双链RNA病毒诱导的牙鲆胚胎细胞差减cDNA文库的构建

Construction of a Subtractive cDNA Library from the Paralichthys olivaceus Embryonic Cells Induced by a Double-Stranded RNA Virus

以紫外线灭活的dsRNA病毒草鱼出血病病毒(GCHV)诱导和模拟诱导的牙鲆胚胎细胞为材料,利用抑制性差减杂交(SSH)技术,成功构建了双链RNA病毒诱导的牙鲆胚胎细胞(FEC)差减cDNA文库。以管家基因αtub lin作为差减指标,经检测,该文库差减效率达210倍,表明经病毒诱导后某些差异表达基因也得到了相应倍数的富集。将获得的cDNA片段连接到pGEM T载体,PCR检测显示差减片段在250bp^2 000bp之间。该差减cDNA文库的构建为从分子水平研究牙鲆培养细胞对dsRNA病毒的免疫反应、以及进一步鉴定和克隆差异表达基因打下了坚实基础。

Using suppression subtractive hybridization (SSH) technique, a subtractive cDNA library was constructed from Japanese flounder (Paralichthys olivaceus) embryonic cells (FEC) induced by UV-inactivated dsRNA virus GCHV (Grass carp hemorrhage virus). A housekeeping gene, α-tublin, was used to estimate the efficiency of subtractive cDNA. In this library, α-tublin was subtracted at about 2^(10) folds, indicating that some differentially expressed genes were also enriched at about the same folds. The length of the subtractive cDNA fragments cloned into pGEM-T vector ranged from 250bp to 2000bp.The results showed that the subtractive cDNA library is successful, which will be very useful for the understanding of antiviral immune response to dsRNA virus and essential for rapid isolation of differentially expressed genes induced by dsRNA virus.