应用蛋白印迹-免疫沉淀-质谱法筛选卵巢癌肿瘤抗原

Screening Tumor Antigens of Ovarian Carcinoma by Western blot, Immunoprecipitation, and Mass Spectrometry

背景与目的肿瘤抗原的筛选对卵巢癌的早期诊断有重要意义。目前,噬菌体抗体库技术(phageantibodylibrary)、核糖体展示技术(ribosomedisplay)和血清学筛选重组cDNA表达文库(serologicalanalysisofrecombinantcDNAexpressionlibraries,SEREX)技术等作为常用的肿瘤抗原筛选方法已得到广泛应用。本研究采用蛋白印迹鄄免疫沉淀鄄质谱方法筛选和鉴定卵巢癌肿瘤抗原,以期建立一种新的筛选肿瘤抗原的方法及技术路线。方法应用蛋白印迹(Westernblot)法筛选出差异明显的卵巢癌患者血清,用该血清与SKOV3细胞裂解物反应,免疫沉淀富集抗原,与正常血清的免疫沉淀样品比较,获得差异蛋白条带,然后进行质谱鉴定、生物信息学分析。结果采用蛋白印迹筛选到标记为7号的卵巢癌患者血清,与其他血清比较有明显的差异,经免疫沉淀、聚丙烯酰胺凝胶电泳与正常血清免疫沉淀样品比较,发现在接近66.2ku的位置处有一差异蛋白条带,经质谱鉴定、生物信息学分析为热休克蛋白(heatshockprotein,HSP)70及细胞角蛋白(cytokeratin)9。结论采用蛋白印迹鄄免疫沉淀鄄质谱法进行卵巢癌肿瘤抗原的筛选和鉴定是一种行之有效的方法。

BACKGROUND & OBJECTIVE:Screening tumor antigens plays a key role in early diagnosis of ovarian carcinoma. Several technologies, such as phage antibody library, ribosome display, and serological analysis of recombinant cDNA expression libraries (SEREX), have been applied widely. This study was to screen and identify tumor antigens of ovarian carcinoma by Western blot, immunoprecipitation, and mass spectrometry, and to establish a new methodological entity. METHODS: Western blot was used to find out the obviously different serum in ovarian carcinoma patients; SKOV3 cells lysate was added in the serum; immunoprecipitation was used to enrich antigens; then, the different protein band was gained when compared with normal serum immunoprecipitates, identified by mass spectrometry, and analyzed with bioinformatics. RESULTS: The serum of patient No. 7 was the obviously different one screened with Western blot. Immunoprecipitation and SDS-PAGE showed a different protein band in the serum appeared in the position of about 66.2 ku when compared with normal serum immunoprecipitates. Through mass spectrometry and bioinformatics analysis, heat shock protein 70 (HSP70) and cytokeratin 9 were identified in the different protein band. CONCLUSION: Western blot, immunoprecipitation, and mass spectrometry is an effectively methodological entity for screening and identifying tumor antigens.