CLONING, EXPRESSION AND LOCALIZATION ANALYSIS OF A SEQUENCE CONSERVED GENE (RGV-12L) FROM RANA GRYLIO VIRUS

Rana grylio virus (RGV) is a member of the genus Ranavirus (family Iridoviridae) which type species is Frog Virus 3 (FV3). Viruses in Ranavirus can infect fish, amphibian and reptiles and result in high economic loss in aquaculture. Based on the previous studies on morphogenesis, cellular interaction, antigenicity comparison, restriction fragment length polymorphism and major capsid protein sequence analysis, RGV has been identified as an iridovirus similar to FV3. In the study, a sequence conserved gene (RGV-12L) of Iridoviridae was cloned and identified from RGV, it was 894 bp in length and encoded a protein of 297aa with a predicted molecular mass of 33 kD, Identities between RGV-12L and other viruses in Ranavirus ranged from 61.2% to 100% based on amino acid comparison. However, only 13.7% to 37.2% identities acquired when compared to viruses in Lymphocystivirus, Megalocytivirus and Chloriridovirus. The sequence had been submitted to GenBank and got a accession number of GQ849011. A recombinant prokaryotic expression plasmid pET32a-12L containing the fragment was constructed, and the plasmid was transformed into E. coli BL21(DE3), and then the bacteria were induced by IPTG at a finally concentration of 1m mol/L and expressed a 53 kD fusion protein. The protein was purified from inclusion bodies under denaturing conditions using a His-Bind Purification Kit (Novagen), mixed with an equal volume of Freund’s adjuvant (sigma) and used to immunize mice by hypodermal injection once every 7 days. After the fifth immunization, mice anti-RGV-12L serum was connected. The serum was used in Western blotting analysis and immunofluorescence observation. The temporal expression pattern RGV-12L was characterized during RGV infection by RT-PCR and Western blotting, the RGV-12L specific-fragment was barely detected at 4h postinfection (p.i.) and obviously at 8h p.i., and its content increased to high level at 48h p.i.. At protein level, a specific protein band was observed from 8h p.i. and increased to high level at 48h p.i.. To further classify the transcripts of RGV-12L, drug inhibition analysis was carried out by a viral DNA replication inhibitor (Cytosine β-D-arabinofuranoside, Arac), the specific protein band was not detected in samples pretreated by Arac but could be detected in samples untreated by Arac. Therefore, the data confirmed that RGV-12L is a late gene during the in vitro infection. The location of RGV-12L in RGV-infected FHM cells was analysed by immunofluorescence, mice anti-RGV-12L serum was used as primary antibody and FITC-conjugated goat anti-mouse serum was used as secondary antibody, the result showed that the FITC green fluorescence was scattered in cytoplasm and nucleus during RGV infection, whereas no any fluorescence signals was detected in the mock-infected cells. Moreover, some compartments were not only immunostained by mice anti-RGV-12L serum but also stained by hoechst33342 in the cytoplasm of RGV-infected cells. The compartments may be viromatrix in which a large number of RGVs were replicated and assembled, the data suggesting that RGV-12L may participate in virus replication, assembly and release. Collectively, current data indicate that RGV-12L was a late gene of iridovirus encoding protein that distributed both the cytoplasm and nucleus.