HSP70-肿瘤肽的纯化及其抗肿瘤免疫效应

Purification and antitumor effect of heat shock protein70-associated tumor peptides

目的 :探索热休克蛋白 70 (HSP70 ) 肿瘤肽分离、纯化方法 ,并观察其抗肿瘤免疫保护效应。方法 :采用低渗匀浆、超速离心、ConA Sepharose亲和层析、ADP Agarose亲和层析和DEAE离子交换的亲和层析 ,从热处理的小鼠肝癌 (HCaF)细胞中分离、纯化HSP70 肿瘤肽 ,并通过主动免疫保护试验观察其抗肿瘤免疫效应。结果 :蛋白得率为每g湿重瘤细胞可纯化 5 0～ 10 0 μgHSP70 肿瘤肽 ;经SDS PAGE和Westernblot检测 ,纯化的HSP70 肿瘤肽具有很高的纯度和特异性 ;HSP70 肿瘤肽主动免疫的小鼠可抵抗HCaF细胞的攻击 ,存活率达 75 %。结论 :采用低压亲和层析可获得高纯度的HSP70 肿瘤肽 ,且可诱导明显的抗肿瘤免疫保护效应。

Objective:To establish a new method of purifing heat shock protein70(HSP70) associated tumor peptides from tumor cell pellet and observe its immunoprotective effect against tumor.Methods:HSP70 associated tumor peptides were purified from tumor cell pellet treated in water bath(42℃),by ConA Sepharose column,ADP Agarose column,DEAE ino exchange chromatography.The purified fractions tested by SDS PAGE,immunoblotted with anti HSP70 mAb,and observed its immunoprotective effect of antitumor by active immunoprotective assays.Results:The final protein recovery was 50～100 μg per 1 g tumor cell(wet weight);The final purified HSP70 associated tumor peptides have a high purity and specificity;Mice immunized with HSP70 associated peptide complexes purified from HCaF were protected from HCaF cells challenge,and its survival rate is 75%.Conclusion:HSP70 associated tumor peptides with high purity was obtained using the low affinity chromatography,and purified HSP70 associated tumor peptides can elicit significantly immunoprotective effect against HCaF.