基于CRISPR/Cas9系统对乳酸乳球菌进行绿色荧光蛋白标记

乳酸乳球菌是治疗剂在体内运送的良好载体,研究其在体内的真实运送情况需对其进行标记。该实验利用CRISPR/Cas9系统对乳酸乳球菌(Lactococcus lactis)NZ9000进行增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)标记,用于研究乳酸乳球菌在体内的运送,评价其作为益生菌的功能。基于该实验室已构建的乳酸乳球菌CRISPR/Cas9编辑质粒pLL25构建重组质粒pYSH,其上携带eGFP及同源臂,电转入乳酸乳球菌NZ9000感受态细胞中,将基因组中的乳酸脱氢酶基因(ldh)替换为绿色荧光蛋白基因,从而使Lactococcus lactis NZ9000获得标记,表达绿色荧光蛋白。对绿色荧光标记的Lactococcus lactis NZ9000突变株,酶标仪定量分析eGFP的表达强度。荧光强度定量分析结果表明,在乳酸乳球菌不同生长阶段,eGFP基因均能稳定表达。

Green fluorescent protein labeling of Lactococcus lactis based on CRISPR/Cas9 system

Lactococcus lactis is a good carrier for the delivery of therapeutic agents in vivo. To investigate its actual delivery in vivo, L. lactis needs to be labeled. In this study, L. lactis NZ9000 was labeled with an enhanced green fluorescent protein(eGFP) using the CRISPR/Cas9 system to study its transport in vivo and evaluate its function as a probiotic. Firstly, a recombinant plasmid pYSH carrying eGFP and homologous arms was constructed based on the existing L. lactis CRISPR/Cas9 editing plasmid pLL25 in our laboratory. Secondly, pYSH was electro transferred into L. lactis NZ9000 competent cells, replacing the lactate dehydrogenase(ldh) gene in the genome with the enhanced green fluorescent protein gene, thus enabling L. lactis NZ9000 to express a green fluorescent protein. Finally, we measured the fluorescence intensity of the green fluorescence-labeled L. lactis NZ9000 mutant. The quantitative results of fluorescence intensity showed that eGFP could be stably expressed in different growth stages of L. lactis NZ9000.