Research On Functional Mechanism Of 3nIFNa And 3nIFNa2

Higher vertebrates utilize immune systems to protect them against the invasion of pathogen microbes,which are classified into innate immune system and adaptive immune system.The innate immune system provides an immediate,but non-specific response,which exists in all plants and animals.Fishes possess both immune systems;but they depend much more on the innate immune system.IFNs are antiviral cytokines and play key roles in both innate and adaptive immune responses in higher vertebrates;The interferon(IFN)is crucial to host resistance to viruses.Triploid crucian carp is much more resistant than its female parent(Red crucian carp(2nRCC))and male parent(The allotetraploid(4nAT)).In this laboratory,we carried out extensive research on the innate immune mechanism of triploid crucian carp,and tried to clarify the mechanism of strong resistance to triploid crucian carp.Our preliminary work has cloned two kinds of interferon of 3nIFNa and 3nIFNa2,and constructed its recombinant expression plasmid,we has successfully expressed 3nIFNa and 3nIFNa2 in eukaryotic cells.The experimental results show that 3nIFNa is a secreting cytokines,and 3nIFNa2 is an intracellular cytokine.In this paper,we investigated the functional mechanisms of 3nIFNa and 3nIFNa2 in innate immune response.The specific contents and results are as follows:1.The triploid crucian carp was injected with SVCV,GCRV virus or PBS buffer for 41h after injection and RNA was extracted from the tissue and reverse transcribed into cDNA as template.The selected tissues included heart,skin,spleen,kidney,intestine,liver and gill.Real-time quantitative PCR showed that the mRNA expression levels of 3nIFNa and 3nIFNa2 in the SVCV and GCRV groups were significantly higher than those in the PBS group.2.Western blot experiments show that 3nIFNa was a protein that secreted cells.Therefore,EPC cells were pretreated with the collected supernatants which contain different concentrations of 3nIFNa for 24 h.After treatment,EPC cells were infected with SVCV or GCRV virus.The supernatant was collected after 24 hours of infection and the virus titer was measured.The results showed that the virus titers in the supernatant media were much lower than those of the control cells.3.EPC cells which overexpressed 3nIFNa and 3nIFNa2 recombinant expression plasmids were infected SVCV or GCRV virus,the supernatant was collected after 24 hours and the virus titer was measured.The results showed that the EPC cytopathic effect of the recombinant plasmid was significantly decreased,and the virus titer of the supernatant was significantly lower than that of the control group.4.Glucosidase digestion experiments showed that 3nIFNa2 had an N-linked glycosylation modification;The glycosylation site is at the 177 amino acid site that an asparagine.Construction of its non-glycosylated mutant 3nIFNa2-N177Q,Western blotting experiments showed that 3nIFNa2-N177Q was an non-secreted protein.Crystalline violet experiments and plaque experiments showed that both 3nIFNa2-N177Q and wild-type 3nIFNa2 had the ability to resist SCVC and GCRV.