Chimeric PPV Virus Like Particles Displaying The Immunoreactive Epitopes Of The South African Territories Serotype 2 Foot And Mouth Disease Virus

Foot and Mouth Disease Virus?FMDV?is divided into 7 serotypes:A,O,C,Asia l,South African Territories?SAT?1,2,3.Among them,SAT FMDVs have obvious regional characteristics,mainly prevailing in the South of the Sahara desert and affecting buffalo and other cloven-hoofed animals.In recent years,SAT2 FMDV was reported to appear in the Middle East after crossing the long-standing regional boundaries and will pose a potential threat to our livestock industry.There are few researches on SAT2 FMDV in China,so it is necessary to understand its protein structure and immunoreactive epitopes for the preparation of vaccine required as the strategic reserve.In this study,B cell epitope:VYTKAAAAIRGDRAALAAKYADTNHTLPPTFNFGYVTVDK and T cell epitope:NVQEGRRKHTDVAFLLDRST?PAT/1/2012,GenBank:JX014256?were selected according to the related references,laying a foundation for the further design and development of epitope-based SAT2FMDV vaccine.Porcine Parvovirus?PPV?can cause miscarriage,sterility,stillbirth,mummified foetus and weak foetus in primary sows and serologically negative perinatal sows.The PPV positive rate in the pig herd is as high as 85%,causing huge losses to the swine industry in China.VP2 accounts for more than 80%of viral capsid and can self-assemble into PPV particles.VP2 protein has four Loop rings located on the surface and can assembled into chimerical virus-like particles?VLPs?with exogenous epitopes,which can induce the body to produce specific antibodies and cytokines.In this study,the screened SAT2FMDV epitopes were embedded in different sites of PPV VP2 protein and assembled into chimeric VLPs which were expected to achieve double effects of being against PPV and SAT2 FMDV after the immunization of animals.In this study,the Bac-to-Bac system was firstly used to construct a recombinant baculovirus containing the optimized VP2 gene of PPV AV30 strain.Then the recombinant baculovirus was infected sf9 and HF cells to express VP2 protein,with a molecular weight of about 64 KDa.Transmission Electron Micrograph?TEM?showed that the expressed VP2 protein self-assembled into VLPs,named VP2-VLPs,which was similar in size and shape to the purified PPV AV30 authentic virion.BALB/c mice were stimulated to produce anti-PPV specific antibodies after immunization of VP2-VLPs.Spleen lymphocytes isolated from the immunized mice proliferated significantly under the stimulation of PPV AV30.The levels of cytokines including IFN-?,IL-2 and IL-4 were significantly increased in the sera from the immunized mice.Furthermore,the effects of the embedded epitopes on the VP2 protein structure were analyzed via bioinformatics approach.Three sites of the VP2 protein were selected as the candidates,i.e.,the Loop2,N-terminal and the region immediately following Loop4.The chimeric proteins carrying the epitopes of B cell and T cell of SAT2 FMDV were named as L₂B,NT₁L₂B,NT₁L₂B₄B,and N?T₁?₂L₂B₄B.The chimeric protein encoding gene was inserted into the vector pFastBac^TMDual.Western Blot and IFA analysis showed that the recombinant chimeric proteins were expressed in HF cells.Observed by TEM,four recombinant chimeric proteins were self-assembled into VLPs,which were similar in size and shape to VP2-VLPs.This study laid a foundation for the preparation of SAT2 FMD vaccine and expected to achieve the purpose of preventing both SAT2FMDV and PPV.