人肾癌组织中热休克蛋白gp96-肽复合物提取、纯化及体外的免疫效应

The immune effect in vitro of HSPgp96-peptide complex derived and purified from human renal cell carcinomar tissues

目的建立从人肾癌组织中提取、纯化热休克蛋白(HSP)gp96肽复合物的方法,并检测其体外免疫效应。方法采取三步蛋白纯化法从人肾癌患者肿瘤组织中提取、纯化HSPgp96肽复合物,SDSPAGE、Westernblot鉴定HSPgp96肽复合物。应用酶联免疫斑点(ELISPOT)法检测HSPgp96肽复合物、单纯多肽产生γ-干扰素效应T细胞频数的体外免疫活性。结果应用该方法提取的蛋白质经SDSPAGE和Westernblot特异性鉴定,证实是HSPgp96肽复合物;蛋白得率为每g湿重肾癌组织可纯化HSPgp96肽复合物40～60μg。γ-干扰素ELISPOT检测,HSPgp96肽复合物组产生γ-干扰素效应T细胞频数最高(287.50±22.34),以后依次为单纯多肽组(135.25±12.80)、阳性对照组组(102.25±14.76)、阴性对照组(35.50±11.10),各组与阴性对照组比较差异有统计学意义(P<0.05);HSPgp96肽复合物组与单纯多肽组、阳性对照组比较差异有统计学意义(P<0.05);单纯多肽组与阳性对照组比较差异无统计学意义(P>0.05)。结论三步蛋白纯化法不仅操作简便、重复性高,而且提取的HSPgp96肽复合物具有高获率、高纯度、高特异性及高免疫效应等优点。

Objective To build the method that can be used to derive and purify HSPgp96-peptide complex from human renal cell carcinomar (RCC) tissues,and research the immune effect of HSPgp96-peptide complex in vitro.Methods Three-step purification of HSPgp96 and Western-blot was used to derive,purify and certify HSPgp96-peptide complex from RCC tissues.PMBC were stimulated with HSPgp96-peptide complex,peptide separately.ELISPOT was used to detect the number of IFN-γ+T cells.Results The HSPgp96-peptide complex had a high purity and specificity.The final protein recovery was 40～60 μg/g tumor tissue (wet weight).The number of developing IFN-γ+T stimulated by HSPgp96-peptide complex was maximum (287.50± 22.34),followed by the number of developing IFN-γ+T stimulated by peptide (135.25± 12.80),PHA (102.25± 14.76) and blank control (35.50± 11.10).HSPgp96-peptide complex had the strongest immune effect in vitro.Conclusion Three-step purification of HSPgp96 was simple andhad a hight reproducibility.HSPgp96-peptide complex obtained by this method had the merits of high purity,high specificity and strong immune effect in vitro.