CONSTRUCTION OF ANTIVIRAL SUBTRACTIVE cDNA LIBRARY OF CULTURED FISH CELLS

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 215 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.