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目的:研究并比较结核分枝杆菌(H37Rv)Ag85B、MPT64DNA.以及两者的融合基因(AM)的免疫原性。方法:雌性C57BL/6小鼠25只,随机分为5组,即A组(PBS)、B组(peDNA3.1)、C组(pcDNA/Ag85B)、D组(pcDNA/MPT64)和E组(pcDNA/AM)。分别于胫前肌注射7.5 g/L利多卡因和质粒混合物(1:4,100μL,含质粒70μg/次),间隔2 wk免疫 1次,共3次。末次免疫后4 wk取血分离血清测定总IgG,同时分离脾淋巴细胞,用PPD刺激后分别做脾淋巴细胞增殖实验(MTT比色法)和测定脾淋巴细胞培养上清中IFN-γ的水平。结果:Ag85B、MPT64和AM质粒DNA,均能诱导小鼠产生较高水平的PPD特异性IgG。免疫小鼠脾淋巴细胞体外经PPD刺激后,能产生特异性淋巴细胞增殖和分泌IFN-γ。peDNA/AM组IFN-γ的分泌水平明显高于pcDNA/Ag85B和pcDNA/MPT64免疫组(P<0.05)。结论:结核分枝杆菌Ag85B-MPT64融合基因疫苗,能在小鼠体内诱导特异性细胞和体液免疫。
Objective: To study and compare the immunogenicity of Mycobacterium tuberculosis (H37Rv) Ag85B, MPT64DNA and their fusion genes (AM). Methods: Twenty-five female C57BL / 6 mice were randomly divided into five groups: group A (PBS), group B (pcDNA / Ag85B), group D (pcDNA / AM). The rabbits were injected with 7.5 g / L lidocaine and plasmid mixture (1: 4, 100 μL, containing plasmid 70 μg / time) respectively in the anterior tibial muscle for 3 times at 2 wk intervals. Spleen lymphocytes were isolated at 4 wk after the last immunization, and splenic lymphocyte proliferation (MTT colorimetric assay) and splenic lymphocyte culture supernatant IFN-γ levels . Results: Ag85B, MPT64 and AM plasmid DNA could induce mice to produce higher levels of PPD-specific IgG. Splenic lymphocytes from mice immunized with PPD stimulated specific lymphocyte proliferation and secretion of IFN-γ. The secretion of IFN-γ in peDNA / AM group was significantly higher than that in pcDNA / Ag85B and pcDNA / MPT64 groups (P <0.05). Conclusion: Mycobacterium tuberculosis Ag85B-MPT64 fusion gene vaccine can induce specific cellular and humoral immunity in mice.