细粒棘球绦虫乳酸脱氢酶B细胞表位的预测及鉴定

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (6): 554-558.

细粒棘球绦虫乳酸脱氢酶B细胞表位的预测及鉴定

闫帅^1,², 严萍¹, 莫筱瑾², 徐斌², 张颋^2,^*(), 胡薇², 詹若挺¹, 叶萍³

1 广州中医药大学中药资源科学与工程研究中心,岭南中药资源教育部重点实验室（广州中医药大学）、国家中成药工程技术研究中心南药研发实验室,广州 510006
2 中国疾病预防控制中心寄生虫病预防控制所,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,卫生部寄生虫病原与媒介生物学重点实验室,上海 200025
3 四川省甘孜藏族自治州疾病预防控制中心,康定 626000

收稿日期:2017-06-02 出版日期:2017-12-30 发布日期:2018-01-10
通讯作者: 张颋
基金资助:
国家自然科学基金（No.81201315）;中国博士后基金（No.2014M551331）;国家科技重大专项（No.2012ZX10004220）

Prediction and identification of B cell epitopes of lactate dehydrogenase of Echinococcus granulosus

Shuai YAN^1,², Ping YAN¹, Xiao-jin MO², Bin XU², Ting ZHANG^2,^*(), Wei HU², Ruo-ting ZHAN¹, Ping YE³

1 Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Traditional Chinese Medicine; Key Laboratory of Chinese Medicinal Resource from Lingnan(Guangzhou University of Traditional Chinese Medicine),Ministry of Education; Joint Laboratory of National Engineering Research Center for the Pharmaceutics of Traditional Chinese Medicines, Guangzhou, 510006, China
2 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO Collaborating Center for Tropical Diseases, National Center for International Research on Tropical Diseases, Key Laboratory of Parasite and Vector Biology of the Chinese Ministry of Health, Shanghai 200025, China
3 Center for Disease Control and Prevention of Garzê, Kangding 626000, China];

Received:2017-06-02 Online:2017-12-30 Published:2018-01-10
Contact: Ting ZHANG
Supported by:
Supported by the National Natural Science Foundation of China(No.81201315);the China Postdoctoral Science Foundation(No.2014M551331) and the National Science and Technology Major Project of China(No.2012ZX10004220)

摘要/Abstract

摘要：

目的预测及鉴定细粒棘球绦虫乳酸脱氢酶（Echinococcus granulosus lactate dehydrogenase,EgLDH）的B细胞表位,为研制敏感性和特异性强的棘球蚴病诊断方法奠定基础。方法通过ESPript 3.0在线网站和IEDB软件对EgLDH的二级结构、β转角、表面可及性、柔韧性、抗原性、亲水性、线性表位等参数进行预测,筛选出具有潜在优势的B细胞表位区域;选取合适的B细胞表位进行体外多肽段合成;通过ELISA方法对合成的B细胞表位进行诊断效能评价,分别检测棘球蚴病患者血清、囊尾蚴病患者血清和健康者血清,评价敏感性、特异性和交叉反应性。用囊液抗原作为对照。结果 EgLDH二级结构中α螺旋占37.16%,β-折叠占9.67%,无规则卷曲占53.17%,存在4个跨膜区。筛选获得7个潜在的B细胞表位区域。体外合成了4个氨基酸长序列的B细胞表位PP-1（215~228）、PP-2（189~200）、PP-4（79~89）和PP-7（21~33）,ELISA结果显示,4个B细胞表位PP-1、PP-2、PP-4和PP-7对棘球蚴病患者血清的敏感性分别为52.9%（18/34）、61.8%（21/34）、82.3%（28/34）和50%（17/34）,PP-4与PP-1、PP-2和PP-7的敏感性差异有统计学意义（χ² = 8.100、5.143、9.091,均P < 0.05）,囊液抗原的敏感性为91.1%（31/34）,与PP-1、PP-2和PP-7的差异有统计学意义（χ² = 9.600、5.780、10.530,均P < 0.05）,与PP-4的差异无统计学意义（χ² = 0.444,P > 0.05）;对健康者血清的特异性分别为100%（21/21）、100%（21/21）、95%（20/21）和95%（20/21）,囊液抗原的特异性为95%（20/21）,与PP-1、PP-2、PP-4和PP-7的差异无统计学意义;对囊尾蚴病患者血清的交叉反应性分别为：0、1/10、2/10和2/10,囊液抗原的交叉反应性为5/10。结论 EgLDH 的B细胞表位PP-4对棘球蚴病患者血清有较强的敏感性和特异性,有望成为潜在的诊断抗原。

