人转铁蛋白纯化制备及其抗原活性验证

Preparation of human transferrin and verification of the antigenic activity

目的采用非亲和纯化方法从人血清中制备获得高纯度转铁蛋白(TRF),并用此为免疫抗原免疫新西兰大白兔,验证TRF蛋白的免疫原性。方法首先采用两步硫酸铵沉淀法从血清中粗纯TRF蛋白,再用两步阴离子交换层析柱进行纯化,最终得到TRF。结果 TRF蛋白的聚丙烯酰胺凝胶电泳法(SDS-PAGE)纯度与外购纯品相当,且高效液相色谱(HPLC)纯度达到96.8%,纯化回收率为78.6%。对于同一批TRF样品,TRF检测试剂盒(免疫法)测得的活性浓度与紫外分光光度计法测得的蛋白浓度比值(Rc)约为0.95,说明制备的TRF与免疫检测试剂盒中的TRF抗体具有良好的抗原反应性。最后用纯化样品免疫新西兰大白兔,经过4次免疫后的抗血清效价滴度达到1∶128 000,说明制备的TRF具有良好的免疫原性。结论制备的高纯度TRF具有良好的抗原反应性及免疫原性,可用于动物免疫以制备抗TRF抗体,为配制TRF检测试剂(免疫法)提供良好的原料基础。

Objective Non-affinity methods were used to purify transferrin(TRF)with high purity from human serum,and the immunogenicity of TRF was evaluated by immunizing New Zealand rabbits.Methods Firstly,TRF was extracted from serum by precipitation with ammonium sulfate and then purified by two-step anion exchange chromatography.Results SDS-PAGE purity of the prepared TRF was similar with the control pure product,and the HPLC purity reached to 96.8%with a yield of 78.6%.For the same batch of TRF sample,the ratio between the activity concentration determinated using TRF kit(immunoturbidimetry method)and the protein concentration determinated using uv-spectrophotometric method was about 0.95,which indicated that the prepared TRF for antigen could react well with the TRF antibody included in the TRF kit.Finally,New Zealand rabbits were immunized using the purified TRF,and the titer of the rabbit anti-serum could reach 1∶128 000 after four time immunization,which also indicated that the prepared TRF had good immunogenicity.Conclusion The TRF with high purity had good antigen reactivity and immunogenicity to prepare anti-TRF antibody by immunizing rabbits,which could provide qualified materials for the production of TRF kit(immunoturbidimetry method).