Construction Of Recombinant Porcine Parvovirus-Like Particles With Somatostatin And Its Immune Efficacy Evaluation

Porcine parvovirus (PPV) is one of the major etiological agent of reproductive failure in pigs. PPV is an extremely durable and highly infectious virus. Pigs of all ages are susceptible to this virus, the clinical signs of PPV are characterized by abortion, fetal death, fetal abnormality, mummification and aciesis, It also causes the dermatitis and diarrhea. PPV is epidemic in most regions of the worldwide, and causes economic losses to the swine industry in the worldwide. VP2 protein is the major capsid protein of virus particles, with emagglutination activity, accounting for 80% of the total viral capsid proteins. VP2 protein carries the major antigenic determinants, which can induce the protective neutralizing antibodies. When expressing VP2 in vitro, it not only can self-assemble to a complete virus-like particles, but also can be used as antigen delivery carriers to display other antigens. So, it is expected to become a good multivalent vaccine vector which has been an important research direction for virus vaccine.Somatostain (SS) is a peptide hormone composed of 14 amino acids and secreted by hypothalamus which acts as inhibitior on growth. Exhaustion or immune neutralization of somatostain is able to improve growth hormone (GH) level and animal growth, peptide chain of somatostain is short and weak immunogenicity, so it need cloning to other vector enhance immunogenicity.In this study, the VP2 capsid protein of Porcine parvovirus was used as a antigen carrier which not only can self-assembles into empty virus-like particles(VLPs),but also has the high immunogenicity and High-level expression when expressed in baculovirus system. PPV-VLPs has been engineered to express exogenous polypeptides, that can induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. Four copies of somatostatin gene was inserted to N-terminus of VP2 to generate Chimeric virus-like particles (CVLPs), which can be used to develop vaccine for preventing Porcine parvovirus infection and promoting the growth of animals. This idea of vaccine development has been little reported. Based on the above mentioned research background, recombinant Baculovirus expressing the fused gene of PPV VP2 and SS was constructed and the immunogenic effect was evaluated. This paper includes:1 The construction and identification for recombinant Baculovirus the fused gene of porcine parvovirus VP2 and somatostatinAccording to the sequence of PPV NJ-a strain gene published in the GenBank, A pair of primers was designed to amplify VP2 gene. The VP2 gene was cloned into baculovirus vector pFast-HT A, and the positive recombinant plasmid was named as pFast-VP2. Four copies of the synthetic somatostatin gene was identified and cloned to N-terminal of VP2 gene in pFast-VP2, obtaining recombinant plasmid pFast-SS4-VP2. Sf-9 cell was transfected with the positve reBacmid-SS4-Vp2, following the pFast-SS4-VP2 was transformed into DH10Bac competent cells and identified with blue-white screening and PCR analysis. When the cytopathic effect (CPE) was obvious, the transfected sf-9 cell was harvested from the cell plate, and then the positive recombinant virus was named as rBac-SS4-VP2 (P1). The P2 and P3 generation recombinant virus was obtained by infecting sf-9 cells with P1 and P2 generation virus sequently. The insertion for the target gene into Baculovirus genome was proved with PCR analysis; The expression for target protein was identified with SDS-PAGE, Western-blot and indirect immunofluorescence (IFA); The virus particle self-assembly was observed through electron microscope.2 The immunogenic efficacy for the recombinant protein SS4-VP2 expressed in BaculovirusIn order to test the immunogenic efficacy for recombinant protein SS4-VP2 obtained in forth chapter,80 four-week-old mice (clean grade) were randomly divided into eight groups with ten of them in each group, respectively. Mice in groupⅠwere injected with purified rSS4-VP2/oil; Mice in groupⅡwere injected with purified SS4-VP2/Alhydrogel; Mice in groupⅢwere injected with purified SS4-VP2/IMS; mice in groupⅣwere injected with purified SS4-VP2/Alhydrogel+ CpG; Mice in groupⅤwere injected with purified rBac-SS4-VP2/Alhydrogel; Mice in groupVI were injected with PPV NJ-a inactivated vaccine. At the same time, theⅦandⅧgroups served as negative and blank controls, were inoculated with the same amount of wild-type Baculovirus and 200μL steriled PBS, respectively. Injections were done with the quantity of Baculovirus and inactivated porcine parvovirus at 5×105TCID50 or 50μg purified recombinant protein via intramuscular (i.m.) route for three times at two weeks interval. The immunized mice were bleeded weekly after primary vaccination, and porcine parvovirus antibodies was detected with an commercial ELISA kit; Neutralizing antibody titer for each group was detected with virus neutralization test assay at two weeks interval after the primary vaccination for five times to evaluate the humoral immune response; Growth hormone and insulin-like cytokine levels in serum were detected the immune activity of somatostatin.3 The preparation of the specific polyclonal antiserum for porcine parvovirus NJ-a strainporcine parvovirus NJ-a strain was concentrated and purified with PEG-NaCl Vmethod to obtain high-purity PPV antigen following cultured with PK-15 cell. Rabbits was vaccined with treated PPV antigen emulsified with Freund's adjuvant, incomplete Freund's adjuvant, and then PPV antigen alone at two week interval. Western-blot and indirect immunofluorescence have demonstrated that the polyclonal antibody can generate the specific reaction with the baculovirus expressing VP2 proteins. Conclusion:the rabbit anti-PPV-specific polyclonal antibodies established basis for further study for protein expression and functional analysis of PPV.