中华皮肤科杂志 ›› 2009, Vol. 42 ›› Issue (11): 774-777.

• 论著 • 上一篇    下一篇

TAP1-EGFP和TAP2-EGFP融合蛋白在恶性黑素瘤细胞系中的表达

李延1,陶娟1,刘业强2,杨井3,涂亚庭4   

  1. 1. 华中科技大学同济医学院附属协和医院皮肤科
    2. 华中科技大学同济医学院协和医院
    3. 华中科技大学同济医学院附属协和医院
    4. 武汉华中科技大学同济医学院附属协和医院皮肤科
  • 收稿日期:2008-11-10 修回日期:2008-12-15 出版日期:2009-11-15 发布日期:2009-11-13
  • 通讯作者: 李延 E-mail:littleagong@gmail.com
  • 基金资助:

    国家自然科学基金资助项目（编号）

Identification and subcellular localization of pTAP-EGFP fusion protein

  • Received:2008-11-10 Revised:2008-12-15 Online:2009-11-15 Published:2009-11-13

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摘要：

目的 将pTAP1-EGFP和/或pTAP2-EGFP转入恶性黑素瘤细胞株，并筛选稳定转染细胞株。比较转染前后TAP表达的变化。将pTAP-EGFP和pDsRed2-ER共转染至A375细胞株，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，并检测转染后TAP1和TAP2表达水平的变化。将pDsRed2-ER和pTAP1-EGFP或pTAP2-EGFP共转染入A375细胞系，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。结果 将pTAP1-EGFP和/或pTAP2-EGFP转染A375细胞株后筛选出稳定克隆。转染了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。共转染了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，证实TAP1-EGFP和TAP2-EGFP融合蛋白能够定位在内质网上。结论 pTAP1-EGFP和/或pTAP2-EGFP质粒转染能显著提高A375细胞系TAP在蛋白水平的表达。我们所构建的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，对TAP的天然构象没有影响，并能够准确定位在内质网上，为研究TAP所诱导的后续免疫效应提供了良好的基础。

关键词: 黑色素瘤, 主要组织相容性复合物, 内质网, 抗原处理相关转运蛋白

Abstract:

Objective To construct a eukaryotic expression vector for transporters associated with antigen processing gene fused with enhanced green fluorescent protein (EGFP) gene and to analyze the expression and subcellular localization of the fusion protein in A375 cells. Methods The monoclone A375 cells with stable expression of TAP were obtained by G418 selection and were identified with checking EGFP expression by FCM and observing under Fluorescence microscope. Each plasmid (pTAP1-EGFP, or pTAP2-EGFP), together with pDsRed2-ER vector, were transiently transfected into A375 cells,and the localizations of expressed proteins were analyzed by confocal microscopy. Results pTAP1-EGFP and pTAP2-EGFP were used to restore the expressions of TAP1 and TAP2 in the antigen presentation pathway-deficient human MM cell line, A375. Transfection of A375 cells with pTAP1-EGFP and pEGFP-TAP1+TAP2 increased the expression of TAP1 and TAP2. The localizations of TAP1-EGFP and TAP2-EGFP overlapped with that of the ER marker, indicating that TAP protein localized subcellularly on the ER. Conclusions The expression vector for TAP-EGFP fusion gene has been constructed successfully and expressed in A375 cells. The expressed fusion protein has similar subcellular localization to pDsRed2-ER. pTAP-EGFP showed efficacy in increasing TAP mRNA and protein in A375 cell lines.