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目的:制备了壳聚糖季铵盐-DNA疫苗(pgD)纳米粒复合物,并对其诱导BALB/c小鼠产生免疫应答的能力做了初步研究。方法:通过复凝聚法制备了HTCC-pgD纳米粒复合物并对其进行表征。60只BALB/c小鼠随机分成5组:①HTCC10对照组;②pcDNAkan空载体对照组;③重组质粒pgD组;④HTCC∶pgD=5∶1组;⑤HTCC∶pgD=10∶1组。各组小鼠后腿肌肉注射,注射剂量为100μg/(只.次),隔2周免疫1次,共免疫2次。末次免疫2周后,通过流式细胞仪检测外周血中CD4+、CD8+百分率,ELISA法检测血清中IgG抗体效价、IFN-γ、IL-4含量这4个方面来研究该纳米粒复合物对小鼠免疫反应的影响。结果:HTCC能与质粒pgD很好结合成纳米复合物,保护DNA免受核酸酶的降解。小鼠经免疫2次后,流式细胞仪检测外周血中CD4+T淋巴细胞亚群结果显示HTCC-pgD纳米粒复合物组的CD4+百分率要高于其他对照组(P<0.05)。血清ELISA检测IgG结果显示与对照组相比,HTCC-pgD纳米粒复合物可诱导小鼠机体产生较高滴度的IgG抗体。HTCC-pgD纳米粒复合物组的小鼠血清中IFN-γ水平与对照组相比有所上调,IL-4水平有下降。结论:通过复凝聚法制备的HTCC-pgD纳米粒复合物可诱导BALB/c小鼠产生特异性的细胞免疫和体液免疫,为HSV-2型DNA疫苗研制提供科学依据。
OBJECTIVE: To prepare chitosan quaternary ammonium salt-DNA vaccine (pgD) nanoparticle complex and to study its ability to induce immune response in BALB / c mice. Methods: HTCC-pgD nanoparticle composites were prepared by complex coacervation and characterized. 60 BALB / c mice were randomly divided into 5 groups: ① HTCC10 control group; ② pcDNAkan empty vector control group; ③ recombinant plasmid pgD group; ④ HTCC: pgD = 5: 1 group; ⑤ HTCC: pgD = 10: 1 group. Mice in each group were intramuscularly injected with a dose of 100μg / (only. Times), immunized once every 2 weeks for 2 times. Two weeks after the last immunization, the percentage of CD4 + and CD8 + in peripheral blood was measured by flow cytometry, and the titer of IgG antibody, IFN-γ and IL-4 in serum were detected by ELISA Effect of immune response in mice. Results: HTCC can well combine with plasmid pgD into nano-composite to protect DNA from nuclease degradation. The results of flow cytometry on CD4 + T lymphocyte subsets in peripheral blood showed that the percentage of CD4 + in HTCC-pgD nanoparticle complex group was higher than that in other control groups (P <0.05) after mice were immunized twice. Serum ELISA detection of IgG results showed that compared with the control group, HTCC-pgD nanoparticle complex can induce higher titers of IgG antibodies in mice. The serum level of IFN-γ in HTCC-pgD nanoparticle complex group was increased compared with the control group, IL-4 level was decreased. CONCLUSION: HTCC-pgD nanoparticle complex prepared by complex coacervation can induce specific cellular and humoral immunity in BALB / c mice and provide a scientific basis for the development of HSV-2 DNA vaccine.