| Trichinella spiralis, the etiologic agent of trichinosis, infects a wide range of mammalian hosts and is one of the most widespread parasitic diseases worldwide. DNA vaccination has become an increasingly attractive approach for vaccination against T. spiralis infection. The parasite can persist in muscle tissues for many years through its ability to produce a variety of production of biologically active proteins to suppress the host immune response. The immunomodulatory functions of excretory-secretory (ES) products during T. spiralis infection and their roles in establishing parasitism in the host have led researchers to explore their potential as a vaccine target. Ubiquitin is a highly conserved76-amino-acid polypeptide in the cytoplasm of eukaryotic cells. Ubiquitin/proteasome-dependent pathway is a highly selective proteolysis pathway. Ubiquitination has been used in DNA vaccination. In this study, plasmids expressing/co-expressing macrophage migration inhibitory factor (MIF), multi cystatin-like domain protein (MCD-1) of T. spiralis (TsMIF and TsMCD-1) were constructed. Their ability to generate a protective immune response against T. spiralis infection was evaluated in BALB/c mice. Results of DNA vaccination showed Thl immune response, and the pVAXl-Tsmif-Tsmcd-1vaccine induce a partial protection against infection. In addition, coexpression with Ub enhanced the Thl cytokines production and CD8+T cells immune response, as well as the protection against T. spiralis infection induced by pVAXl-Tsmif-Tsmcd-1vaccine.According to GenBank sequences of Tsmif(AY050661) and Tsmcd-1(DQ777102), five primers were designed and used to clone two gene segments of MIF(Tsmif and Tsmif") and one gene segment of MCD-1(Tsmcd-1) with RT-PCR from adult worm of Trichinella spiralis. Three recombinant plasmids of pVAX1-Tsmif, pVAXl-Tsmcd-1and pVAX1-Tsmif-Tsmcd-1were constructed respectively. Digestion identification showed two segments of3000bp and345bp,3000bp and1221bp,3000bp and1602bp, respectively. Comparisons of sequences of Tsmif and Tsmif " with the GenBank sequence of Tsmif (AY050661) indicated nucleotide and amino acid sequence homologies of99.7%respectively. Comparisons of the sequence of Tsmcd-1with the GenBank sequence of Tsmcd-I (DQ777102) indicated nucleotide and amino acid sequence homologies of99%and100%.Three recombinant plasmids of pET28(b)-Tsmif, pET28(b)-Tsmcd-1and pET28(b)-Tsmif-Tsmcd-1were constructed and expressed in Escherichia. coli BL21. SDS-PAGE showed that the recombinant protein of rTsMIF existed in supernant as a soluble protein; recombinant proteins of rTsMCD-1and rTsMIF-TsMCD-1mainly existed in inclusion bodies. Western blot showed bands of16.5kDa,50kDa and63kDa respectively, suggesting that these three recombinant proteins could react with serum of mice infected with Trichinella spiralis. To produce anti-serum, purified recombinant proteins were mixed with FCA (freund’s complete adjuvant) and subcutaneously injected BALB/c mice respectively. A rTsMIF-TsMCD-1-based indirect enzyme-linked immunosorbent assay (rTsMIF-TsMCD-1-ELISA) was developed for detecting specific antibodies against TsMIF and TsMCD-1. The conditions of rTsMIF-TsMCD-1-ELISA were determined as follows:2.3μg/ml of rTsMIF-TsMCD-1used to coat ELISA plate,1:128of sera as detecting samples and serum sample being incubated at37℃for90minutes.DNA vaccination of three recombinant plasmids expressing/co-expressing TsMIF and TsMCD-1were performed by intramscular injection in BALB/c mice respectively, including pVAX1-Tsmif, pVAX1-Tsmcd-1, or pVAX1-Tsmif-Tsmcd-1, with controls of PBS and pVAXl. Groups of20mice were immunized twice at two-week intervals. Fourteen days past the second vaccination, challenge was carried out by oral infection with200L1infectious T. sipralis larvae per animal. Thirty-five days later,10mice in every group were killed to detect LPG (larvae per gram) and reduction ratio of muscle larvae. Detection of in vivo target gene