应用血清蛋白质组分析筛选人胃癌相关抗原

Screening Human Gastric Carcinoma-associated Antigens by Serologic Proteome Analysis

背景与目的:寻找胃癌特异性或相对特异性标志物是胃癌研究亟待解决的课题。本研究利用血清蛋白质组分析方法,建立人胃癌组织蛋白质双向凝胶电泳图谱以及分别与胃癌患者血清和非癌人群血清的免疫印迹图谱,初步筛选人胃癌相关抗原。方法:运用2-DE分离人胃癌组织的总蛋白质;一个样品跑3块凝胶,1块考马斯亮染显色作为平行胶,另2块分别以胃癌患者自身血清和非癌人群血清作一抗进行免疫印迹分析,获得免疫印迹图谱;对图谱进行比较分析,识别差异反应的蛋白质点,根据差异反应蛋白质点位置在平行胶上找出匹配的蛋白质点;对差异反应的蛋白质点应用MALDI-TOF-MS进行质谱分析鉴定,获得相应的肽质指纹图谱,通过数据库搜索鉴定出差异反应的蛋白质。结果:人胃癌组织2-DE分离后分别与胃癌患者自身血清和非癌人群血清进行免疫印迹,获得分辨率较高的反应图谱;图像分析找出差异反应的蛋白质点14个,质谱鉴定获得hnRNPK、hnRNP H1、hnRNP H2、CK18、Fkbp52与Hsp90复合物、DBP、SBP1、eEF-1、ACTB、CKB、PSMC3、DDAH-1、NADH脱氢酶硫-铁蛋白1等13个蛋白质。结论:获得分辨率较高的人胃癌组织蛋白质组与胃癌患者自身血清及非癌人群血清反应的免疫印迹反应图谱,初步筛选了13个人胃癌相关抗原。为进一步筛选可应用于临床早期诊断、治疗、预后评估及疾病检测的人胃癌分子标志物奠定了基础。

BACKGROUND ＆ OBJECTIVE： It is urgent to find the specific or relatively specific biomarkers in the study of gastric carcinoma. This study was to screen human gastric carcinoma-associated antigens by serologic proteome analysis （SERPA）. METHODS： Total protein were isolated from 10 specimens of human gastric carcinoma by 2-dimensional electrophoresis （2-DE）. The immunoblotting imaging films were obtained and the differential reacting protein spots were recognized. Then the differential reacting proteins were obtained in the replica gel by matching analysis, and identified by peptide mass fingerprint （PMF） based on matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： Fourteen differentially expressed proteins （react only to the patient＇s serum） were found. Of the 14 proteins, 13 were identified as human gastric carcinoma-associated antigens, including heterogeneous nuclear ribonucleoprotein K （hnRNP K）, hnRNP H1, hnRNP H2, cytokeratin 18 （CK18）, crystal structure of Fkbp52 C-terminal domain complex with the C-terminal peptide meevd of hot-shock protein 90 （Hsp90）, serum vitamin D-binding protein precursor （DBP）, selenium-binding protein 1 （SBP1）, eukaryotic translation elongation factor 1 delta （eEF-1）, ACTB protein, brain creatine kinase （CKB）, proteasome 26S ATPase subunit 3 （PSMC3）, dimethylarginine dimethylaminohydrolase 1（DDAH-1）, and NADH dehydrogenase Fe-S protein 1. CONCLUSIONS： Thirteen human gastric carcinoma-associated antigens were found by SERPA. The results provide foundation for screening molecular biomarkers for the diagnosis, therapy, prognostic evaluation, and prevention of human gastric carcinoma.