Screening And Identification Of T And B Lymphocyte Epitopes On Capsid Protein Of Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV-2) is one of most important viral pathogens which are severely jeopardizing swinery health, and is widespread in swine population. PCV-2 usually co-infects pigs with other pathogens, and is the main pathogen of porcine circovirus associated disease (PCVAD). PCVAD has complicated symptoms, and is difficult to treat. Integrated control with timely detection and effective prevention is needed to deal with PCVAD. So far, PCV-2 vaccine has been successful in clinical trials in some foreign countries, and some vaccines have been put into application in China recently. Other candidate vaccines are under active study, it is important to discover new PCV-2 vaccine with assured effect and promoted immunizing potency for the control of PCVAD.Three pairs of specific primers were designed according to published information of distribution of B-cell epitopes and the prediction results of nuclear localization signal (NLS) in the capsid protein of PCV-2 to amplify truncated sequences of Cap gene with the intention of finding the dominant linear B-cell epitope and facilitating further study on NLS such as finding functional epitopes. Three truncated sequences of PCV-2 Cap gene named F2-1, F2-2 and XF2-2 were amplified, and the sequence of F2-1 includes 12 bp deleted NLS sequence which covers the mainly functional sequence for nuclear localization, F2-2 and XF2-2 are NLS deleted. The three sequences are subcloned to pET-32a(+) to construct pET-F2-1, pET-F2-2 and pET-XF2-2 fusion expression vectors. The recombinant fusion expression vectors were transformed into Rosetta(DE3) E.coli and expressed by induction of IPTG. Three recombinant fusion proteins were obtained, and the molecular weights of His-F2-1, His-F2-2 and His-XF2-2 fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa. Immunoreactivity between each expressed protein and anti-PCV-2 sera was detected by ELISA, and better immunoreactivity was detected between His-XF2-2 and anti-PCV-2 sera.Expressed fusion proteins were purified and utilized to immunize BALB/c mice to produce monoclonal antibodies (mAbs). Seven mAbs were screened out, and the mAbs binding regions on Cap were determined by ELISA additivity tests and cross reactions. N-terminal part 55-96 aa was confirmed as binding regions of FmAb-7 and FmAb-13 mAbs. 97-141 aa was confirmed as binding regions of FmAb-2, FmAb-3, FmAb-8 and FmAb-10 mAbs, and 142-211 aa was confirmed as binding regions of FmAb-15 mAb. Immunoreactivity values between His-XF2-2 and seven mAbs were compared, and mAbs against peptide of 97-141 aa show best reactivity. The overlapping peptides covering 97-141 aa region were synthesized, and each peptide contains 15 residues with 5 residues overlapping with the adjacent peptides. PEPSCAN indicates that the dominant linear B-cell epitope locates at 122-136 aa with the help of dot-ELISA and peptide ELISA. Indirect ELISA and sandwich ELISA were established with His-XF2-2 and FmAb-8 as coating antigen to detect antibody and antigen of PCV-2, and good performances were obtained.Capsid protein gene of PCV-2 and porcine interferon-γgene (PoIFN-γ) were amplified from the constructed recombinant cloning vectors named pMD18-PCV-2Cap and pMD18-PoIFN-γby PCR, and subcloned into pBudCE4.1 expression vector for mammalian cell to construct co-expressing vector pBud-IFN-PCV as DNA vaccine. BALB/c mice were immunized with DNA vaccine Prime and recombinant truncated Cap protein expressed in Rosetta(DE3) Boost strategy. Candidate epitopes with nine residues were predicted with online cytotoxic lymphocyte(CTL) epitope prediction methods and synthesized. Splenocytes were collected from immunized mice, and were stimulated by the synthesized peptides. CTL epitopes were screened out and identified by enzyme linked immunospot assay (ELISPOT) and intracellular cytokine staining (ICS). Splenocytes secreting IFN-γcould be detected after the stimulation of R116 G V G S S A V I, T137 Y D P Y V N Y S, R159 Y F T P K P V L and PMA+Ionomycin positive control. The amounts of IFN-γsecreting CD8+ T lymphocytes stimultated by R116 G V G S S A V I, T137 Y D P Y V N Y S and R159 Y F T P K P V L was extremely different with amounts of IFN-γsecreting CD8+ T lymphocytes stimultated by other peptides analyzed by statistical analysis (P<0.01).In this study, PCV-2 Cap gene including NLS was successfully expressed in prokaryotic expression system. Seven strains of mAbs against Cap were screened out, and a dominant linear B-cell epitope was identified by PEPSCAN. Three CTL epitopes on Cap were screened out with mice as animal model, and in the three CTL epitopes, T137 Y D P Y V N Y S and R159 Y F T P K P V L are H-2Kd restricted, R116 G V G S S A V I is H-2Dd restricted. Identification of dominant B-cell epitope and preparation of mAb against it will contribute the more accurate detection of antibody and antigen. The identification of CTL epitopes on Cap may pave the way for understanding of cell-mediated immune responses and further study on new multi-epitope vaccine.