利用PiggyBac转座子系统快速控制GPI锚定蛋白的表达

Quick Conversion From GPI-Positive to GPI-Negative Cells Using piggyBac Transposon System

DOI：10.3969/j.issn.1673-1689.2017.09.006

中文关键词: GPI锚定蛋白 PiggyBac转座子 PIGK

英文关键词: GPI-anchor proteins,piggyBac（PB）,PIGK

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中文摘要:

GPI锚定蛋白的合成是一个发生在内质网中多步骤、多基因参与的过程。PIGK是GPI锚定蛋白转酰胺酶复合物的一个催化亚基，负责把GPI前体转移到蛋白质上。作者在人体胚胎肾细胞（HEK 293）中获得了PIGK敲除的细胞株，敲除PIGK的细胞不能表达GPI锚定蛋白。利用piggyBac（PB）转座子系统，在细胞中重新插入PIGK基因，细胞膜表面的GPI锚定蛋白恢复表达。实验成功构建了HEK293pB-PIGK细胞株并命名为G36，通过引入PB转座酶或FLP重组酶，可以控制GPI锚定蛋白的表达。本系统可以快速调控细胞表面蛋白质的表达并能够用于基础研究和工业应用。

英文摘要:

The biosynthesis of GPI is carried out in the endoplasmic reticulum（ER） through multiple steps and many genes are involved in the reactions. PIGK is one of the subunits of the catalytic domain of the GPI transmidase complex，responsible for the transfer of GPI moiety on protein. In this study，we generated a PIGK-knockout（KO） HEK 293 cell line，which showed no surface expression of GPI-anchored proteins（GPI-APs）. By mediation of piggyBac（PB） transposon system，we rescued the PIGK gene in PIGK-KO cells，which showed the recovery of the surface expression of GPI-APs. We successfully established a HEK 293 cell line，HEK293pB-PIGK，named G36，which can be converted from GPI-AP positive to negative cells by the introduction of PB transposase or FLP recombinase. Our system is useful to modulate surface proteins quickly and can be applied for both basic and applied researches.

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