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目的 探索肿瘤热休克蛋白 70抗原肽复合物 (HCA70 )分离、纯化的方法。方法 采用低渗、反复冻融匀浆、ADP -Agarose亲和层析结合DEAE离子交换层析 ,从体外培养的小鼠肺腺癌细胞 (LA - 795)中纯化HAC70 ;通过SDS -PAGE电泳、Westernblot检测、小鼠移植瘤动物实验证实所获得蛋白是否为HAC70。结果 获得蛋白分子量为 70kD左右 ,与抗HSP70特异单抗结合 ,用此蛋白主动免疫的小鼠可抵抗LA795细胞的攻击 ,获得了高纯度的HAC70。结论 采用ADP -Agarose亲和层析结合DEAE离子交换层析可获得高纯度的HAC70。
Objective To explore the isolation and purification of tumor heat shock protein 70 peptide complex (HCA70). Methods HAC70 was purified from mouse lung adenocarcinoma cells (LA - 795) cultured in vitro by hypotonic and repeated freeze - thaw homogenate, ADP - Agarose affinity chromatography and DEAE ion exchange chromatography. SDS - PAGE electrophoresis, Western blot assay, mouse xenograft animal experiments confirmed that the obtained protein is HAC70. As a result, the molecular weight of the protein was about 70 kD, which was bound to anti-HSP70 specific monoclonal antibody. The mice immunized with this protein could resist the attack of LA795 cells and obtain high-purity HAC70. Conclusion High purity HAC70 was obtained by ADP-Agarose affinity chromatography coupled with DEAE ion exchange chromatography.