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目的 确定一套CD2 2 6单克隆抗体 (McAb)识别CD2 2 6分子胞膜外区结构域的部位。方法 应用计算机软件分析CD2 2 6分子蛋白质序列疏水性 ,确定其亲水性区域。采用PCR方法扩增CD2 2 6分子基因序列 ,分别缺失相应亲水性区域 ,构建CD2 2 6截短体重组真核细胞表达载体pcDNA3 PTA1T1和pcDNA3 PTA1T2。测序正确后 ,转染COS7细胞 ,抗CD2 2 6分子多克隆抗体间接免疫荧光染色 ,流式细胞术检测CD2 2 6分子截短体的表达。表达成功后 ,采用间接免疫荧光染色和流式细胞术分析 ,用一套CD2 2 6McAb检测与截短突变体的免疫反应性 ,从而确定CD2 2 6McAb识别CD2 2 6分子结构域的部位。结果 PCR扩增出CD2 2 6分子相应目的片段序列 ,定向克隆入pcDNA3真核表达载体 ,DNA序列测定正确。转染COS7细胞后 ,流式细胞术检测CD2 2 6截短体分子有较高水平的表达。CD2 2 6McAbLeoA1、NewE1和FMU1～ 7均可识别CD2 2 6全长分子 ;LeoA1、NewE1、FMU1、FMU2、FMU4和FMU5与截短突变体PTA1T1和PTA1T2均无反应性 ;FMU3可结合PTA1T1分子 ,不结合PTA1T2分子 ;FMU6和FMU7均可结合PTA1T1及PTA1T2分子。结论 CD2 2 6McAbLeoA1、NewE1、FMU1、FMU2、FMU3、FMU4和FMU5识别CD2 2 6分子胞膜外区D1结构域 ,其中FMU3识别的位点位于V1和V2环之间 ;FMU6?
Objective To determine a set of CD2 2 6 monoclonal antibody (McAb) recognizing the extracellular domain of CD2 26 molecule. Methods The software was used to analyze the hydrophobicity of CD2 2 6 protein sequence to determine its hydrophilic region. The CD2 2 6 gene sequence was amplified by PCR, and the corresponding hydrophilic regions were deleted respectively to construct recombinant eukaryotic expression vectors pcDNA3 PTA1T1 and pcDNA3 PTA1T2 of CD2 2 6 truncation. After sequencing, the transfection of COS7 cells, indirect immunofluorescence staining of polyclonal anti-CD26 antibody, and the detection of CD226 truncated expression by flow cytometry. After successful expression, the immunoreactivity with the truncated mutant was detected by a set of CD226McAbs using indirect immunofluorescence staining and flow cytometry analysis to determine where CD226McAb recognizes the CD226 domain. Results The corresponding fragment of CD2 2 6 was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3. The DNA sequence was determined correctly. After transfection of COS7 cells, the expression of CD2 2 6 truncated molecule was detected by flow cytometry. CD2 26McAbLeoA1, NewE1 and FMU1 ~ 7 all recognize CD2 2 6 full-length molecules; LeoA1, NewE1, FMU1, FMU2, FMU4 and FMU5 are not reactive with the truncated mutant PTA1T1 and PTA1T2; FMU3 can bind to PTA1T1 molecule, Combined with PTA1T2 molecule; both FMU6 and FMU7 can bind PTA1T1 and PTA1T2 molecules. Conclusion CD2 26McAbLeoA1, NewE1, FMU1, FMU2, FMU3, FMU4 and FMU5 recognize the D1 domain of the extracellular domain of CD2 26, of which the FMU3 recognition site is located between V1 and V2 loops.