Zhang Ning, Hou Xilin, Yu Liyun, Wang Guihua, Zhao Zhigang, Gao Yu
College of Life Science and Technology, Heilongjiang BaYi Agricultural University, Daqing 163319, China.
Wei Sheng Wu Xue Bao. 2010 Sep;50(9):1232-8.
We studied the colonization ability and the distribution of the recombinant Lactobacillus casei in mouse intestine.
We used Green fluorescent protein (GFP) gene as reporter in constructing the recombinant plasmid pLA-GFP,which was electrotransformed into the host cells L. casei. Six-week-old female SPF BALB/c mice were orally fed with the recombinant L. casei of approximately 10(9). Groups of at least three mice per condition were killed at 1. 5 h, 3 h, 12 h, 1 d, 3 d, 5 d, 6 d, 7 d,and its duodenum, jejunum, ileum, caecum intestinal tract rinse solution was sampled separately. The recombinant bacteriain intestinal tracts were examined by plate culture count.
The molecular weight of the recombinant protein was about 69 kDa in the result of western blot. The GFP fusion protein on the cell surface was confirmed by fluorescence microscopy and flow cytometric analysis. A portion of the recombinant L. casei was able to adhere and colonize in different regions of murine intestinal tract, and the planting peak was appeared on day 6 postinoculation. The ratio of the seventh day to the first day of the recombinant L. casei adhered to the intestinal mucosa in the duodenum,jejunum, ileum, and caecum was 16.49%, 25.08%, 47.71%, and 41.03%, respectively.
The recombinant L. casei stably expressing GFP could colonize mouse intestine. The field planting rule was ileum > caecum > jejunum > duodenum. Our findings indicated that L. casei used as a deliver vector in oral vaccine is feasible, but the impact on intestinal immune mechanism in mice is needed more research.
研究重组干酪乳杆菌在小鼠肠道中的定植能力及分布情况。
以绿色荧光蛋白（GFP）基因作为报告基因构建重组质粒pLA-GFP，将其电转化至干酪乳杆菌宿主细胞。给6周龄雌性SPF级BALB/c小鼠口服约10⁹的重组干酪乳杆菌。每种条件下至少3只小鼠为一组，分别在1.5小时、3小时、12小时、1天、3天、5天、6天、7天处死，分别采集其十二指肠、空肠、回肠、盲肠的肠道冲洗液。通过平板培养计数检测肠道中的重组菌。
蛋白质免疫印迹结果显示重组蛋白分子量约为69 kDa。通过荧光显微镜和流式细胞术分析证实细胞表面存在GFP融合蛋白。部分重组干酪乳杆菌能够在小鼠肠道的不同区域黏附定植，接种后第6天出现定植高峰。重组干酪乳杆菌在十二指肠、空肠、回肠、盲肠黏附于肠黏膜的第7天与第1天的比例分别为16.49%、25.08%、47.71%和41.03%。
稳定表达GFP的重组干酪乳杆菌能够在小鼠肠道定植。定植规律为回肠＞盲肠＞空肠＞十二指肠。我们的研究结果表明，干酪乳杆菌作为口服疫苗的递送载体是可行的，但对小鼠肠道免疫机制的影响还需要更多研究。
