梅毒螺旋体TrkA蛋白的表达、纯化及多克隆抗体制备  

Expression and purification of Treponema pallidum TrkA and preparation of polyclonal antibody

作　　者：曹二龙 唐愈菲 宁韶辉 CAO Er-long;TANG Yu-fei;NING Shao-hui(Department of Pathogenic Biology of Laboratory Medicine,Shaoyang University,Shaoyang 422600,Hunan,China;Laboratory Department of the Second Affiliated Hospital of Shaoyang University)

机构地区：[1]邵阳学院医学检验学院病原生物学教研室,湖南邵阳422600 [2]邵阳学院附属第二医院检验科

出　　处：《中国病原生物学杂志》2022年第6期660-664,共5页Journal of Pathogen Biology

基　　金：湖南省自然科学基金项目(No.2020JJ5522);湖南教育厅科研项目(No.18C0810)。

摘　　要：目的 分析梅毒螺旋体钾离子转运蛋白TrkA的生物学特性,表达、纯化并制备TrkA蛋白的抗血清。方法 从NCBI中查到梅毒螺旋体钾离子转运蛋白TrkA的氨基酸序列,采用生物信息学方法分析TrkA蛋白的基本理化性质、信号肽、定位、跨膜区、糖基化位点、磷酸化位点、二级结构、三级结构和B细胞表位。按照梅毒螺旋体TrkA的DNA序列设计引物,PCR扩增TrkA基因,双酶切后与质粒pET28a相连,连接产物pET28a-TrkA转转化入大肠埃希菌BL21,培养后使用IPTG诱导重组TrkA蛋白表达,利用Ni^(2+)亲和层析法纯化TrkA蛋白。取TrkA蛋白皮下接种小鼠,免疫3次,制备小鼠血清,ELISA法检测血清TrkA特异性IgG滴度。结果 生物信息学预测TrkA蛋白共有236个氨基酸,相对分子质量26.7ku,为胞内蛋白。该蛋白二级结构中α螺旋占45.34%,β折叠占20.76%,无规卷曲占27.97%,β转角占5.93%。Phyre2网站预测TrkA蛋白三级结构中85.5%氨基酸处于最合理区。TrKA蛋白含有6个连续B细胞表位和4个非连续表位。成功构建重组质粒pET28a-TrkA并获得高纯度的重组TrkA蛋白。该蛋白免疫小鼠能够诱导产生特异性IgG抗体,血清抗体滴度为1∶25 600。结论 生物信息学分析梅毒螺旋体TrkA为亲水性蛋白,含有B细胞歌表位,纯化的重组TrkA蛋白具有良好的抗原性,能诱导小鼠产生高滴度抗体。Objective To analyses the biological information of Treponema pallidum TrkA,construct the recombinant prokaryotic expression plasmid,expresses and purify the recombinant TrkA protein,and tests its immunological activity.Methods The amino acid sequence of T.pallidum TrkA protein was obtained from NCBI website.Bioinformatics methods were used to analyze the basic physical and chemical properties,signal peptide,localization,transmembrane region,glycosylation site,phosphorylation site,secondary structure,tertiary structure and B cell epitope of TrkA protein.PCR primers were designed according the DNA sequence of TrkA which were used to amplify the TrkA gene.The TrkA were double digested then was ligated to the plasmid pET28 a.The recombinant plasmid pET28 a-TrkA was transformed into E.coli BL21,and the recombinant TrkA protein was induced by IPTG which was purified by Ni^(2+) affinity chromatography.Mice were immunized with recombinant TrkA protein and the content of total IgG were determined.Results TrkA protein is an intracellular protein with 236 amino acids with a relative molecular weight of 26.7×10~3.The content of α-helix is as high as 45.34%.The content of random coil was 27.97%,β-sheet was 20.76% and β-turn was 5.93%.The tertiary structure of TrkA protein predicted by phyre2 website and 85.5% amino acids in the most reasonable region.TrkA protein contains 6 continuous B cell epitopes and 4 discontinuous epitopes.The recombinant plasmid pET28 a-TrkA was successfully constructed and the recombinant protein was purified.The specific IgG was induced by TrkA protein.The serum antibody titer in serum was 1∶25 600.Conclusion The purified recombinant T.pallidum TrkA protein has good immunogenicity and can induce mice to secrete high titer antibodies.

关 键 词：梅毒螺旋体 钾离子转运蛋白 生物信息学分析 免疫原性 

分 类 号：R377[医药卫生—病原生物学]