Construction, Preparation And Immune Effect Evaluation Of PPRV Virus-like Particles

Peste des petits ruminants(PPR) is commomly known sheep plague, also named pseudo rinderpest. It is an acute or subacute and highly contagious disease of small ruminants, caused by peste des petits ruminants virus(PPRV). PPR was included in the single OIE(Office International Des Epizooties) list of notifiable errestrial animal diseases. It is classified as 1st animal disease in our country. The China’s first sample of PPRV apperared in Tibet in July 2007. In 2014, PPRV broke out and spread over 20 provinces and areas in China, causing huge economic losses. In addition, PPRV can transmit by air, the most susceptible animals is small ruminants, especially wild small ruminants is not restricted by national borders, it is easy to lead to the global spread, so, it is particularly important to effective prevention.Attenuated vaccine is only allowed to use with OIE permission to prevent PPRV, however, PPRV is sensitive to heat, it is difficult to keep in the process of protection and transportation of the vaccine in cold chain status,in addition, attenuated vaccine also exists strong a risk of virulence return, this limits the PPR attenuated vaccine widely use in the field, so it is urgent need to develop a safe, effective and good thermal stability vaccine candidate.Compared with attenuatedvaccine and inactivated vaccine, virus-like particles(VLPs) vaccines become one of the security and stability of vaccine candidates. Virus-like particles are contrusted by one or several viral structural proteins which can self-assemb into particulates closely resembling the original virus, and mimic the morphology of the natural virus. With no genetic material in VLPs, they are replication and infection incopetent. VLPs display antigenic epitopes in the correct conformation and in a highly repetitive manner, and leading to highly immunogenic. VLPs can take up by professional antigen-presenting cells effectively, stimulate innate immunity and further elicits adaptive immune and inflammatory responses against infection. Therefore, VLPs represent a major advancement in the development of subunit vaccines with enhanced immunogenicity. Several viruses VLPs have been developed as effective vaccine candidates, such as influenza、hepatitis and PCV II VLPs, which have been progress to clinical trial or approve to commercial production. Therefore, in this study, we use baculovirus-insect cell expression system to construct and produce different PPRV strain VLPs, to selected virus like particles with good immunogenicity to immune animal,laying the foundation for the development of safe and effective peste des petits ruminants virus like particles vaccine candidate.The main research contents are as follows:Chapter one: Prokaryotic expression of M, F, H genes of peste des petits ruminants virus and preparation of polyclonal antibodiesTaking Peste des petits ruminants virus strain(Nigeria 75/1) as the study object, the research designed the primers for the major epitope region of M, F and H proteins by amplifying the PPRV M, F, H gene segment of the size of 1005 bp, 1245 bp and 1653 bp with the major epitope region coding sequence except transmembrane region and signal peptide sequence with the method of RT-PCR(Reverse Transcription-Polymerase Chain Reaction), then M was cloned to prokaryotic expression vector of p ET-30a(+), F and H gene were cloned to prokaryotic expression vector of p GEX-4T-1, constructed the recombinant plasmid of p ET-30a(+)-M, p GEX-4T-F and p GEX-4T-H, and transformed the expression plasmids into Escherichia coli Transetta(DE3)strain. The optimization of the induced protein expression condition is hours induction expression in 0.4 m M IPTG at 37℃, the SDS-PAGE result shows that the molecular weight of expressed M, F and H proteins were identified as61 k D、66 k D and 60 k D, which are consistent with the expected value. Recombinant proteins mainly were expressed in the form of inclusion body and they can be recognized with sheep anti-PPRV polyclonal antibody by Western bolt. Purified recombinant M, F and H proteins by Ni-NTA affinity chromatography were immunized to Kun Ming mice by subcutaneous injection. Blood were collected and produced the polyclonal antibodies of mouse anti-M, F and H protein after three times’ immunization by three weeks intervals, Laying the foundations for the later identification for recombinant baculovirusChapter two: Constructing virus-like particles of peste des petits ruminantsvirus Nigeria 