expression was performed by RT-PCR and Western blot, indicating that target genes were expressed in injected muscle but not in contralateral noninjected muscle, and coexpression protein of TsMIF and TsMCD-1was found in mice vaccinated with pVAX1-Tsmif-Tsmcd-1. Sera collected0,10,20and28days after the first vaccination were assayed for the presence of specific antibodies against recombinant TsMIF-TsMCD-1protein. Results showed that vaccination with pVAXl-Tsmif failed to induce specific antibodies and pVAX1-Tsmcd-1or pVAXl-Tsmif-Tsmcd-1vaccine induced specific antibodies of IgG, IgM and IgA, but no IgE. Levels of specific antibodies in mice vaccinated with pVAX1-Tsmif-Tsmcd-1were less than pVAX1-Tsmcd-1(P<0.05). Both IgG2a and IgG2b showed higher proportion than IgG1(P<0.05) in these two groups, showing a Thl humoral immune response. Concentrations of five serum cytokines were detected and showed higher levels of Thl cytokines (IFN-y), no Th2cytokines (IL-4, IL-5). The lower levels of TGF-β1might be related to suppressing predominant cytokines (IFN-y). The failure to detection of IL-17displayed the absence of inflammation. Fourteen days past the second vaccination, the percentages of CD4+T cells (CD4+%) and CD8+T cells (CD8+%) in peripheral blood lymphocytes were determined, showing more CD4+%in all of the three recombinant plasmids groups than PBS control (P<0.05), especially pVAX1-Tsmif-Tsmcd-1vaccine (P<0.01). The percentage of CD8+T cells in mice vaccinated with pVAX1-Tsmif and pVAX1-Tsmif-Tsmcd-1respectively was more than PBS control (P<0.05), but no significant difference was observed in pVAXl-Tsmif vaccination. Ratio of CD4+%to CD8+%in either of the three recombinant plasmids groups was higher than PBS control (P<0.05), indicating the enhancement of proliferation of mouse T cells. Challenge infection demonstrated that no reduction of LPG was observed in mice vaccinated with pVAX1-Tsmif or pVAX1-Tsmcd-1, but immunization with pVAX1-Tsmif-Tsmcd-1reduced worm burdens by23.17%versus controls (P<0.05) suggesting a partial immune protection against T. spiralis.To confirm the influence of ubiquitination on DNA vaccination, the Ub gene of mouse was cloned with RT-PCR and three recombinant plasmids of pVAX1-Ub-Tsmif, pVAX1-Ub-Tsmcd-1and pVAX1-Ub-Tsmif-Tsmcd-1were constructed respectively. These recombinant Ub-coexpression plasmids were transformed in BHK cells, and Western blot analysis failed to detect target bands respond to anti-serum of recombinant protein TsMIF-TsMCD-1. However, after adding the proteasome inhibitor MG-132into transformed cells, bands of22kDa,57kDa and68kDa were observed respecitvly, which were consistent with the sizes of coexpression proteins of Ub-TsMIF, Ub-TsMCD-1and Ub-TsMIF-TsMCD-1, respecitvly. These results showed that proteins coexpressing mouse ubiquitin (8.57kDa) and target protein (TsMIF, TsMCD-1, and TsMIF-TsMCD-1) were expressed in BHK cells transformed with the Ub-coexpression plasmids; ubiquitination promoted the degradation of target proteins in BHK cells and MG-132could inhibit the degradation. Later, DNA vaccination with these recombinant Ub-coexpression plasmids was performed in BALB/c mice, with the controls of PBS, pVAXl and pVAX1-Ub. After detection of specific antibodies response, serum cytokines and CD4+or CD8+T cells, as well as the protection against T. spiralis challenge infection, the immunity efficacies of these Ub-coexpression plasmids were evaluated. The results displayed that Ubiquitination reduced humarol immune responses, but promoted Thl cytokines production, CD8+T cells prolifecation and the specific CTL response in vaccinated mice. Importantly, a stronger reduction of LPG was induced in mice vaccinated with pVAXl-Ub-Tsmif-Tsmcd-1. These results also suggested that the stronger Thl cytokines and CD8+T cells immune response induced by ubiquination might play an active role in immune protection against T. spiralis. |
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