Zhang Fengbo, Ma Xiumin, Zhu Yuejie, Wang Hongying, Liu Xianfei, Zhu Min, Ma Haimei, Wen Hao, Fan Haining, Ding Jianbing
State Key Laboratory Incubation Base of Xinjiang Major Diseases Research, The First Affiliated Hospital of Xinjiang Medical University Urumqi 830054, Xinjiang, P.R. China.
Department of Immunology, Xinjiang Medical University Urumqi 830011, Xinjiang, P.R. China.
Int J Clin Exp Pathol. 2014 Aug 15;7(9):5655-64. eCollection 2014.
This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus.
Genomic DNA and total RNAs were extracted from four different developmental stages of protoscolex, germinal layer, adult and egg of Echinococcus granulosus, respectively. Fluorescent quantitative PCR was used for analyzing the expression of egG1Y162 gene. Prokaryotic expression plasmid of pET41a-EgG1Y162 was constructed to express recombinant His-EgG1Y162 antigen. Western blot analysis was performed to detect antigenicity of EgG1Y162 antigen. Gene sequence, amino acid alignment and phylogenetic tree of EgG1Y162 were analyzed by BLAST, online Spidey and MEGA4 software, respectively.
EgG1Y162 gene was expressed in four developmental stages of Echinococcus granulosus. And, egG1Y162 gene expression was the highest in the adult stage, with the relative value of 19.526, significantly higher than other three stages. Additionally, Western blot analysis revealed that EgG1Y162 recombinant protein had good reaction with serum samples from Echinococcus granulosus infected human and dog. Moreover, EgG1Y162 antigen was phylogenetically closest to EmY162 antigen, with the similarity over 90%.
Our study identified EgG1Y162 antigen in Echinococcus granulosus for the first time. EgG1Y162 antigen had a high similarity with EmY162 antigen, with the genetic differences mainly existing in the intron region. And, EgG1Y162 recombinant protein showed good antigenicity.
本研究旨在克隆、鉴定和分析细粒棘球绦虫egG1Y162基因的特征。
分别从细粒棘球绦虫原头蚴、生发层、成虫和虫卵四个不同发育阶段提取基因组DNA和总RNA。采用荧光定量PCR分析egG1Y162基因的表达。构建pET41a-EgG1Y162原核表达质粒以表达重组His-EgG1Y162抗原。进行蛋白质免疫印迹分析以检测EgG1Y162抗原的抗原性。分别使用BLAST、在线Spidey和MEGA4软件分析EgG1Y162的基因序列、氨基酸比对和系统发育树。
egG1Y162基因在细粒棘球绦虫的四个发育阶段均有表达。并且，egG1Y162基因在成虫阶段表达最高，相对值为19.526，显著高于其他三个阶段。此外，蛋白质免疫印迹分析显示EgG1Y162重组蛋白与细粒棘球绦虫感染的人和犬的血清样本有良好反应。而且，EgG1Y162抗原在系统发育上与EmY162抗原最接近，相似度超过90%。
本研究首次在细粒棘球绦虫中鉴定出EgG1Y162抗原。EgG1Y162抗原与EmY162抗原具有高度相似性，遗传差异主要存在于内含子区域。并且，EgG1Y162重组蛋白显示出良好的抗原性。
