In this study, CRISPR/Cas9 system was used to label Lactobacillus casei LC2 W with red fluorescent protein (RFP) for investigating the distribution and colonization of Lactobacillus casei in intestinal tract and evaluating its function as probiotics. First, the plasmid pLCNICK-1628-RFP was constructed and was introduced into the receptor cells of LC2 W. Then, the LC2 W-1628 gene in genome of LC2 W was deleted and the RFP gene was inserted into the genome of LC2 W. Therefore, LC2 W could express RFP. The results showed that RFP could be expressed stably in Lactobacillus casei LC2 W by measuring the fluorescence intensity.%本实验利用CRISPR/Cas9系统对干酪乳杆菌(Lactobacillus casei)LC2W进行红色荧光蛋白(red fluorescent protein,RFP)标记,用于研究干酪乳杆菌在肠道内的分布和定植状况,评价其作为益生菌的功能.首先,基于本实验室已有的干酪乳杆菌CRISPR/Cas9编辑质粒pLCNICK-1628构建重组质粒pLCNICK-1628-RFP,电转入干酪乳杆菌LC2W感受态细胞中,使干酪乳杆菌基因组中的LC2W-1628基因被红色荧光蛋白基因替换,从而使干酪乳杆菌LC2W能表达出红色荧光蛋白.得到红色荧光标记的干酪乳杆菌LC2W突变株后,测定了其荧光强度-OD600标准曲线,发现RFP在干酪乳杆菌LC2W中能稳定表达.
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