非液相等电聚焦蛋白质分离技术获取人热休克蛋白-多肽复合物

Heat shock protein peptide complexes processed with the technique of isoelectric focusing

目的:利用非液相等电聚焦蛋白质分离技术(FS-IEF)从肝细胞癌(HCC)患者的癌组织的裂解液内提纯热休克蛋白多肽复合物(HSP-PCs)。制备热休克蛋白(HSP)肿瘤疫苗。方法:制备HCC细胞蛋白,经等电聚焦蛋白质分离获得目的蛋白,SDS-PAGE电泳和Western-Blot鉴定为热休克蛋白多肽复合物。利用紫外吸收法测定其浓度。结果:利用FS-IEF从HCC肿瘤细胞裂解液中成功分离提纯出HSP-PCs,分离的富含分子伴侣的细胞裂解物(CRCL)经鉴定为CRT、HSgpP96、HSP90和HSP70。这些分子伴侣蛋白沿pH梯度集中分布于4～7号收集管中,pH跨度为4.4～5.2。1g肝癌组织经液相等电聚焦分离后可产生1000μg的热休克蛋白疫苗。结论:通过非液相等电聚焦蛋白质分离技术可获得纯化的所需量的HSP-PCs。

Objective：By free so1ution-isoelectric focusing（FS-IEF） to obtain purified heat shock protein peptide complexes（HSP-PCs） from clarified tumor homogenate of patient with hepatocellular carcinoma and to utilize these to prepare the tumor vaccine.Methods：Preparation of the mixed was separated by isoelectric focusing aim protein.The molecular weight and identity of the glycoprotein were confirmed by SDS-PAGE gel electrophoresis and Western Blot respectively.The concentration was measured by ultraviolet absorption spectrometry.Results：Heat shock protein peptide complexes were purified successfully from clarified tumor homogenate of hepatocellular carcinoma by free so1ution-isoelectric focusing.Western Blot showed that the isolated CRCL contained four kinds of chaperone proteins：HSP96、HSP90、HSP70 and CRT.The pH values ranged from 4.4 to 5.2（lane 4~7）.These chaperone proteins clustering along the pH gradient were very typical,1 g HCC could generate 1 000 μg HSP-vaccine by FS-IEF.Conclusion：Free solution-isoelectric focusing（FS-IEF） may be utilized to obtain urified heat shock protein peptide complexes（HSP-PCs） for the requirements of clinical research.