WO2018166079A1 - 嗜吞噬细胞无形体蛋白aph1384的应用 - Google Patents

嗜吞噬细胞无形体蛋白aph1384的应用 Download PDF

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WO2018166079A1
WO2018166079A1 PCT/CN2017/087912 CN2017087912W WO2018166079A1 WO 2018166079 A1 WO2018166079 A1 WO 2018166079A1 CN 2017087912 W CN2017087912 W CN 2017087912W WO 2018166079 A1 WO2018166079 A1 WO 2018166079A1
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牛华
贺美玲
吴淑燕
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苏州大学
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Abstract

本发明提供了嗜吞噬细胞无形体蛋白APH1384作为粒细胞无形体病诊断抗原的应用。

Description

嗜吞噬细胞无形体蛋白APH1384的应用 技术领域
本发明涉及免疫学领域，尤其涉及一种嗜吞噬细胞无形体蛋白APH1384的应用。
背景技术
嗜吞噬细胞无形体是一种专性细胞内寄生的以蜱虫为传播媒介的革兰氏阴性菌，能导致粒细胞无形体病，如人粒细胞无形体病和犬粒细胞无形体病的发生。美国CDC的数据显示，从2000年到2012年人粒细胞无形体病的感染例数由348例增至2389例，呈快速增长的趋势。此外，粒细胞无形体病在澳大利亚、欧洲多国、日本及韩国的人群和家畜中发生局部流行。在国内，由中国疾控中心组织的血清流行病学调查表明，我国的农业从业者中粒细胞无形体病的血清阳性率高达13.9％。与欧美粒细胞无形体病患者相比，我国无形体病患者病情更为严重，死亡率可高达8.1％。我国的病人会发展成其他严重的症状，如其中41.2％会发展成多器官衰竭。另外，据中国疾控中心调查，在犬和羊的嗜吞噬细胞无形体检测中，血清阳性率分别高达10.05％和3.82％。目前，粒细胞无形体病已在世界范围内流行，成为重要的公共卫生问题。因此加强粒细胞无形体病诊断方法的优化，对医学、公共卫生及畜牧业领域具有十分重要的现实意义。
目前，粒细胞无形体病血清学诊断方法是以嗜吞噬细胞无形体来源的特异性种属蛋白为诊断抗原，检测被感染对象血清中抗该病原菌的特异性抗体，从而判断受试者是否感染嗜吞噬细胞无形体。血清学诊断方法主要包括间接免疫荧光技术(Indirect fluorescent antibody assays,IFA)，免疫印记技术(WesternBlotting和DotBlotting,WB和DB)及酶联免疫吸附实验(ELISA)。对于人或动物大批量血清样本的检测主要使用ELISA和DB方法。
IFA方法是使用嗜吞噬细胞无形体感染的细胞制备成抗原片，对临床血清标本中抗嗜吞噬细胞无形体的特异性抗体进行检测。通过孵育标记有荧光素的二抗，在荧光显微镜下观察抗原片上的荧光强度判断血清是否为阳性。该技术是目前临床上用于检测粒细胞无形体病感染的金标准，但是操作复杂，判断结果主观，且抗原片的价格昂贵，因此不适合临床上大批量血清样本的检测。而WB，DB和ELISA检测方法以嗜吞噬细胞无形体特异性重组蛋白或蛋白抗原表位多肽为诊断抗原，检测人或动物血清样本中是否存在抗嗜吞噬细胞无形体的特异性抗体。其中，ELISA方法简单，可自动化，可以用于大量临床样本的检测。但目前，这三种血清学检测方法所使用的诊断抗原均为嗜吞噬细胞无形体外膜蛋白P44，而以P44作为 单一诊断抗原的检测方法存在漏检的缺陷，其检测灵敏度为80-90％。总之，IFA法检测成本高，需要价格昂贵的抗原片，且判定结果主观，操作时间相对较长，不适合大量临床样本的检测；而WB，DB和ELISA法虽然操作简单，成本低，但目前他们均以P44作为单一诊断抗原，存在漏检的缺陷，检测灵敏度有待提高。
