弓形虫ME49虫株α-微管蛋白对神经瘤母细胞自噬的影响

Effect of α-tubulin of Toxoplasma gondii ME49 strain on autophagy of neuroblastoma cells

为研究刚地弓形虫α-微管蛋白(TgTUBA1)与神经瘤母细胞(N2a)自噬的关系,利用TgTUBA1基因的CDS序列设计引物并引入HA标签序列,提取弓形虫ME49虫株总RNA并反转录获得cDNA,以cDNA为模板进行PCR扩增,获得目的片段,并克隆至pcDNA3.1(+)载体上。将构建的真核表达质粒pcDNA3.1(+)-HA-TUBA1转染至N2a细胞内,通过透射电子显微镜和荧光显微镜观察自噬小体,并通过Western-blot检测LC3Ⅰ/Ⅱ的表达变化。结果显示,成功构建了pcDNA3.1(+)-HA-TUBA1真核表达载体,Western-blot试验检测到50 ku大小的特异目的条带。与空载体组相比,pcDNA3.1(+)-HA-TUBA1转染N2a细胞48 h后,N2a细胞皱缩、突触减少;观察到特征性的自噬小体;自噬相关蛋白LC3Ⅰ/Ⅱ表达上调。本研究证明了TgTUBA1蛋白可以促进N2a细胞自噬现象的发生,这为宿主细胞抗弓形虫感染提供了新的研究思路。

In order to study the relationship between Tg TUBA1 of Toxoplasma gondii and the autophagy of neuroblastoma(N2 a) cells,the CDS sequence of Tg TUBA1 gene was used to design primers with HA tag sequence,and the total RNA was extracted from T.gondii ME49 strain and used to synthesize c DNA by using reverse transcription.Then target fragment was amplified by PCR,and cloned into the pc DNA3.1(+) vector.An eukaryotic expression plasmid pcDNA3.1(+)-HA-TUBA1 was constructed and was transfected into N2 a cells.Afterwards,autophagosomes were observed by transmission electron microscopy and immunofluorescence microscopy.The expression of LC3 I/II was detected by Western-blot.The results showed that the eukaryotic expression plasmid pc DNA3.1(+)-HA-TUBA1 was successfully constructed and a specific band about 50 ku was detected by Western-blot.And after 48 hours of pcDNA3.1(+)-HA-TUBA1 transfection,compared with the control group,transfected N2 a cells were shrinkage and their synapses were decreased.Moreover,characteristic autophagosomes were found and the expression of LC3Ⅰ/Ⅱ was up-regulated.Therefore,Tg TUBA1 protein promoted the autophagy of N2 a cells,which provides a new idea for the research of host cells against T.gondii infection.