细粒棘球绦虫半胱氨酸蛋白酶抑制剂基因的克隆、表达及诊断价值的初步评价

doi:10.11843/j.issn.0366-6964.2018.03.014

摘要/Abstract

摘要：

探究细粒棘球绦虫半胱氨酸蛋白酶抑制剂的基本特性并初步评价其诊断价值，旨在为细粒棘球绦虫的防控提供依据。本研究原核表达出Eg-cystatin重组蛋白并对其进行生物信息学、免疫印迹分析，用荧光免疫定位的方法检测该蛋白质的分布情况，并以绵羊细粒棘球蚴阳性血清评价Eg-cystatin重组蛋白的诊断价值。结果如下：Eg-cystatin生物信息学分析结果显示，该蛋白质含有一个N端信号肽以及cystatin结构域，是典型的2型半胱氨酸蛋白酶抑制剂，进化树分析显示Eg-cystatin属于绦虫cystatin Ⅱ型，并且与其他绦虫的cystatin相似性为72.20%~80.66%，免疫荧光定位发现该蛋白质主要分布在原头蚴的表皮和顶突钩，生发层，成虫虫体内部和虫卵。基于Eg-cystatin建立的间接ELISA方法的特异性为79.1%，敏感性为83.3%，与剖检符合率为81.25%。Eg-cystatin在成虫和幼虫时期均有表达，提示该基因与虫体生命活动息息相关，但Eg-cystatin蛋白不适合作为绵羊细粒棘球蚴病的诊断抗原候选。

Abstract:

This experiment was conducted to study the characterization and diagnostic value of cystatin of Echinococcus granulosus and provide basis for prevention and control of Echinococcus granulosus. We cloned and expressed Eg-cystatin, and presented a bioinformatic characterization, Western blotting. Immunofluorescence localization was performed to determine the distribution of cystatin from E. granulosus, and explore its potential for diagnosis of cystic echinococcosis (CE) in sheep based on indirect ELISA. The results showed that Eg-cystatin is belong to a typical type Ⅱ cystatin with an N-terminal signal peptide and the cystatin-like domain (G, Q-X-V-X-G, P-W). Phylogenetic analysis indicated that Eg-cystatin belongs to the cestode cystatin clade, and shares 72.20%-80.66% identity with cystatins from other cestodes. Native Eg-cystatin was located on the tegument and hooks of protoscoleces (PSCs), the whole germinal layer, and the inner body and eggs of adult worms. Indirect ELISA exhibited low specificity (79.1%) and sensitivity (83.3%), and the diagnostic accordance rate was 81.25% compared with the results of necropsy. Eg-cystatin was widely distributed in the larva and adult worm of E. granulosus, suggesting that it is closely related to the life activity of this parasite. However, rEg-cystatin is not a suitable antigen for the detection of sheep CE.

中图分类号:

S852.734

吴茂迪, 宋星桔, 闫敏, 阳爱国, 郭莉, 王凝, 谢跃, 古小彬, 杨光友. 细粒棘球绦虫半胱氨酸蛋白酶抑制剂基因的克隆、表达及诊断价值的初步评价[J]. 畜牧兽医学报, 2018, 49(3): 572-579.

WU Mao-di, SONG Xing-ju, YAN Min, YANG Ai-guo, GUO Li, WANG Ning, XIE Yue, GU Xiao-bin, YANG Guang-you. Cloning, Expression and Preliminary Evaluation Diagnostic Value of Echinococcus granulosus Cystatin Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(3): 572-579.

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