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。
Objective To construct a novel tuberculosis vaccine candidate LIΔinlB1-Ag85C by knocking out the inlB1 gene of Listeria ivanovii （LI） recombinant strain LI-Ag85C, and study the biological characteristics of the attenuated strain in vitro and in vivo. Methods Targeting plasmid carrying inlB1 upstream and downstream sequences was constructed and electroporated into LI-Ag85C competent cells. Afterward inlB1 gene was knocked out by homologous recombination. Recombinant attenuated strain LIΔinlB1-Ag85C and parental strain LI-Ag85C were tested in growth characteristics, hemolyticability, the adhesion and invasion tendency to HepG2 in vitro and the median lethal dose （LD50） for C57BL/6 mice in vivo. Results Genome sequence of the attenuated tuberculosis vaccine candidate LIΔinlB1-Ag85C was as expected. The attenuated strain and the parental strain showed the similar growth curve in vitro. The adhesion rates of the two strains were 6.66% and 7.46%, respectively, and the invasion rate of them were 0.031% and 0.042% respectively. LIΔinlB1-Ag85C seemed having a lower adhesion and invasion rates to HepG2 cells, however the difference had no significance. The hemolytic ability of recombinant strain was the same as to the parental strain. The LD50 of LIΔinlB1-Ag85C and LI-Ag85C for C57BL/6 mice were 3.2×10^8 CFU/per mouse and 6.7×10^7 CFU/per mouse, respectively. LIΔinlB1-Ag85C showed a significantly decrease in animal virulence. Conclusion A novel tuberculosis vaccine candidate LIΔinlB1-Ag85C based on attenuated Listeria ivanovii was successfully constructed with a significant decrease in toxicity.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2017年第6期814-818,共5页
Journal of Sichuan University(Medical Sciences)
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
关键词
绵羊李斯特菌
结核疫苗
inlB1基因敲除
减毒
Listeria ivanovii （LI） Tuberculosis vaccine inlB1 gene knock-out Attenuation
