摘要目的 制备高亲和性的羊布鲁杆菌M5-90 BP26抗原单克隆抗体,并对抗体进行鉴定.方法 将质粒pET-28a-BP26转化感受态大肠埃希菌(E.coli)BL21 (DE3),构建原核表达质粒pET-28a-BP26,用镍离子金属螯合亲和层析法(Ni-NTA)纯化、重组BP26蛋白(rBP26).将纯化后的rBP26蛋白与弗氏完全佐剂等体积混合成乳化抗原,接每只100 μg/0.1 ml在BALB/c小鼠皮下多点注射进行初次免疫；2周后,按每只50μg/0.1ml 在小鼠皮下多点注射进行再次免疫.再次免疫后2周,小鼠尾静脉取血测效价,检测免疫效果；在细胞融合前3天,将乳化抗原按每只小鼠50μg腹腔注射加强免疫.将小鼠骨髓瘤SP2/0细胞与脾细胞按1∶5比例在聚乙二醇( PEG) 1450作用下进行细胞融合,置于37℃、5％CO2培养箱中培养；10 d后取细胞培养上清液,用间接酶联免疫吸附法(ELISA)测定,筛选分泌抗rBP26抗体的杂交瘤细胞；再经过反复3次克隆,筛选出单克隆全阳性的杂交瘤细胞建株,用杂交瘤细胞株制备BP26抗原单克隆抗体,并以初始杂交瘤细胞克隆导命名.利用羊布鲁杆菌M5 -90疫苗株制备膜蛋白提取物(NMP),采用免疫印迹法(Western)及斑点间接酶联免疫吸附法(Dot-ELISA)对筛选的单克隆抗体进行免疫学特性鉴定,用单克隆抗体亚型试剂盒进行抗体分型和Kappa (κ)和Lambda(λ)轻链鉴定.结果 成功地获得2株能稳定分泌抗rBP26抗原的高亲和性杂交瘤3C3和5A5单克隆抗体,并能与羊布鲁杆菌M5-90疫苗株上的NMP结合,构建成M5-90 BP26抗原单克隆抗体,抗体亚型分别为IgG1和IgG2b,轻链均为κ链.结论 鉴定结果表明,成功制备2株羊布鲁杆菌BP26抗原的高亲和性单克隆抗体,抗体具有识别M5-90疫苗株的膜蛋白构象表位的能力,优势表位的识别可为羊布鲁杆菌M5-90疫苗株的改造提供实验依据.

abstractsObjective To prepare high specific monoclonal antibodies(mAbs) against BP26 of Brucella(B.)melitensis.Methods A recombinant plasmid pET-28a-BP26 was constructed and transformed into competent Escherichia coli BL21 (DE3),and then the bacteria were induced by 1 mmol/L isopropylthio-β-D-galactoside (IPTG).After induction,the recombinant BP26 protein (rBP26) was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PGAE) and nickel ion affinity chromatography(Ni-NTA).Mice were inoculated with rBP26 antigens for three times at 2-week intervals.The first subcutaneous injection contained 100 μg rBP26 with 0.1 ml complete Freund adjuvant.The second subcutaneous injection was 50 μg rBP26 with 0.1 ml incomplete Freund adjuvant.The antibody titers to rBP26 were determined 2 weeks after each reimmunization.Three days before cell fusion,the mice with the highest titer were intraperitoneally injected with 50 μg rBP26 in 0.1 ml PBS.Pre- and post-immunization sera were collected and used as negative or positive controls for screening mAbs.Mice with the highest titer were sacrificed and spleen cells were isolated.The spleen cells of rBP26 immunized mice were fused with SP2/0 myeloma cells in a ratio of 5 ∶ 1 by polyethylene glycol(PEG) 1450.Antibody-producing hybridomas were primarily screened by an indirect enzyme-linked immunosorbnent assay(ELISA) with rBP26.Reactive hybridomas were subcloned for 3 times,then the strains of hybridoma cells secreting antibodies against BP26 were obtained.Supernatant of cloned hybridoma cultures was collected for mAb analyses.These mAbs were named by the hybridoma clone number and tested their reactivity to membrane proteins extracted(NMP) from B.melitensis vaccine strain(M5-90) by Western blotting and Dot-ELISA.mAbs isotyping and kappa(κ) or lambda(λ) light chain was identified by Mouse Monoclonal Antibody Isotyping Kit.Results A total of two mAbs reactive to rBP26 of B.melitensis were selected from antibody screening hybridomas by indirect-ELISA.The two mAbs were named 3C3 and 5A5,and identified as IgG1 (κ) and IgG2(κ),respectively.They could react with NMP from M5-90.Conclusions Results of identification show that two mAbs against rBP26 can be produced.The two mAbs can recognize natural BP26 protein,giving the experimental materials for further research on identification of its epitopes.