细粒棘球绦虫磷酸甘油酸变位酶的表达、定位和诊断价值的初步评价

doi:10.12140/j.issn.1000-7423.2020.02.010

摘要/Abstract

摘要：

目的克隆、表达细粒棘球绦虫磷酸甘油酸变位酶（EgPGAM）,分析其免疫反应性及其在原头节、包囊及成虫中的分布情况,对EgPGAM的诊断价值进行初步评价。方法提取细粒棘球蚴原头节RNA,逆转录为cDNA,PCR扩增EgPGAM基因,测序。通过多种生物信息学软件分析EgPGAM的理化性质、保守结构域、抗原表位和同源性等。利用Mega 5.0软件进行序列特征分析,采用邻接法构建系统进化树。将EgPGAM基因连接至pET32a表达载体,转入大肠埃希菌（E. coli）BL21（DE3）中进行诱导表达,镍柱纯化,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析其表达情况并进行蛋白质免疫印迹（Western blotting）分析。免疫荧光定位分析EgPGAM在原头节、包囊及成虫中的分布。采用ELISA评价EgPGAM的诊断价值。结果 EgPGAM长756 bp,共编码251个氨基酸,编码蛋白的相对分子质量（M_r）约28 000,等电点为8.29,不含信号肽,无跨膜区。EgPGAM含有12个高保守的氨基酸位点、3个保守的催化活性位点、1个底物结合位点和7个B抗原表位。EgPGAM的氨基酸序列与多房棘球绦虫、猪带绦虫、华支睾吸虫、秀丽隐杆线虫、人和羊的PGAM序列相似性分别为99%、94%、78%、45%、57%和4%。SDS-PAGE分析结果显示,EgPGAM在E. coli BL21（DE3）中大量表达,主要以可溶形式存在;Western blotting分析结果显示,EgPGAM可与感染细粒棘球蚴的绵羊血清发生反应。免疫荧光定位显示,EgPGAM主要分布在原头节表皮层和顶突沟,包囊生发层和成虫薄壁组织。建立的间接ELISA敏感性为55.6%（15/27）,特异性为86.4%（89/103）。结论 EgPGAM广泛分布于细粒棘球蚴和细粒棘球绦虫中,具有较好免疫反应性,但敏感性较低,不适合作为细粒棘球蚴病的候选诊断抗原。

关键词: 细粒棘球绦虫, 磷酸甘油酸变位酶, 原核表达, 免疫定位, 酶联免疫吸附试验

Abstract:

Objective To clone and express Echinococcus granulosus phosphoglycerate mutase (EgPGAM), analyze its immunoreactivity and distribution in protoscoleces, hydatid cysts and adult worms, and preliminarily evaluate the diagnostic value.Methods Protoscolex RNA was extracted and reverse transcribed to cDNA, from which EgPGAM gene was amplified by PCR and sequenced. The physicochemical properties, conserved domains, antigen epitopes and homology of EgPGAM were analyzed by bioinformatics softwares. Mega 5.0 software was used to analyze the characteristics of the sequence, and the phylogenetic tree was constructed with the neighbor joining method. The EgPGAM gene was inserted into the pET32a plasmid, and transferred into Escherichia coli BL21(DE3) for induction of expression. The expressed proteins were purified by nickle column, and further analyzed by SDS-PAGE for verifying the expression products, and Western blotting for immunoreactivity. The distribution of EgPGAM in protoscoleces, hydatid cysts and adults was analyzed by immunofluorescence assay. The diagnostic value of EgPGAM was evaluated by indirect enzyme-linked immunosorbent assay(ELISA).Results The EgPGAM gene is 756 bp in length, encoding 251 amino acids. The encoded protein has a relative molecular weight Mr about 28 000, an isoelectric point of 8.29, containing no signal peptide or transmembrane region. EgPGAM protein contained 12 highly-conserved amino acid sites, 3 conserved catalytic sites, one substrate-binding site and 7 B epitopes. The EgPGAM had 99%, 94%, 78%, 57%, 45% and 4% amino-acid sequence homology with PGAMs of E. multilocularis, Taenia solium, Clonorchis sinensis, Caenorhabditis elegans, Homo sapiens and Ovis aries, respectively. SDS-PAGE showed that EgPGAM was highly expressed in E. coli BL21(DE3), mainly in soluble form. Western blotting showed reactions of EgPGAM with serum samples from E. granulosus-infected sheep. Immunofluorescence assay revealed that the EgPGAM was localized mainly on the tegument and hooks of protoscoleces, the germinal layer of the hydatid cyst and the cyst wall parenchyma of adults. The sensitivity and specificity of EgPGAM-based indirect ELISA were 55.6%(15/27) and 86.4% (89/103), respectively.Conclusion EgPGAM was widely distributed in the larvae and adult worms of E. granulosus. The recombinant EgPGAM had apparent immunoreactivity, but with low sensitivity in the indirect ELISA test. Therefore, EgPGAM is not a suitable candidate of diagnostic antigen for echinococcosis.

Key words: Echinococcus granulosus, Phosphoglycerate mutase, Prokaryotic expression, Immunofluorescence localization, Enzyme-linked immunosorbent assay

中图分类号:

R383.33

宋宏宇, 梁雨晴, 华瑞其, 史媛, 阳爱国, 郭莉, 袁东波, 谢跃, 杨光友. 细粒棘球绦虫磷酸甘油酸变位酶的表达、定位和诊断价值的初步评价[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(2): 194-201.

Hong-yu SONG, Yu-qing LIANG, Rui-qi HUA, Yuan SHI, Ai-guo YANG, Li GUO, Dong-bo YUAN, Yue XIE, Guang-you YANG. Expression, localization and preliminary evaluation of diagnostic value of Echinococcus granulosus phosphoglycerate mutase[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(2): 194-201.

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