关键词: 细粒棘球绦虫, 乳酸脱氢酶, B细胞表位, 预测, 诊断抗原

Abstract:

Objective To predict and identify B cell epitopes of lactate dehydrogenase of Echinococcus granulosus(EgLDH), in order to advance the development of highly specific and sensitive diagnostic method for echinococcosis.Methods ESPript 3.0 online program and IEDB software was used to predict secondary structure, β-fold, Emini Surface Accessibility, Karplus & Schulz Flexibility, Kolaskar & Tongaonkar Antigenicity, Parker Hydrophilicity and Bepipred Linear Epitope of EgLDH.Based on the prediction, potential epitopes were synthesized in vitro to assess their diagnostic efficacy using ELISA.The sensitivity, specificity and cross-reactivity were evaluated using sera from patients with echinococcosis, cysticercosis and healthy participants.Cystic fluid antigen was the control.Results The predicted secondary structure of EgLDH was composed of 37.16% α-helix, 9.67% β-fold and 53.17% random coil, with four transmembrane domains.Seven potential B cell epitopes were predicted, of which four were synthesized, i.e.peptides PP-1（215-228）, PP-2 （189-200）, PP-4 （79-89） and PP-7 （21-33）, with a sensitivity of 52.9%（18/34）, 61.8%（21/34）, 82.3%（28/34） and 50.0%（17/34）, respectively, to sera from patients with echinococcosis.There were significant differences between PP-4 and PP-1, PP-2 as well as PP-7（χ² = 8.100, 5.143, 9.091, P < 0.05）.The sensitivity of cystic fluid antigen was 91.1% （31/34）, which was significantly different from those of PP-1, PP-2 and PP-7 （χ² = 9.600, 5.780, 10.530, P < 0.05）, but not PP-4 （χ² = 0.444, P > 0.05）.The specificity to normal serum was 100% （21/21）, 100% （21/21）, 95% （20/21） and 95% （20/21）, respectively, all comparable to that of cystic fluid antigen （95%, 20/21）.The cross-reactivity to cysticercosis was 0, 1/10, 2/10 and 2/10, respectively, while that of the cystic fluid antigen was 5/10.Conclusion The PP-4 epitope of EgLDH B-cell shows a high sensitivity and specificity to sera from patients with echinococcosis and may be a potential diagnostic antigen for hydatid disease.

Key words: Echinococcus granulosus, Lactate dehydrogenase, Epitopes, Prediction, Diagnosis

中图分类号:

R383.3

闫帅, 严萍, 莫筱瑾, 徐斌, 张颋, 胡薇, 詹若挺, 叶萍. 细粒棘球绦虫乳酸脱氢酶B细胞表位的预测及鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 554-558.

Shuai YAN, Ping YAN, Xiao-jin MO, Bin XU, Ting ZHANG, Wei HU, Ruo-ting ZHAN, Ping YE. Prediction and identification of B cell epitopes of lactate dehydrogenase of Echinococcus granulosus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(6): 554-558.

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多肽段Peptides	不同类型棘球蚴病患者血清敏感性/% Sensitivity of different types of echinococcosis patients serum /%			特异性/%Specificity/%	交叉反应性 Cross-reactivity
多肽段Peptides	囊型 Cystic echinococcosis	泡型 Alveolar echinococcosis	合计 Total	特异性/%Specificity/%	交叉反应性 Cross-reactivity
PP-1	58.8(10/17)	47.1(8/17)	52.9(18/34)	100(21/21)	0
PP-2	47.1(8/17)	76.5(13/17)	61.8(21/34)	100(21/21)	1/10
PP-4	76.5(13/17)	88.2(15/17)	82.3(28/34)^a	95(20/21)	2/10
PP-7	47.1(8/17)	52.9(9/17)	50.0(17/34)	95(20/21)	2/10
囊液抗原 Cystic fluid antigen	100 (17/17)	82.3(14/17)	91.1(31/34)	95(20/21)	5/10