红细胞RhD膜蛋白提取与固相化方法的研究  

RhD membrane protein extraction and immobilization method

作　　者：章昊[1] 徐华[2,3] 周华友 张印则[1,3] 

机构地区：[1]深圳市血液中心,广东深圳518035 [2]陕西省血液中心 [3]Rh血型系统基础与临床研究关键技术攻关合作组 [4]南方医科大学南方医院

出　　处：《中国输血杂志》2017年第9期975-977,共3页Chinese Journal of Blood Transfusion

摘　　要：目的提取红细胞RhD膜蛋白并将其固化至乳胶微球以获得固相化RhD蛋白靶分子。方法采集3份志愿者全血标本,分别使用Yared MA法提取浓缩红细胞、试剂盒法提取浓缩红细胞及血影细胞膜蛋白的方法提取标本红细胞RhD膜蛋白,通过蛋白质定量检测及免疫印迹试验评价不同方法的提取效果。将不同方法提取出的RhD蛋白固化至乳胶微球,并通过流式细胞仪分析其抗原活性。结果 RhD膜蛋白浓度(mg/m L)分别为(n=3):0.24±0.04(Yared MA法提取浓缩红细胞)、4.82±0.09(试剂盒法提取浓缩红细胞)和4.31±0.39(试剂盒法提取血影细胞)与浓缩红细胞上的RhD膜蛋白浓度(mg/m L)。免疫印迹试验:Yared MA法从浓缩红细胞提取的RhD蛋白仅有1条RhD特异性条带;试剂盒法从浓缩红细胞提取的RhD蛋白无特异性条带,从血影细胞提取的RhD蛋白有2条RhD特异性条带。流式分析(n=3):Yared MA法提取的浓缩红细胞样品与试剂盒法提取的血影细胞样品、浓缩红细胞样品的荧光强度分别为:98.00±12.53、92.67±10.69、50.00±13.89。结论 Yared MA法提取出的RhD膜蛋白固化于乳胶微球可获得RhD蛋白纯度及抗原性较佳的固相靶分子。Objective To extract RhD membrane protein from red blood cells and solidify them on latex beads to obtain the solid-phase target RhD antigen.Methods To extract red blood cells RhD membrane protein of 3 blood donor samples by Yared MA method for extraction of concentrated red blood cells,kit method for extraction of concentrated red blood cells and ghost cells respectively,and to evaluate extractive effect by protein quantitative analysis and western blot.To analyze the activity of RhD antigen solidified on latex beads by flow cytometer.Results The concentration of membrane protein extracted by Yared MA method,ghost cells and packed red cells membrane protein extract by reagent kit was 0.24±0.04,4.31±0.39 and 4.82±0.09 mg/m L（n = 3） respectively.Western blot result showed membrane protein get by Yared MA method had one RhD specific band,and ghost cells membrane protein get by reagent kit had two bands,but packed red cells membrane protein had none band.Flow analysis results showed that the fluorescence intensity of samples RhD specificity get by Yared MA method,ghost cells and packed red cells membrane protein extract by reagent kit was 98.00 ± 12.53,92.67 ± 10.69 and 50.00±13.89（n = 3） respectively.Conclusion To solidify RhD membrane protein get by Yared MA method could obtain high purity immobilization RhD antigen target.

关 键 词：RhD膜蛋白 固相化 RhD蛋白靶分子 Yared MA法 

分 类 号：R446.62[医药卫生—诊断学] R457.11[医药卫生—临床医学]