Study On Recombinant Baculovirus-Mediated Genetic Engineering Vaccine Of The Highly Pathogenic Porcine Reproductive And Respiratory Syndrome

Porcine reproductive and respiratory syndrome (PRRS), which caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a serious infectious disease of swine. It is characterized by severe reproductive failure in sows, and respiratory distress in piglets. Vaccine inoculation is the main method to prevent these diseases. Currently, the commercial vaccines to prevent PRRS are mainly attenuated and inactivated vaccines, but the immune effect is not satisfactory or unassured. For the neutralizing antibodies caused by the classical strains can not neutralize the highly pathogenic PRRSV mutantion, so it is necessary to find a new kind of vaccine based on the highly pathogenic PRRSV mutantion.The SFV replicon vector is a eukaryotic expression vector which based on SemLiki Forest virus, it has both the function of self-replication, and high level expression of foreign genes, and the same characteristics to induce the host cell apoptosis as virus. The baculovirus is one of the vectors which has been widely used in recent years, it has been widely used in gene therapy and vaccine development. Recent studies indicate that baculovirus can transfer gene expression cassette drived by mammalian promoter into mammalian cells, and the infected cells can produce large amounts of target protein, but the baculovirs can't replicate and express its genes. At the same time, baculovirus can mediate foreign gene expression in-vivo in mammalians, when made suitable modification or pre-processing.Based on the high transduction efficiency of the recombinant baculovirus and the self-replication and the characteristic of inducing the host cell's apoptosis of the SFV replicon, we used a baculovirus vector in which the vesicular stomatitis virus glycoprotein G (VSV-G) was presented in the viral envelope, so that it can eliminate the complement sensitivity of baculovirus. Then we get a recombinant baculovirus BV-G-CMV/SFV-ORF3/4/5/6 which co-expressing the ORF3, ORF4, ORF5 (modified), and ORF6 of highly pathogenic PRRSV mutants WUH3. The main research works were as following:1. Construction of the "suicidal" DNA vaccine pSCA1-CMV/SFV-ORF3/4/5/6.Amplified the genes ORF3, ORF4, ORF5 (modified) and ORF6 of the highly pathogenic PRRSV WUH3 and the 26s of the SFV replicon, then we got four gene expression cassettes which under the control of 26s about the four genes.Then we inserted these four gene expression cassettes into the plasmid pSCA1, and got a "suicidal" vaccine pSCAl-CMV/SFV-ORF3/4/5/6 which co-expressing the ORF3, ORF4, ORF5 (modified), and ORF6 of the highly pathogenic PRRSV WUH3.2. Construction of the recombinant baculovirus BV-G-CMV/SFV-ORF3/4/5/6.Digested with restriction enzymes SpeⅠand SphⅠ, we got a fragment CMV/SFV-ORF3/4/5/6 from pSCAl-CMV/SFV-ORF3/4/5/6, then inserted this fragment into the vector pFastBac-VSV/G which digested with the same restriction enzyme, we got a shuttle plasmid pFastBac-VSV/G-CMV/SFV-ORF3/4/5/6. Using the Bac-to-Bac(?) system (Invitrogen) following the manufacturer's instructions, we got a recombinant baculovirus vector containing the SFV replicon under the transcriptional control of CMV-IE enhancer/promoter BV-G-CMV/SFV-ORF3/4/5/6 which co-expressing the ORF3, ORF4, ORF5, and ORF6 of the highly pathogenic PRRSV WUH3.3. Protective immunity induced by the recombinant baculovirus BV-G-CMV/SFV-ORF3/4/5/6 in a mouse model.To investigate the specific immune response induced by recombinant baculovirus, several groups of mice were subjected to intramuscular with different doses of recombinant baculovirus ranging from 107 to 109 pfu. The results indicate that the recombinant baculovirus can induce a high cellular immune response and specific neutralizing antibody titer under dose-dependent pattern. The highest level of PRRSV-specific immune responses was observed in mice immunized with 109pfu at 6 weeks after the first immunization, in which the ELISA antibody level were reach to 1:4480 (anti-GP5 protein) and 1:11200 (anti-PRRSV), and the neutralizing antibodies level were reach to 1:39 (CH-1a) and 1:45 (WUH3), and the production of IFN-γin supernatant from cultured speen lymphocytes was found more than 1065pg/mL by ELISA assay. Compared with the "suicidal" DNA vaccine, the data showed a significant difference (P<0.01). These results indicate that the recombinant baculovirus is a promising candidate vaccine against PRRSV infection.