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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。
Objective To predict B cell epitope of murine Mincle protein and prepare its polyclonal antibody. Methods Based on the amino acid sequence of mouse Mincle protein, signalP3.0 and TMHMM Server online software were used to analyze its signal peptide and transmembrane structure. DNAstar software was used to predict the secondary structure. The flexibility, hydrophilicity, Sexual and surface antigenicity index; PET30a (+) / Mincle protein was fused and purified by a nickel column, and then the rabbit polyclonal antibody was obtained. Results The Mincel protein was a transmembrane protein. N-terminal amino acids 1-22 were probably located on the surface of the protein and possibly epitopes. The recombinant vector PET30a (+) / Mincle was successfully constructed and expressed in E. coli BL21 The fusion protein was purified by Ni-affinity chromatography and the recombinant protein with high purity was obtained. The rabbit polyclonal antibody with the titer of 1: 128 000 was prepared by immunizing the rabbit with purified protein. Western-blot showed that the antibody was highly specific. Conclusions The 1-22 amino acids of N-terminal, extracellular domain of Mincle protein can be used as the main epitope region of Mincle protein to prepare Mincle polyclonal antibody.