Immnunological Characterization And Antiviral Activity Of Goose Type ? Interferon And Type ? Interferon

Interferon (IFN) is a type of effective antiviral immune protein. IFN will bind their cognate receptors to stimulate STATs to activate the transcription and expression of antiviral molecules during the foreign viral infection process. This study firstly identified the type? and type ? interferon receptor (IFNR) genes, then the IFNa, IFNy and their cognate receptors tissue expression level were analyzed. The recombinant goose IFNa and IFNy proteins were successfully expressed, their immune activity and antiviral function were identified from the multiple aspects such as the immune and related immune cytokines regulation, viral infection and antigen location, as well as its IFN antiviral effects. Results are as follows:1. Molecular cloning of interferon receptorsIn this study, Sichuan white goose IFNAR1 (1616 bp), IFNAR2 (1525 bp), IFNGR1 (1322 b), IFNGR2 (1438 bp) gene completed sequences were cloned by RACE method. Their sequences were all submitted to NCBI and gene accession numbers were acquired as the IFNAR1 (KM504097), IFNAR2 (KM504096), IFNGR1 (KM457284), IFNGR2 (KM461716). The structural domains and molecular evolutionary tree analysis showed that avian IFNRs maintain similar molecular structure and close evolution relationship.2. Tissue distribution and immunological characterization of interferon and its cognate receptorsTissue distribution of goose (1 week) IFN and its receptors were examined by built RT-qPCR. It showed that IFNa, IFNy and IFNR genes were highly expressed in the immune-related tissues. Goose IFNa, IFNy showed the upregulation trend at the different level in PBMCs treated by different immune agonists in vitro. The study showed the goose type? and type ? IFN immune activity in response of TLRs agonists.3. The effects on the goose IFN expression by H9N2, GPV, TMUV infection.The geese were aritificially infected by the H9N2, GPV, TMUV, the antigen signals and IFN transcriptional level were detected by the IHC and RT-qPCR, respectively. AIV H9N2 antigen signal can be obviously detected in spleen, bursa of Fabricius, small intestine, rectum, lung, trachea, and liver. In GPV infected group, GPV signals were strongly detected in liver, lungs, small intestine, and rectum. In TMUV infected group, positive TMUV and CD8 signals can be detected in liver, brain, spleen, small intestine. In geese infected by GPV and TMUV, IFNy was expressed at higher level than control group in several immune tissues (spleen, thymus, Harderian gland) and non-immune tissues (small intestine, liver, lung), its expression is related to the distribution and intensity of CD8 and antigen positive signals. In addition, goose PBMCs incubated with H9N2, GPV or TMUV can produce the dramatic upregulation of IFNa and IFNy.4. The subcellular localization studies of goose type ? IFN and type ? IFN proteins.Based on EGFP skeleton vector, recombinant plasmids pEGFP-IFNa, pEGFP-IFNy were successfully constructed and effectively expressed in BHK21 cells, which were around 46 kDa and 44 kDa, respectively. Morphologies of IFNa appeared in the dot-strings and rings patterns diffusely distributed along the periphery of the nucleus. Morphologies of IFNy showed patterns of dots, spots, diffuse stars, located in the cell nucleus and perinuclear compartment.5. Construction of eukaryotic plasmids and the expression of recombinant proteinsPCR and restriction enzyme digestion results showed that pcDNA3.1-IFNa and pcDNA3.1-IFNy were successfully constructed. Plasmids were then transfected into the BHK21 cells cultured in vitro, western blot analysis confirmed that IFNa and IFNy were effectively expressed in the BHK21 cells transfected by eukaryotic plasmids, the recombinant goose IFNa and IFNy protein were about 20 kDa and 18 kDa, respectively. The results also showed interferon protein can be expressed in cells at higher lever at post transfection 24 hours.6. The antiviral activity of recombinant type? IFN and type ? IFN proteins.Using the western blot, RT-qPCR and FACS analysis, the viral EGFP, viral copies, viral titers were detected. The results showed that exogenous goose IFNa and IFNy protein can effectively inhibit DPV propagation and replication in DEFs, which proved that goose? IFN and type ? IFN proteins exhibit antiviral function. Moreover, goose IFNs can upregulate the duck IFNs and ISGs transcriptional level in duck-origin cells, indicating that they also have the biological activity in duck-origin cells. It provided a basis for the further studies about goose IFN antiviral function and its application.