GFP用于单核细胞增生李斯特菌PrfA调控毒力基因actA转录表达的研究

PrfA是单核细胞增生李斯特菌(LM)中迄今为止发现的惟一个调控绝大多数毒力基因转录表达的蛋白因子.为了研究PrfA转录调控毒力基因表达的分子机制,将无启动子的绿色荧光蛋白(GFP)基因与毒力基因actA的启动子融合,连接到穿梭载体pLSV16质粒上,构建成表达融合载体pLSV16-PactA-gfp,然后将其电转化入LM野生株P14、PrfA高表达突变株P14a和prfA基因等位缺失突变株A42中表达.利用荧光显微镜和荧光酶标仪检测上述3株细菌中绿色荧光蛋白的不同表达强度,从而评价actA基因依赖于PrfA的转录活性强弱.结果显示,绿色荧光蛋白在P14a中发出的荧光强度最高,P14次之,A42最弱,两两比较均有显著差异(P＜0.01),表明毒力基因actA的转录水平高低与PrfA的活性成正相关,其转录表达依赖于PrfA的调控;该试验同时也显示GFP能方便、有效地用于研究PrfA调控LM不同毒力基因的转录表达水平.

Use of GFP to Study the Expression of the Virulence Gene actA Regulated by PrfA in Listeria monocytogenes

PrfA is the only regulatory protein identified to date to be necessary for the regulation of the expression of most of the virulence genes in L. monocytogenes.To study the molecular mechanism of the PrfA-dependent virulence genes expression,a new expression vector pLSV16-PactA-gfp was constructed by incorporation of a promoter of virulence gene actA into upstream of a promoterless green fluorescent protein gene gfp,and then electroporated into L.monocytogenes P14 (wild type),P14a (PrfA high expression mutant),A42(prfA deletion mutant).The expression level of actA regulated by PrfA was therefore evaluated with the fluorescence intensity of GFP protein.The results showed that the highest fluorescence intensity was observed in P14a,and the following was in P14,where the weakest in A42 as well.It suggested that the expression of actA was dependent on PrfA regulation.GFP reporter system could facilitate the research on the regulation of the expression of the PrfA-dependent virulence genes in Listeria monocytogenes.