技术服务

结核分枝杆菌荧光标记菌株技术服务

荧光蛋白可以用于标记蛋白,在特定激发光激发下会发出特定的荧光,广泛应用于活细胞实时观察。双分子荧光互补技术是将荧光蛋白合理拆分后与待研究的两个目的蛋白分别进行连接,在结核分枝杆菌细胞内科研究蛋白-蛋白相互作用。而荧光蛋白标记结核分枝杆菌特定蛋白后,可以研究该蛋白的定位,测定结核分枝杆菌氧化还原水平,研究结核分枝杆菌在动物细胞中的动态变化等,有助于结核分枝杆菌免疫学领域的研究。

Figure 7. M.tb localizes to LC3-positive compartments. (A) GFPLC3expressing macrophages were infected with mCherry-M.tb (MOI 50

or 10, 6 h) and then fixed and observed by confocal microscopy. At high MOI there are GFP-LC3 rings surrounding (arrowhead) around mCherry-M.tb and co-localization indicated by yellow pixels (arrow) that are not seen in macrophages challenged at low MOI. (B) LAMP1 immunostaining of GFP-LC3 cell line macrophages infected with M.tb Edrman (MOI 50 or 10, 5 h) demonstrates co-localization of LC3 and lysosomal markers (arrowheads).

doi:10.1371/journal.pone.0027972.g007

Figure 2. Differences in expression of rv2390c9::GFP in Mtb present in vaccinated versus mock-treated mice. Erdman(rv2390c9::GFP, smyc9::mCherry) was inoculated into vaccinated or mock-treated C57BL/6J WT mice for up to 56 days. (A) shows 3D confocal images from a 14, 28, or 42 day infection. All bacteria are marked in red (smyc9::mCherry), reporter signal is shown in green (rv2390c9::GFP), nuclei are marked in grayscale (DAPI), and phalloidin staining of f-actin is shown in blue. Scale bar 10 mm. (B) shows quantification of the GFP/mm3 signal for each bacterium measured from multiple 3D confocal images, at the indicated time points. Each point on the graph represents a bacterium or a tightly clustered group of bacteria (mock-treated – filled symbols, vaccinated – open symbols). Horizontal lines mark the median value for each group. p-values were obtained with a Mann-Whitney statistical test.

doi:10.1371/journal.ppat.1004394.g002