摘要
【目的】研究重组干酪乳杆菌（Lactobacillus casei）在小鼠肠道内定植能力及分布规律。【方法】利用绿色荧光蛋白（GFP）基因作为报告基因,构建pgsA基因与GFP的融合基因载体pLA-GFP,电转化到乳酸杆菌中,得到阳性重组菌。将重组菌以每只109mL-1的量,口服接种SPF级BALB/c小鼠,分别于口服后的1.5h、3h、12h、1d、3d、5d、6d、7d之后取其十二指肠、空肠、回肠、盲肠的肠道冲洗液,通过平板菌落计数法检测肠道内的重组干酪乳杆菌。【结果】Western blot结果显示约69kDa的融合蛋白在乳酸菌中得到了正确的表达;重组菌在蓝紫光激发下,发出绿色荧光。小鼠口服重组菌后能在肠道黏膜的不同部位以一定的比例存活并附着在肠黏膜表面,口服6d后达到定植高峰期,7d后在十二指肠、空肠、回肠和盲肠定植率分别占第1天的16.49%、25.08%、47.71%、41.03%。【结论】GFP在干酪乳杆菌中得到了稳定的表达,且在小鼠肠道内具有良好的定植能力,定植规律回肠〉盲肠〉空肠〉十二指肠,这为研究乳酸杆菌作为口服疫苗抗原递送载体及其对小鼠肠道免疫机理提供试验基础。
[Objective]We studied the colonization ability and the distribution of the recombinant Lactobacillus casei in mouse intestine.[Methods]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 109.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.[Results]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.[Conclusion] 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.
出处
《微生物学报》
CAS
CSCD
北大核心
2010年第9期1232-1238,共7页
Acta Microbiologica Sinica
基金
国家人事部留学回国人员科技项目择优资助项目~~
关键词
干酪乳杆菌
绿色荧光蛋白
肠道
定植
Lactobacillus casei
Green fluorescent protein
Intestinal tract
Colonization
