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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。
The double-stranded RNA virus-induced embryonic cells of the Japanese flounder were constructed successfully by using suppression subtractive hybridization (SSH) technique using the induced embryonic cells of Japanese flounder (Paralichthys olivaceus) induced and induced by UV-inactivated dsRNA virus Grass carp hemorrhagic disease virus (GCHV) FEC) subtractive cDNA library. The differential expression of housekeeping gene αtubin was used as a subtractive marker. The differential subtraction efficiency of this library was 210-fold, indicating that certain differentially expressed genes were also enriched by virus induction. The obtained cDNA fragment was ligated into pGEM T vector, and the PCR test showed that the subtracted fragment was between 250bp and 2000bp. The construction of the subtractive cDNA library laid a solid foundation for molecular immune responses to dsRNA virus in cultured flounder Paralichthys olivaceus and further identification and cloning of differentially expressed genes.