摘要
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 the tumor antigen p185 oncoprotein. Methods: The p185 oncoprotein was purified by using an affinity chromatography column. In this experiment, an λ-globulin containing the anti-p185 oncoprotein monoclonal antibody was obtained form 520C9 hybridoma ascite with saturated ammonium sulfate (SAS)precipitation. The antibody activity was tested and then coupled with CNBr-activated Sepharose 4B as solid phase medium to pack the affinity chromatography column. The tunor cells were fully disrupted by cell lysis buffer medium and then cell membrane lysate was obtained. The p185 oncoprotein antigen was binded with the anti-p185 antibody when the cell lysate run through the column. The gradient salt solution(0. 5 ~ 1.0 mol/L NaCl) as elute buffer to dissociate the p185 antigen from the solid phase medium and elution fraction was collected. The p185 antigen protein was identified by using ELISA、SDS-PAGE and Western Blot assay. Results:The results showed that the protein 0D280 peak in the elution solutuon collected from affinity chromatography was positive correlation with the p185 protein immunoreactivity. The molecular weight of the purified protein was 185 kD and the protein showed good immunoreactivity to antiP185 monoclonal antibody by SDS-PACE and Western Blot. The recover rate of p185 proein from the lysate of tumor cells is about 1. 129 ×10-3. Conclusion: The tumor antigen p185 oncoprotein was purified successfully from transgene 3T3/neu cells.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2000年第7期381-383,共3页
Chinese Journal of Immunology
基金
国家自然科学基金!39640009
关键词
亲和层析法
肿瘤抗原
P185蛋白
纯化
p185 protein Onconprotein purification Affinity chromatography Tumor antigen
