mCherry红色荧光标记乳酸菌的融合表达系统构建及应用

为开发新型荧光蛋白标记乳酸菌以填补国内研究空白,利用pSIP载体,构建了以红色荧光蛋白mCherry为标记,并以乳酸菌胆盐水解酶基因bsh为报告基因的乳酸菌融合表达系统。在4种不同启动子(P_(sppA)、P_(ldhL)、P_(32)和P_(slpA))调节下,相继实现了融合基因的诱导型和组成型表达,表达的融合重组蛋白mCherry-BSH同时检测出红色荧光活性和胆盐水解酶BSH活性。mCherry红色荧光标记的乳酸菌融合表达系统的成功构建不仅为研究乳酸菌在生物体内的分布、定植及存活情况从而揭示其益生功能的作用机理提供有利条件,也为更多活性蛋白在乳酸菌宿主中的表达、细胞定位、功能鉴定的研究奠定基础。

Construction and application of mCherry red fluorescent protein fusion expression system in lactic acid bacteria

In order to enrich the library of domestic research about new red fluorescent marker in lactic acid bacteria (LAB), we described a new fusion expression system in Lactobacillus plantarum WCFS1 based on the pSIP vector. This system contained red fluorescent protein mCherry as a marker and bile salt hydrolase gene (bsh) as a reporter gene. Moreover, in this study, four different promoters (PsppA, PldhL, P32 and PslpA) were used to regulate the expression of the fusion protein mCherry-BSH, completing the inducible and constitutive expression in lactic acid bacteria. The recombinant protein mCherry-BSH presented activity of red fluorescence and bile salt hydrolase (BSH). The successful construction of the fusion expression system in LAB using a red fluorescent protein mCherry provides favorable conditions for the distribution, intestinal colonization and survival rate of lactic acid bacteria, so as to reveal the function mechanism of its probiotic characteristics; and the system also could lay the foundation for researches on protein expression, cellular localization and properties identification of active protein in lactic acid bacteria.