| Porcine circovirus diseases (PCVD), which caused by porcine circovirus type 2 (PCV2), is a new economically important infectious disease, including postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC) and reproductive disorders. Among them, PMWS has been considered to have a severe economic impact on pig industry worldwide. It most commonly affects pigs between 5 and 12 weeks of age, which display clinical signs of wasting, enlarged lymph nodes, respiratory distress, anaemia, diarrhoea, jaundice and occasionally pallor. In addition, because of the immunosuppression, diseased animals are more susceptible to opportunistic pathogens and usually develop severe clinical signs.PCV2, a member of the family circoviridae, is a small, non-enveloped, single-stranded DNA virus containing a circular genome of 1767/1768 bp. The genome contains three major open read frames (ORFs):ORF1 encodes several forms of non-structural replicase proteins involve in viral replication; ORF2 encodes a major capsid protein (Cap) approximately 30kD in size, which contains several linear B-cell epitopes and neutralizing epitopes and has been recognized as the major immunogen that could induce protective immunity against viral challenge; and ORF3 encodes a 105-amino acid protein which appears to be involved in virus-induced apoptosis. PCV2 has been divided into two major genotypes:PCV2a and PCV2b. PCV2a contains 2A,2B,2C,2D and 2E subtypes and PCV2b contains 1A, 1B and 1C subtypes. Recently, PCV2b has become the prevailing virus in many countries and is thought to be more pathogenic than PCV2a.Vaccination was considered as an effective tool for prevention of PCVD. Commercially available vaccines include inactive PCV2 vaccine, recombinant baculovirus expressed Cap subunit vaccine and inactive chimeric PCV1-PCV2 vaccine. In this study, genomes of 31 PCV2 isolated strains were obtained and phylogenetic analysis was done to explore the molecular epidemiology of PCV2 in eastern china. For developing new genetically engineering vaccines against PMWS, different truncated Cap subunits were expressed in prokaryotic E. coli expression system and the antigenic characteristics of these proteins were systemically analyzed. In addition, recombinant E. coli and baculovirus expressing Cap and somatostatin fusion protein were constructed and immunogenicity of recombinant proteins was analyzed in pig vaccinantion challenge model, respectively. The contents of this paper contain six parts as following:1. Molecular epidemiological investigation of PCV2 strains isolated from Eastern China After clinical detection, viral isolation and determination, we obtained sequences of 31 PCV2 isolates from different farms of 11 provinces of eastern China and analyzed the genetic characterization of them with other 105 Eastern China-derivate PCV2 isolated during 2001-2009. The results showed that these PCV2 isolates could be divided into 2 groups, PCV2b (108 of 1A/1B,19 of 1C) and PCV2a (1 of 2A,2 of 2D,6 of 2E). Among the 9 PCV2a isolates, eight were found before the year 2005. Meanwhile, three major heterogenic regions were observed in amino acid positions 53-91,121-151 and 190-210; a few specific substitution patterns were found in each subgroup and a few recombinants of different genotypes existed in nature. These results indicated that both of PCV2a and PCV2b strains existed in eastern china during 2001-2009, and genotype PCV2b predominated in the PMWS occurrences after 2006. It may provide useful information for prevention and control of PCVD in the future.2. Prokaryotic expression of different Cap subunits of PCV2 and identification of their immunogenic characteristics Five different fragments of the Cap protein gene were amplified by PCR and cloned into pET-32a vector, respectively. SDS-PAGE and Western blot analysis indicated the proteins (Cap-A:51-150aa; Cap-B:51-200aa; Cap-C: 51-234aa; Cap-D:101-200aa; Cap-E:101-234aa) were expressed successfully. The ELISA plates were coated with purified proteins for testing their reactivity with 14 IFA negative and 10 IFA positive serums. The results shown that S/N value of Cap-C was higher than other subunits, which suggests that Cap-C possesses the best reactionogenicity and could be used as ELISA antigen. Meanwhile, mice were inoculated with these five subunit proteins mixing with Freund's adjuvant, respectively. Cap-C could induce the strongest antibody response, while Cap-D induces the lowest antibody level. It implies that Cap-C is the most immunogenic antigen and could be used as diagnose antigen and subunit vaccine candidate. 3. Protective immunogenicity of prokaryotic Cap subunit vaccine of porcine circovirus type 2 Cap-C protein was mixed with different adjuvant and their immunogenicity was tested in mice. The results indicated that antibody responses in different adjuvant vaccinated groups were significantly higher than that of without adjuvant, but no significant difference was observed between different adjuvant vaccinated groups. Thus Cap-C subunit vaccine was made with CP940 adjuvant, and it was used for pig experiment. The results showed that high level of PC V2-specific antibody could be induced after the second vaccination. After challenge, pigs in the vaccinated group had no clearly clinical signs, although some of them had increased rectal temperatures for short time. The relative daily weight gain (RDWG) in Cap-C group was similar to that in empty control group. But it