Study On Recombinant Vaccine Of Goatpox Virus Vectored Antigen Genes Of Peste Des Petits Ruminants Virus And Foot And Mouth Disease Virus

Foot and mouth disease (FMD), peste des petits ruminants (PPR) and capripox (CP) are threehighly contagious and economically important diseases affecting goats, sheep and other smallruminants, caused by foot and mouth disease virus (FMDV), peste des petits ruminants virus (PPRV)and capripoxvirus (CPV) respectively. The three diseases are published as class A of infectiousdiseases by the world organization for animal health (OIE). At present, the prevention and control ofthese three diseases mainly rely on the inactivated vaccine and attenuated vaccine. The vaccines havethe deficiency of short immune duration, poor thermal stability and reversion of virulence in thepractical application. Therefore, it becomes the objective need to develop more effective and safer newgeneration vaccine.Virus vector is one of the hotspots in the field of modern biology research. A number of viralvectors have been successfully constructed, and become a new vaccine candidate. Capripoxvirusvector is one of these virus vectors. Among three viruses infected goat and sheep, fusion protein (F)and hemagglutinin (H) of PPRV are major antigen inducing organism to produce neutralizing antibody;P1-2A of FMDV can form viral nucleocapsid in the presence of3C, which is the main inducement toproduce neutralizing antibody, and the VP1fragment of P1protein occupies the most concentratedarea of antigenic determinants. Therefore, VP1gene or P12A3C fragment of FMDV and H gene or Fgene of PPRV is the main candidate antigen gene of recombinant vaccines and subunit vaccines. In thisstudy, according to DNA recombination technique, the transferring vectors containing antigen genes ofPPRV or/and FMDV were constructed to develop recombinant capripoxvirus. Finally, severalrecombinant capripoxviruses were generated by inserting the antigen genes of FMDV or/and PPRV tocapripoxvirus genome in the process of homologous recombination between the transferring vectorand the virus. As a live vector transporting exogenous protein, the target of recombinant capripoxvirusis to develop an efficient vaccine that can effectively prevent the PPR, FMD and CP. The results wereas following:1. A transferring plasmid named ptkpp was constructed to use in the recombination ofcapripoxvirus. Furthermore, its function was improved by lengthening homologous flanks, insertingreport gene (GFP) and pressure selection gene (gpt) and introducing multiple cloning site (MCS). Thetransferring plasmids named pftkpgigp and pftkpgigpi were constructed eventually and these plasmidscan be used repeatedly in the capripoxvirus recombination. Moreover, the function of promoter p11and p7.5in these plasmids were demonstrated to ensure their proper work in practice.2. The antigen genes of PPRV or/and FMDV were inserted into the transferring plasmid pftkpgigpor pftkpgigpi and the six recombinant plasmids were constructed and were named pftkpgigp-H,pftkpgigp-F, pftkpgigp-VP1, pftkpgigp-P12A3C, pftkpgigp-H-i-P12A3C and pftkpgigp-F-i-P12A3Crespectively according to the foreign gene. These plasmids can be used in the following experiments.3. The above six plasmids were transfected into the lamb testis (LT) cells infected capripoxvirus previously using Lipofectamine respectively. Consequently, six recombinant virus suspension of firstgeneration were obtained after homologous recombination. However, four recombinantcapripoxviruses were generated by gpt drug selection and GFP selection repeatedly and were identifiedby cytopathic effect (CPE) and PCR method. These recombinant viruses were namedrGPV/FMDV-VP1, rGPV/PPRV-F, rGPV/PPRV-H and rGPV/PPRVF-FMDVP12A3C respectivelyaccording to its exogenous gene contained.4. The transcription and expression of the exogenous genes in the rGPVs was demonstrated byRT-PCR method and immunofluorescence assay at the level of mRNA and protein. The results showedthat all foreign genes were transcribed successfully and their products could be reacted with thespecific antibody, demonstrating the immunogenic activity of the expressing products.5. The goats were inoculated to rGPV with a dose of105TCID50intradermally and high levels ofanti GPV, FMDV and PPRV antibody were induced respectively. There was no significant difference atthe titers of anti-GPV antibody between rGPV and GPV vaccine; however, there was significantdifference at the titers of anti-FMDV antibody between rGPV and FMDV vaccine. Furthermore, therewas no significant difference at the titers of anti-PPRV antibody between rGPV and PPRV vaccine afterbooster immunization.