Wang Yan-chun, Yuan Sheng-ling, Tao Hao-xia, Wang Ling-chun, Zhang Zhao-shan, Liu Chun-jie
State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing, 100071, China,
World J Microbiol Biotechnol. 2015 Feb;31(2):345-52. doi: 10.1007/s11274-014-1786-x. Epub 2014 Dec 12.
To investigate the surface display of the anthrax protective antigen (PA) on attenuated Bacillus anthracis, a recombinant B. anthracis strain, named AP429 was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and the anthrax PA. Crerecombinase action at the loxP sites excised the antibiotic marker. Western blot analysis, fluorescence-activated cell sorting and immunofluorescence analysis confirmed that PA was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Notwithstanding extensive proteolytic degradation of the hybrid protein SLHs-PA, quantitative ELISA revealed that approximately 8.1 × 10(6) molecules of SLHs-PA were gained from each Bacillus cell. Moreover, electron microscopy assay indicated that the typical S-layer structures could be clearly observed from the recombinant strain micrographs.
为了研究炭疽保护性抗原（PA）在减毒炭疽芽孢杆菌表面的展示情况，构建了一种重组炭疽芽孢杆菌菌株AP429，方法是将一个翻译融合体整合到染色体中，该融合体包含编码表层蛋白EA1细胞壁靶向结构域和炭疽PA的DNA片段。Cre重组酶在loxP位点的作用切除了抗生素标记。蛋白质免疫印迹分析、荧光激活细胞分选和免疫荧光分析证实，PA在无抗生素标记的重组菌株的表层成功表达。尽管杂合蛋白SLHs-PA发生了广泛的蛋白水解降解，但定量ELISA显示，每个芽孢杆菌细胞可获得约8.1×10⁶个SLHs-PA分子。此外，电子显微镜检测表明，从重组菌株的显微照片中可以清楚地观察到典型的表层结构。
