Expression of a Biologically Active GFP-alpha(S1)-Casein Fusion Protein in Lactococcus lactis

概要

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In this study, we successfully developed a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced green fluorescent protein fused to alpha(S1)-casein (GFP-alpha(S1)Cas). A modified lactic acid bacterial vector (pNZ8148#2) was constructed by inserting genes for GFP and alpha(S1)-casein, a major cow's milk allergen, and the resulting vector, pNZ8148#2-GFP-alpha(S1)Cas, was applied to the expression of recombinant GFP-alpha(S1)Cas protein (rGFP-alpha(S1)Cas) in NZ9000. After inducing expression with nisin, the production of rGFP-alpha(S1)Cas was confirmed by confocal laser microscopic analysis, and the expression conditions were optimized based on fluorescent analysis and western blotting results. Moreover, the in vitro treatment of splenocytes isolated from alpha-casein (a parts per thousand yen70 % alpha(S)-casein)-immunized mice with rGFP-alpha(S1)Cas resulted in increased IL-13 mRNA expression. The observed allergic activity is indicative of the Th2-cell mediated immune response and is similar to the effects induced by exposure to alpha-casein. Our results suggest that the expression of rGFP-alpha(S1)Cas in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to specific milk allergen.