重组羊奇异变形杆菌ompA乳酸乳球菌的构建及其在小鼠体内的定植规律检测

Construction of recombinant Lactococcus lactis-expressing sheep Proteus mirabilis ompA and detection of its colonization in mice

构建了合羊奇异变形杆菌外膜蛋白A（ompA）编码基N的重组栽体pNZ8149-ompA，并将其电转入乳酸乳球菌NZ3900中。利用Westenblot检测目的蛋白的表达，流式细胞仪及间接免疫荧光技术检测目的蛋白在乳酸乳球菌中的定位。将重组乳酸乳球菌灌胃BALB／c小鼠，分别于口服后1，2，3，4，5，6，7d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乳酸乳球菌在肠道的定植情况。Westernblot检测到目的蛋白于49000处出现特异性条带；流式细胞仪检测到重组乳酸乳球菌表面荧光强度明显强于空质粒对照组；间接免疫荧光试验检测到重组乳酸乳球菌菌体表面出现绿色荧光。灌服小鼠后，重组乳酸乳球菌在小鼠各肠段的定植比例不同，定植能力于口服后6d达到高峰，7d后在重组菌在十二指肠、空肠、回肠和盲肠的定植率分别占第1d的15．23％，24．19％，48．57％，40．07％。本试验证明羊奇异变形杆菌ompA能够稳定表达于乳酸乳球菌表面，且重组乳酸乳球菌在小鼠肠道内具有良好的定植能力，其定植能力为回肠〉盲肠〉空肠〉十二指肠。这为研究乳酸乳球菌作为羊奇异变形杆菌口服疫苗抗原递送栽体提供了试验基础。

The recombinant plasmid pNZ8149-ompA containing sheep Proteus mirabilis outer membrane protein A（ompA） gene was constructed, and electro-transformed into Lactococcus lactis- NZ3900. The expression of the target protein was detected by Westen blot, and the localization of the target protein in Lactococcus lactis was detected by indirect immunofluorescence assay and flow cytometry. BALB/c mice were perfused with recombinant Lactococcus lactis and the intestinal flushing fluid of duodenal,jejunum, ileum, and intestinal tract of the mouse were collected after oral administration of 1,2,3,4,5,6,7 d. intestinal colonization by Lactococcus lactis was detection in plate colony counting method. Specific bands appeared at 49 000 detected by Western blot;green fluorescence were detected on the surface of recombinant Lactococcus lactis by indirect immunofluoreseenee assay; recombinant Lactococcus lactis surface fluorescence intensity was stronger than the empty plasmid control group detected by flow cytometry. Recombinant Lactococcus lactis can survive in a certain proportion in different parts of the intestinal tract of mice and reach a peak of colonization after oral administration of 6 d. After 7 d, the colonization rates of the recombinant Lactococcus lactis in the duodenum,jejunum,ileum and cecum were 15.23%,24.19%,48.57%, 40.07% respectively compared those of the first day. The result showed that the sheep Proteus mirabilis ompA can be expressed on the surface of Lactococcus lactis stably,and the recombinant bacteria had a good colonization ability in mouse, as follow ileum〉 cecum〉 jejunum〉 duodenum.