绵羊李斯特菌载体结核疫苗株inlB1基因缺失减毒株的构建及评价

发布时间：2018-11-09 16:06

【摘要】：目的对以绵羊李斯特菌(Listeriaivanovii,LI)为载体的结核疫苗候选株LI-Ag85C进行基因减毒,初步评价其体内外生物学特性。方法构建含有inlB1基因上、下游同源序列的打靶质粒,电转原始菌LI-Ag85C感受态细胞,同源重组敲除inlB1基因;测定减毒株LIΔinlB1-Ag85C和原始株LI-Ag85C的体外生长曲线;测定两株菌对于肝癌细胞系HepG2细胞的黏附、侵袭能力的影响,比较两株菌溶血能力的差异,两株菌对于小鼠的半数致死量(median lethal dose,LD_(50))差异。结果敲除inlB1基因的重组结核疫苗候选株LIΔinlB1-Ag85C的基因序列符合预期结果。减毒株与原始株的体外生长情况基本一致;对于HepG2细胞的黏附率分别为6.66%和7.46%,侵袭率分别为0.031%和0.042%,减毒株的黏附、侵袭能力均低于原始株,但差异无统计学意义;减毒株的溶血活性较原始株无明显变化;对小鼠的LD_(50)值分别为3.2×10~(8 )CFU/只和6.7×10~(7 )CFU/只,减毒株LD_(50)相较于原始株明显提高。结论成功构建inlB1基因缺失的新型结核疫苗候选株LIΔinlB1-Ag85C,其毒力较原始株降低。
[Abstract]:Objective to study the gene reduction of TB vaccine candidate strain LI-Ag85C using Listeria ovis (Listeriaivanovii,LI) as vector and to evaluate its biological characteristics in vitro and in vivo. Methods the target plasmids containing the upstream and downstream homologous sequences of inlB1 gene were constructed, the LI-Ag85C receptive cells of the original strain were electrotransferred, the homologous recombination knocked out the inlB1 gene, and the growth curves of the attenuated strain LI 螖 inlB1-Ag85C and the original strain LI-Ag85C were measured in vitro. The effects of the two strains on the adhesion and invasion of hepatoma cell line HepG2 were determined, and the hemolytic ability of the two strains was compared. The (median lethal dose,LD_ (50) of the two strains to mice was different. Results the gene sequence of recombinant TB vaccine candidate strain LI 螖 inlB1-Ag85C with knockout of inlB1 gene was in line with the expected results. The growth in vitro of attenuated strain and original strain was basically the same. The adhesion rates of HepG2 cells were 6.66% and 7.46%, and the invasion rates were 0.031% and 0.042%, respectively. The adhesion and invasion ability of the attenuated strains were lower than those of the original strains, but the difference was not statistically significant. The LD_ (50) value of attenuated strain was 3.2 脳 10 ~ (8) CFU/ only and 6.7 脳 10 ~ (7) CFU/, respectively, and the LD_ (50) value of attenuated strain LD_ (50) was significantly higher than that of original strain. Conclusion the virulence of novel TB vaccine candidate strain LI 螖 inlB1-Ag85C, with inlB1 gene deletion is lower than that of original strain.
【作者单位】：四川大学华西公共卫生学院卫生检验与检疫系;
【基金】：国家自然科学基金面上项目(No.31570924) 四川省科技厅国际合作项目(No.2017HH0080)资助
【分类号】：R392

【相似文献】