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- 학과정보 : 동물학과 (검색결과 1,395 건)
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대장균 Heat-labile toxin B와 Classical swine fever virus E2 단백질의 Chimeric 항원에 대한 생쥐의 점막면역반응
The Escherichia coli heat-labile enterotoxin(LT) is a very potent mucosal immunogen. LT also has strong adjuvant activity towards co-administrated unrelated antigens and is currently investigated for mucosal vaccines. However, direct use of LT holotoxin as an adjuvant has been restricted by its toxicity which was mediated by ADP-ribosylation activity in A subunit. But, according to many current reports, the nontoxic recombinant B(LTB) subunit of LT also acts as a mucosal adjuvant when intranasally or orally immunized with unrelated antigens. So, the purpose of this study was to demonstrate whether the LTB subunit acts as a mucosal adjuvant in the genetically fused chimeric antigens with epitope genes of envelope glycoprotein E2(gp51-54) of the classical swine fever virus(CSFV), and to characterize the mucosal immune response to the chimeric antigen which contain LTB subunit at cellular and gene expression level. In this study, LTB subunit gene, E2 epitope genes and chimeric genes were subcloned to pET28a vector and recombinant proteins were expressed in BL21(DE3) by 1 mM IPTG induction. Recombinant proteins were purified by nickel chelating affinity chromatography. Purified LTB subunit antigen, E2 epitope antigens and chimeric antigens were orally immunized to Balb/c mice and antibody responses in serum and fecal solution were assayed by enzyme-linked immunosorbent assay(ELISA). Differentiation and modulation patterns of B and T lymphocytes by chimeric antigen were analyzed by FACS analysis. Expression level of Cytokines related to isotype switching of B cell, Th1 responses and Th2 responses was analyzed by realtime quantitative RT-PCR. From the result of antibody assay, It was revealed that total serum Ig level of orally immunized chimeric antigens containing 5' flanked LTB subunit(LE2, LE2 and LE3) was higher than the total antibody level of control, rLTB, E2 epitope and chimeric antigens which contain 3' flanked LTB subunit. Importantly, total serum Ig and fecal S-IgA level of LE1 were significantly increased when it compared with rLTB and E2 epitope. Analysis of IgG subclass responses shown that chimeric protein, LE1, stimulate both IgG1 and IgG2a responses but relative IgG1 level is more higher. This may indicate that chimeric antigen stimulates TH2 dominant responses rather than mixed Th1/Th2 response that is in the case of LT holotoxin. From results of FACS analysis, increase of IgA expressed B-cell population and CD8^(+) T-cell population were detected in small intestine and lymph node of chimeric antigen immunized mice and, the increase of CD3^(+)/CD69^(+) T-cell population were detected from small intestine, lymph node and thymus. However suppressor T-cell population were decreased in these tissues. These results revealed that the mechanism of mucosal immune modulation of LTB subunit in the chimeric antigen was different from other data which CD8^(+) T-cell down-regulation were reported. So, The function of LTB subunit in the chimeric antigen may increase binding to G_(M1) and induce to the processing pathway of endogenous MHC class I, then stimulate cell mediated immunity. In the Peyer's patch of the chimeric antigen immunized mouse, the inducer site of mucosal immunity, expression level of IL-4, IFN-gamma and TGF-beta were significantly increased when they were compared with rLTB and E2 epitope immunized mice. However, in other tissues, IFN-gamma expression was down-regulated and this expression pattern was not related to rLTB mediated cytokine expression patterns. These results suggest that the LTB subunit In chimeric antigen has mucosal adjuvant effect as a carrier, and the chimeric antigen which contain LTB subunit effectively stimulate mucosal immune system in mice by inducing S-IgA secretion, CTL mediated immune response and modulation of Th2 dominant responses. These studies have highlighted the potential of the LTB in chimeric protein as a effective adjuvant capable of trigerring mucosal and systemic immunity. The findings should allow the development of mucosal vaccination strategies in the absence of residual toxicity associated with the use of holotoxin.
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