【摘要】 草鱼呼肠孤病毒(Grass carp reovirus, GCRV)可引发草鱼(Ctenopharyngodon idellus)出血病,导致高死亡率。草鱼吻端成纤维细胞(Grass carp snout fibroblast cells, PSF)是GCRV的敏感细胞系。JAM-A (Junctional adhesion molecule A)为免疫球蛋白超家族成员,是多种病毒的细胞受体。本研究在前期克隆到草鱼3种jam-a基因,命名为gcjam-a1,gcjam-a2和gcjam-a3。在获取ORF序列的基础上,利用qRT-PCR分析了3种gcjam-a在草鱼胚胎及幼鱼不同发育时期及PSF细胞中受GCRV(GD108株)感染前后的表达模式。结果显示,检测的13个胚胎及幼鱼发育时期中,gcjam-a1在未受精卵中高表达,在受精卵至出膜前的胚胎表达水平均较低;从出膜后1～3 d表达量开始上升;出膜6～15 d均呈高水平表达。gcjam-a2与gcjam-a3在草鱼胚胎及幼鱼发育各阶段表达水平较低。在无病毒感染的PSF细胞中,gcjam-a只有少量表达。受GCRV-GD108感染后,病毒S7基因在PSF细胞中的拷贝数随时间呈显著上调趋势,gcjam-a的表达量也有不同程度的上调,mRNA上调水平为gcjam-a1>gcjam-a2>gcjam-a3。本研究证实了3种gcjam-a基因在PSF细胞中的表达均与GCRV-GD108感染相关,其中,gcjam-a1的表达水平受GCRV-GD108感染影响最大,同时,它在孵化出膜后表达上升,推测它可能与病毒的感染更相关。gcjam-a1可作为下一步GCRV与宿主互作研究中的候选分子。更多还原

【Abstract】 Grass carps are seriously threatened by GCRV(grass carp reovirus) that can cause high mortality to fingerling and yearling grass carps. Grass carp snout fibroblast cells(PSF) are highly sensitive to GCRV. Junctional adhesion molecule A(JAM-A), an immunoglobulin superfamily member, acts as a viral cell receptor. In our previous study, the cDNA sequences of grass carp jam-a1, jam-a2, and jam-a3(named gcjam-a1, gcjam-a2, and gcjam-a3) were cloned. Based on this, q RT-PCR was used to analyze the expression pattern of gcjam-as at different embryonic and juvenile development stages and in GCRV-GD108-infected PSF cells. The results showed that the expression pattern of gcjam-a1, 2, and 3 differed during the embryonic development stages. m RNA expression of gcjam-a1 could be detected in unfertilized eggs and at a lower level from the fertilized egg stage to 1 dph(day post hatch). However, the m RNA was highly expressed at 1～3 dph and high levels were maintained from 3 dph to the end of the experiment(15 dph). The expression of gcjam-a2 and gcjam-a3 was very low at different embryonic development stages compared to that of gcjam-a1. gcjam-as were only slightly expressed in non-infected PSF cells. After GCRV-GD108 infection, the expression of S7 in PSF cells increased significantly, and the expression of gcjam-as in PSF cells also increased to different levels after GCRV-GD108 infection. Upregulation of the gcjam-as was in the order: gcjam-a1> gcjam-a2>gcjam-a3. The results showed that the expression of jam-as was related to GCRV infection in PSF cells and that the expression of jam-a1 was most influenced by GCRV-GD108 infection. It is also expressed in early embryonic development, suggesting that jam-a1 is the most relevant to GCRV infection. This study will lay the foundation for further research on GCRV receptors.更多还原