炭疽芽孢杆菌突变株AP431与A16R的生物特性比较研究  

Comparative study on biological characteristics of Bacillus anthracis mutant strain AP431 and its parent strain A16R

作　　者：聂伯尧 苏润雨 陶好霞[2] 袁盛凌[2] 刘纯杰[2] 杨百亮[1] 王艳春[2] 

机构地区：[1]天津农学院动物科学与动物医学学院,天津300384 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《军事医学》2016年第5期379-383,共5页Military Medical Sciences

摘　　要：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 describe the differences in biological characteristics of the Bacillus anthracis mutant strain,a recombinant B. anthracis,named AP431,and its initial strain A16 R. Methods AP431 and A16 R strains were cultured in the same condition to contrast their growth rate with drawing growth curves. Their biochemical characteristics were detected using API 50 CH,accompanied by 50 biochemical tests for the study of carbohydrate metabolism of microorganisms. Expression and location of protective antigens were determined using Western blotting and flow cytometry,respectively. A continuously mixed co-culture experiment was conducted to reveal the competitive relationship between them. Bactericial test of hydrogen peroxide was conducted to compare the susceptibility to peroxide between AP431 and A16 R. The virulence of these strains was evaluated in a C57 B / L mouse model. Results Compared with the initial strain A16 R,AP431 had a slower growth rate but presented the same biochemical characteristics,and it showed a weaker resistance to peroxide. The level of protective antigens in the lysate,cell wall and medium was significantly higher than that of A16 R. Animal challenging test showed that there was a significant decrease in virulence in the AP431 group. Conclusion The novel mutant AP431 has obvious advantages in the expression of protective antigens and biosafety. B. anthracis AP431 has the potential to serve as a platform of live attenuated vaccines.

关 键 词：芽孢杆菌 炭疽 生物特性 A16R 毒力 突变 

分 类 号：R378.72[医药卫生—病原生物学]