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目的：从转染HER2／neu基因的3T3／neu细胞中纯化p185蛋白，研究其免疫原性和作为肿瘤疫苗的可能性。方法：采用溴化氰活化的Sepharose4B为基质，通过交联520C9单克隆抗体（杂交瘤腹水中沉淀γ-球蛋白），用亲和层析的技术纯化p185蛋白。经梯度盐溶液解离与抗体结合的p185蛋白，收集到蛋白洗脱峰，用ELISA检测p185的免疫反应性，并经SDSPAGE和WesternBlot进一步鉴定。结果：经过亲和层析所收集的蛋白吸收峰与p185活性峰重合，SDS-PAGE和WesternBlot进一步证实纯化蛋白的分子量约为185kD，是HER2/neu编码的肿瘤抗原。从肿瘤细胞中纯化p185肿瘤抗原的收获率约为1．129×10-5）mg／mg蛋白浓度的细胞裂解上清。结论：从转基因细胞中成功获取p185肿瘤抗原，将用于后续研究。
OBJECTIVE: To purify p185 protein from 3T3/neu cells transfected with the HER2/neu gene and study its immunogenicity and its potential as a tumor vaccine. Methods: The p185 protein was purified by affinity chromatography using cross-linked 520C9 monoclonal antibody (hybrid ascites-precipitated gamma-globulin) using cyanogen bromide activated Sepharose 4B as a matrix. The p185 protein bound to the antibody was dissociated by gradient salt solution, and the elution peak of the protein was collected. The p185 immunoreactivity was detected by ELISA and further identified by SDSPAGE and Western Blot. RESULTS: The protein absorption peaks collected by affinity chromatography coincided with the p185 activity peak. SDS-PAGE and Western Blot further confirmed that the molecular weight of the purified protein was approximately 185 kD, which is HER2/neu-encoded tumor antigen. The p185 tumor antigen was purified from tumor cells at a yield of about 1.129 x 10-5 mg/mg protein concentration of the cell lysate supernatant. Conclusion: Successful acquisition of p185 tumor antigen from transgenic cells will be used for subsequent studies.