The Study On Constructing Equine Influenza H3N8 Virus-like Particles And Tetherin Diversity

Equine Influenza(EI) is a acute and highly contact respiratory infectious disease that is caused by equine influenza virus and infects horses, donkeys and mules, etc., In recent years, with the flourishing development of horsemanship and gradual emergence of horse-breeding industry, the public safety issues of EI are widely concerned in international and domestic.Tetherin protein is regarded as a natural innate immunity factor that is widely used in restricting envelop retrovirus. As a kind of retrovirus, it is proved that EI can be limited budding by Tetherin. However, Tetherin has the diversity of gene level in different varieties or even in the same variety of different individuals. The sequence difference ranges from 0.5% to 4%. On the other hand, sequence difference will impact Tetherin limiting role on EI virus. By collecting horse blood samples, extracting RNA, transcribing to DNA,connecting to pLB vector to measure sequences in the company, remaining existing mutation sequences, and connecting mutation sequences to the pCMV-10 vector, the eukaryotic expression vectors of Tetherin mutation sequence was successfully constructed. A total of 124 Tetherin sequences were obtained from 18 horses, including 26 mutation sequences. Mutation types included replacement, deficiency and early termination. There were some mutation sites with reference values. The eukaryotic expression vector of the mutation provided reference and support for studying site functions of Tetherin and the relation between Tetherin's diversity and functions in the future.Virus-like Particles(VLPs) is the research hotspot in recent years and widely applied in vaccine production, module vector and foundational studies of the function of some specific proteins, and the mechanism of virus infection to cell. However, the study on applying mammalian cell expression system to construct EI virus VLPs was never reported before. Moreover, the mammalian cell expression system can decorate VLPs in highest level and make VLPs have the native conformation with extremely high pixel just like the natural virus particles. Particularly in the basic study, the VLPs constructed by the expression system are equipped with stronger simulation and process reproducibility. The experiment selected four main structure proteins, including HA, NA, M1 and M2 in EI H3N8(A/equine/Miami/1/63), to construct eukaryotic expression vectors firstly and to collect VLPs in cell supernatant by cotransfection on 293 T cells. Supernatant was measured by HA test and the HA titer was 23 titer. Next, the home-made EI antibody was used for western-blot test and interacted with VLPs in the ultra-purified supernatant. The obvious HA belt was observed by western-blot test. The experiment proved that the mammalian cell expression system was used to construct VLPs of EI successfully. The successful EI VLPs can be used for subsequent VI virus study in laboratoriess. What's more, some mechanisms should be further discussed and this study could help a lot in the basic research.