Preliminary Antiviral Mechanism Study Of Alligator Sinensis Viperin

Viperin,a protein molecule with high efficiency and broad spectrum antiviral activity and closely related to endoplasmic reticulum and induced by IFN,belongs to the family of interferon stimulating gene family.Viperin has been proved to be able to stimulate in the presence of interferons and different viruses.Under the induction of expression and play antiviral effects,involved in cell signal regulation and other biological related effects.It plays an important role in innate immunity.With the deepening research,the mechanism of antiviral function of Viperin is more and more specific and other biological functions are constantly being discovered.Therefore,since the discovery,related research of Viperin has been aroused more and more attention.Alligator sinensis is now under a national level animal protection,dating back 200 million years of history,they have an important historical evolution in history.Research of anti-virus and immune function of Chinese alligator,contributes to the control and prevention of natural reserves and wild Chinese alligator diseases and ensures the scientific and sustainable development of alligator breeding.In this study,we amplified the Viperin gene of Alligator sinensis,analyzed it by bioinformatics methods and verified the antiviral function of Viperin and reveal the key anti-viral function domain of Viperin.The perfect understanding of its immune system will have important implications for the study of human innate immunity.In order to study the sequence structure and biological function of Viperin Alligator sinensis,the peripheral blood total RNA of Alligator sinensis was extracted and the cDNA was obtained by reverse transcription.The primers were designed and the cDNA was amplified by PCR and ligated into pMD-19T vector.The recombinant plasmids were confirmed by PCR and the positive plasmids were sequenced.Bioinformatics analysis of the sequenced sequences includes amino acid sequence analysis,antigenic analysis,phylogenetic tree analysis,homology analysis,protein structure and function prediction.The sequence was submitted to the GenBank database under an accession number KY464129.The complete ORF sequence of the Viperin gene of Alligator sinensis is 1011bp,encoding a total of 336 amino acids.The first 18 amino acids are signal peptides.The molecular weight of the mature protein was 38 kDa and the isoelectric point was 8.72.Alligator sinensis Viperin contains N-terminal domain,SAM domain and C-terminal domain.The tertiary structure showed that the protein mainly consisted of 8 a-helices and 8?-sheets,which was consistent with the structure of classical Viperin,all of which are TIM barrel structures.By homology and phylogenetic tree analysis,Alligator sinensis Viperin had the highest homology with C.crocodyns,accounting for 96.4%of the total,with the same branch in the phylogenetic tree.The expression of Viperin in different tissues of Alligator sinensis was measured and analyzed.The expression of Viperin in each tissue was found to be in an descending order of blood,small intestine,liver,kidney,heart and muscle.The expression levels of Viperin in blood of different age groups also differed,and the results laid the foundation for the study of the regulation mechanism and function of Viperin gene in Alligator sinensis.The Viperin gene fragment was ligated with pET-28a.The prokaryotic expression and purification of the protein was performed to immunize rabbits.The serum was collected in 4th immune time and the polyclonal antibody of Viperin was successfully produced.The complete gene and truncated Viperin were respectively ligated into the eukaryotic expression vector pcDNA3.1/myc-His A,transfected into chicken fibroblast cells.NDV-infected chicken fibroblasts transfected with Chinese alligator Viperin to detect Viperin protein anti-virus ability and found that intact Viperin protein has the highest antiviral ability,N-terminal truncated Viperin protein also has certain antiviral ability,while C-terminal truncated Viperin protein has almost no antiviral ability,the inhibition rate test result shows that intact Viperin has an inhibitory rate of 83.75%for chicken fibroblast NDV infected with 100%cytopathic effect,23.75%inhibition rate for N-terminal truncated Viperin,and 2.3%for C-terminal truncated Viperin.It proved that C-terminal protein plays a pivotal role in the antiviral ability of Alligator sinensis Viperin,and the localization of Viperin in the cytoplasm through indirect immunofluorescence assay laid the foundation for future study on antiviral mechanism of Viperin.In summary,this study studied the bioinformatics characteristics of Viperin alligator sinensis,explored the expression of Viperin in vitro preliminary study of anti-viral function,found that alligator Viperin have the same antiviral features as other species,which lays the foundation for more in-depth study of Viperin's function and innate immune related research,and also provides a theoretical guarantee for the prevention and scientific management of Alligator sinensis.