| Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens, causing reproductive failure in pregnant sows and respiratory distress in piglets and growing pigs worldwide. PRRSV infection can cause a delayed and defective adaptive immune response. Nucleotide sequence analysis has shown that PRRSV can be divided into European (Type â… ) and North American (Type â…¡) genotypes. The viral ORFs la and lb encode non-structural polypeptides with replicase associated activities whereas ORFs2-7translated from a nested set of subgenomic RNA (sgRNA) encode the viral structural proteins. Among them, NSP1protein is one of the important non-structural proteins, which could inhibit the activity of IFN-β promoter and expression of type I interferons. In this study, six monoclonal antibodies (MAbs) against PRRSV NSP1protein were screened, and two linear epitopes (54-59aa and157-163aa) and one antigenic domain discontinuous epitope (185-232aa) were identified in this protein. Seven T-cell epitopes were identified in the viral ORF3-7coding proteins of North American-type PRRSV by IFN-y ELISPOT analysis. Two plasmid DNA-based vaccines encoding B and T cell epitopes were designed and constructed. The immunogenicity of these recombinant plasmids was examined in mice model. The results indicated that the recombinant plasmid expressing SynBT2genes could induce effective immune responses. It provides the foundation for developing the new vaccine against PRRSV.The contents of this research contain four parts as following:1. Preparation and identification of monoclonal antibodies against NSP1protein of PRRSVBasing on the gene sequence of PRRSV SY0608in GenBank, the specific primers were designed and the gene was amplified by PCR. The recombinant plasmid pET-32a-NSP1was constructed and then transformed into E.coli BL21strain. The recombinant NSP1protein was induced by IPTG at18℃or37℃. The results of SDS-PAGE and Western blot analysis indicated that the recombinant NSP1protein was expressed mainly in the intact form at37℃with the molecular weight of64KDa. Some of them could be cleaved into two subunits. The sizes of recombinant NSP1α protein and NSP1β were approximately39KDa and26KDa respectively, and these two proteins could also be expressed correctly at18℃which confirmed by SDS-PAGE and Western blot. The recombinant NSPl proteins showed a strong reaction with the PRRSV-positive sera. To prepare monoclonal antibody specific to NSP1protein, BALB/c mice were immunized with the purified NSP1inclusion body. Six monoclonal antibodies (MAbs) against PRRSV NSP1were screened, named1H7,2G5,3C7,3E11,4D4and4H2. Western blot and IFA results indicated that4of6MAbs recognized the recombinant NSPl a and2MAbs recognized NSP1(3. Among them, MAbs2G5,3E11and4D4could recognize European type PRRSV strain in PAM cells, but other MAbs could not react with this strain. The titer of the antibody could be secreted stability during20passages of the fusion cells in vitro. The MAbs against NSP1will be useful for mapping the epitopes of the protein and developing diagnosis of PRRSV in the future.2. Identification of B-cell epitopes in the NSP1protein of PRRSVBased on the gene sequence of PRRSV NSP1,36pairs of primers were designed and the gene fragments were amplified by PCR. Fragments were cloned into pET-32a vector. The recombinant proteins were expressed by Escherichia coli system and identified by Western blot. Epitope mapping results indicated that MAb4H2recognized the linear epitopes E(54)EPLRW(59)in NSP1α, MAbs(2G5,3E11and4D4)recognized the epitopes H (157) VLTNLP (163) in NSP1α, MAbs3C7and1H7reacted with the epitopes185aa to232aa in NSP1β. The peptides SP1(54-59aa) and SP2(157-163aa) were synthesized and used as ELISA antigens. Peptide-based ELISA results indicated that the synthetic peptides SP1and SP2had immune reactivity with the MAbs4H2and2G5(3E11,4D4), respectively. Protein sequence alignment of NSP1indicated that E (54) EPLRW (59) was conserved in all North American PRRSV strains, whereas European type strains has variable amino acids in this region. The epitope H (157) VLTNLP (163) was relatively conserved among all PRRSV strains, except for a L162â†'S162change in European type strains. The epitope185-232aa was variable among North American PRRSV strains. These results will facilitate future investigations into the function of NSP1of PRRSV and diagnostic methods for PRRSV infection. 