Studies On The Cloning, Expression And Antiviral Activity Of Typeâ… IFN In Chinese Giant Salamander(andrias Davidianus)

Chinese giant salamander(Andrias davidianus), a rare animal in China, is the largest extant amphibian species in the word. In the evolution history of vertebrate, the Chinese giant salamander represents a transitional form that links the aquatic animals to terrestrial organisms, so it is of an important value in scientific researches of the origin, evolution biodiversity and gene mutation of species. Moreover, the Chinese giant salamander has high nutritional value and medicinal value. In the recent years, with the rapid development of the artificial breeding and cultivation of Chinese giant salamander, a severe infectious disease caused by the Chinese giant salamander iridovirus has been spreading throughout China and caused huge economic losses. A better understanding of the antiviral defense mechanism in the Chinese giant salamander to the virus infection is essential for the efficient control of the disease. Interferons are secreted cytokines with a broad-spectrum of anti-viral, anti-tumor and immunomodulatory activity. Except for the model organism Xenopus Laevis, there are limited information about antiviral functions of TypeⅠIFN in other amphibians. In this study, the full-length c DNA and genomic DNA sequence of TypeⅠIFN were cloned, identified and characterized from Chinese giant salamander and its antiviral activity against GSIV was examined in vitro. The results reported herrin provide a fundamental base for the further study of immune defense system and antiviral infection mechanism in Chinese giant salamander. The results are as follows:1.Based on the template of total RNA extracted from the Chinese giant salamander kidney, the full-length c DNA sequence of gs IFN was cloned by RACE PCR. The full-length c DNA of gs IFN is 1113 bp and encodes a putative protein of 186 amino acids with 19 aa sinal peptide. The deduced amino acid sequence ha s the C-terminal CAWE motif, which is mostly conserved in the higher vertebrate TypeⅠIFNs. Based on the template of liver genomic DNA, the full-length genomic DNA sequence of gs IFN was determined using Genome Walking. In genomic arrangement, the gs IFN consisted of 5622 bp, including 5 exons and 4 introns.2.The q RT-PCR results indicated that the transcripts of gs IFN were detected in all selected organs. The highest expression levels of gs IFN were observed in the blood, kidney and spleen. After stimulation of PBLs with 5 μg/ml poly I:C, gs IFN m RNA expression increased at 4 h post-exposure, and then reached the peak level at 12 h(2.8-fold). After stimulation of PBLs with 25 μg/ml of poly I:C a significant up-regulation of gs IFN was observed at 24 h(3.6-fold). After the PBLs were exposed to GSIV the expression of gs IFN was significantly enhanced at 12 h(13.7-fold). The gs IFN expression level was significantly increased in freshly isolated peripheral blood leucocytes after poly I:C and GSIV stimulation, suggesting the importance of this cytokine in the antviral immune response of the Chinese giant salamander.3. Constructing the expression plasmid of p EGFP-N1-IFN and transfecting the Chinese giant salamander muscle cell line(GS-M) cells to over-express the gs IFN in GS-M cells, the gs IFN could induce the expression of the downstream Mx gene and significantly suppressed the CPE formed in gs IFN over-expressed cells after GSIV infection. In addition, in gs IFN over-expressed cells the transcription levels of the GSIV MCP and IE-ICP46 genes were reduced 4 and 7-fold at 24 h, respectively, compared to non-transfected cells. At 48 h, GSIV MCP and IE-ICP46 transcripts decreased about 8 and 12-fold, respectively, compared to the normal cells. The virus titer in gs IFN over-expressed cells was about 1.5 magnitudes lower than that in the normal cells. The immunofluorescence assay indicated that the MCP synthesis was inhibited in gs IFN over-expressed cells after infection. This result demonstrated that the gs IFN has an inhibitory effect on GSIV replication and showed a significant antiviral activity.