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目的制备稳定、特异的汉坦病毒核蛋白重组抗原和单克隆抗体。方法构建汉坦病毒(HTNV)-76118株核蛋白原核表达载体(pBV)220-S1.3和(pET)28a-S1.3,大肠埃希菌诱导表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western-blot分析显示,在48kD附近有目的蛋白表达,且有较好的抗原活性。用纯化的包涵体抗原免疫Balb/c小鼠,取脾细胞与SP2/0骨髓瘤细胞融合,经镍合氨基三乙酸(Ni-NAT)亲和纯化的核蛋白包被酶标板,进行ELISA筛选阳性克隆株,并建立细胞系,选2株高效分泌抗核蛋白单克隆抗体(McAb)的阳性杂交瘤细胞,常规制备腹水,进行单抗鉴定。结果成功构建了重组核蛋白原核表达载体pBV220-S1.3和pET28a-S1.3,获得了高纯度重组核蛋白(rNP)及其高效价单克隆抗体2E6和4D8。结论重组核蛋白抗原及其单克隆抗体在流行性出血热的监测、诊断和科研中有重要价值。
Objective To prepare stable and specific recombinant hantaan nucleoprotein antigens and monoclonal antibodies. Methods The prokaryotic expression vector (pBV) 220-S1.3 and (pET) 28a-S1.3 of Hantavirus (HTNV) -76118 were constructed and induced by Escherichia coli. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot analysis showed that in the vicinity of 48kD target protein expression, and has good antigen activity. Balb / c mice were immunized with the purified inclusion body antigen. The spleen cells were fused with SP2 / 0 myeloma cells and coated with Ni-NAT affinity-purified nucleoprotein coated ELISA plates for ELISA Positive clones were screened and cell lines were established. Two positive hybridomas secreting monoclonal antibodies against McAb (McAb) were selected and ascites was routinely prepared for monoclonal antibody identification. Results The recombinant prokaryotic expression vectors pBV220-S1.3 and pET28a-S1.3 were successfully constructed, and highly purified recombinant nucleoprotein (rNP) and its high titer monoclonal antibodies 2E6 and 4D8 were obtained. Conclusion Recombinant nucleoprotein antigens and their monoclonal antibodies have important value in the monitoring, diagnosis and research of epidemic hemorrhagic fever.