细粒棘球绦虫囊化原头蚴AQP基因转录动态及异源表达实验研究

Transcription profile and heterologous expression of Echinococcus granulosus aquaporins genes in the encysted protoscolex

目的探索细粒棘球蚴水通道蛋白(Echinococcus granulosus aquaporins,EgAQPs)在棘球蚴液形成过程中的作用。方法收集体外培养不同时期的棘球蚴标本和BALB/c小鼠体内接种的继发棘球蚴标本,RT-qPCR方法检测12个EgAQPs基因在体内、体外培养不同发育时期的转录表达。以细粒棘球蚴cDNA为模板,扩增EgAQP、EgAQP1、EgAQP3、EgAQP4-1、EgAQP4-4和EgAQP9-16个基因编码区片段,同时,通过化学合成方法扩增另外6个EgAQPs(EgAQPAn.G、EgAQPFA-CHIP、EgAQP4-2、EgAQP4-3、EgAQP4-5、EgAQP9-2)基因编码区片段,并在非洲爪蟾卵母细胞中检测这12个EgAQPs基因的透水功能,Western blot检测EgAQPs在卵母细胞膜上的表达。结果体外培养的原头蚴及感染小鼠的继发棘球蚴体积均随培养时间的延长而逐渐增大,棘球蚴囊内透明液体增多。RT-qPCR结果显示12个EgAQPs基因在体内、体外培养不同发育时间的转录表达水平均较低。注射EgAQPs基因的非洲爪蟾卵母细胞的体积变化率和透水系数与阴性对照组差异无统计学意义,这12个EgAQPs基因在卵母细胞中未表现出水通道功能。Western blot检测12个EgAQPs基因在卵母细胞总蛋白、胞质蛋白与胞膜蛋白上的表达,结果均未观察到蛋白在预期的水通道蛋白理论分子量处表达。结论12个EgAQPs基因在原头蚴囊化形成棘球蚴过程中转录表达水平均较低,这些基因在爪蟾卵母细胞的异源表达均未表现出通透水的功能。

Objective To explore the role of Echinococcus granulosus aquaporins(EgAQPs)in the accumulation of hydatid fluid.Methods The secondary hydatids cultured in vitro and inoculated in BALB/c mice were collected respectively,and the relative transcription expression levels of 12 EgAQPs genes from the hydatid cysts cultured in vitro and in mice were detected by RT-qPCR.Six gene coding region fragments of EgAQP,EgAQP1,EgAQP3,EgAQP4-1,EgAQP4-4 and EgAQP9-1 were amplified using cDNA templates.The other 6 genes coding region fragments of EgAQPs(EgAQPAn.G,EgAQPFA-CHIP,EgAQP4-2,EgAQP4-3,EgAQP4-5 and EgAQP9-2)were amplified with chemical synthesis.The water permeability of these 12 EgAQPs genes were detected in Xenopus laevis(X.laevis),and the expression of EgAQPs on the oocytes membranes was detected by Western blotting.Results The volume of hydatid cultured in vitro and in vivo was increased gradually with the extension of culture time,and there was more and more transparent hydatid fluid in hydatid cyst.The results of RT-qPCR showed that the relative transcription levels of EgAQPs genes in the hydatid cysts were very low or even undetectable except for EgAQP3 and EgAQP9-1 at the beginning of culture in vitro.There were no significant differences in relative volume change rates and water permeability coefficients of X.laevis oocytes injected with EgAQPs genes when compared with the negative control,and these EgAQPs genes did not show water channel functions in the oocytes.Western blot assay displayed that there was no expression of EgAQPs genes in total,cytoplasmic and membrane protein on the oocytes.Conclusion The transcription levels of EgAQPs genes are quite low in the encystation process of protoscolex,and their heterologous expression do not show water channel functions in X.laevis oocytes.