The Study Of Antiviral Activity Against FMDV By IFNα/TFNγ/sIFITM3 And Construction And Immunogenicity Study Of Foot-And-Mouth Disease Recombinant Pseudotyped Baculovirus

Foot-and-mouth disease (FMD) is a highly contagious disease, it can cause disease of cloven-hoofed animals. The disease is caused by the foot-and-mouth disease virus (FMDV). The typical symptoms of the disease are fever, lameness, blisters produced on the hoof, tongue, mouth, breast of the sick animals. Although being the high disease incidence, the disease has a low mortality rate. Young animals have a high mortality rate. The disease not only reduces the causative ability of the animal, decreases the milk yield of female animals, causes the miscarriage of the pregnant female,result in great economic losses, but also produce a lot of the negative impact in the international economic and trade. Now the main strategies for the prevention and treatment of the disease mainly rely on the inactivated vaccine. However, the disease has seven different FMDV serotypes and rapid antigenic variation, there is no cross-protection between different serotypes, there only is a part of cross-immunity between different subtypes and the vaccine has a security risk, therefore more and more researchers pay attentions to the development of safe and efficient novel biological agents.Interferon (IFN) is a pleiotropic cytokine, it can interact with the corresponding interferon receptors, which activate the signaling pathway, then induce a large number of transcription of the interferon-stimulated genes (ISGs). ISGs express a series of corresponding functional protein which realize a variety of functions at last. IFITM3 is induced by IFN, it is a transmembrane protein family which play an important role in anti-viral, anti-tumor and immune surveillance.The study is carried out on the FMDV new biological research. In this study, we use the baculovirus containing VSVG as the vector, get the recombinant baculovirus which express IFN-a/y or sIFITM3, then evaluate the antiviral effect at the cellular level. At the same time, we build the recombinant pseudotype baculovirus expressing IFN-y and FMDV immunogenic gene, and detect its immune effect. The main study are as follows:1.The expression of IFNα/IFNγ/sIFITM3 mediated by baculovirus and the antivirus effect against FMDV.In recent years, the baculovirus is more and more applied to research and clinical production, not only because of its advantage in expression and modified, but also there is no security issue. Baculovirus do not infect the vertebrate cell, thus insert the G protein gene of vesicular virus into the baculovirus vector to promote the fusion of the membrane, which can mediate recombinant baculovirus’ access into cell and expression efficiently. This study insert IFNa/IFNy/sIFITM3 of swine into baculovirus vector pFB1-VGVG, then get three transfer vector of recombinant pseudotype baculovirus. The transfer vector are transformed into DH10Bac competent, at last get the recombinant bacmid by resistance and blue-white screening. The recombinant bacmid is transfected into sf9 cell by Lipofection Reagent to get the recombinant pseudotype baculovirus Ac-VSVG-IFNα/IFNγ/sIFITM3. We also construct the recombinant pseudotype baculovirus which expressing the enhanced green fluorescent protein (eGFP). In vitro, Ac-VSVG-eGFP is transducted into IBRS-2 cell, and it is best at 37℃。 Western blot show that the interest protein is successfully expressed in IBRS-2 cell. Plague reduction assay show that:the IBRS-2 cell transducted by Ac-VSVG-IFNα/IFNγ/sIFITM3 at 1 multiplicity of infection(MOI) does and 0.1MOI does can completely inhibit 100PFU FMDV; IBRS-2 cell transducted by Ac-VSVG-IFNα/IFNγ/sIFITM3 at 0.01～0.001MOI also can reduce the plague numbers significantly. The result show that Ac-VSVG-IFNα/IFNγ/sIFITM3 have a significant effect. Ac-VSVG-IFNγ is transducted into IBRS-2 cell with Ac-VSVG-IFNα/sIFITM3 respectively, the plague reduction assay show that it has significant synergistic antivirus effect. Ac-VSVG-IFNα/IFNγ transduce IBRS-2 cell,the transcriptional level of ISGs in IBRS-2 cell remarkable enhance detected by fluorescence quantitative PCR.2.Construction and immunogenicity of the recombinant virus vaccine mediated by baculovirus which expressing FMDV P1 and IFN-λThe results of the above studies show that Ac-VSVG-IFNγ has prominent anti-proliferation of FMDV. In order to obtain a better FMDV vaccine candidate strains, we construct a recombinant pseudotype baculovirus expressing IFN-γ and FMDV immunogenic gene. The P1 gene of FMDV is inserted into the transfer vector of recombinant pseudotype baculovirus, Ac-VSVG-IFNγ-2P1 is obtained by the method descried above. The IBRS-2 is transducted by Ac-VSVG-IFNγ-2P1 at 1MOI and 0.1 MOI does, which can completely inhibit the proliferation of 100PFU gengma strains and Es strains. Swine is immuned by intramuscular injection at 7.5×107 PFU/head does, booster immune once after four weeks. Neutralization test and ELISA test show that the recombinant pseudotype baculovirus Ac-VSVG-IFNγ-2P1 can effectively induce FMDV antibody production after immunization, and the antibody levels have been increasing, antibody levels significantly improve after the second immunization.It is indicating that the recombinant baculovirus has a good prospect.