Fine Localization Of Epitopes On Capsid Proteins Of Porcine Circovirus And Its Indirect ELISA Technique

Porcine circovirus (PCV), a member of the family Circoviridae, is a small, non-enveloped virus, with a circular, single-stranded DNA genome. PCV includes nonpathogenic Porcine circovirus type 1 (PCV1) and pathogenic Porcine circovirus type 2 (PCV2). The circular PCV2 viral genome contains 1767-1768 nucleotides while the PCV1 genome contains 1759 nucleotides. However, the genomic organization of both PCV1 and PCV2 is similar, containing 11 potential open reading frame (ORF). Recently, the ORF1-encoding replicase (Rep) involved in viral replication, the ORF2-encoding capsid (Cap) involved in host immune response and the product encoded by ORF3 associated with pathogenicity of PCV2 were verified. PCV2 was believed to be the primary causative agent of porcine circovirus-associated diseases (PCVD), especially postweaning multisystemic wasting syndrome (PMWS) which caused economically great loss in swine industry. Many researchers have developed ORF2-based vaccines for prevention of PCV2-associated diseases. However, the mechanism by which those vaccines against PCV2 induced immune response was unclear and remained to be understood for developing more effective vaccines to PCV2. In this study, the antigenic characterization of the capsid protein of PCV1 and PCV2 was identified, and an enzyme-linked immunosorbant assay for serodiagnosis of PCV2 infection was developed using the recombinant capsid protein of PCV2 as antigen.The nuclear localization signal-defected capsid protein genes of PCV2 and PCV1 were amplified with specific primers and cloned into prokaryotic vector pGEX-4T-l and pET28a(+), respectively, to construct recombinant plasmid pGEX-PCV2-dCap and pET-PCVl-dCap. The recombinant PCV2 dCap protein fused with glutathione S-transferase (GST-dCap) was expressed as a soluble form, and the expression conditions were optimized to be that recombinant bacteria containing pGEX-PCV2-dCap were induced by a final concentration of 0.1 mM of IPTG for 4 h at 37℃. While the 6×His-tagged PCV1 dCap (His-dCap) was expressed as inclusion bodies after induction by a final concentration of 1 mM of IPTG for 4 h at 37℃. The recombinant PCV2 GST-dCap protein with a mass of 48kD and PCVI His-dCap protein with a mass of 28kD were purified respectively by GST affinity chromatography and Ni-NTA affinity chromatography. And the 22-kD PCV2 recombinant dCap protein was obtained through cleavage from GST-dCap with thrombin. The yield of recombinant PCV2 GST-dCap protein and PC VI His-dCap was about 6.14 mg per liter cell culture and 36 mg per liter cell culture, respectively. Western blot analysis indicated that the recombinant PCV2 GST-dCap protein and dCap were recognized by swine anti-PCV2 serum, while PCV1 His-dCap protein was recognized by anti-His tag monoclonal antibody, but not by the swine anti-PCV2 serum.Thereafter, the recombinant PCV2 GST-dCap, purified PCV2 pariticles and PCV1 His-dCap were used to immunize Balb/c mice for production of monoclonal antibodies (mAb) against PCV Cap. After screening by ELISA and IFA, cloning by limiting dilution and identification by western-blotting assay, five hybridomas, named 1B3,1B9,8A12,8B12 and 8C12 whose supernatant only recognize PCV2 native capsid protein, four hybridomas, named 3F6,5A3,5E11, and 6E1 whose supernatant could recognize the native capsid proteins of PCV2 and PCVI and one hybridomas, named 4C3 whose supernatant could recognize recombinant PCV2 GST-dCap and PCV1 His-dCap, were developed using PCV2 GST-dCap as immunogen; And using the purified PCV2 particles as immunogen, four hybridomas named 1E3,2B1,6H9 and 7F5, whose supernatant could react with the native capsid proteins of PCV2 and PCV1 but not with recombinant PCV1 His-dCap were obtained; While using the PCV1 His-dCap as immunogen, ten hybridomas named 1A11,4A2,1D11,2D4,2F5,4F5,3F11,2G9,3G7 and 3H1, respectively whose supernatant could only recognize PCV1 native capsid protein and one hybridomas named 4H7 whose supernatant could recognize both PCV2 capsid and PCVI capsid were developed. Among these