张段玲(1988-),女,硕士,研究方向:动物传染病。
国家林业局疫源疫病监测项目《我国境内不同种属猴免疫球蛋白同源性分析及其单克隆抗体的制备》。
目的制备抗食蟹猴、恒河猴等非人灵长类实验动物免疫球蛋白二级抗体,开展对其传染病血清学快速诊断方法的建立。方法采用饱和硫酸铵盐析、Agarose-Protein G亲和层析技术,从食蟹猴血清中提纯IgG。经SDS-PAGE电泳鉴定,采用常规法免疫C57BL/6小鼠,三次免疫后取脾细胞与Sp2/0-Ag14骨髓瘤细胞通过PEG4000融合制备杂交瘤细胞,利用间接ELISA、Western blot等方法进行筛选、鉴定。结果得到5株阳性杂交瘤,分别命名为2B6、2B7、2D9、3B2、5E4,并且5株杂交瘤分泌的抗体均与恒河猴的IgG或血清发生交叉反应,而与其他物种如东北虎、犬等动物的IgG或血清无交叉反应。结论5株杂交瘤产生的单克隆抗体(McAb)具有较好免疫活性,且能长期、稳定地分泌抗体。此项研究工作为后续研究食蟹猴、恒河猴传染病血清学诊断方法奠定基础。
Objective In order to prepare immunoglobulin G (IgG) secondary antibody of non-human primates such as Macaca fascicularis and Macaca mulatta and establish a quick serological method to diagnose their diseases. Methods Macaca fascicularis IgG was isolated and purified from Macaca fascicularis serum by precipitated use saturated ammonium sulfate and affinity chromatography technology with Agarose-Protein G. After identified by SDS-PAGE electrophoresis murine monoclonal antibody(McAb)were produced by conventional immune method and hybridoma technology.Monoclonal antibody was produced when C57BL/6 mice was immunized three times and its spleen cells were fused with SP2/0-Ag14 myeloma cells. Indirect ELISA and Western blot methods were used for identification. Result5 strains of myeloma cells acquired which were named 2B6, 2B7, 3B2, 5E4, 2D9.The antibodies they secret can all react well with both IgG and their serum of Macaca fascicularis and Macaca mulatta. There are no cross reaction with other species such as east-northern tigers and dogs. Conclusion The 5 strains of monoclonal antibodies can secrete antibody chronically and steadily. The study can be valuable in establishing a quick serological method in diagnosing the infectious disease.
张段玲,张彦龙,白素英.小鼠抗食蟹猴IgG单克隆抗体的制备[J].中国比较医学杂志,2012,(5):68~71.
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