| Porcine Bocavirus (PBoV), is a member of Family Parvoviridae, Subfamily Parvovirinae. It was first found in swine with post-weaning multisystemic wasting syndrome (PMWS), in Sweden in 2009. After that, PBoV was reported all over the world. Till now, PBoV infection was discovered in Sweden, China, the US, Canada, Mexico, Romania, Hungary, Uganda, Korea, and Japan.PBoV is a non-lipid enveloped, single-stranded DNA virus, exhibiting icosahedral symmetry structure. The genome is around 5kb, and the diameter is 25-30nm. The whole genome mainly contains 3 open reading frames (ORFs), ORF1, ORF2 and ORF3. ORF1, which encodes a non-structural protein NS1, is located at 5’-end of the genome. It is believed that, ORF1 contains conserved motifs, and is related to rolling-circle replication, helicase, and ATPase activities. ORF2, which encodes capsid proteins VP1 and VP2, is related to virus antigenicity. ORF3, encodes non-structural protein NP1 with unknown function. It is reported that, NP1 is essential to replication of Canine Minute Virus (CMV). Another report indicated that, NP1 gene could block IFN production, suggesting that it may participate in immune-evasion mechanism. According to the differences of nucleotide sequences, PBoV can be classified into three genotypes, PBoV1, PBoV2, and PBoV3. The pathogenicity of PBoV is still not clear. The seroprevalence of PBoVs in PMWS swine, was reported to be much higher than non-PMWS swine, indicating that PBoV may be related to PMWS. Moreover, PBoV usually co-infected pigs with PCV2, PTTV, PRRSV, and PBoV is often detected in lymph nodes, serum, intestine, suggesting that PBoV might cause other disorders. However, as there is no evidence on PBoV pathogenicity, further methodology needs to be developed. Although the PCR, RT-PCR, indirect immunofluorescence assays have been developed to examine PBoVs antigens, there is no precise method to detect the antibodies. Thus, to obtain the antigen of the virus is very important for studying the immunogenicity, early diagnosis of the virus, and even the development of the vaccine. At present, gene expression systems include:bacterium, yeast, insect cell, and mammal cell expression system. The bacterium expression system is easy to carry out, but the protein could not be modified with glycosyl. Thus limited proteins could be expressed with this system. For the yeast system, the proteins that are expressed by the system are different from the natural proteins. For the mammal system, the productivity is relatively poor. On the contrary, the proteins expressed by insect cell expression system, are not only structurally the same as the natural proteins, but also in high production. The mammal expression system, which is also called baculovirus expression system, could abundantly express exogenous gene, through recombinant baculovirus in insect cells. The target protein possesses almost the same antigenicity as the natural protein.Objective In the present study, in order to obtain the PBoV-LPs, PBoV VP2 gene was expressed by the baculovirus expression system in insect cells. It will be helpful to study the PBoV sero-epidemiology, the cross-reactivity with other bacaviruses, and the pathogenecity, and also, to develop the PBoV vaccine in the future.Methods1. PBoV VP2 gene was amplified by PCR. The VP2 gene DNA was abundantly amplified by constructing the cloning vector.2. The Baculovirus transfer vector pVL1393-VP2 was constructed. Then the linearized baculovirus DNA and the pVL1393-VP2 transer vector, were both tranfected into Sf9 cells, so that the recombinant baculovirus could be obtained, which was designated as Ac[VP2]. The Ac[VP2] was then abundantly amplified in Sf9 cells.3. The Ac[VP2] was then transfected into Tn5 cells to express the proteins. At first, supervise the expression process of the proteins, by collecting the culture samples day by day, and then the samples were examined through the SDS-PAGE electrophoresis, so that the optimum expressing time can be accertained. Then to abundantly express the proteins according the right culture period.4. Collected the supernatant after 7 days p.i.. The supernatants were concentrated by ultracentrifugation, and