mAbs, mAb 5A3,3F11,3G7,2G9 and 1A11 belong to IgG2b, mAb 7F5 and 2F5 belong to IgG2a, the other mAbs belong to IgGl subtype. All the mAbs haveκ-light chain of immunoglobulin. Neutralization assay indicated that mAb 3F6,1E3,6H9 and 7F5 to PCV2 Cap had the neutralizing ability to PCV2, and mAbs 1E3 and 7F5 also showed neutralizing ability to PCV1, while all the mAbs to PCVI His-dCap had no neutralizing ability to PCVI and PCV2.Utilizing those monoclonal antibodies to PCV Cap protein, swine anti-PCV2 or-Cap sera as well as synthesized overlapping peptides from PCV2 Cap, mapping and localization of B-cell epitopes on capsids of PCV was performed. Five linear B-cell epitopes on PCV Cap were identified and localized respectively at aa 231-233 (231LNP233) recognized by PCV2-specific mAbs 8A12,8B12 and 8C12, aa 195-202 (195HVGLGTAF202) recognized by PCV2-specific mAb 1B3 and 1B9, aa 175-192 (175QPNNKRNQLWLRLQTAGN192) recognized by mAb 4C3 to recombinant PCV Cap protein, aa 156-162 (156YHSRYFT162) recognized by PCVl-specific mAb 3G7, and aa 92-103 (92LPFQYYRIRKAK103) recognized by PCVl-specific mAbs 3F11,2G9 and 2D4. While mAb 3F6,1E3,4H7 and 4F5 againt PCV2 and PCV1 Cap recognized two epitopes of aa 156-162 and aa 175-192. Thus, the epitope,231LNP233, was specific for PCV2 Cap; 175QPNNKRNQLWLRLQTAGN192 and 156YHSRYFT162 was the common epitopes of PCV1 Cap and PCV2 Cap and 92LPFQYYRIRKAK103 was specific for PCV1 Cap. Analysis of substitution with alanine showed that the proline at aa 233 was a vital and type-specific residue for the epitope 231LNP233 and PCV2 capsid protein. Epitope mapping of the capsid of PCV2 was also achieved by reaction of synthesized peptides with swine anti-PCV2 sera. The resulting immunoactive regions of the capsid of PCV2 were located between amino acids (aa) 25-42,55-72,95-112,115-142, 156-192,216-233, respectively. Further analysis of the kinetic curves of antibodies to those epitopes in swine anti-PCV2 or-Cap polyclonal antibody revealed that the immune responses to these antigenic determinants in individual pig were different, and the four epitopes, aa 115-132, 156-162,175-192 and 230-233 were immunodominant in PCV2 capsid, however, the corresponding antibody was detectable after 28 days post-inoculation. And the epitope 231LNP233 was showed to have the potential as serological marker for PCV2.Using the PCV2 GST-dCap as antigen, an alternative indirect enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of PCV2 infection was developed. The optimal antigen concentration and serum sample dilution were set at 1.24μg/ml and 1:400; The optimal dilution of the conjugate was 1:2000; The 0.05 M Tris-HCl buffer at pH 8.5 was the best coating buffer; The 5%skimmed milk was the optimal blocking buffer; The phosphate buffer containing 0.05% tween-20 was the best diluent; The optimal reaction time for serum samples or conjugate and chromatogenic substrate was 30 min and 10 min, respectively. A S/P ratio of 0.16 was set as a negative-positive cutoff. Repeatability tests revealed that the coefficient of variation of positive sera within and between runs were 1-13% and 5-22%. Cross-reactivity assay showed that this assay was PCV-specific. This assay was validated by comparison with an indirect immuno-fluorescence assay (IIF), a PCV2-based ELISA, a PCV2-specific peptide-based ELISA and a commercial PCV2 ELISA kit. The diagnostic sensitivity (DSN), specificity (DSP) and accuracy of the PCV2 CAP ELISA were 95.3%,93.9% and 95.1%, compared with IIF on 1080 field serum samples, and 93.3%,84.2%and 91.1%, compared with the PCV2-based ELISA on 79 field sera, and 93.8%,88.9%and 92.4%by comparison with the PCV2 peptide-based ELISA on 92 field sera, and 91.1%,90.2%and 90.5%by comparison with the commercial PCV2 ELISA kit on 85 field sera, respectively. The kinetic curve of PCV2 antibody after infection or vaccination was ploted by this ELISA, indicating that this assay could detected the seroconversation to PCV2. These data indicated that this ELISA is highly specific and sensitive for large scale surveys of PCV2 infection at low cost and the evaluation of the efficiency of various vaccines against PCV2.