| Porcine circovirus diseases, has been recently associated with a number of disease Syndromes, which have been collectively named porcine circovirus diseases (PCVD) Postweaning multisystemic wasting Syndrome (PMWS), porcine dermatitis and nephropathy Syndrome (PDNS), porcine respiratory disease complex (PRDC) and reproductive disorders are the most relevant ones. Among them, PMWS can cause severe immunosuppression in swine and have a severe economic impact on pig industry worldwide. PCV2ORF2encodes a main structure protein——Cap protein, which contains several B-cell epitopes, and has good immunogenicity that could induce protective immunity against viral challenge; In this study, A blocking ELISA method for PCV2was developed based on a Cap protein and its specific monoclonal antibody. For developing new genetically engineering vaccines against PMWS, recombinant erkaryotic expression plasmids were constructed based on the modified ORF2gene, and different Cap subunits were expressed in prokaryotic E. coli expression system. The immunogenicity of these recombinant plasmids and proteins were systemically analyzed. The contents of this paper contain four parts as following:1. Indentification of liner B-cell epitope on Cap of PCV2Nine 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; Cap-F:101-234aa; Cap-G:101-234aa; Cap-H:101-234aa; Cap-I:101-234aa;) were expressed successfully. Among them,CapH and Capl could be recognized by the monoclonal antibody (5H7),which is against the recombinant PCV2Cap. It indicated that5H7MAb recognized the linear epitope Y (156) HSRYFT (162), which is located on156-162aa of Cap.The results of comparing the Cap gene sequence with22derivate PCV2isolates showed that the sequence of Y (156) HSRYFT (162) on Cap was conserved in all the reference strains. These results may facilitate future investigations into the function of Cap and provide useful information for diagnostic methods for PCV2infection.2. Development of blocking ELISA for the detection of antibody to PCV2Cap by using peroxidase-labelled momoclonal antibodyA blocking ELISA method was developed based on PCV2Cap protein and its specific monoclonal antibody (5H7). The reaction conditions for each step were optimized as:The coating antigen was2ug/ml, and the coating time was2h at37℃; The sealing buffer was1%BSA, and the sealing time was3h at37℃; Sera samples diluted by1fold and incubated2h at37℃; MAb-HRP dilution was3000fold and incubated0.5h at37℃; The TMB substrate was added and incubated at37℃for15min before terminated with stop solution. The results of33PCV2negative serum samples were statistically analyzed, and the cutoff value of blocking ELISA was determined:samples presenting a percentage inhibition (PI) of≥40.30%were considered as positive; samples with a calculated percentage inhibition of≤28.46%were rated as negative and those presenting a blocking effect between28.46%and40.30%were considered doubtful. For100serum samples, the results of indirect ELISA and blocking ELISA indicated that, the sensitivity and specificity of the blocking ELISA were84%and94%respectively, and the coinsidence is89%. In the repeatability test, both the intra-batch and inter-batches variation coefficients were less than10%. Recombinant antigen had no cross reaction with the antibodies to FMDV, PRV, PRRSV, M. suis and EMCV. This method was used to detect311clinical serum samples from different areas of China, resulting a positive rate of85.53%. These results suggested that the established blocking ELISA is specific, sensitive and reproducible. And it could be used to detect the antibody to PCV2in the future.3. Modification of PCV2ORF2gene and construction and identification of the recombinant eukaryotic expressing plasmids.In order to improve the immunity of PCV2ORF2DNAvaccine, ORF2gene was engineered with the codon usage optimization for mammalian cell expression based on the native ORF2gene of PCV2strain. Additional modifications included:inserting a kozak sequcence in the ORF2initiation codon upstream, mutating the first amino acid C, which is downstream of the ATG, to G, and making a link between two modified ORF2with FMC V2A. After amplification of the gene and insertion into pVAXl plasmid, the recombinant pVAX-Cap, pVAX-SynCap, pVAX-SynCap (m) and pVAX-SynCap-2A-SynCap were constructed and confirmed by restriction enzyme ananlysis and sequencing. To detect the expression efficiency of the ORF2genes, BHK-21cells were transfected individually with pVAX-Cap, pVAX-SynCap, pVAX-SynCap (m) and pVAX-SynCap-2A-SynCap, pVAX-Cap and pVAXl used as controls, and Western blot was performed at48h post-transfection,β-actin used as a control. The results showed that Cap-specific protein bands with expected molecular sizes could be detected in lysates of cells transfected with recombinant plasmids, but not in lysates of cells transfected with the empty vector. Furthermore, the expression of Cap in the cells transfected with pVAX-SynCap, pVAX-SynCap (m) and pVAX-SynCap-2A-SynCap was increased, comparing to that in the cells tranfected with pVAX-Cap. It indicated that the modified ORF2gene exhibit greater expression efficiency in mammalian cells than the native ORF2did. It could be used for candidate ofDNAvaccine of PCV2.4. Protective immunogenicity of the eukaryotic expressing plasmids and the recombinant attenuated S.typhimurium strain based on the modified Cap gene.The immunogenicity of the plasmids DNA, pVAX-Cap, pVAX-SynCap, pVAX-Syn Cap (m) and pVAX-SynCap-2A-SynCap were examed. The results showed that the mice immunized with recombinant plasmids pVAX-SynCap (m) and pVAX-SynCap developed significantly higher titers of antibodies to PCV2and produced stronger lymphocyte proliferation responses compared to the mice immunized with other recombinant plasmids. Then, the recombinant plasmid pVAX-SynCap (m) was transformed into an attenuated S.typhimurium strain SL3263by electroporation, resulting in a strain SL-pVAX-SynCap (m). The pig experiment results showed that the pigs vaccinated with the recombinant S.typhimurium and recombinant plasmid developed specific anti-PCV2antibody by ELISA and neutralizing assay. Meanwhile, no humoral immune response could be detected in other groups. After challenge, piglets inoculated with pVAX-SynCap (m) and SL-pVAX-Syn Cap (m) showed lighter clinical signs, lower viremia and less gross lesion of lungs, comparing with those in other groups. It indicated that SL-pVAX-SynCap (m) could markedly increase the immune responses and provide protective efficiency against virulent PCV2challenge in pigs.In summary, the blocking ELISA method for detection of antibody to PCV2Cap were developed; It is confirmed that, the modified ORF2gene exhibited greater expression efficiency in mammalian cells than the native ORF2did. These results may be useful for developing novelDNAvaccines and dignostic mathods against PCVD. |
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