S)3-CBD, Tpp47-(G4S)3-CBD三种融合蛋白的表达和纯化结果，蛋白大小分别为34.2, 34.9, 66.1kDa，图中标记蛋白加上了5.2kDa的载体大小。
图6 A 金黄色葡萄球菌，B经过CTC处理的金黄色葡萄球菌，C经过热处理后制备完成的红色金葡菌载体，D 金黄色葡萄球菌的透射电镜（TEM）图片，金葡呈葡萄串状，E红色金葡菌载体的TEM图片显示，载体状态分散且单一，F红色金葡菌载体也具有红色荧光，荧光图片显示载体分散，G粒径分析显示红色金葡菌载体在处理后有皱缩，粒径为609.2±97.3nm，比报道的粒径800nm略小。
图7 1-119例临床样本为梅毒特异抗体血清学阳性样本，样本在临床有TPPA和TP-ELISA结果，协同凝集检测结果显示119例样本皆为阳性结果，判读标准为+++，密集且明显的凝集；++，分散但明显的凝集;+较少的、分散但明显的凝集；—，无明显凝集产物。
图8 120-209例临床样本为梅毒特异抗体血清学阴性样本，样本在临床有TPPA和TP-ELISA结果，协同凝集检测结果显示119例样本皆为阴性结果。
图9 采用第108例临床检测结果进行2倍梯度稀释，在1/32倍时，检测结果判读处于弱阳性。空白对照（B）和阴性对照（N）无凝集产物产生。
图10 方法的可重复性和可再现性如图所示，探针的稳定性由左至右分别为4℃，1天，14天和30天检测结果。
具体实施方式
本发明所述的梅毒特异性抗体的检测探针，是将梅毒螺旋体三种抗原Tpp15,Tpp17, Tpp47通过(G4S)3柔性linker同CBD分别融合，得到的蛋白Tpp15-(G4S)3-CBD，Tpp17-(G4S)3-CBD, Tpp47-(G4S)3-CBD同时标记在红色灭活的金葡菌载体的表面。
本发明上述检测探针的制备方法，是将梅毒螺旋体抗原和噬菌体裂解酶CBD通过基因重组，构建表达质粒，在大肠杆菌表达系统进行蛋白表达，可溶蛋白纯化后同新型金葡菌载体共同孵育，可得到检测梅毒特异抗体的协同凝集探针。
具体地，本发明的梅毒特异抗体检测探针制备按照以下步骤：
S1，将梅毒螺旋体抗原Tpp15, Tpp17, Tpp47通过(G4S)3柔性linker同噬菌体裂解酶（lysin） PlyV12 CBD通过基因重组并在大肠杆菌中表达融合蛋白，获得蛋白Tpp15-(G4S)3-CBD、Tpp17-(G4S)3-CBD,、Tpp47-(G4S)3-CBD，冷冻保存备用；
S2，Tpp15-(G4S)3-CBD、Tpp17-(G4S)3-CBD、 Tpp47-(G4S)3-CBD标记在红色灭活的金黄色葡萄球菌载体上。
本发明的梅毒特异抗体检测探针制备方法为图1。梅毒特异抗体检测探针对抗体检测的原理如图2。梅毒特异抗体新型探针、制备方法和应用技术路线总览如图3。
实施例1，检测探针的制备过程。
本发明的梅毒特异抗体检测探针制备按照以下步骤：
S1，将梅毒螺旋体抗原Tpp15, Tpp17, Tpp47通过(G4S)3柔性linker同噬菌体裂解酶（lysin） PlyV12 CBD通过基因重组并在大肠杆菌中表达融合蛋白，蛋白生产和纯化的过程为常规基因重组和蛋白表达纯化过程。结果如图4,5所示。获得蛋白Tpp15-(G4S)3-CBD、Tpp17-(G4S)3-CBD,、Tpp47-(G4S)3-CBD，冷冻保存备用；
S2，Tpp15-(G4S)3-CBD、Tpp17-(G4S)3-CBD、 Tpp47-(G4S)3-CBD标记在红色灭活的金黄色葡萄球菌载体上。
S2中，具体步骤如下：
(1) 取红色灭活的1*1010 Particle/ml金黄色葡萄球菌0.1M Tris-Cl（pH8.0）悬液，CBD-(G4S)3-TpN15，CBD-(G4S)3-TpN17，CBD-(G4S)3-TpN47按1:1:1的摩尔比加入悬液，37℃,180rpm孵育30min。所用金黄色葡萄球菌形态如图6所示。
(2) 4℃，4000rpm，5min收集沉淀，上清可回收再利用，相同体积0.1M Tris-Cl（pH8.0）缓冲液洗三遍后，相同体积0.1M Tris-Cl（pH8.0）重悬；
(3) 加入质量体积比1%的脱脂奶粉后，轻微颠倒溶解，37℃,180rpm孵育30min。
