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目的系统评价所构建的炭疽芽孢杆菌突变株AP431的生物特性,并与出发菌株A16R对比,为后续研究提供数据基础。方法绘制生长曲线比较突变株AP431和A16R的生长性能;糖酵解实验分析二者生化特性的差异;免疫印迹和流式细胞术检测菌株的保护性抗原PA蛋白表达情况;连续传代共培养分析其竞争关系;过氧化氢灭菌实验和抑菌圈实验分析菌株对活性氧的耐受性;以C57小鼠为动物模型比较二者的毒力大小。结果与A16R相比,突变株AP431生长稍慢,生化特性基本一致。突变株AP431的PA蛋白在S层呈现表达良好,并且在培养基上清、细胞内和外膜上的表达水平均比A16R高。共培养实验发现AP431的生存竞争能力明显减弱。过氧化氢敏感实验表明,突变株AP431对过氧化氢的敏感性比A16R显著增高。动物毒力实验结果显示,分别在两种注射剂量下,突变株的致死率显著降低,与A16R组相比差异显著(P<0.01)。结论与A16R相比,炭疽菌突变株AP431保护性抗原PA表达水平明显提高,毒力明显降低,可作为新的疫苗候选株进行进一步研究。
Objective To systematically evaluate the biological characteristics of the constructed Bacillus anthracis mutant AP431 and compare with the original strain A16R to provide the data basis for the follow-up study. Methods The growth curves were compared with the growth performance of mutant AP431 and A16R. The differences of biochemical characteristics between the two strains were analyzed by glycolysis assay. The expression of PA protein was detected by Western blotting and flow cytometry. Competitive relationship; hydrogen peroxide sterilization experiments and inhibition zone experiments of strains resistant to reactive oxygen species; C57 mice as animal model to compare the size of the virulence. Results Compared with A16R, mutant AP431 grew slightly slower and had the same biochemical characteristics. The PA protein of mutant strain AP431 showed good expression in the S layer and higher expression level in the culture supernatant, intracellular and epicardial than A16R. Co-culture experiments found AP431 significantly reduced the competitiveness of survival. Hydrogen peroxide sensitive experiments showed that the mutant AP431 hydrogen peroxide sensitivity was significantly higher than the A16R. The results of animal virulence experiments showed that the lethality of the mutants was significantly reduced at the two doses, which was significantly different from that of the A16R group (P <0.01). Conclusion Compared with A16R, anthrax strain AP431 protective antigen PA expression levels significantly increased virulence was significantly reduced, as a new vaccine candidate strains for further study.