细粒棘球绦虫EgBAL蛋白的生物信息学分析及原核表达

doi:10.16431/j.cnki.1671-7236.2025.02.030

摘要/Abstract

摘要： 【目的】探究细粒棘球绦虫(Echinococcus granulosus)胆盐活化脂肪酶(EgBAL)的生物信息学特征并进行原核表达，检测EgBAL重组蛋白的免疫原性，为囊性棘球蚴病疫苗抗原筛选提供理论依据。【方法】从NCBI数据库获取细粒棘球绦虫EgBAL基因序列(GenBank登录号：HM748917)，设计特异性引物，以细粒棘球绦虫原头蚴cDNA为模板，通过PCR扩增并克隆EgBAL基因。使用Mega 7.0软件，通过邻接法(NJ)构建系统进化树，运用生物信息学软件对EgBAL蛋白理化性质和结构特征进行预测分析。构建含EgBAL基因重组pET-22b质粒，并转化大肠杆菌BL21(DE3)感受态细胞诱导表达EgBAL重组蛋白，通过SDS-PAGE检测蛋白表达情况，并进行纯化。利用Western blotting分析重组蛋白的免疫原性。【结果】细粒棘球绦虫EgBAL基因片段大小为1 020 bp。系统进化树显示，EgBAL蛋白与中华树鼩EgBAL蛋白处于同一分支，进化距离较近，相似性高达99.60%。生物信息学分析显示，EgBAL基因全长1 014 bp，编码337个氨基酸，EgBAL蛋白分子质量为37 089.59 u，理论等电点为4.77，不稳定指数为44.72，为不稳定蛋白，脂肪系数为66.11，亲水性为－0.429，为亲水性蛋白，无跨膜区域及信号肽。EgBAL蛋白具有14个潜在B细胞抗原表位，二级结构包括α-螺旋、延伸链、β-转角和无规则卷曲,分别占27.90%、12.76%、12.76%和56.08%。此外，EgBAL蛋白含有35个磷酸化位点，其中包括12个丝氨酸磷酸化位点、16个苏氨酸磷酸化位点和7个酪氨酸磷酸化位点。本研究成功构建重组pET-22b-EgBAL质粒，诱导表达得到EgBAL重组蛋白大小约38 ku。Western blotting结果显示，该重组蛋白可被细粒棘球绦虫感染的昆明小鼠和犬的阳性血清识别，具有良好的免疫原性。【结论】本研究成功克隆了细粒棘球绦虫EgBAL基因、表达了EgBAL重组蛋白，并初步证实重组EgBAL蛋白具有较好的免疫原性，提示其有作为囊型棘球蚴病疫苗或诊断候选抗原的潜力，为进一步研究EgBAL蛋白的功能提供了科学依据。

关键词: 细粒棘球绦虫; EgBAL基因; 生物信息学分析; 原核表达

Abstract: 【Objective】 The aim of this study was to explore the bioinformatics characteristics of bile salt activated lipase (EgBAL) in Echinococcus granulosus and perform prokaryotic expression to detect the immunogenicity of EgBAL recombinant protein,provide theoretical basis for antigen screening of cystic echinococcosis vaccine. 【Method】 EgBAL gene sequence (GenBank accession No.:HM748917) of Echinococcus granulosus was obtained from NCBI database,specific primers were designed,and EgBAL gene was amplified and cloned by PCR using the cDNA of Ecanococcus granulosus as template.Phylogenetic tree was constructed by NJ method using Mega 7.0 software.The physical and chemical properties and structural characteristics of EgBAL protein were predicted and analyzed by bioinformatics softwares.Recombinant pET-22b plasmid containing EgBAL gene was constructed and transformed into Escherichia coli BL21(DE3) competent cells to induce expression of EgBAL recombinant protein.The protein expression was detected by SDS-PAGE and purified.The immunogenicity of the recombinant protein was analyzed by Western blotting. 【Result】 The fragment size of the EgBAL gene in Echinococcus granulosus was 1 020 bp.The phylogenetic tree showed that the EgBAL protein was in the same branch as the EgBAL protein of the Tupaia belangeri with a close evolutionary distance and a similarity as high as 99.60%.Bioinformatics analysis revealed that the EgBAL gene was 1 014 bp in length,encoding 337 amino acids.The molecular weight of EgBAL protein was 37 089.59 u,with a theoretical isoelectric point of 4.77,an instability index of 44.72,indicating an unstable protein,a fat coefficient of 66.11,a hydrophilicity of －0.429,a hydrophilic protein with no transmembrane region or signal peptide.EgBAL protein had 14 potential B-cell epitopes,and the secondary structure included alpha helix,extended chain,beta turn and random coil,accounting for 27.90%,12.76%,12.76% and 56.08%,respectively.In addition,EgBAL protein contained 35 phosphorylation sites,including 12 serine phosphorylation sites,16 threonine phosphorylation sites and 7 tyrosine phosphorylation sites.In this study,the recombinant pET-22b-EgBAL plasmid was successfully constructed,and the size of EgBAL recombinant protein was about 38 ku.Western blotting results showed that the recombinant protein could be recognized by the positive sera of Kunming mice and dogs infected with Echinococcus granulosus,and had good immunogenicity. 【Conclusion】 This study successfully cloned the EgBAL gene of Echinococcus granulosus,expressed the recombinant EgBAL protein,and preliminarily confirmed the recombinant EgBAL protein had good immunogenicity,suggesting its potential as a vaccine or diagnostic candidate antigen for cystic echinococcosis.These findings provided a foundational scientific basis for further investigation into the functional role of the EgBAL protein.

Key words: Echinococcus granulosus; EgBAL gene; bioinformatics analysis; prokaryotic expression

中图分类号:

S852.73⁺4

赵文卿, 薄新文, 普娜, 张玉霞, 陈旭珂, 张艳艳, 孙艳, 齐萌, 王正荣. 细粒棘球绦虫EgBAL蛋白的生物信息学分析及原核表达[J]. 中国畜牧兽医, 2025, 52(2): 801-810.

ZHAO Wenqing, BO Xinwen, PU Na, ZHANG Yuxia, CHEN Xuke, ZHANG Yanyan, SUN Yan, QI Meng, WANG Zhengrong. Bioinformatics Analysis and Prokaryotic Expression of EgBAL Protein of Echinococcus granulosus[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 801-810.

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