论文部分内容阅读
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 isolate the gene of LI-Ag85C, a candidate strain of Listeriaivanovii (LI), and to evaluate its biological characteristics in vitro and in vivo. Methods The targeting plasmid containing the upstream and downstream homologous sequences of inlB1 gene was constructed. The LI-Ag85C competent cells and the inlB1 gene were knocked out by homologous recombination. The in vitro growth curve of the attenuated strain LIΔinlB1-Ag85C and the original strain LI-Ag85C The effects of two strains on the adhesion and invasion of hepatocellular carcinoma cell line HepG2 were assayed. The difference of hemolytic ability between the two strains was compared. The median lethal dose (LD 50) of the two strains was different. Results The gene sequence of LIΔinlB1-Ag85C, a candidate candidate tuberculosis vaccine candidate knocking out inlB1 gene, was in accordance with the expected results. The in vitro growth of the attenuated strain was basically the same as that of the original strain. The adhesion rates of HepG2 cells were 6.66% and 7.46%, respectively. The invasion rates were 0.031% and 0.042%, respectively. The adhesion and invasion ability of attenuated strains were lower than that of the original strain , But the difference was not statistically significant. The hemolytic activity of the attenuated strain had no significant change compared with the original strain. The LD_ (50) values of the mice were 3.2 × 10 ~ (8) CFU / 6.7 × 10 ~ (7) CFU / only, attenuated strain LD_ (50) significantly increased compared with the original strain. Conclusion The new strain of tuberculosis vaccine LIΔinlB1-Ag85C with inlB1 gene deletion was successfully constructed and its virulence was lower than that of the original strain.