TAP1-EGFP和TAP2-EGFP融合蛋白在恶性黑素瘤细胞系中的表达

Identification and subcelhtlar localization of transporter associated with antigen processing （TAP） 1-EGFP and TAP 2-EGFP fusion proteins in malignant melanoma

目的探讨pTAP1-EGFP和（或）pTAP2-EGFP转入恶性黑素瘤细胞株后TAP表达的变化，观察TAP在A375中表达后的亚细胞定位。方法在恶性黑素瘤细胞株A375中转入pTAP1-EGFP和（或）pTAP2-EGFP，G418筛选稳定转染细胞株，检测转染后TAP1和TAP2表达水平的变化。将pDsRed2-ER和pTAP1-EGFP或pTAP2-EGFP共转染入A375细胞内，激光共聚焦显微镜下观察TAP1-EGFP和TAP2-EGFP融合蛋白的亚细胞定位。流式细胞仪检测转染前后细胞表面HLA—I的表达。结果将pTAP1-EGFP和（或）pTAP2-EGFP转染A375细胞株后筛选出稳定克隆。转染pTAP1—EGFP和（或）pTAP2-EGFP后能明显增加A375细胞TAP1和TAP2在蛋白水平的表达，并能增加细胞表面HLA—I的表达。共转染pDsRed2-ER和pTAP1-EGFP或pTAP2-EGFP后，在激光共聚焦显微镜下观察，发现TAP1-EGFP和TAP2-EGFP融合蛋白的绿色荧光能够与pDsRed2-ER的红色荧光重叠。结论构建的pTAP1-EGFP和（或）pTAP2-EGFP质粒在A375细胞系中表达成为TAP1-EGFP和TAP2-EGFP融合蛋白后，能准确定位在内质网上，为研究TAP诱导的后续免疫效应提供基础。

Objective To construct an eukaryotic expression vector for TAP genes fused with enhanced green fluorescent protein （EGFP） gene, and to analyze the expression and subcellular localization of the fusion protein in A375 human malignant melanoma cells transfected with the eukaryotic expression vector. Methods A375 cells were cotransfected with the combination of plasmid （p） TAP1-EGFP or pTAP2-EGFP and pDsRed2-endoplasmic reticulum （ER）, or with pEGFP-TAPI and -TAP2, or monotransfected with pTAP1-EGFP or pTAP2-EGFP alone. The monoclonal A375 cells stably expressing TAP were obtained by G418 selection. Then, the distribution and expression of fusion proteins were assessed in A375 cells by using fluorescence microscopy and Western blot, respectively. Flow cytometry was used to measure the expression of HLA class I on A375 cells. Results Transfection of A375 cells with pTAP1-EGFP or pTAPI-EGFP and pTAP2-EGFP significantly increased the expression of TAP 1 and TAP 2 in as well as HLA class I antigen on A375 cells. The green fluorescence of TAP1-EGFP and TAP2-EGFP overlapped with the red fluorescence of ER marker in cotransfected cells, indicating that TAP was localized subcellularly on the ER. Conclusions The expression vector for TAP-EGFP fusion gene has been constructed successfully and expressed in A375 cells, and the expressed fusion protein is subcellularly localized to ER. This study will provide a basis for the research into subsequent immune response following induction of TAP expression.