重组酶Exo的纯化及多克隆抗体制备

目的:纯化Exo重组酶融合蛋白并制备相应抗体。方法:用阴离子交换柱对蛋白进行初步纯化,然后用Ni-NTA介质填充的层析柱分离纯化含His标签的融合蛋白,用谷胱甘肽琼脂糖4B介质填充的层析柱分离纯化GST融合蛋白;二次纯化的蛋白利用硝酸纤维素膜结合法制备抗原蛋白并免疫实验动物。结果:ELISA结果显示血清抗体效价可达到1∶12 800,说明通过Western免疫印迹自制的多克隆抗体能特异地与Exo重组蛋白相互作用。结论:该蛋白纯化方法操作简单,制备的抗原纯度高,多克隆抗体特异性好。

Purification and Polyclonal Antibody Preparation of Recombinase Exo

Objective： To purify recombinase Exo and prepare its polyclonal antibody.Methods： Exo proteins were initially purified with anion-exchange column,then His-tag fusion proteins were purified with chromatographic column filled with Ni-NTA and GST-fusion proteins were purified with chromatographic column filled with glutathione Sepharose 4B amboceptor.Antigens for immuning animals were prepared by NC membrane combined techniques with these two-step purified proteins.Results： ELISA results showed that all titers of prepared antibodies can be attainable as high as 1∶12 800,thus,suggested that polyclonal antibodies prepared by Western blotting like method can specifically combine Exo antigen.Conclusion： The protein purification process for polyclonal antibodies preparation is easily operated compared to other methods.The purity of our prepared antigens is relatively high and the affinity of our polyclonal antibodies is strong.