| Porcine reproductive and respiratory syndrome (PRRS) is characterized by severe reproductive failure and a high rate of late abortion and early farrowing in sows, and respiratory disease and mortality in young pigs. A typical, but not often observed hallmark of the disease is"blue ears". It is caused by PRRSV which can replicate in macrophages, inducing prolonged viremia, and may cause persistent infections that last for months after infection,neutralizing antibodies to PRRSV appeared approximately four weeks after infection and had relatively low titers throughout the course of infection. Cell-mediated immune (CMI) response to PRRSV determined by lymphocyte blastogenesis and adaptive cytokine production was delayed which take difficult to PRRS prevention and control. Currently, vaccine immunization is the principal method to control the disease. However, the neutralizing antibodies induced by traditional vaccines could not block the early stages of PRRSV infection, thus the acquired cellular immune responses palyed a key role on control of the disease. This requires us develop the vaccine and adjuvant that could induce effective and sustainable cellular immune responses.The ORF6 gene of PRRSV encoded the non-glycosylated M protein is the most conserved structural protein of virus. The M protein could induce the most strong T lymphproliferation response, which play the role on the PRRSV infected CMI response. Inerleukin-18 is a pleiotropic cytokine, which participated in priming cellular immune response. IL-18 could induce Th1 cells and NK cells to produce IFN-γ, promoted T and B lymphocyte differentiation and enhaned FasL mediated cytotoxicity.The ORF6 gene and IL-18 gene with linker were amplified, then clone the target genes into the pMD-18T vector and sequencing. The homology analysis of ORF6 gene indicated, compared with representative North-Amercian stains as VR-2332 and CH-1a, the higher homology of nucleotide which can achieve 94.3%. The flexibility, secondary structure and antigenicity analysis of recombinant protein IL18-linker-M were performed using the ProtScale and HNN program on www.expasy.org and DNAStarTM software, which showed the structure and biological effects of both proteins will not change.On the basis, three recombinant plasmids that expressed IL-18 and M protein of PRRSV with different expression patterns were constructed, pEGFP-ORF6, pEGFP-IL18 and pEGFP-IL18-ORF6. The recombinant plasmids were transfected into Mare-145 cells in vitro, 48 hours later we directly observe the EGFP (enhanced green fluorescent protein) by fluorescence microscope to determine the expression of targer genes. The cells tradnsfected with recombinant plasmids were collected and lysised, then the cell lysate were analysis by Western blot. According to the fluorescence observation and Western bolt assay indicated that the recombinant plasmids could expressed correctly in the eukaryotic cells.The immunogenicity efficiency of recombinant plasmids were further evaluated in the piglets by intramuscular injection the piglets which were PRRSV natural host anmial. The results indicated the pEGFP-ORF6 and pEGFP-IL18-ORF6 immune groups produce specific antibodies in the 14 days, and the production of antibodies increased with time, but the antibody levels between two groups were not significant (p<0.05). Neutralizing antibody analysis showed the pEGFP-N1 induce low titer neutralizing antibodies in 42 days. However the pEGFP-IL18-ORF6 could enhance cell-meditated immune response and elicit effective Th1 type immune response by stimulate PRRSV-special T lymphocyte proliferation and promote the secretion levels of IFN-γand IL-2. It indicated the IL-18 effectively play the utility of molecular adjuvant which could enhance the cell-mediated immune response and Th1 type immune response. In conclusion, it provides the theoretical basis for the development of more effective PRRS vaccine and cytokine used as the molecular adjuvant in vaccine research. |
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