多房棘球绦虫EM13蛋白生物信息学分析及原核表达载体的构建

Bioinformatics analysis of the Echinococcus multiloculariss antigen EM13 and construction of a prokaryotic expression vector

目的 构建多房棘球绦虫(Echinococcus multilocularis)Em13原核表达载体并进行生物信息学分析。方法 在NCBI数据库中检索并下载EM13蛋白的氨基酸序列,使用生物信息学软件预测EM13蛋白质的基本理化性质及功能。通过PCR方法扩增Em13基因。双酶切目的基因和载体质粒pET28a,经T4连接酶连接,将连接产物转化入大肠埃希菌BL21(DE3)感受态细胞。结果 EM13蛋白由426个氨基酸组成,包括63个酸性氨基酸和56个碱性氨基酸,分子质量为47 535.12,等电点为5.64,为亲水性蛋白,不含有信号肽序列和跨膜结构域,含F-BAR和SH3结构域。磷酸化位点预测显示有29个丝氨酸磷酸化位点、16个苏氨酸磷酸化位点、7个酪氨酸磷酸化位点以及25个翻译后修饰位点。其二级结构α-螺旋所占的比例为58.45%,β-转角占2.58%,无规则卷曲占35.45%。抗原表位预测显示EM13含有5个潜在的优势B细胞表位、24个潜在优势细胞毒性T淋巴细胞表位和14个潜在优势辅助性T细胞表位。序列比对显示与多房棘球绦虫EM13蛋白序列同源性最高的来自细粒棘球绦虫EG13。构建了Em13/pET28a重组质粒,测序后与NCBI中录入的序列进行比对,结果一致。结论 EM13蛋白具有较多的抗原结合位点,且构建了Em13/pET28a原核表达载体,为EM13蛋白作为泡性棘球蚴病的特异性诊断抗原或疫苗候选抗原的鉴定奠定了基础。

Objective The prokaryotic expression vector of Echinococcus multilocularisEml3 was constructed and analyzed by bioinformatics.Methods The amino acid sequence of the EM13 protein was searched and downloaded from the NCBI database,and the basic physicochemical properties and functions of the EM13 protein were predicted using bioinformatics software.The Em13 gene was amplified by PCR.The target gene and vector plasmid pET28a were double-enzymatically cleaved,ligated by T4 ligase,and the ligation product was transformed into E.coli BL21(DE3)competent cells.Results EM13 protein consists of 426 amino acids,including 63 acidic amino acids and 56 basic amino acids,with a molecular weight of 47535.12 and an isoelectric point of 5.64.It is a hydrophilic protein,does not contain signal peptide sequences and transmembrane domains,and contains F-BAR and SH3 domains.Phosphorylation site prediction showed that EM13 protein had 29 serine phosphorylation sites,16 threonine phosphorylation sites,7 tyrosine phosphorylation sites and 25 post-translational modification sites.The proportion ofα-helix in its secondary structure was 58.45%,β-turn was 2.58%,and random curl was 35.45%.Antigenic epitope prediction showed that EM13 contained 5 potentially dominant B-cell epitopes,24 potentially dominant cytotoxic T-lymphocyte epitopes,and 14 potentially dominant helper T-cell epitopes.Sequence alignment showed that the highest homology with the EM13 protein sequence of E.multilocularis was E.granulosus EG13.The Em13/pET28a recombinant plasmid was successfully constructed,sequenced and compared with the sequences recorded in NCBI with consistent results.Conclusion EM13 protein has more antigen-binding sites and the construction of the Eml3/pET28a prokaryotic expression vector,which lays the foundation for the characterization of the EM13 protein as a specific diagnostic antigen of Alveolar echinococcosis or avaccine candidate antigen.