为制备单核细胞增生李斯特菌(LM)减毒的prfA基因重组菌,本研究通过构建重组质粒pKSV7-△prfA-gfp,电转化至LM TA感受态细胞中,将绿色荧光蛋白基因gfp插入LM的prfA基因第25 bp～93 bp之间,在42℃和10 μg/mL氯霉素的双重选择压力下,筛选LM重组菌(rLM-△prfA-gfp),并对其生物学特性进行鉴定.试验结果表明,重组菌与亲本菌相比具有相似的体外生长特性,溶血素基因(hly) mRNA的转录水平降低；对小鼠的毒力显著减弱,其LD50由105.53升高到109.79,对肝、脾、肾的损伤不明显；免疫保护力试验显示重组菌与LM TA灭活疫苗的保护率分别为90％和35％,表明rLM-△prfA-gfp比传统疫苗具有更高的保护力.本研究构建的rLM-△prfA-gfp具有良好的遗传稳定性,并且具有较好的免疫原性,为LM活疫苗载体和分子标记疫苗的研制奠定了基础.%Listeria monocytogenes is a potential live bacterial vaccine carrier to significantly stimulate the T-cell-mediated immune response. To attenuate the bacteria, a recombinant L. monocytogenes (rLM-ΔprfA-gfp) was generated by insertion of the gfp gene into prfA gene (25 bp to 93 bp) resulting in partial deletion of the prfA gene in L. monocytogenes TA wild strain via homologous recombination with pKSV7-ΔprfA-gfp transformation. The results showed that 50% lethal doses (LD50) and prfA mRNA level of rLM-ΔprfA-gfp were 4.26 logs higher and 0.46 times lower than the parent strain, respectively, but their growth properties were similar in vitro. Furthermore, BALB/c mice were immunized with rLM-ΔprfA-gfp or the LM TA inactived vaccine strains and challenged with virulent strain L. monocytogenes TA and the protection rate were 90% and 35%, respectively, indicating that the rLM-ΔprfA-gfp provided the higher protection in mice than LM TA vaccine strains. The present study could facilitate the research on L. monocytogenes live vaccine vector and molecular Marker vaccines.
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