3. Evaluation of potential T-cell epitopes in ORF3-7structural proteins of genotype-â…¡ PRRSVIn order to determine T-cell epitopes in the structural proteins of PRRSV, the T-cell epitopes were predicted in GP3, GP4, GP5, membrane (M) and nucleocapsid (N) proteins of PRRSV using bioinformatics and synthesized based on the sequence of PRRSV. And2428-30days old pigs free of PRRSV were challenged with a highly pathogenic PRRSV SY0608strain and raised separately and observed for70days. To determine peptide-induced IFN-y secretion, PBMC were isolated at42and63days post challenge for stimulation with peptides and determined by IFN-y ELISPOT assay, respectively. The results showed that7distinct regions appeared to contain immunodominant T-cell epitopes, which were named as GP3-3(106-114aa) in GP3, GP4-3(8-16aa) in GP4, GP5-1(119-127aa) and GP5-2(151-159aa) in GP5, M-1(43-51aa) and M-4(106-116aa) in M, N-1(25-33aa)in N proteins. Among them, GP4-3, GP5-1and GP5-2were the same as those identified by other researchers previously. The sequence alignment revealed that the epitopes represented by peptide GP3-3, M-1, M-4and N-1was fully conserved in all of the examined genotype â…¡ PRRSV isolates and the epitopes represented by peptides GP4-3, GP5-1and GP5-2showed1-4amino acid replacements. These epitopes should be considered for incorporation into a multivalent vaccine against PRRSV.4. Development and immunogenicity of recombinant plasmid containing B-and T-cell epitopes domain of structure proteins of PRRSVIn order to construct a new DNA vaccine against PRRSV, the main B and T cell epitope genes of PRRSV were selected and composed together by two types (BT1and BT2). To minimize interference between adjacent epitopes, each epitope was separated from its neighboring one by GPGPG. To increase the expression efficiency of the genes in vivo, the genes were engineered with the codon usage optimized for mammalian cell expression and were chemically synthesized. Then those genes were cloned into an expression vector pVAX1, and the recombinant pVAXl-SynBT1, pVAX1-SynBT2were constructed and confirmed by restriction enzyme analysis and sequencing. Meanwhile, two control plasmids pVAX1-SynB and pVAX1-SynT only expressing the same numbers of B and T cell epitope genes of PRRSV were also constructed. HEK-293A cells were transfected individually with the recombinant plasmids, and Western blot results showed that SynBTl, SynBT2and SynB proteins were expressed effectively.70BALB/c mice were divided as7groups each with10, and group1-4were inoculated intramuscularly with the four recombinant plasmids respectively by three times at3-week intervals. Group5was injected with plasmid pVAX1-SynGP5/GP4-5containing the GP5gene and the epitope GP4-5of PRRSV as positive control. The results showed that the mice inoculated with the recombinant plasmids pVAXl-SynBT1, pVAX1-SynBT2and pVAX1-SynGP5/GP4-5developed PRRSV-specific antibodies and cellular immune response at9weeks post primary inoculation. The mice immunized with the recombinant plasmids pVAX1-SynBT2developed significantly higher titers of anti-PRRSV ELISA antibody and neutralizing antibody compared to the mice immunized with other recombinant plasmids. It was also found that lymphocytes of the mice immunized with pVAX1-SynBT2was primed for significant higher levels of PRRSV specific IFN-y and IL-4upon stimulation with purified PRRSV antigen in vitro than those of mice immunized with other recombinant plasmids. It suggested that the recombinant plasmid expressing SynBT2genes could induce effective immune responses. |
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