两种棘球绦虫的水通道蛋白基因克隆及功能特性的比较

目的 探索棘球绦虫水通道蛋白(aquaporin, AQP)在棘球蚴、泡球蚴囊液形成中的作用,为阐明棘球蚴、泡球蚴囊液的形成过程以及筛选棘球蚴病新的药物治疗靶点提供理论依据。方法 利用RT-PCR扩增棘球蚴、泡球蚴AQPs基因,构建AQPs体外转录载体,并在非洲爪蟾(Xenopus laevis)卵母细胞中进行异源表达,以验证其水通道功能。利用生物信息学方法分析两种棘球绦虫水通道蛋白跨膜结构域特点及差异,并对棘球绦虫和人AQPs进行多序列比对,分析棘球绦虫AQPs序列结构与人AQPs的差异。结果 通过RT-PCR成功克隆了棘球蚴EgAQP4和EgAQP9、泡球蚴EmAQP4和EmAQP9四个基因,注射了这些基因cRNA的卵母细胞与注射DEPC水的对照组卵母细胞的体积变化率(V/V0)差别无统计学意义(F=1.143,P＞0.05),透水系数差别亦无统计学意义(F=1.416,P>0.05),这四个AQPs基因在非洲爪蟾卵母细胞中未表现出水通道的功能。跨膜结构域预测及多序列比对结果表明,与经典的水通道蛋白相比,EgAQP4和EmAQP4虽然N和C末端均位于胞质侧,但跨膜结构域数目为4个,NPA基序替换为NPM和NPT;而EgAQP9和EmAQP9虽然具有2个保守的NPA基序,但跨膜结构域数目为5个,N末端位于胞外侧、C末端位于胞质侧。棘球绦虫AQPs蛋白结构存在的这些差异,可能与其在卵母细胞中未表现水通道蛋白的功能有关。结论 两种棘球绦虫基因组中都存在编码AQPs的基因,但这些AQPs基因的功能验证试验未见到水通道功能。该结果可能正好说明了囊液的水分子既不能通过AQPs进入生发层细胞而致使细胞肿胀坏死,也不能以AQPs为通道从生发层细胞转移出囊壁,致使囊液累积、棘球蚴包囊或泡球蚴囊泡体积长大。

Comparison of gene clone and functional characteristics of aquaporins of Echinococcus granulosus and Echinococcus multilocularis

We explored the roles of aquaporins (AQPs) of Echinococcus granulosus and E. multilocularis in the formation of hydatid fluid, which may provide a foundation for better understanding the mechanism of formation of hydatid fluid and selecting new targets of chemotherapy for echinococcosis. AQPs genes of E. granulosus (EgAQPs) and E. multilocularis (EmAQPs) were cloned by the method of RT-PCR. Heterologous expression vectors of AQPs were constructed and heterogeneously expressed in Xenopus laevis oocytes in order to identify the water channel activities of these AQPs. The characteristics and differences of transmembrane regions between EgAQPs and EmAQPs were analyzed by bioinformatics method. In addition, multiple sequence alignment was carried out to analyze the structure difference between human AQPs and AQPs of Echinococcus spp. Results showed that two EgAQPs genes (EgAQP4 and EgAQP9) and two EmAQPs genes (EmAQP4 and EmAQP9) were isolated successfully by the method of RT-PCR. The swelling rate (V/V0) of AQPs cRNA-injected oocytes was not significantly different from DEPC water-injected oocytes in control (F=1.143, P＞0.05), as well as osmotic water permeability coefficient (F=1.416, P>0.05). These four AQPs genes could not perform their water channel activities in X. laevis oocyte. Results of transmembrane regions prediction and multiple sequence alignment showed that compared with the conventional AQPs, although N and C termins of EgAQP4 and EmAQP4 were located in cytoplasm, the number of transmembrane regions was four, and NPA motifs were replaced with NPM and NPT; although NPA motifs were conserved in EgAQP9 and EmAQP9, the number of transmembrane regions was five, and N termin was extracellular, but C termin was cytoplasmic. AQPs of Echinococcus could not perform their water channel activities in X. laevis oocyte may be caused by these structure differences between AQPs of Echinococcus and human AQPs. Although AQP encoding genes were identified in the genome of E. granulosus and E. multilocularis, the water channel activity could not be found in the functional verification test of these AQPs genes. All these results may suggest that neither water can enter the germinal layer to cause vesicle lysis nor can water be transported through these AQPs so that the accumulation of hydatid fluid can increase the volume of hydatid cyst or vesicles.