Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells
Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells
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DOI：
10.1128/aem.01325-06
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发表时间：
2006-11-01
影响因子：
4.4
通讯作者：
Chatel, Jean-Marc
中科院分区：
文献类型：
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作者：
Guimaraes, Valeria Dellaretti;Innocentin, Silvia;Chatel, Jean-Marc
使用食品级细菌乳酸乳球菌作为粘膜水平的DNA递送载体是一种有吸引力的DNA疫苗接种策略。前期实验表明，重组L.表达单核细胞增生李斯特菌inIA基因的乳酸菌可将功能性基因递送到哺乳动物细胞中。在这里，我们探讨了潜在的使用非侵入性L。乳酸菌菌株作为DNA递送载体。我们构建了两个大肠杆菌-L。乳酸穿梭质粒pLIG：BLG 1和pLIG：BLG 2，含有牛β-乳球蛋白（BLG）cDNA的真核表达盒。用pLIG：BLG 1转染Cos-7细胞后获得最大的BLG表达，然后将其用于转化L.乳酸菌MG1363。由此产生的L。乳酸菌MG1363（pLIG：BLG 1）不能表达BLG。L.通过在与（i）纯化的pLIG：BLG 1、（ii）MG 1363（pLIG：BLG 1）、（iii）MG 1363（pLIG）和纯化的pLIG：BLG 1的混合物和（iv）MG 1363共孵育3小时后检测Caco-2人结肠癌细胞中的BLG来分析作为DNA递送载体的乳酸菌。仅在与MG 1363（pLIG：BLG 1）共孵育的Caco-2细胞中检测到BLG cDNA和BLG表达。在共孵育后24和48 h之间，Caco-2细胞中BLG cDNA水平降低。Caco-2细胞在24 h开始表达BLG，并在24 - 72 h之间增加。与MG 1363（pLIG：BLG 1）共孵育后48小时，Caco-2细胞开始分泌BLG。我们的结论是，乳球菌可以提供BLG的cDNA到哺乳动物上皮细胞，证明他们的潜力，提供在体内的DNA疫苗。
The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inIA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine P-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.