| Chinese giant salamander Ranavirus (CGSRV) belongs to Iridoviridae, Ranavirus, which is a new pathogeny of the cultured Chinese giant salamander in recent years. The characteristics of this disease are high morbidity and mortality. Therefore, the disease poses a serious threat to the healthy culture of Chinese giant salamander. The prevention and control of this virosis have become a research hot point. RNA interference (RNAi) is a significant technology, has been used to analyse gene expression regulation and function in organisms.RNAi uses the small interfering RNA (siRNA) that shares the identical sequence with the target gene as sequences specific destruction trigger to silence the mRNA. Therefore, RNAi could target the viral mRNA to specifically silence viral gene cloning and viral protein synthesis. RNAi is a result of biological evolution, which takes part in a variety of eukaryotes functions, including anti-virus, genomic rearrangements, chromatin remodeling, maintenance of stem cells, construction of brain morphology, decision of development timing and so on.The goal of this study is to research the main function gene expression and proliferation of CGSRV by RNAi. Using the method of chemosynthesis, the specific siRNAs of major capsid protein gene, methyltransferases gene and DNA polymerase gene were achieved and were transfected into Epithelioma Papillosum Cyprini (EPC) cell before CGSRV infection. The effects of siRNAs on CGSRV proliferation were directly detected by observing the cytopathogenic effect (CPE) each 12 hours. The genes expression after RNAi was detected by real-time fluorescence quantitative PCR. And the virulence of released virus was detected by CGSRV titration assay. Both analysis of the expression rate of specific genes and the virulence of released virus were to indentify the influence by RNAi.At first, the specific siRNAs of major capsid protein gene, methyltransferases gene and DNA polymerase gene were designed and synthesized. Three or four siRNAs were designed for each genes and the negative control siRNA (NC-FAM) was signed with fluorescence. NC-FAM was transferred into EPC cell in different concentrations by using siRNA-Mate Reagent. The optimized conditions were obtained in 24-well plates with 80 nM of siRNA concentration and 200 uL of transfection volume. The inoculative virus concentration was 1000 times-fold dilution of virus suspension with 150 μL of inoculation volume. Through observing the CPE after RNAi every 12 h, the result preliminarily showed that the best interference effect of siRNAs were siR-MCP-3, siR-MCP-4, siR-MT-1, siR-DP-1 and siR-DP-2; then siR-MCP-1, siR-MCP-2, siR-MT-2 and siR-DP-3 were slightly worse and the siR-MT-3 had the weakest interference effect. The relatively expression rates at 24h, 36h,48h,60h,72h and 84h of the MCP gene, MTases gene and DNA polymerase gene after RNAi were detected by real-time fluorescence quantitative PCR. The result showed the relatively expression rates decreased at differenct degrees:the best interference rate of the MCP gene, MTases gene and DNA polymerase gene was 75%, 78%and 81% respectively; and the siRNAs were siR-MCP-4, siR-MT-1 and siR-DP-1 correspondingly. TCID50 assay was aslo carried out to detecte the virulence of released virus. The result showed TCID50 decreased 1294-time,1109-time, 1000-time after interfering MCP, MTases and DNA polymerase gene by RNAi respectively.Above all, the specific siRNAs designed in this research could reduce expression of the MCP gene, MTases gene and DNA polymerase gene and decrease virulence of released virus to a certain extent. This conclusion can provide reaearchers scientific basis to gene therapy for Chinese giant salamander ranavirus disease. |
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