| Summary: | 博士 === 國立臺灣海洋大學 === 水產養殖學系 === 95 === Mx proteins are interferon induced, anti-viral proteins, expressed in response to treatment with double stranded RNA or virus infection. Here we report the cloning, sequencing and antiviral property of three forms of Mx genes, MxI, MxII and MxIII from grouper (Epinephelus coioides) kidney cDNA library that was treated by poly I:C, LPS, or fomalin inactivated YGNNV and GIV. Multiple comparison of grouper Mx amino acid sequences revealed the conservation of Mx putative GTP-binding domain, dynamin family signature and leucine zipper motif. Phylogenetic analysis revealed that these grouper Mxs along with that of other fish form separate branches independent of avian and mammalian homologues, reflecting a distant evolutionary relationship from birds and mammals.
On tissue specific expression for Mx, Northern blot demonstrates the constitutive expression of Mx transcripts in grouper tissues such as, heart, spleen, kidney and gill and induced expression in brain, spleen, kidney and gill in response to the poly I:C. But, poly I:C failed to up-regulate the Mx expression in liver and intestine. On real time PCR for Mx, the results of YGNNV infection is similar to poly I:C treatment. Surprising, GIV infection induces the expression of MxI gene, and is higher than YGNNV infection. However, the expression of Mx genes is not induced in spleen by GIV infection.
We had established a new cell line from grouper brain (GB3 cell line) and prepared stable clones expressing Flag-epitope tagged grouper MxI, MxII and MxIII. Transient transfection and Immunostaining shows that all the three grouper Mx proteins are localized in the cytoplasm. To examine the antiviral activity of grouper Mx proteins, these stable clones were infected by a nodavirus, yellow grouper nervous necrosis virus (YGNNV) and the results showed that all the three Mx isoforms have the efficiency of reducing the titre of virus from 10 to 100 fold. Moreover, through immunocytochemistry we demonstrated that Mx protein can inhibit the YGNNV propagation in GB3 cells. Taken together, this study demonstrates that grouper Mx proteins have efficient inhibitory activity against nodavirus, the most endangered virus of fish and this information would be helpful to design effective DNA vaccines that can confer an early non-specific antiviral protection.
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