摘要
目的重组表达类鼻疽菌Ⅲ型分泌系统BipD蛋白,制备多克隆抗体并鉴定其免疫学性质。方法利用pET-28a表达系统在Eschericahia coli(E.coli)BL21(DE3)中重组表达BipD蛋白,通过His Trap亲和层析纯化获得rBipD蛋白,免疫BALB/c小鼠获得rBipD多克隆抗体;分别用兔抗类鼻疽菌血清和类鼻疽菌感染阳性患者血清进行Western blot实验检测rBipD的免疫反应性;通过Western blot及免疫荧光染色检测鉴定rBipD的免疫原性;以rBipD建立间接ELISA检测临床类鼻疽患者血清抗体。结果成功构建pET-28a-BipD重组质粒并转化至E.coli BL21(DE3)中诱导表达、纯化获得了相对分子质量约为36×10^(3)的rBipD蛋白,纯度约为95.4%。rBipD蛋白具有良好的免疫原性和免疫反应性。免疫小鼠可产生特异性抗体,制备鼠抗rBipD多克隆抗体,效价达1∶512000。5.0μg/mL rBipD蛋白可与类鼻疽患者血清发生免疫反应,而不与结核患者血清发生免疫反应,差异有统计学意义(P<0.01)。结论成功制备了具有免疫学活性的rBipD蛋白及其多克隆抗体,为其用于临床免疫学诊断和类鼻疽菌感染免疫机制研究提供了良好的工具。
Objective To express recombinant Burkholderia pseudomallei(B.pseudomallei)typeⅢsecretion system BipD protein,prepare its polyclonal antibodies and verify their immunological traits.Methods The recombinant pET-28a-BipD plasmid was generated,and the pET-28a-BipD-carried E.coli BL21(DE3)bacteria were induced with isopropyl-β-d-thiogalactoside(IPTG)to express recombinant BipD(rBipD)protein.The rBipD was obtained by affinity chromatography using His Trap column,then mixed with Fredrick’s adjuvant to immunize BALB/c mice by intraperitoneal injection in order to obtain anti-rBipD polyclonal antibodies.The immunoreactivity of rBipD was detected by Western blot assay using rabbit anti-melioidosis serum and the serum from melioidosis patients.The immunogenicity of rBipD was evaluated using Western blotting and immunofluorescence staining.Finally,rBipD was used to establish an indirect ELISA to detect serum antibodies of clinical melioidosis patients.Results The recombinant plasmid pET-28a-BipD was successfully constructed and transformed into E.coli BL21(DE3)to induce rBipD expression with IPTG treatment.The obtained rBipD had a relative molecular weight of 36×10^(3) and a purity of 95.4%,and had good immunogenicity and immunoreactivity.It could induce the production of specific antibodies after immunizing mice,and mouse polyclonal antibodies against rBipD were prepared with the titer of 1∶512000.rBipD of 5.0μg/mL produced specific immune response with the serum of melioidosis patients,but had no specific reaction with the serum of tuberculosis patients,with statistical difference(P<0.01).Conclusionr BipD with immunological activity is successfully prepared and purified,and its polyclonal antibodies are also developed,which provide a good tool for clinical immunological diagnosis and study of immune mechanism of B.pseudomallei infection.
作者
南栋琪
文远
陈建高
饶承龙
吴潘
张子元
王施韦
闫晶敏
李倩
毛旭虎
NAN Dongqi;WEN Yuan;CHEN Jiangao;RAO Chenglong;WU Pan;ZHANG Ziyuan;WANG Shiwei;YAN Jingmin;LI Qian;MAO Xuhu(Department of Clinical Microbiology and Immunology,Faculty of Pharmacy and Medical Laboratory,Army Medical University(Third Military Medical University),Chongqing,400038,China;State Key Laboratory of Trauma and Chemical Poisoning,Army Medical University(Third Military Medical University),Chongqing,400038,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2024年第15期1713-1720,共8页
Journal of Army Medical University
基金
国家自然科学基金面上项目(32270190)。
关键词
类鼻疽伯克霍尔德菌
BipD蛋白
重组表达
抗体制备
Burkholderia pseudomallei
BipD protein
recombinant expression
polyclonal antibodies preparation
