基于WIPI1蛋白自噬检测方法及其在PCV2感染细胞自噬研究中的应用

WIPI1-based detection of autophagy in PK-15 cells and its application in PCV2-induced autophagy

多种病毒感染能够诱导细胞自噬,WIPI1蛋白是一种新发现的细胞自噬标记分子。为在PK-15细胞中建立基于红色荧光蛋白(Ds Red)与WIPI1融合表达蛋白的自噬检测方法,本研究以猪圆环病毒2型(PCV2)感染为模式病毒,以药物处理作为阳性对照验证该方法的适用性。采用亚克隆策略构建了表达猪源WIPI1与Ds Red融合蛋白的真核重组表达质粒p Red-WIPI1,western blot检测显示Ds Red-WIPI1融合蛋白能够在PK-15细胞内表达,但不激活自噬标志分子LC3-II形成。然而,激光共聚焦分析显示,PCV2感染或自噬诱导剂Thapsigargin(Tg)及饥饿处理均能够显著促进WIPI1在细胞内形成点状聚集,并且促进Ds Red-WIPI1与EGFP-LC3的共定位。此外,荧光定量PCR结果显示,PCV2感染或Tg处理均能够显著上调wipi1的m RNA水平。本研究在稳定表达EGFP-LC3的PK-15细胞中建立了基于WIPI1蛋白的细胞自噬检测方法,该方法可以用于深入探索相关病毒感染诱导细胞自噬及其机制。

Many viruses have the ability to induce autophagy in infected cells. The protein WIPI1 has recently been described as a new autophagy marker. This study was attempted to develop an autophagy detection method based on the DsRed-WlPI1 fusion protein in PK-15 cells that could be used to detect autophagic responses in the cells infected with PCV2 or treated with chemical antophagy-inducer. Subcloning method was used to fuse the wipil gene to the 3＇end of DsRed gene in the pcDNA3.1 backbone （named pRed-WIPI1）. Western blot showed that the fusion protein was expressed in PK-15 cells without inducing autophagy. PCV2 infection, treatment with thapsigargin （Tg） and starvation could increase the formation of DsRed-WIPI1 punctuation, co-localization of DsRed-WIPI1 with EGFP-LC3 as shown by confocal imaging. In addition, real-time PCR detection showed that PCV2 infection and Tg treatment significantly up-regulated the mRNA level of wipil. In conclusion, we have developed a WIPI1-based method for monitoring autophagy in PK-15 cells stably expressing EGFP-LC3, which can be used to examine the autophagic responses and mechanisms after virus-infection.