细粒棘球绦虫胰岛素样生长因子1受体蛋白的生物信息学分析及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.11.034

摘要/Abstract

摘要： 【目的】对细粒棘球绦虫的胰岛素样生长因子1受体(insulin-like growth factor 1 receptor of Echinococcus granulosus sensu stricto，EgIGF-1R)蛋白进行生物信息学分析，制备EgIGF-1R的多克隆抗体并定位其在幼虫阶段的表达。【方法】使用Mega 11.0软件对EgIGF-1R(GenBank登录号:XP_024354248.1)及其同源基因的氨基酸序列进行ClustalW多重比对，并构建系统进化树。通过生物信息学方法分析EgIGF-1R蛋白的理化特征、跨膜区域、蛋白结构等生物学信息。选择EgIGF-1R的优势肽段免疫新西兰大白兔制备其多克隆抗体，并利用ELISA方法检测抗体效价。通过免疫组织化学、免疫荧光试验定位检测EgIGF-1R在细粒棘球绦虫原头蚴和囊泡阶段的表达情况。【结果】氨基酸序列比对结果显示，EgIGF-1R与日本血吸虫、智人IGF-1R同源基因氨基酸序列相似性分别为32.02%和31.57%。进化树分析结果显示，EgIGF-1R与多房棘球绦虫、布氏姜片虫及日本血吸虫的亲缘关系较近；与智人、小鼠等物种的亲缘关系较远。生物信息学分析结果显示，EgIGF-1R蛋白含有1 680个氨基酸；EgIGF-1R蛋白分子质量为1.839×10⁵ u，等电点为8.59；具有2个跨膜区域，分别位于第1 034－1 056和1 164－1 186位氨基酸，是一种跨膜蛋白；其含有18个酪氨酸磷酸化位点、123个丝氨酸磷酸化位点和60个苏氨酸磷酸化位点。EgIGF-1R蛋白二级结构中α-螺旋、β-折叠、无规则卷曲占比分别为21.19%、16.61%和62.20%；3D结构中N-端和C-端相距较远。EgIGF-1R与人源胰岛素样生长因子1及人源胰岛素的对接分数分别为－1 739.46和－1 099.10 kJ/mol。ELISA结果显示，EgIGF-1R多克隆抗体效价为1∶64 000。免疫组织化学和免疫荧光试验结果显示，EgIGF-1R在原头蚴的顶突、吸盘和被膜表达，在囊泡的生发层表达。【结论】EgIGF-1R蛋白具有结合人源胰岛素生长因子1等细胞因子的结构域，表达于细粒棘球绦虫的原头蚴和囊泡等幼虫阶段，本研究制备的EgIGF-1R多克隆抗体为研究EgIGF-1R在细粒棘球绦虫致病机制中的作用奠定了基础。

关键词: 细粒棘球绦虫; 胰岛素样生长因子1受体; 生物信息学分析; 多克隆抗体

Abstract: 【Objective】 This study aimed to perform bioinformatics analysis on the insulin-like growth factor 1 receptor of Echinococcus granulosus sensu stricto (EgIGF-1R) protein,prepare polyclonal antibodies against EgIGF-1R,and locate its expression in the larval stage. 【Method】 ClustalW multiple alignment was performed on the amino acid sequences of EgIGF-1R (GenBank accession No.:XP_024354248.1) and its homologous genes using Mega 11.0 software,and the phylogenetic tree was constructed.The physicochemical characteristics,transmembrane regions,protein structure and other biological information of EgIGF-1R protein were analyzed by bioinformatics methods.The dominant peptide of EgIGF-1R was selected to immunize New Zealand White rabbits to prepare its polyclonal antibody,and the antibody titer was detected by ELISA method.The expression of EgIGF-1R in the protoscolex and vesicle stages of Echinococcus granulosus sensu stricto was precisely detected by immunohistochemistry and immunofluorescence assays. 【Result】 The amino acid sequence alignment results showed that EgIGF-1R shared 32.02% and 31.57% similarity with amino acid sequence of homologous genes of Schistosoma japonicum and Homo sapiens IGF-1R,respectively.The phylogenetic tree analysis results showed that EgIGF-1R was closely related to Echinococcus multilocularis, Fasciolopsis buski,and Schistosoma japonicum.The genetic relationship with species such as Homo sapiens and Mus musculus was relatively distant.Bioinformatics analysis results showed that EgIGF-1R protein contained 1 680 amino acids.The molecular weight of EgIGF-1R protein was 1.839×10⁵ u,with an isoelectric point of 8.59.It had two transmembrane regions,located at amino acids 1 034-1 056 and 1 164-1 186,and was a transmembrane protein.It contained 18 tyrosine phosphorylation sites,123 serine phosphorylation sites,and 60 threonine phosphorylation sites.The proportions of alpha helix,beta sheet,and random coil in the secondary structure of EgIGF-1R protein were 21.19%,16.61%,and 62.20%,respectively.The N-terminus and C-terminus were far apart in 3D structure of EgIGF-1R protein.The docking fractions of EgIGF-1R with human insulin-like growth factor 1 and human insulin were －1 739.46 and －1 099.10 kJ/mol,respectively.The ELISA results showed that the titer of EgIGF-1R polyclonal antibody was 1∶64 000.The results of immunohistochemistry and immunofluorescence assays showed that EgIGF-1R was expressed in the rostrum,sucker and tegument of protoscolices and in the germinal layer of vesicles. 【Conclusion】 EgIGF-1R protein had structural domain that binded to human cytokines like IGF-1,and was expressed in the larval stages of Echinococcus granulosus sensu stricto,including the protoscolices and vesicles.The EgIGF-1R antibody prepared in this study provided a foundation for investigating the role of EgIGF-1R in the pathogenic mechanism of Echinococcus granulosus sensu stricto.

Key words: Echinococcus granulosus sensu stricto; insulin-like growth factor 1 receptor; bioinformatics analysis; polyclonal antibody

中图分类号:

夏衣旦木·吐尼牙孜, 廖霞, 李婧, 贾雨桐, 赵金龙, 杜韫, 方梓屹, 林仁勇, 吕国栋. 细粒棘球绦虫胰岛素样生长因子1受体蛋白的生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2025, 52(11): 5393-5402.

XIAYIDANMU Tuniyazi, LIAO Xia, LI Jing, JIA Yutong, ZHAO Jinlong, DU Yun, FANG Ziyi, LIN Renyong, LYU Guodong. Bioinformatics Analysis and Polyclonal Antibody Preparation of Insulin-like Growth Factor 1 Receptor of Echinococcus granulosus sensu stricto[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5393-5402.

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