was significantly high compared to the challenge-control group. The pathological lesions, viral load and viremia presented in Cap-C group were similar to those in the commercial inactive PCV2 vaccine (iPCV2) group, while milder than those in challenge-control group. It indicated that the recombinant Cap-C protein expressed in E. coli might be an attractive candidate vaccine for preventing the disease associated with PCV2 infection.4. Construction and identification of recombinant baculovirus expressing Cap protein of porcine circovirus 2 fused with somatostatin VLPs vaccine is a new kind of subunit vaccine, which combines many of the advantages of whole-virus vaccines and recombinant subunit vaccines. Meanwhile, it could be used as carriers for foreign antigen fragments or epitopes. Baculovirus expression system is frequently used for VLPs production. Somatostatin (SS) contains 14 amino acids, which regulate the release of growth hormone (GH) together with growth hormone release factor (GHRF). It have been shown that fusion expression of SS with carrier protein and inoculate animals with the recombinant protein could induce SS-specific antibody, neutralize endogenous SS and promote animal growth. In this study, the ORF2 gene of a PCV2b strain and ORF2-SS gene was amplified by PCR and cloned into the donor vector pFastBacTMHTB of Bac-to-Bac baculovirus expression system. After PCR, enzyme digestion and sequencing analysis, the purified positive recombinant plasmid pF-Cap/pF-Cap-SS were transformed into E.coli competent cells DH10Bac containing baculovirus shuttle vector. The white colonies were selected and the recombinant bacmid rBac-Cap/rBac-Cap-SS were verified by PCR. After transfection of the recombinant bacmids into Sf9 insect cells, recombinant baculovirus rAc-Cap/rAc-Cap-SS were obtained and confirmed by Western blot and immunofluorescence assay with antibody against Cap protein and rabbit anti-SS antibodies. It was further demonstrated by electron micrography after discontinuous sucrose gradient centrifuge that the baculovirus-derived Cap-SS protein self-assembles into virus-like particles (VLPs) that are morphologically and antigenically similar to natural PCV2. Viral plaque assay indicated that viral titer was 5×10' pfu/mL.5. Immunogenicity of recombinant baculovirus expressed Cap-SS subunit vaccine Recombinant BCap/BCap-SS proteins expressed in baculovirus expression system were tested in animal model, aiming at protective and growth promotion efficacy of these subunit vaccines. The results of mice experiment showed that both of BCap/BCap-SS could induce similar level of PCV2-spectific antibody responses (1:1600), and body weight gain of BCap-SS group was higher than other two groups. Piglets experiment results showed that high level of PCV2-specific antibody could be induced after the second vaccination (1:1024). Meanwhile, at 14 and 28 day post primary vaccination, RDWG of BCap-SS group was higher than other groups. After challenge, pigs in the vaccinated group had no clearly clinical signs, although some of them had increased rectal temperatures for a short time. The RDWG in challenge control group was significantly lower than other groups. In addition, the pathological lesions, viral load and viremia presented in vaccinated groups were similar, while milder than those in challenge-control group. It indicated that the recombinant baculovirus expressed BCap-SS protein might be a novel vaccine candidate for preventing PCVD and improving pig growth.6. Construction and immunogenicity of prokaryotic E.coli expressing Cap-C protein fused with somatostatin SS gene was inserted into the 3'-terminal of the coding region of Cap-C and was cloned into pET-32a vector. Highly purified recombinant proteins were obtained after crude purification and dialyze. Results of SDS-PAGE and Western blot analysis indicated the protein (Cap-SS) was expressed successfully and possessed good antigenicity. The protective and growth promotion efficacy of Cap-SS protein was tested in piglet vaccination challenge model, with Cap-C based subunit vaccine as a control. The results showed that high levels of PCV2-specific antibody could be induced after the second vaccination. At 28 day post primary inoculation, RDWG in Cap-SS group was higher than those in other groups. Meanwhile, SS concentration was decreased at 14 dpi, comparing to those in other groups. After challenge, pigs in the vaccinated group had no clearly clinical signs, although some of them had increased rectal temperatures for short time. The RDWG in challenge control group was significantly lower than other groups. In addition, the pathological lesions, viral load and viremia presented in vaccinated groups were similar each other, while milder than those in challenge-control group. It indicated that the prokaryotic Cap-SS protein might be a novel vaccine candidate for preventing PCVD and improving pig growth.In summary, it has been confirmed that both of PCV2a and PCV2b strains existed in eatern china during 2001-2009, PCV2b is the predominated genotype. Prokaryotic expressed Cap-C (51-234aa) is the most immunogenic fragement and could induce protective immunity in pigs. Recombinant baculovirus and E. coli expressed Cap-SS fusion proteins not only induce protective immunity but also promote growth in pigs. These results may useful for developing novel subunit vaccines against PCVD.
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