75/1 strain and its immunogenicity study in miceThe MP plays an important role not only in virus entry and membrane fusion but also in virus release and assembly, and it maintains the basic form of PPRV. Thus, in the present study, we chose three proteins as basic elements to construct the peste des petits ruminants virus( PPRV) vaccine strain Nigeria 75/1 VLPs. M、F and H genes fragment were amplified by RT-PCR and then cloned into p Fast BacTMDual vector containing double promoter, to build recombinant donor plasmid p Fast BacTMDual-2M、p Fast BacTMDual-2F、p Fast BacTMDual-2H witch contain double purpose genes. The shuttle plasmid r Bacmid-2M, r Bacmid-2F, r Bacmid-2H were extracted after donor plasmids were transformed into DH10 BacTM cells for homologous recombination, then transfected to Sf9 cells to obtain recombinant baculovirus rp FB-2M、 rp FB-2F、rp FB-2H. The expression of membrane protein(M)、fusion protein(r F) and hemagglutinin protein(r H) were identified byindirect immunofluorescence assay(IFA). It also showed that the positive sera could recognize non-denatured full-length M、F、H protein by IFA. Virus-like particles were constructed with three recombinant baculovirus co-infecting Sf9 cells and then virus-like particles were purified after enlarge cultivation. By electron microscopy, the PPRV VLPs was round or elliptical, and with diameter was approximately 80-100 nm, obvious surface spikes were observed. The sample after purification is identified by Western-blot, showing 38 k D、59k D and 68 k D protein band, indicating that virus-like particles were constructed successfully with matrix membrane protein and two capsule membrane glycoprotein. These results suggest that when expressed in Sf9 insect cells, the MP, H and F of PPRV could self-assemble into PPR VLPs, which named it VLPs75/1.Protective neutralizing antibody can be induced in mice after VLPs were immuned to mice for three times.Chapter three: Constructing virus-like particles of peste des petits ruminantsvirus Tibet30 strain and its immunogenicity study in mice and goatsIn this study, VLPs of PPRV were generated in a baculovirussyetem through expression of PPRV matrix(M) protein, hemaglutin(H) protein, fusion(F) protein and nucleocapsia(N) protein by co-infection in insect cells.Analysis of Western-blot, immunofluorescence, electron microscopy demonstrated expression of the four proteins in insect cells. The PPR virus-like particles(VLPs) were purified by sucrose density gradient centrifugation and showed morphology similar to the native PPR virus particles under the electron microscope. The PPRV VLPs was also indentified by the immunogold electron microscopy. Immunization with VLPs in mice and goats elicited robust neutralization and potent celluar immune response. Mouse studies demonstrated that he PPR VLPs also elicited significantly more IFN-γsecreting CD4+ and CD8+ T cells than the PPR VLPs, but PPR VLPs elicited significantly more IL-4-secreting CD4+ and CD8+ T cells than the PPRV. Moreover, VLPs recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. VLPs induced a strong, specific Ig G2 a response but not an Ig G1 response, suggesting the activation of Th1 class immunity;in contrast,Th2 class immunity was observed with PPRV. VLPs can induce B cells and DC recruitment and/or activation, T lymphocyte proliferation and activation, therefore, the PPRV VLPs has potential to be a new type of animal vaccine which is safe and effective.Chapter four: Constructing virus-like particles of peste des petits ruminantsvirus Tibet30 strain in Hign Five cells and its immunogenicity study in mice and goatsIn order to compare the production of PPRV VLPs in High Five cell and Sf9 cell by using Bac-to-Bac system, in this study, we amplified the PPRV M, F, H gene segment of China/tibet/geg/07-30 strainand constructed recombinant baculovirus rp FB-H-F-M. Theexpression of membrane protein(M)、fusion protein(r F) and hemagglutinin protein(r H) were identified byindirect immunofluorescence assay(IFA). IFA 、 Western blot and hemagglutination test showed High Five cells is not only more efficient packaging virus-like particles, but also have a higher production of PPRV VLPs.We enlarge the cultivation of virus-like particles by ueing recombinant baculovirus to infect High Five cells,then the PPRV VLPs were purified.. Protective neutralizing antibody can be induced in mice after VLPs were immuned to mice for three times. At the same time, PPRV VLPs can induce cellular immunity in mice with higher secretion IFN-γ and IL-4 compared control group.