人CD226(PTA1)分子胞膜外区截短体真核表达载体的构建、表达及其识别结构域的分析

Construction and expression of CD226(PTA1)truncated gene eukaryotic expression vectors and identification of the epitopes localization recognized by CD226 McAbs

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 identify the localization of the epitopes recognized by CD226 McAbs in the extracellular region of CD226. Methods Analysing the hydrophobicity of CD226 with software on internet website Expasy. Deleting part of the CD226 gene encoded hydrophilicity region using PCR and cloning the PCR products into pcDNA3. The recombinant expression vector was sequenced and transfected into COS7. The expression of truncated CD226 was identified by indirect immunofluorescence staining and flow cytometry with anti-CD226 polyclonal antibody. All nine clones of CD226 McAbs were used to react with the COS7 parent as well as CD226 gene transfected COS7 cells or truncated mutants, and analysed by flow cytometry. Results The PCR products were in accordance with expectation and the sequencing results were completely correct. Truncation mutated CD226 gene could be expressed on COS7 cells. Leo A1, New E1, FMU1, FMU2, FMU4 and FMU5 could bind wild type of CD226, but could not bind PTA1T1 and PTA1T2. FMU3 could bind wild type of CD226 and PTA1T1, but not PTA1T2. FMU6 and FMU7 could bind wild type of CD226, PTA1T1 and PTA1T2. Conclusion The epitopes recognized by Leo A1, New E1, FMU1, FMU2, FMU4 and FMU5 were located in domain 1 of CD226, the epitopes of FMU6 and FMU7 were in domain 2 and the epitope of FMU3 is located between V1 and V2 loop. [