发明内容
为解决上述检测方法的不足，本发明的目的是提供一种嗜吞噬细胞无形体蛋白APH1384的应用，该蛋白抗原性较强，能弥补P44漏检的缺陷，可提高粒细胞无形体病检测的灵敏度，从而有利于临床上快速精准地诊断粒细胞无形体病的发生。
本发明提供了一种嗜吞噬细胞无形体蛋白APH1384作为粒细胞无形体病诊断抗原的应用。
进一步地，嗜吞噬细胞无形体蛋白APH1384的氨基酸序列如SEQ ID No.1所示。
进一步地，嗜吞噬细胞无形体蛋白APH1384的抗原表位的氨基酸序列如SEQ ID No.2所示。
借由上述方案，本发明至少具有以下优点：
本发明提供了一种粒细胞无形体病血清学诊断抗原APH1384，该蛋白可弥补现有粒细胞无形体病诊断抗原P44漏检的缺陷，提高了粒细胞无形体病检测的灵敏度，有利于临床上快速精准地诊断粒细胞无形体病的发生。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是APH1384蛋白的SDS-PAGE电泳检测结果；
图2图示了APH1384抗原性的WB技术检测结果；
图3图示了APH1384多肽抗原性的DB技术验证结果。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1  APH1384蛋白(嗜吞噬细胞无形体种属特异性蛋白)的制备
利用生物信息学技术，以嗜吞噬细胞无形体基因组编码的610个未知功能蛋白为靶标，筛选出嗜吞噬细胞无形体种特异性蛋白。对其中一些蛋白(包括APH1384)进行了克隆表达 和抗原性鉴定。研究结果发现APH1384具有强的抗原性。以下为APH1384详细的克隆表达和抗原性鉴定步骤。根据Gene Bank中aph1384基因序列设计含有限制性核酸内切酶位点的特异性引物：aph1384-F-TCCGAATTCATGTTTGATATATTTAATGATGCTG；aph1384-R-GCACTCGAGTTATTCGGACGCAGAGGCT。以嗜吞噬细胞无形体的全基因组DNA为模板，PCR扩增aph1384基因的DNA片段。将扩增的目的基因DNA片段和原核表达载体pET28a(+)分别用限制性核酸内切酶双酶切，并使用T4DNA连接酶进行连接。连接产物转化至克隆宿主菌E.coli TOP10中，提取质粒进行测序，将阳性质粒转化至表达宿主菌E.coli BL21(DE3)中进行蛋白表达。37℃条件下培养含重组质粒的E.coli BL21(DE3)，加入IPTG诱导APH1384蛋白的表达，诱导3.5h后，离心收集菌体，变性裂解处理后通过SDS-PAGE电泳检测APH1384蛋白的表达水平，结果如图1所示。图1显示在21.5kDa大小处出现目标蛋白条带，而未加IPTG对照组，无目标蛋白条带。采用镍柱亲和层析的方法纯化APH1384蛋白，其氨基酸序列如SEQ ID No.1所示。
以纯化的嗜吞噬细胞无形体种属特异性蛋白APH1384为抗原，使用6份嗜吞噬细胞无形体阳性血清通过WB技术检测APH1384的抗原性。首先将纯化的重组APH1384蛋白上样于SDA-PAGE胶，蛋白转至硝酸纤维素膜，将膜切为条状，封闭30min，与一抗阳性血清(1:2000)进行孵育，PBS洗涤3次，与二抗(HRP标记的羊抗人IgG，1:5000)进行孵育，PBS洗涤4次，显影。结果见图2，其中图2中的1-6分别代表嗜吞噬细胞无形体阳性人血清，APH1384即为嗜吞噬细胞无形体种属特异性蛋白。结果显示6份阳性血清均能与APH1384反应。APH1384蛋白与嗜吞噬细胞无形体阳性血清呈现较强阳性反应，说明APH1384具有较强的抗原性。
实施例2  APH1384多肽的制备
利用生物信息学方法预测获得APH1384的抗原表位，其氨基酸序列如SEQ ID No.2所示。化学合成APH1384抗原表位多肽，利用DB技术对APH1384多肽的抗原性进行验证，具体操作如下：