(4) 4℃，4000rpm，5min收集沉淀，相同体积0.1M Tris-Cl（pH8.0）缓冲液洗三遍后，1/10体积0.1M Tris-Cl（pH8.0）重悬备用。
实施例2 梅毒特异抗体方法的验证实验
检测梅毒特异抗体结果采用TPPA法进行验证。TPPA的结果为临床实验室的检测结果，由宜昌市第一人民医院暨三峡大学人民医院检验科实验室数据系统直接导出。
样本收集由宜昌市第一人民医院暨三峡大学人民医院检验科完成，共收集梅毒血清学阳性样本119例，阴性样本90例，其中阴性样本含老人、小孩和孕妇血清样本各30例，样本的收集通过了医院伦理委员会的审核并且获得了患者的知情同意。
通过和临床样本的检测结果的比较评价梅毒特异抗体检测新型探针和方法的特异性。结果如图7,8所示。
采用临床第108号阳性样本，分别进行1/2, 1/4, 1/8, 1/16, 1/32的梯度稀释，同时和空白和阴性对照进行平行测试，评价梅毒特异抗体检测新型探针和方法的灵敏度。结果如图9所示。
进行三次重复实验评价可重复性；探针在4℃保存1, 14, 30天分别进行测试评价稳定性。结果如图10所示。
实施例3 金葡菌载体体外检测的安全性
在前期的工作中，为了进一步确定具有双重性质的新型金黄载体的应用潜能，我们在体内和体外分别进行了该新型生物纳米粒子的培养实验和安全性评价。在体外LB平板和LB液体培养基的培养实验中，该新型生物纳米粒子表现出很好的生物安全性。而在Balb/c小鼠模型中，1*109 个新型生物纳米粒子的腹部注射却让小鼠在36小时内100%致死，此实验指出了该新型生物纳米粒子在体内应用的局限性，也为其他改造金黄色葡萄球菌的实验提供了很好的动物实验依据。
本方法的梅毒特异抗体检测方法简单、快速、低廉、准确、体外应用安全、而且不需要特殊仪器，实际操作起来和TPPA的方法流程几乎相同，容易被临床实验室检验人员接受协同凝集的结果判读只需要5min种，可以实现现场和床边检测。
Claims (5)
1.一种梅毒特异性抗体的检测探针，是将梅毒螺旋体三种抗原Tpp15, Tpp17, Tpp47通过(G4S)3柔性linker同噬菌体裂解酶 PlyV12 CBD分别融合，得到的蛋白Tpp15-(G4S)3-CBD，Tpp17-(G4S)3-CBD, Tpp47-(G4S)3-CBD同时标记在红色灭活的金葡菌载体的表面。
2.根据权利要求1所述的检测探针，其特征在于，所述金葡菌载体被四唑氯化物染料染至红色；所述四唑氯化物染料具体为5-氰基-2，3-二(4-甲基苯基)四唑氯化物。
3.权利要求1所述检测探针的制备方法，其特征在于，是将梅毒螺旋体抗原和噬菌体裂解酶CBD通过基因重组，构建表达质粒，在大肠杆菌表达系统进行蛋白表达，可溶蛋白纯化后同红色灭活的金葡菌载体共同孵育，得到检测梅毒特异抗体的探针。
4.权利要求3所述的制备方法，其特征在于，包括以下步骤：
S1，将梅毒螺旋体抗原Tpp15, Tpp17, Tpp47通过(G4S)3柔性linker同噬菌体裂解酶PlyV12 CBD通过基因重组并在大肠杆菌中表达融合蛋白，获得蛋白Tpp15-(G4S)3-CBD、Tpp17-(G4S)3-CBD、Tpp47-(G4S)3-CBD，冷冻保存备用；
S2，将Tpp15-(G4S)3-CBD、Tpp17-(G4S)3-CBD、 Tpp47-(G4S)3-CBD标记在红色灭活的金黄色葡萄球菌载体上。
5.根据权利要求4所述的制备方法，其特征在于，S2中，具体步骤如下：
(1) 取红色灭活的1*1010Particle/ml金黄色葡萄球菌0.1M Tris-Cl悬液，CBD-(G4S)3-TpN15，CBD-(G4S)3-TpN17，CBD-(G4S)3-TpN47按1:1:1的摩尔比加入悬液，37℃，180rpm孵育30min；
(2) 4℃，4000rpm，5min收集沉淀，上清可回收再利用，相同体积0.1M Tris-Cl缓冲液洗三遍后，相同体积0.1M Tris-Cl重悬；
(3) 加入质量体积比1%的脱脂奶粉后，轻微颠倒溶解，37℃，180rpm孵育30min；
(4) 4℃，4000rpm，5min收集沉淀，相同体积0.1M Tris-Cl缓冲液洗三遍后，1/10体积0.1M Tris-Cl重悬备用。
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