Cloning,Expression And Antiviral Activity Analysis Of Chinese Alligator IFN-?

Interferon(IFN)is an important class of cytokines with a variety of biological activities,including antiviral,involved in immune system cells and so on.IFN is now divided into three subtypes:type ?,type ? and type ?,which exert diverse immune functions in the body.Type ? IFN plays a crucial role in linking innate and adaptive immunity,with IFN-? being the longest recorded cytokine in clinical use.The IFN-?proteins of different species show high homology,and the IFN-a gene contains no introns and has two conserved disulfide bonds.Chinese alligator is a first-class protected animal in China.It has special significance in evolutionary history and has strong immunity.Therefore,it has high research value and economic value.However,there are few studies on immune related to reptiles and Chinese alligator.In this study,the IFN-? gene of Chinese alligator was cloned,and the sequence was analyzed by bioinformatics.The expression of IFN-? in different tissues and hibernating and non-hibernating alligators was determined.The protein was obtained by prokaryotic expression and eukaryotic expression.The antiviral activity was analyzed and the research laid the foundation for further research in the future.The blood of Chinese alligator cultured in a Chinese alligator ecological park in Nanjing was used as the material to extract mRNA for reverse transcription to obtain cDNA.The specific primers were designed for PCR to obtain the IFN-? sequence of Chinese alligator,which was sequenced correctly and cloned into pMD-19T vector.Analysis of the sequence indicated that the complete ORF of the IFN-? gene of Chinese alligator was 672 bp,encoding 223 amino acids,and the first 26 amino acids were signal peptides.The molecular weight of the mature Chinese alligator IFN-? protein is 26 ku,and the isoelectric point is 9.51.The tertiary structure prediction showed that the protein contained 5 segments of ?-helix,which was consistent with the type I interferon structure.The analysis of its homology found that the sequence of Chinese alligator IFN-? and the lyrics of the Alligator mississippiensis IFN-? was the highest.The phylogenetic tree analysis showed that the genetic distance between the Chinese alligator and the Alligator mississippiensis was the closest,and it was clustered together with other crocodiles.One branch,and closer to the turtles and birds,form a sister group,fish is farthest from the Chinese alligator.The expression levels of IFN-? in different tissues of Chinese alligator were determined by real-time PCR.The order from high to low was blood,pancreas,lung,small intestine,liver,kidney and muscle.The expression level of IFN-? in hibernation was lower than that in non-hibernation period.The prokaryotic expression vector pET32a-IFN was correctly constructed and introduced into the E.coli expression system,and the polyclonal antibody was prepared by immunizing several mice with purified recombinant protein.the titers of polyclonal antibodies were 1:25600 and 1:51200.In order to study the antiviral activity of Chinese alligator IFN-?,DF1 cells and Vero cells were cultured,and the titer of vesicular stomatitis virus(VSV)was measured,and the antiviral activity of purified prokaryotic expression of Chinese alligator IFN-? protein was determined,the anti-VSV activity was 2.11×104 U/mL on DF1 cells,and the anti-VSV activity on Vero cells was 1.58×104 U/mL.The antiviral potency of the protein was verified from another perspective by neutralization experiments.The eukaryotic expression plasmid pcDNA3.1-caIFN was constructed and transfected into cells.The indirect immunofluorescence assay proved that IFN-? was successfully expressed and the protein was localized in the cytoplasm.At 48 h,the expression level of Chinese alligator IFN-?was higher than that at 24 h.The eukaryotic expression plasmid pEGFP-N1-caIFN was constructed and transfected into Vero cells to prove that the eukaryotic expression of the Chinese alligator IFN-? protein can inhibit the transcription of VSV virus mRNA and inhibit the proliferation of the virus.Through the above research,it provides a theoretical basis for the breeding and protection of Chinese alligator,and lays a foundation for further understanding of the immune function of Chinese alligator and reptile.Thus,the Chinese alligator can be used as a bridge to understand the relationship between lower vertebrate immune function and higher vertebrate immune function.