猪ARAF基因真核表达及其对PK15细胞凋亡的调控作用

Eukaryotic expression of porcine ARAF gene and its regulatory role on apoptosis of PK15 cells

为研究猪ARAF(v-raf murine sarcoma 3611 viral oncogene homo)lo基g因在PK15细胞的表达、定位及其对细胞凋亡的调控作用,本文以PK15细胞为研究对象,RT-PCR扩增猪ARAF基因CDS序列,经TA克隆至载体pGEM-T-Easy,随后加酶切位点和保护性碱基设计引物,PCR扩增后,构建重组真核表达载体pc DNA3.1-EGFP-ARAF,重组载体经脂质体法转染PK15细胞,荧光显微镜观察转染效果,qPCR与Westernblot分别检测A R A F基因mRNA和蛋白表达水平,间接免疫荧光试验观察猪A R A F的亚细胞定位;同时,流式细胞术及免疫印迹试验探究ARAF对PK15细胞凋亡的影响。结果显示,构建的重组真核表达载体转染成功,荧光显微镜检查可见转染组和空载组细胞均表达绿色荧光蛋白。转染组细胞ARAF基因mRNA及蛋白表达量较空载组和空白对照组显著升高(P<0.01)。激光共聚焦显微镜观察到该基因表达蛋白主要定位于PK15细胞胞质中。流式细胞术及免疫印迹试验检测时可见转染组PK15细胞凋亡率显著增加;相较空载组和空白对照组,转染组细胞中凋亡相关蛋白BAD(B-cell lymphoma-2 antagonist of cell d)e、aBtchl-2(B-cell lymphoma-2)蛋白的表达量无显著性变化,而BAX(B-cell lymphoma-2 associated)X、Cleaved caspase-3的蛋白表达量显著升高(P<0.01),显示高表达ARAF基因可促进PK15细胞的凋亡。上述研究结果为进一步深入探讨ARAF基因对PK15细胞凋亡的调控机制研究奠定了重要基础。

To study the expression and localization of porcine ARAF(V-Raf Murine Sarcoma 3611 viral oncogene homolog)gene in PK15 cells and its regulatory role on cell apoptosis,PK15 cells were used as the research subject.The CDS sequence of porcine ARAF gene was amplified by RT-PCR,and cloned into PGEM-T-Easy vector by TA cloning.Then,PCR amplification was performed using a pair of primers with restriction sites and protective bases,and the recombinant eukaryotic expression vector pc DNA3.1-EGFPARAF was constructed.The recombinant vector was transfected into PK15 cells by liposome method,and the transfection efficiency was observed by a fluorescence microscope.The m RNA and protein expression levels of ARAF gene were detected by q PCR and Western blot,respectively.The subcellular localization of ARAF was observed by an indirect immunofluorescence assay.Simultaneously,flow cytometry and western blot were performed to evaluate the effect of ARAF on PK15 cell apoptosis.The results showed that the recombinant eukaryotic expression vector was successfully constructed and transfected into PK15cells.Fluorescence microscopy showed that green fluorescent protein was expressed in both of the pc DNA3.1-EGFP-ARAF and pc DNA3.1-EGFP transfection groups.The m RNA and protein expressions of ARAF gene in pc DNA3.1-EGFP-ARAF transfection group were significantly higher than those in pc DNA3.1-EGFP transfection and blank control groups(P<0.01).Laser confocal microscopy observation revealed that the expressed protein was mainly located in the cytoplasm of PK15 cells.Flow cytometry and western blot assay were used to detect the apoptosis of PK15 cells in different transfection groups.The apoptosis rate of PK15 cells in pc DNA3.1-EGFP-ARAF transfection group was significantly increased.Compared with thepc DNA3.1-EGFP transfection and blank control groups,the expression levels of apoptosis-related proteins BADand Bcl-2were not significantly changed in the transfection group,while the expression levels of BAX and Cleaved caspase-3 were significantly increased(P<0.01),suggesting that high expression of ARAF gene promoted apoptosis of PK15 cells.This study laid an important foundation for further exploring the regulatory mechanism of ARAF gene on PK15 cells apoptosis.