【摘要】 目的用人工合成的钠-钙交换体α1和α2重复序列作为抗原,制备并纯化抗α1和α2重复序列抗体。方法通过主动免疫大鼠,获得抗血清。采用酶联免疫吸附测定法(SAELISA)测定抗体滴度,并将高滴度抗血清(≥1∶640)上样至HiTrapProteinGHP进行亲和层析对抗体进行纯化。纯化效果用非变性聚丙烯酰胺凝胶电泳检测,并采用Bradford蛋白质定量法对纯化后抗体进行定量。结果大鼠经主动免疫后,抗α1抗血清滴度从免疫前的1∶(25.5±1.5)升高到1∶(905.1±2.2)(P<0.01);抗α2抗血清滴度从免疫前的1∶(26.4±1.5)升高到1∶(1076.3±2.0)(P<0.01)。纯化后的抗α1和抗α2重复序列抗体电泳时都仅有一条带出现,分子量160kDa左右,与IgG标准品相一致。用Bradford蛋白质定量法测得抗α1和α2抗体浓度分别为1.0mg/ml和1.2mg/ml。结论本研究获得了高纯度、高滴度的抗α1抗体,为下一步对其进行功能研究奠定了基础。更多还原

【Abstract】 Objective To raise and purify antibodies against the synthesized α-1 repeat and α-2 repeat. Methods The rats were actively immunized with synthesized α-1 and α-2 repeats.Titer of the antibodies was determined by SA-ELISA.To diminish the undesired effects of other proteins than immunoglobulin,especially serum albumin,the positive antisera with high titer (≥1∶640) were applied on the HiTrap protein G HP column which was an affinity chromatography column for purification of IgG from serum.The purity of the antibodies was tested by PAGE in nonreducing condition and the concentrations of the antibodies were determined by the protein quantification method described by Bradford. Results ① Titers of antibodies against α-1 repeat and α-2 repeat in both immunized groups differed largely among different rats and increased from 1∶(25.5±1.5) and (26.4±1.5) before immunization to 1∶(905.1±2.2) and (1 076.3±2.0) (P<0.01) after the second booster immunization,respectively.②From the results of PAGE in nonreducing condition,only one band with the same molecular weight as standard IgG could be seen in the samples of the purified antibodies against α-1 repeat and α-2 repeat.③The concentrations of the antibodies against α-1 repeat and α-2 repeat were about 1.0 mg/ml and 1.2 mg/ml respectively by the Bradford assay. Conclusion Antibodies against the synthesized α-1 repeat and α-2 repeat are successfully raised and purified,providing a basis for further investigation on their function.更多还原