稳定表达马红球菌VapA蛋白细胞系的建立及其对巨噬细胞自噬的影响

Establishment of macrophage cell line stably expressing VapA protein of Rhodococcus equi and exploration of the effect within macrophage autophagy

通过构建稳定表达VapA蛋白的J774A.1巨噬细胞系,探究马红球菌VapA蛋白对巨噬细胞自噬的影响。构建vapA基因的重组慢病毒载体pCDH-CMV-MCS-EF1-Puro-vapA,将其转染J774A.1细胞,经过嘌呤霉素筛选重组细胞,利用RT-PCR、Western-blot和免疫荧光鉴定重组细胞系中VapA蛋白的表达;利用RT-PCR检测自噬相关基因LC3-Ⅱ、p62、Beclin1、Atg5及Atg7的表达情况,明确VapA蛋白对巨噬细胞自噬的影响。结果显示,本研究成功构建了稳定表达VapA蛋白的J774A.1巨噬细胞系;与对照组相比,Vap A蛋白稳转细胞系中自噬相关基因LC3-Ⅱ、Beclin1、Atg5及Atg7的m RNA表达量显著降低,p62无显著差异。上述结果表明,本研究通过VapA蛋白的体外细胞模型发现,VapA在转录水平抑制巨噬细胞自噬,为VapA蛋白在巨噬细胞内的功能研究及马红球菌的致病机制研究提供了理论基础。

The aim of this study was to construct a stable macrophage cell line1 which could express Vap A protein from Rhodococcus equi and explore the effect of Vap A protein in macrophages autophagy.A vap A gene recombinant vector p CDH-CMV-MCS-EF1-Puro-vap A was constructed,then it was transfected J774 A.1 cells.Puromycin was used to select the macrophage cell lines which could expressing Vap A protein.RT-PCR and Western-blot and immunofluorescence were used to confirmed the expression of Vap A in macrophages.We used RT-PCR to accessed the Vap A effects on autophagy by detecting expression of LC3-Ⅱ,p62,Beclin1,Atg5 and Atg7 on transcriptional level.We successful constructed the J774 A.1 macrophage cell line which could stably express Vap A protein.The m RNA expressions of LC3-Ⅱ and Beclin1 and Atg5 and Atg7 of Vap A protein stable expression macrophage cell line were significant decreased compared with untreated J774 A.1,meanwhile p62 did not show significant difference.The results showed that Vap A protein could inhibit macrophage autophagy at the transcriptional level on cell model in vitro.This research provided a basis for the Vap A protein function study in cell and its pathogenesis of R.equi.