弓形虫H4和Rab2基因真核表达载体的构建及其对N2a细胞凋亡和自噬的影响

Construction of eukaryotic expression vectors with Toxoplasma gondii H4 and Rab2 genes and their effects on the apoptosis and autophagy of N2a cells

本研究旨在探索弓形虫Rab2和H 4基因在神经细胞凋亡和自噬过程中的作用,并进一步研究雷帕霉素(rapamycin,RAPA)对其的影响。本试验以弓形虫M e49虫株c DN A为模板,扩增H4和Rab2基因,并构建到真核表达载体pcDNA3.1 (+)上,转染小鼠神经母细胞瘤细胞(N2a),同时设置RAPA给药组,检测目的基因及凋亡和自噬相关蛋白的表达情况。结果显示,重组质粒成功构建,弓形虫Rab2和H4基因在N2a细胞中表达,导致细胞凋亡相关蛋白Bcl-2下调和细胞自噬相关蛋白LC3Ⅰ/Ⅱ上调,但在RAPA组,Bcl-2却有上调趋势。此外还发现H4基因对于Rab2表达有促进作用。结果表明,H4和Rab2的表达可以促进N2a细胞的凋亡和自噬,而RAPA具有抑制细胞凋亡的作用,从而保护神经系统,这为弓形虫病的预防治疗提供了新思路。

Toxoplasmosis is a fatal disease mediated by Toxoplasma gondii invasion in the central nervous system(CNS). We aimed to find out the role of Rab2 and H4 genes of T.gondii in the process of neuronal apoptosis and autophagy, and the effect of rapamycin(RAPA). By using the c DNA of T.gondii Me49 strain as a template, H4 and Rab2 genes were amplified and were linked to the eukaryotic expression vector pc DNA3.1(+), then were transfected into mouse neuroblastoma N2 a cells(N2 a) by lipofectamine2000.We set up a RAPA group and detected the expression of the target genes, the apoptotic and autophagic proteins. In this study, the recombinant plasmids of pc DNA3.1(+)-HA-H4 and pc DNA3.1(+)-HA-Rab2 were constructed successfully, and were expressed in N2 a cells, where the expression levels of both genes were significantly higher than the control group(P<0.01).In the transfected group, the expression of Bcl-2 gene had been on a downward trend, and the expression of LC3 gene had been on an upward trend, but the expression of Bcl-2 increased after adding with PAPA. Moreover, co-transfection of pc DNA3.1(+)-HA-Rab2 and pcDNA3.1(+)-HA-H4 showed that H4 gene could promote Rab2 expression.Specifically, we found that H4 and Rab2 proteins can promote the apoptosis and autophagy of N2a cells,which can be inhibited the apoptosis by the RAPA. Because of the CNS protection by adding PAPA,this study might provide a new idea for the prevention and treatment of animal toxoplasmosis.