鼠Mincle蛋白B细胞抗原表位预测及多克隆抗体制备  

B-cell antigen epitope prediction and polyclone antibody preparation of mouse Mincle protein

作　　者：郑小莉[1] 张艺[2] 谢晓东[3] 罗波[1] 刘佳佳[4] 

机构地区：[1]泸州医学院基础医学院生物化学教研室,646000 [2]泸州医学院基础医学院医学机能实验室,646000 [3]泸州医学院基础医学院教务处实习科,646000 [4]泸州医学院基础医学院基础医学院免疫学教研室,646000

出　　处：《免疫学杂志》2011年第8期683-687,共5页Immunological Journal

基　　金：四川省卫生厅项目(100208)

摘　　要：目的预测鼠Mincle蛋白的B细胞抗原表位及制备其多克隆抗体。方法以鼠Mincle蛋白氨基酸序列为基础,利用signalP3.0和TMHMM Server在线软件分析其信号肽和跨膜结构,利用DNAstar软件预测其二级结构、蛋白骨架区的柔韧性、亲水性、表面可能性及表面抗原性指数;融合表达PET30a(+)/Mincle蛋白并通过镍层析柱纯化后,免疫兔获得Mincle多克隆抗体。结果 Mincel蛋白是一种跨膜蛋白质,N末端第1-22号氨基酸可能位于蛋白的表面并可能为抗原表位;成功构建了重组载体PET30a(+)/Mincle,转化大肠杆菌BL21后表达包涵体融合蛋白,利用镍亲和层析得到了无生物活性的高纯度重组蛋白,纯化蛋白免疫兔,制备了滴度达1:128 000的多克隆抗体;Western-blot检测显示抗体特异性高。结论 Mincle蛋白N端即胞外区的第1-22氨基酸可作为Mincle蛋白的主要抗原表位区以制备Mincle多克隆抗体。In this study,we aim to predict the B-cell antigen epitope and prepare the polyclone antibody of mouse Mincle protein.First,by using signalP3.0,TMHMM server and DNAstar software,1-22 amino acids of Mincle were predicted to be the possible B-cell antigen epitopes.Secondly,the recombinant PET30a（＋）/Mincle plas-mid was constructed and then transformed into BL21.Fusion protein PET30a（＋）/Mincle was induced with IPTG and the expression production was purified by Ni-NTA His Bind column.The purified PET30a（＋）/Mincle protein was utilized to immunize rabbit and high quality antibody（the titer over 1：128 000） was gained.From all result,we can presume that the 1th-22nd amino acids of Mincle may suitable to be B-cell antigen epitope to gain poly-clone antibody of mouse Mincle.

关 键 词：鼠Mincle蛋白 B细胞抗原表位 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]