【摘要】 紫外线灭活的草鱼出血病病毒 (GCHV)能诱导鲫囊胚培养细胞 (CAB)产生高滴度的干扰素 ,从而诱导宿主细胞基因表达的改变并处于抗病毒状态。提取灭活病毒诱导未经病毒诱导的CAB细胞mRNA ,利用抑制性差减杂交技术 ,成功构建了鱼类培养细胞抗病毒基因差减cDNA文库。以鲫管家基因α tubulin和 β actin作为差减指标 ,检测差减cDNA文库的差减效率分别高达 2 15和 2 7倍 ,表明经过病毒诱导后的细胞中 ,某些差异表达基因的富集效率也接近 2 15倍。鱼类抗病毒基因差减cDNA文库的建立对快速分离、克隆鱼类抗病毒相关基因和认识鱼类细胞抗病毒免疫的分子机理有重要意义。更多还原

【Abstract】 The vertebrate interferon (IFN) system provides an initial line of defense against viral infection by inducing the synthesis of proteins that inhibit virus replication, but the normal cells do not, as a rule, contain or synthesize IFN. UV-inactivated GCHV is very effective for inducing blastulae embryonic cells of crucian carp (Carassius auratus L.)(CAB) to secrete interferon, which in turn act in both autocrine and paracrine fashion to induce the expressions of interferon responsive genes including antiviral genes, and finally contribute to form antiviral states in host cells. However, to date little was known about these proteins and genes. In order to analyze the change of gene expressions and isolate these related genes induced or activated by virus infection in CAB cells, an antiviral subtractive cDNA library of CAB was constructed by using Suppression Subtractive Hybridization (SSH) techniques. Typically, GCHV was purified and inactivated under UV irradiation for 5 min, and then CAB cells were treated with UV-inactivated GCHV. The mRNAs were extracted from virally infected CAB cells and mock-infected cells, respectively, and used for constructing subtractive cDNA library. In the present paper, the quality of cellular RNAs and adaptor ligation efficiency were analyzed, and two housekeeping genes, α-tubulin and β-actin, were used to estimate the efficiency of subtractive cDNA. In the antiviral subtractive cDNA library, the two housekeeping genes were subtracted very efficiently at appropriate 2 15 and 27 folds, respectively, indicating that some differentially expressed genes including antiviral genes induced by viral infection were also enriched at the same folds. The subtractive cDNAs were ligated into the pGEM-T EASY vector (Promega) and following transfection into competent E.coli cells. 16 colonies were randomly selected from the plasmid library, and PCR anlysis showed that the inserted cDNA fragments were between 0.2—2kb in length. The results suggected that the antiviral subtractive cDNA library is very successful, which will be essential for rapid isolation of fish antiviral genes and helpful for elucidation of molecular mechanism of fish innate defense against virul infections in the future.更多还原