表达荧光蛋白卡介苗菌株的构建与应用及其qPCR定量方法的建立

Construction and use of fluorescent protein-labeled BCG and establishment of a qPCR technique

目的构建稳定表达红、绿色荧光蛋白的卡介苗(Bacille Calmette-Guérin,BCG)菌株,并建立快速定量BCG细菌数的qPCR方法。方法电转化构建稳定表达红、绿色荧光蛋白的BCG菌株,比较其与野生菌株的生长曲线。将此荧光蛋白标记的BCG感染人源单核巨噬细胞系THP1细胞,用共聚焦显微镜观察感染吞噬效果。基于BCG 16SrRNA序列设计特异性引物,应用qPCR定量检测BCG细菌基因组DNA,根据细菌平板计数菌落数与qPCR循环数绘制标准曲线,建立qPCR定量BCG细菌数的方法。结果构建的荧光蛋白标记BCG菌株荧光信号强,生长曲线与野生菌株基本一致,共聚焦显微镜观察细菌感染细胞后呈现良好荧光效果。建立的qPCR检测方法能定量检测BCG细菌数量,重复试验中组内和组间的变异系数均小于3%。结论构建的卡介苗菌株稳定表达荧光蛋白。建立的qPCR方法重复性好,可用于对BCG菌株的快速计数。

Objective To generate BCG strains expressing red or green fluorescent proteins and to establish a qPCR technique for rapid quantification of BCG bacterial counts. Methods Plasmid transformation was used to generate BCG strains expressing red or green fluorescent proteins by electroporation, and the growth curve of transformed BCG strains was compared to parental BCG strains. Human mononuclear macrophage cell line THP1 cells were infected with the fluorescent protein-labeled BCG strains, which were observed under a confocal microscope to determine bacterial infection. Specific primers were designed based on the BCG 16 SrRNA sequence and used to quantitatively detect BCG bacterial genomic DNA with qPCR. A qPCR quantification technique was established to quantify the number of BCG bacteria based on the standard curve between BCG genomic DNA and colony formation unit(CFU) counting using a plate culture. Results The transformed fluorescent protein-labeled BCG strains produced strong fluorescent signals, and the growth curve did not differ significantly from that of parental strains. Under a confocal microscope, the fluorescent strains were readily evident in THP1 cells after infection. The technique for bacterial quantification with qPCR that was established was able to quantify the number of bacteria. In repeated experiments, the coefficient of variation between technical and biological repeats was less than 3%. Conclusion The transformed BCG strains stably expressed red or green fluorescent protein. The qPCR technique established here has good reproducibility and can be used to conduct rapid quantitative research to count BCG strains.