日本血吸虫SjGrpE蛋白的表达纯化及多克隆抗体制备  

Expression, purification and polyclonal antibody preparation of the Schistosoma japonicum SjGrpE protein

作　　者：肖非[1] 胡智 谭潇[1] 黄泽智[1] XIAO Fei;HU Zhi;TAN Xiao;HUANG Ze-Zhi(Department of Pathogenic Biology,Shaoyang University,Shaoyang 422600,China;Shaoyang Wugang Zhanhui Hospital,Hunan Province,China)

机构地区：[1]邵阳学院病原生物学教研室,邵阳422600 [2]湖南省邵阳市武冈市展辉医院

出　　处：《中国血吸虫病防治杂志》2020年第4期355-360,共6页Chinese Journal of Schistosomiasis Control

基　　金：湖南省教育厅课题(19C1641)。

摘　　要：目的分析日本血吸虫核苷酸交换因子(SjGrpE)蛋白生物学特性,表达与纯化重组SjGrpE蛋白并测定其免疫原性。方法采用生物信息学方法预测SjGrpE蛋白的氨基酸组成、分子量、亲/疏水性、跨膜区、信号肽、定位、磷酸化位点、泛素化位点、糖基化位点、二级和三级结构及B细胞表位。以日本血吸虫cDNA为模板,PCR扩增SjGrpE基因,将其双酶切后连接到pET28a载体得到重组质粒pET28a-SjGrpE。将pET28a-SjGrpE转化大肠埃希菌BL21,用IPTG诱导目的蛋白表达,并用镍离子亲和层析法纯化蛋白。将重组SjGrpE蛋白免疫小鼠,分离血清并鉴定获得的抗多克隆抗体。结果SjGrpE蛋白分子量约为24.3 kDa,是一种亲水性蛋白,无跨膜区、无信号肽,定位在线粒体;该蛋白含有18个磷酸化位点和2个泛素化位点,无糖基化位点,含有5个B细胞表位。SjGrpE基因全长为660 bp,成功构建重组质粒pET28a-SjGrpE并纯化获得重组SjGrpE蛋白。重组SjGrpE蛋白能够刺激小鼠分泌高滴度抗体。结论成功制备重组SjGrpE蛋白,该蛋白具有良好免疫原性,为后续研究其作为日本血吸虫病疫苗候选分子的价值奠定了基础。Objective To investigate the biological properties of Schistosoma japonicum SjGrpE protein,and to express and purify the recombinant SjGrpE protein and test its immunogenicity.Methods The amino acid composition,molecular weight,hydrophilicity and hydrophobicity,transmembrane region,signal peptide,localization,phosphorylation site,ubiquitination site,glycosylation site,secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses.The SjGrpE gene was amplified using PCR assay using S.japonicum cDNA as a template,double enzyme-digested and linked to the pET28 a vector to yield the recombinant plasmid pET28 a-SjGrpE.The recombinant plasmid pET28 a-SjGrpE was transformed into Escherichia coli BL21,and then IPTG was employed to induce the expression of the target protein,which was purified by nickel ion affinity chromatography.After mice were immunized with the recombinant SjGrpE protein,mouse sera were collected,and the polyclonal antibody against the SjGrpE protein was characterized.Results SjGrpE protein,which was identified as a hydrophilic protein,was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides,and locate in the mitochondrion.SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites,but had no glycosylation sites.In addition,Sj GrpE protein contained 5 B-cell epitopes.The full length of SjGrpE gene was approximately 660 bp.The recombinant pET28 a-Sj GrpE plasmid was successfully generated,and the recombinant Sj GrpE protein was obtained following the affinity chromatography,which stimulated mice to secrete high-titer antibodies.Conclusion The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity,which provides a basis for evaluating its value as a vaccine candidate for S.japonicum infections.

关 键 词：日本血吸虫 GrpE蛋白 免疫原性 生物信息学分析 

分 类 号：R383.24[医药卫生—医学寄生虫学]