梅毒螺旋体Hsp40蛋白的表达、纯化及免疫学活性分析  

Expression,purification,and analysis of the immunological activity of the Hsp40 protein of Treponema pallidum

作　　者：伍仙 何树光[1] WU Xian;HE Shu-guang(The First Hospital Affiliated ivith the Hunan College of Traditional Chinese Medicine(Hunan Provincial Hospital of Traditional Chinese Medicine),Zhuzhou,Hunan,China 412000)

机构地区：[1]湖南中医药高等专科学校附属第一医院(湖南省直中医医院),湖南株洲412000

出　　处：《中国病原生物学杂志》2021年第6期676-680,共5页Journal of Pathogen Biology

基　　金：湖南省自然科学基金项目(No.2018JJ6116);湖南中医药科研计划重点项目(No.2021050)。

摘　　要：目的表达梅毒螺旋体(Tp)Hsp40蛋白并分析其免疫学活性。方法利用软件预测TpHsp40蛋白的B细胞表位,构建重组质粒pET28a-TpHsp40,转化大肠埃希菌BL21 star(DE3),培养后利用IPTG诱导重组TpHsp40蛋白的表达。收集菌体,破碎、离心后用Ni2+层析柱进行纯化。收集80、120和200 mmol/L咪唑洗脱蛋白,经脱盐、去内毒素后取纯度良好的一组TpHsp40蛋白免疫小鼠,制备抗血清,采用ELISA检测抗体效价并分型。取小鼠脾脏,制成脾细胞悬液,采用CCK-8法检测淋巴细胞增殖情况,ELISA法测定淋巴细胞分泌的细胞因子水平。结果生物信息学分析TpHsp40含有多个B细胞表位。构建的重组质粒转染DE3后经IPTG诱导表达分子质量单位约为42.4 ku的重组TpHsp40蛋白,经亲和层析纯化后免疫小鼠,能够刺激产生高滴度(105log10)的血清抗体。且能够诱导小鼠淋巴细胞增殖(SI值为2.41)和IFN-γ、TNF-α高表达,但不能够诱导Th2型细胞因子表达。结论重组表达的梅毒螺旋体TpHsp40蛋白能够诱导小鼠产生Th1型应答,有望成为Tp疫苗的新靶点。Objective To express the Hsp40 protein of Treponema pallidum and to analyze its immunological activity.Methods B-cell epitopes of the Hsp40 protein of T.pallidum were predicted using software,and the recombinant plasmid pET28 a-TPHsp40 was constructed and transformed into Escherichia coli BL21 STAR(DE3).After culture,expression of the recombinant TPHsp40 protein was induced with IPTG.The bacteria were collected,sonicated and centrifuged,and then the protein was purified using Ni2+chromatography.The target protein was eluted with 80,120,and 200 mM imidazole and desalted,and endotoxins were removed.A group of mice was immunized with the T.pallidum Hsp40 protein of the highest purity to obtain an antiserum.The antibody titer and type were analyzed using ELISA.The proliferation of lymphocytes was detected using a CCK-8 assay,and the cytokine secretions of lymphocytes were analyzed using ELISA.Results T.pallidum Hsp40 contained multiple B-cell epitopes.A recombinant plasmid was successfully constructed,and a recombinant TPHsp40 protein was obtained using affinity chromatography.The T.pallidum Hsp40 protein stimulated the secretion of high titers of antibodies in mice.Moreover,it induced the proliferation of murine lymphocytes,and treated mice had a significantly higher level of Th1 cytokines than that in the control.However,the expression of Th2 cytokines was not induced.Conclusion The recombinant TPHsp40 protein induced a Th1 response in mice,and it could be a new target for vaccines against T.pallidum.

关 键 词：梅毒螺旋体 Hsp40蛋白 表达纯化 免疫学活性 

分 类 号：R377[医药卫生—病原生物学]