CAJ | 학술논문

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中表达后的业细胞定位.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HIA-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.
목적 탐토pTAP1-EGFP화(혹)pTAP2-EGFP전입악성흑소류세포주후TAP표체적변화,관찰TAP재A375중표체후적업세포정위.방법 재악성흑소류세포주A375중전입pTAP1-EGFP화(혹)pTAP2-EGFP,G418사선은정전염세포주,검측전염후TAP1화TAP2표체수평적변화.장pDsRed2-ER화pTAP1-EGFP혹pTAP2-EGFP공전염입A375세포내,격광공취초현미경하관찰TAP1-EGFP화TAP2-EGFP융합단백적아세포정위.류식세포의검측전염전후세포표면HIA-Ⅰ적표체.결과 장pTAP1-EGFP화(혹)pTAP2-EGFP전염A375세포주후사선출은정극륭.전염pTAP1-EGFP화(혹)pTAP2-EGFP후능명현증가A375세포TAP1화TAP2재단백수평적표체,병능증가세포표면HLA-Ⅰ적표체.공전염pDsRed2-ER화pTAP1-EGFP혹pTAP2-EGFP후,재격광공취초현미경하관찰,발현TAP1-EGFP화TAP2-EGFP융합단백적록색형광능구여pDsRed2-ER적홍색형광중첩.결론 구건적pTAP1-EGFP화(혹)pTAP2-EGFP질립재A375세포계중표체성위TAP1-EGFP화TAP2-EGFP융합단백후,능준학정위재내질망상,위연구TAP유도적후속면역효응제공기출.
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-TAP1 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 pTAP1-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 cuccessfully and expressed in A375 cells,and the expressed fusion protein is subcelluiarly localized to ER.This study will provide a basis for the research into subsequent immune response following induction of TAP expression.