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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 establish a method for extracting and purifying heat shock protein (HSP) gp96 peptide complex from human renal cell carcinoma and to detect its in vitro immune response. Methods HSPgp96 peptide complex was extracted and purified from tumor tissue of human renal cell carcinoma by three-step protein purification method. HSPgp96 peptide complex was identified by SDSPAGE and Western blot. The immunocompetence of HSPgp96 peptide complex and pure polypeptide in vitro was detected by ELISPOT assay. Results The protein extracted by this method was identified by SDSPAGE and Western blot. The protein was confirmed to be HSPgp96 peptide complex. The yield of protein was 40 ~ 60μg purified per gram of wet weight renal carcinoma tissue. Interferon ELISPOT assay showed that HSPgp96 peptide complex had the highest frequency of IFN-γ-producing T cells (287.50 ± 22.34), followed by the polypeptide group (135.25 ± 12.80), the positive control group (102.25 ± 14.76) Negative control group (35.50 ± 11.10), the difference between each group and the negative control group was statistically significant (P <0.05); HSPgp96 peptide complex group and the simple polypeptide group, positive control group, the difference was statistically significant (P <0.05 ). There was no significant difference between pure polypeptide group and positive control group (P> 0.05). Conclusion The three-step protein purification method not only has the advantages of simple operation and high repeatability, but also has the advantages of high yield, high purity, high specificity and high immunogenicity for the extracted HSPgp96 peptide complex.