PD-L2剪切异构体与EGFP融合蛋白的表达及其亚细胞定位研究

Expression of the fusion proteins of PD-L2 splice isoforms with EGFP and its subcellular localization

目的 探讨PD L2的剪切异构体在哺乳动物细胞的表达和亚细胞定位。方法 以RT PCR方法克隆人PD L2基因的常规剪切体和新型剪切异构体的cDNA ,构建其与EGFP融合的表达载体 ,分别转染K5 6 2细胞进行表达 ,经抗PD L2抗体染色后以流式细胞仪和激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果 从活化的人白细胞中克隆到常规剪切体PD L2Ⅰ和新型剪切异构体PD L2Ⅱ的cDNA ,后者删除了编码胞外区Ig C结构域的外显子 3。进而构建了PD L2Ⅰ EGFP和PD L2Ⅱ EGFP融合蛋白表达载体 ,分别转染K5 6 2细胞。流式细胞仪分析显示 ,转染前者的细胞中既可检测到EGFP的表达又可在细胞膜上检测到PD L2表达 ;而转染后者的细胞中只能检测到EGFP的表达 ,在其细胞膜上不能检测到PD L2表达。共聚焦显微镜观察显示 ,PD L2Ⅰ EGFP主要分布于细胞膜表面 ,PD L2Ⅱ EGFP则主要分布于细胞内。结论 常规剪切体PD L2Ⅰ能正确定位于细胞膜表面 ,而新型剪切异构体PD L2Ⅱ则位于细胞内 ,不能运输至细胞膜 ,提示不同PD

Objective To investigate the expression and subcellular localization in mammalian cells of the fusion proteins of PD-L2 splice isoforms with enhanced green fluorescent protein (EGFP). Methods The (cDNA) of the conventional splice form and an alternative splice variant of human PD-L2 gene were cloned by RT-PCR and then inserted respectively into the vector pEGFP-N1 to construct the expression vectors for PD-L2-EGFP fusion proteins. The transient expression and subcellular localization of the fusion proteins in the transfected K562 cells was analyzed by flow cytometry and confocal microscopy. Results The cDNA of the conventional splice form PD-L2Ⅰ and an alternative splice variant PD-L2Ⅱ of human PD-L2 gene were cloned from activated human leukocytes. The cDNA of PD-L2Ⅱ was generated by splicing out the exon 3 encoding the extracellular Ig-C domain. The expression vectors for PD-L2Ⅰ-EGFP and PD-L2Ⅱ-EGFP fusion protein were then constructed and K562 cells were transfected with these vectors respectively. Flow cytometry analysis revealed that both EGFP and PD-L2 could be detected in the cells transfected by the vector for PD-L2Ⅰ-EGFP, whereas only EGFP but no PD-L2 could be detected in the cells transfected with the vector for PD-L2Ⅱ-EGFP. Furthermore, confocal microscopy observation showed that the PD-L2Ⅰ-EGFP was distributed predominantly on the surface of the plasma membrane while PD-L2Ⅱ-EGFP was located in the intracellular region. Meanwhile, only EGFP expression could be detected in K562 cells transfected with empty vector and its distribution in the cells was diffuse. Conclusion The conventional splice form PD-L2Ⅰ is predominantly located on the surface of the plasma membrane, whereas the novel alternative spliced isoform PD-L2Ⅱ can not be transported to the cell surface and is intracellularly distributed. These results suggest that different splice variants of PD-L2 may be related with the modulation of function. [