then purified by CsCl gradient centrifugation. To confirm whether the protein was delivered from the VP2 gene of PBoV, the N-terminal 5 aa sequence of p61.5 was checked by microsequencing.5. The VLPs were observed under the electron microscope. As the particles did not appear to be empty, the nucleic acids were extracted, and then were examined to see whether nucleic acids were packaged in the VLPs.6. To obtain the anti-PBoV-LPs hyperimmune serum, the PBoV-LPs were inoculated into a rabbit. To examine whether the PBoV-LPs were cross-reactive with HBoVs, the rabbit anti-PBoV IgG titers were detected by ELISA using HBoV1-LPs, HBoV2-LPs, HBoV3-LPS, HBoV4-LPs, and PCV2-LPs as antigen, respectively.7. The seroprevalence of PBoV was detected in swine and wild boars samples. The virus DNA was examined in samples of wild boars and pigs.Results1. The SDS-PAGE electrophoresis result indicated that, the proteins that were expressed from Tn5 cells were around 61.5KDa. The p61.5 protein inside the Tn5 cells were clearly detected on day 3, and reached a peak on day 5 p.i., And then, the amount of the protein began to reduce gradually. On the contrary, the p61.5 protein was first detected in the supernatants on day 3 p.i., and then increased gradually. This result suggested that, p61.5 protein was released into the cell culture supernatants, after being expressed inside the cells.2. The protein purified by CsCl gradient centrifugation, was also around 61.5KDa, with an average density of 1.300g/cm3. By the protein microsequencing, the p61.5 protein was proved to be identical to the VP2 of PBoV. This result indicated that, the p61.5 protein was precisely delivered from the PBoV VP2 gene.3. The nucleic acids extraction experiments result showed that, no nucleic acids were packaged inside the PBoV-LPs.4. The pre-immunized rabbit serum was used as the control. The PBoV-LPs were inoculated into a rabbit. Finnaly, high titers of anti-PBoV IgG antibody, was detected in the serum. This result indicated that the PBoV-LPs had a good immunogenicity, and could be a candidate reagent for the vaccine development in the future.5. To examine whether PBoV-LPs were cross-reactive with HBoVs, an ELISA method was established and carried out. The ELISA results indicated that the PBoV-LPs could cross-react with the HBoVs, while the PBoV-LPs did not react with rabbit anti-PCV2-LPs, and also the PCV2-LPs did not react with the rabbit anti-PBoV-LPs IgG antibody. These data suggested that, the antigenicities of PBoVs and PCV2 were somewhat different.6. Using PBoV-LP as the antigen, the seroprevalence of PBoV infection was detected in swine and wild boars in Japan. The results showed that, both pigs and wild boars were exposed to PBoV. The serum positivity of PBoV in pigs was higher than 90%, while the positivity in wild boars was 59.5%.7. The PBoV DNA was examined in all the samples by nest PCR.7 samples were detected positive for PBoV DNA. Among the 7 samples,4 samples were from Hyogo,2 samples from Ibaragi, and 1 sample from Nagasaki. All the 7 strains shared nucleotide sequence identity of 80.2-92.1%, and all of them belonged to group PBoV G3, and could be further divided into four subgroups.Conclusion1. In the present study, we successfully produced PBoV-LPs, and we found that, the particles possessed almost the same biological characteristics with the natural VP2 proteins, such as the diameter, molecular weight, density and the immunogenicity.2. An ELISA method was established. We used PBoV-LPs as the antigen. And wee found that, PBoV could cross-react with the HBoVs, but not reacted with PCV2.3. By using PBoV-LPs as the antigen, the seroprevalence of PBoV was detected among swine and wild boars in Japan. And we found that, PBoV widely circulated in Japan with different positive rates. Among all the serum samples,7 samples were examined positive for PBoV DNA, all of which belonged to the same group, and could be further divided into 4 subgroups.In conclusion, the PBoV-LPs we have produced will be useful for antigenicity and sero-epidemic research, as well as for the development of vaccine for PBoVs in the future. |
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