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【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】 To study the colonization ability and distribution of recombinant Lactobacillus casei in mice intestine. 【Method】 The green fluorescent protein (GFP) gene was used as a reporter gene to construct the fusion gene vector pLA-GFP of pgsA gene and GFP and electroporated into Lactobacillus to obtain a positive recombinant strain. The recombinant bacteria were orally inoculated with SPF-grade BALB / c mice at an amount of 109 mL-1 for 1.5 h, 3 h, 12 h, 1 d, 3 d, 5 d, 6 d and 7 d after oral administration, , Jejunum, ileum, cecum intestinal flushing fluid by plate colony counting method to detect the intestinal tract of recombinant Lactobacillus casei. 【Result】 The result of Western blot showed that the fusion protein of about 69 kDa was correctly expressed in lactic acid bacteria. The recombinant bacterium emitted green fluorescence under the excitation of blue-violet light. Mice oral recombinant bacteria can survive in different parts of the intestinal mucosa and attached to the intestinal mucosal surface, reached the peak of colonization 6d after oral administration, in the duodenum, jejunum, ileum and cecum colonization rate after 7d Accounting for 16.49%, 25.08%, 47.71% and 41.03% of the first day. 【Conclusion】 GFP was stably expressed in Lactobacillus casei and had good colonization ability in the intestine of mice. The law of ileum colonization was> cecum> jejunum> duodenum. This study was to investigate the effect of Lactobacillus on oral vaccine antigen delivery Carrier and provide experimental basis for the intestinal immune mechanism in mice.