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目的:探讨壳聚糖纳米包被的结核杆菌早期分泌性抗原(6 kd early secretory antigenic target,ESAT-6)与fms样酪氨酸激酶3配体(fms-like tyrosine kinase 3 ligand,FL)重组DNA疫苗接种C57BL/6小鼠对其Th1和Th2型细胞因子生成的影响。方法:制备壳聚糖(nano-chitosan,CS)纳米颗粒,然后用CS纳米粒包被本室构建的结核杆菌ESAT-6与FL重组质粒(nanoESAT-6-FL)、ESAT-6质粒(nano-ESAT-6)、FL质粒(nano-FL)和pIRES载体(nano-pIRES),并进行电镜和Western blot鉴定。此后将上述质粒分别接种小鼠,同时给小鼠接种未用壳聚糖纳米颗粒包被的上述对应质粒,并设PBS、CS纳米粒及BCG作为对照。接种后9周,取小鼠脾细胞进行培养,并用结核菌纯化蛋白衍生物(purified protein derivatives tuberculin,PPD)给予刺激72 h,然后用ELISA试剂盒检查小鼠脾细胞上清液中的Th1型细胞因子(IFN-γ、IL-12)和Th2型细胞因子(IL-4、IL-10)的分泌水平。结果:成功制备出壳聚糖纳米包被的结核杆菌ESAT-6和FL重组质粒(包括其他几种质粒),并证实被壳聚糖纳米包被的质粒可在真核细胞中表达。用壳聚糖纳米包被的ESAT-6-FL重组质粒免疫小鼠,其脾细胞上清液中的IFN-γ和IL-12水平显著高于未用壳聚糖纳米包被的相应质粒免疫组和其他对照组,而IL-4和IL-10的水平与未用壳聚糖纳米包被的相应质粒免疫组和其他实验组对照组相比均无明显升高。结论:壳聚糖纳米化的ESAT-6-FL重组质粒疫苗接种小鼠能够显著提高小鼠体内Th1型细胞因子的水平。提示用壳聚糖纳米包被结核杆菌ESAT-6-FL重组DNA疫苗能提高其细胞免疫效果。
OBJECTIVE: To investigate the effect of fos-like tyrosine kinase 3 ligand (FL) recombination with 6 kd early secretory antigenic target (ESAT-6) coated with chitosan nano-coated Effect of DNA Vaccine C57BL / 6 Mice on Th1 and Th2 Cytokine Production. METHODS: CS-nanoparticles were prepared. Then CS-coated nanoparticles were used to coat ESAT-6 and FLS recombinant plasmid (nanoESAT-6-FL) -ESAT-6), FL plasmid (nano-FL) and pIRES vector (nano-pIRES), and identified by electron microscopy and Western blot. After that, the above plasmids were inoculated into mice respectively, and the mice were inoculated with the corresponding plasmids which were not coated with chitosan nanoparticles, and PBS, CS nanoparticles and BCG were used as a control. Nine weeks after inoculation, the spleen cells of mice were cultured and stimulated with purified protein derivatives tuberculin (PPD) for 72 h, and then the Th1 type of mouse spleen cell supernatant was examined by ELISA kit Secretion of cytokines (IFN-γ, IL-12) and Th2-type cytokines (IL-4, IL-10) Results: The chitosan-coated Mycobacterium tuberculosis ESAT-6 and FL recombinant plasmids (including several other plasmids) were successfully prepared and confirmed that the chitosan-coated plasmids could be expressed in eukaryotic cells. Immunization of mice with the chitosan-coated ESAT-6-FL recombinant plasmid resulted in significantly higher levels of IFN-γ and IL-12 in the spleen cell supernatant than the corresponding plasmids without chitosan nano-coating Group and other control group, while the levels of IL-4 and IL-10 were not significantly increased compared with the corresponding plasmid-immunized group and the other experimental group without chitosan nano-coating. CONCLUSION: Chitosan nanosized ESAT-6-FL recombinant plasmid vaccinated mice can significantly increase the level of Th1 cytokines in mice. These results suggest that the chitosan nano-coated Mycobacterium tuberculosis ESAT-6-FL recombinant DNA vaccine can improve the cellular immune effect.