【作者】 王弦；

【导师】 宋礼华； 徐振山；

【作者基本信息】 安徽医科大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 目的从转染Her-2/neu基因的T6-17细胞上清中纯化p185蛋白作为阳性抗原对照,利用生物素-亲和素酶联免疫法(Biotin-Avidin ELISA,BA-ELISA法)检测乳腺癌患者血清中p185蛋白,探讨血清p185蛋白在乳腺癌诊断中的意义。方法采用溴化氰活化的Sepharose 4B为基质,通过交联A18 mAb,用亲和层析的技术纯化p185蛋白。将收集的T6-17细胞上清循环过亲和层析柱,经醋酸溶液解离与抗体结合的p185蛋白,收集到蛋白洗脱峰,浓缩后用ELISA检测p185的免疫反应性,并经SDS-PAGE和Western blot进一步鉴定。利用自备的生物素化Her-2/neu单克隆抗体,并利用亲和层析法从T6-17细胞培养上清中纯化的p185蛋白作为阳性对照,采用BA-ELISA方法对乳腺癌和正常对照血清进行p185蛋白检测,同时对乳腺癌患者组织进行p185蛋白免疫组化染色,并对实验结果进行统计学分析。结果从转基因细胞上清中成功获取p185肿瘤抗原;用BA-ELISA方法检测了122例乳腺癌患者血清,血清p185阳性数为39例,检测了110例正常人血清,血清p185阳性数为0例;122例乳腺癌患者中免疫组化p185阳性数为43例,其中血清p185升高38例。结论初步建立了乳腺癌患者血清中p185蛋白的BA-ELISA检测方法,结果显示患者血清中p185蛋白对乳腺癌具有重要的诊断意义,血清p185蛋白和组织p185蛋白呈高度相关。更多还原

【Abstract】 Objective Use p185 by affinity chromatography from the culture supernatant of T6-17 which transfected the Her-2/neu gene as the positive control, to detected p185 in breast cancer patients’ serum by BA-ELSIA, and discuss the significance of serum P185 in breast cancer diagnosis. Methods The p185 oncoprotein was purified by using an affinity chromatography column. In this experiment, the anti-p185 oncoprotein monoclonal antibody was obtained from A18 hybridoma ascite with octoic acid-saturated ammonium sulfate precipitation. The antibody activity was coupled with CNBr-activated Sepharose 4B as solid phase medium to pack the affinity chromatography column. The p185 oncoprotein antigen was binded with the anti-p185 antibody when the Culture supernatant run through the column. The gradient salt solution(0.2M HAC PH2.5)as elute buffer to dissociate the p185 antigen from the solid phase medium and elution fraction was collected. After be concentrated, the p185 antigen protein was identified by using ELISA、SDS-PAGE and Western blot. Use the biotinylation Her-2/neu mcAb made by ourselves, and P185 by affinity chromatography from the culture supernatant of T6-17 as the positive control, to detect the p185 in breast cancer patients’serum and normal control serum by BA-ELSIA, meanwhile perform IHC in these patients tissues to detect P185,then analyze these experimental results by statistics software. Results The oncoprotein p185 was purified successfully from the Culture supernatant of transgene T6-17 cells; detect 122 cases of breast cancer patients’ serum by BA-ELISA method, 39 cases are serum p185 positive; detect 110 cases of normal human serum by BA-ELISA method, 0 case is serum p185 positive; detect 122 cases of breast cancer patients’ tissues by IHC, 43 cases are p185 positive, in which 38 cases serum p185 increased. Conclusion The BA-ELISA method to detect p185 in breast cancer patients’serum has taken shape initially, the serum p185 is significant to breast cancer in diagnosis, and the serum p185 level is highly relevant to tissue expression level of p185.更多还原