摘要
目的预测及鉴定细粒棘球绦虫乳酸脱氢酶(Echinococcus granulosus lactate dehydrogenase,Eg LDH)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Eg LDH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Eg LDH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(χ~2=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(χ~2=9.600、5.780、10.530,均P<0.05),与PP-4的差异无统计学意义(χ~2=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。
Objective To predict and identify B cell epitopes of lactate dehydrogenase of Echinococcus granulosus(Eg LDH), 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 Eg LDH. 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 Eg LDH was composed of 37.16% α-helix, 9.67% β-fold and53.17% 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(χ~2= 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(χ~2= 9.600, 5.780, 10.530, P < 0.05), but not PP-4(χ~2= 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 Eg LDH B-cell shows a high sensitivity and specificity to sera from patients with echinococcosis and may be a potential diagnostic antigen for hydatid disease.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2017年第6期554-558,共5页
Chinese Journal of Parasitology and Parasitic Diseases
基金
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)~~