将APH1384多肽抗原稀释液(浓度分别为1μg/μl、0.1μg/μl、0.01μg/μl)加至硝酸纤维素膜，37℃干燥20-30min，封闭30min，一抗即人阳性血清(1:2000)孵育1h，二抗孵育1h(1:5000)，显影。结果见图3，图3中的N代表阴性血清，P1-P3各代表一份嗜吞噬细胞无形体阳性血清，结果显示，阴性血清不与APH1384反应，而3份阳性血清均与APH1384多肽结合，证明APH1384多肽具有抗原性。
实施例3  APH1384多肽的应用
以合成的APH1384多肽为包被抗原，使用ELISA法检测人血清中特异性抗APH1384抗 体，具体操作步骤如下：
(1)包板：将实施例2合成的APH1384多肽用PBS稀释至20μg/ml，4℃过夜或室温下2h包板(96孔板，每孔50μl)；
(2)封闭：使用PBS清洗96孔板三次，加入小牛血清白蛋白(浓度为1％)的封闭液，每孔200μl，37℃，2h；
(3)一抗孵育：使用封闭液按1:1000的比例稀释待检血清，每孔加入稀释液100μl，37℃孵育1h；
(4)二抗孵育：使用PBS清洗四次(每孔200μl)，加入二抗(HRP标记的羊抗人IgG，1:5000稀释)，37℃孵育1h；
(5)显色：使用PBS清洗3-5次，加入配制好的显色液，每孔100μl，37℃避光10-15min后即加入终止液(浓度为1M的稀盐酸)，每孔100μl，以终止反应。在终止反应后的15分钟内，在450nm的波长处测定各孔溶液的吸光值；
(6)结果判定：计算Cutoff值(Cutoff值＝阴性对照的平均值+3*标准方差)，然后将检测孔中的吸光值与Cutoff值比较。当检测孔中吸光值大于Cutoff值时，表示血清中抗嗜吞噬细胞无形体抗体阳性；当检测孔中吸光值小于Cutoff值时，表示血清中抗嗜吞噬细胞无形体抗体阴性。
实施例4  APH1384多肽的应用
以合成的APH1384多肽为抗原，使用DB法检测人血清中特异性抗APH1384抗体，具体操作步骤如下：
(1)点样：PBS(0.01M，PH 7.4)稀释APH1384多肽抗原至适宜的浓度(分别为1μg/μl、0.1μg/μl、0.01μg/μl)，使用移液器将1μl稀释液加至硝酸纤维素膜，置37℃温箱中干燥20-30min；
(2)封闭：加5ml封闭液(5％脱脂奶粉)于温育盒中，常温封闭30min；
(3)一抗孵育：弃封闭液，加封闭液稀释的人血清标本(1:2000)，室温孵育1h，用PBS震洗3次，每次10min；
(4)二抗孵育：加入封闭液稀释的HRP-羊抗人IgG二抗(1:5000)，室温孵育1h后用PBS震洗3次，每次10min；
(5)显影：ECL化学发光法显影，观察1μg/μl浓度的斑点是否显色，以此来判断血清抗嗜吞噬细胞无形体抗体阴阳性。阴性对照血清在膜片上不出现有颜色的斑点。待检血清如出现肉眼可见的斑点，则为抗嗜吞噬细胞无形体抗体阳性；待检血清如不出现肉眼可见的斑点，则为抗嗜吞噬细胞无形体抗体阴性。
本发明的APH1384蛋白或合成的多肽抗原作为粒细胞无形体病的诊断抗原，可通过多种血清学方法检测多物种血清中的特异性抗体以诊断此物种是否感染嗜吞噬细胞无形体。血清学方法包括ELISA、DB、WB，但不仅限于这三种方法，多物种血清包括人、狗、猫、马等。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Figure PCTCN2017087912-appb-000001
Figure PCTCN2017087912-appb-000002

Claims (3)

  1. 嗜吞噬细胞无形体蛋白APH1384作为粒细胞无形体病诊断抗原的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述嗜吞噬细胞无形体蛋白APH1384的氨基酸序列如SEQ ID No.1所示。
  3. 根据权利要求1所述的应用，其特征在于：所述嗜吞噬细胞无形体蛋白APH1384的抗原表位的氨基酸序列如SEQ ID No.2所示。
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