Study On The Expression And Immunogenicity Of VP2 Protein Of Porcine Bocavirus Mediated By Recombinant Baculovirus

Porcine Bocavirus (PBoV) belong to the member of the family parvovirinae and has a single-stranded DNA genome of approximately 5kb which contains three major open reading frames (ORFs). The ORF1 and ORF3 encode non-structural protein NS1 and NP1 respectively. The ORF2 encodes the overlapping structural proteins VP1 and VP2. ORF3 is the distinctive ORF of the Bocavirus, but its function remains unknown. According to the molecular characterization of canines parvovirus, NP1 is essential for virus DNA replication, and VP1/VP2 is implicated in participating in viral entry progress. Bocavirus infection have been reported to be associated with reproductive failure, as well as gastrointestinal and respiratory tract diseases, especially in young age groups. Porcine bocavirus was first observed in Swedish pigs with a background of post-weaning multisystemic wasting syndrome (PMWS) and co-infection with porcine circovirus type 2. Investigation of the prevalence of porcine bocavirus found that it was almost twice as prevalent in PMWS affected than non-PMWS affected pigs sampled in Sweden. Moreover, Zhai et al reported that PBoV was significantly more prevalent(69.7%) in samples collected from the respiratory tracts of weaning piglets with respiratory tract symptoms than in other samples from such animals, indicating that PBoV might cause swine respiratory tract diseases. As a newly recognized viral pathogen, the etiological role for PBoV in PMWS and its pathogenic potential are largely unknown. Hence, study on this virus and its association with swine respiratory tract diseases are newly developing field.Therefor, we constructed a recombinant baculovirus that expressed porcine bocavirus VP2 genein in this study. Electron microscopy confirmed that the recombinant protein was able to form virus-like particles (VLPs) after sucrose density gradient centrifugation. To detecte the humoral immunity and cellular immunity level, different doses were immune to 5-6 week-old BALB/c mice. Then its immunogenicity was evaluated. The specific contents are as follows:1.Construction of recombinant baculovirus (rBV-VP2)PBoV VP2 gene with restriction sites was amplified by PCR. VP2 gene was cloned into the baculovirus transfer vector pFastBac-HTB, that build the transfer vector pFastBac-HTB-VP2. Transformed E. coli DH10Bac,we can obtain the recombinant shuttle vector Bacmid-VP2 by blue and white colonies and resistance screening. Then genom was extracted and transfected on Sf9 insect cells to obtain recombinant baculovirus rBV-VP2.2.Expression of VP2 recombinant proteins and observation of virus-like particlesThe recombinant baculovirus rBV-VP2 was transduct on sf9 insect cells. Western blotting and indirect immunofluorescence analysis showed that VP2 gene inserted in the recombinant baculovirus was expressed efficiently. It was confirmed that the expression of the purified protein after phosphotungstic acid negative staining can self-assemble into virus-like particles (Virus-like particles, VLPs) by electron microscopy,whose size is 22 to 27nm. The size and shape are very similar to the natural virus particles of bocavirus genus.3. Study of VP2 recombinant proteins immunogenicityIn order to determine whether the recombinant VP2 protein in mice caused by the immune response, the recombinant proteins emulsified with Freund’s adjuvant were immuned 6-week-old BALB/c mice in different doses. The test results showed that the ELISA antibody titer reached 1:634 and 1:1007 on day 21 after the first immuned, and rapidly reached 1:1260 and 1:4031 on day 21 after the second immuned. Two sets of immune experimental group were significantly higher than the PBS control group (P <0.01).The IFN-γ level that tested by IFN-γ ELISA assay kit reached 515,657pg/ml, which were higher than the PBS control group (P<0.01) significantly. Lymphocyte proliferation test results also were confirmed that the immuned mice were able to induce higher specific lymphocyte proliferative response. Therefore, VP2 protein is able to generate a strong humoral and cellular immunity, which can be used as a candidate subunit vaccine against the PBoV.As we know, this is the first report to demonstrate that the PBoV VP2 protein is capable of forming virus-like particles. Importantly, the VLPs can induce significant immune responses in mice. This study provides a foundation for the application of VP2 protein in the clinical diagnosis of PBoV or in the vaccination against porcine bocavirus-associated diseases in the future.