壳聚糖纳米化的结核杆菌ESAT-6和FL重组DNA疫苗对小鼠产生Th1和Th2型细胞因子的影响  

Effects on production of Th1 and Th2 cytokines in mice immunized by recombinant plasmid DNA vaccine encoding Mycobacterium tuberculosis ESAT-6 and FL genes encapsulated with nano-chitosan

作　　者：卢燕来[1] 冯旰珠[1] 蒋青桃[1] 邱文[1] 赵聃[1] 张婧[1] 王迎伟[1] 

机构地区：[1]南京医科大学微生物与免疫学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2014年第5期580-586,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省高校自然科学研究面上项目(12KJB310007);南京医科大学科技发展基金重点项目(2011NJMU263);南京医科大学基础医学院优势学科教师培养基金项目(11JC005)

摘　　要：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 Th1 and Th2 cytokines production in the C57BL/6 mice vaccinated by recombinant ESAT-6 （6KD early secretory antigenic target） and FL （fms-like tyrosine kinase 3 ligand） plasmid DNA vaccine encapsulated with nano-chitosan. Methods：The chitosan nanoparticles were prepared,and recombinant plasmid DNA vaccines,namely pIRES-ESAT-6-FL,pIRES-ESAT-6,pIRES-FL and pIRES plasmids,were encapsulated by the nano-chitosan. Then,the size and morphology of nano-ESAT-6-FL plasmid were examined by transmission electron microscope and the expressions of corresponding protein were detected using Western blot. C57BL/6 mice were immunized with nano-ESAT-6-FL,nano-ESAT-6,nano-FL,and nano-pIRES, respectively. At the same time,the corresponding plasmids without nano-chitosan encapsultion,and PBS,nano-chitosan and BCG were also immunized with C57BL/6 mice. Nine weeks after vaccination,the splenocytes separated from the immunized mice were stimulated with PPD for 72 h in vitro,and the Th1 cytokines （IFN-γ and IL-12） and Th2 cytokines （IL-4 and IL-10） in splenocyte supernatant were measured using ELISA kit. Results：Nano-ESAT-6-FL and other chitosan-encapsulated plasmids were well prepared,and nano-ESAT-6-FL plasmid expressed the corresponding protein in glomerular mesangial cells （GMC）. The mice immunized with nano-ESAT-6-FL plasmid group（nano-ESAT-6-FL） had significantly higher levels of Th1 cytokines （IFN-γ and IL-12） in splenocyte supernatants than those of the mice treated with control plasmids,but as for IL-4 and IL-10,the changes of the two cytokines in mice with mentioned-above plasmids did not show obvious differences. Conclusion：Recombinant ESAT-6-FL plasmid DNA vaccination encapsulated by nano-chitosan could markedly improve production of Th1 cytokines,suggesting that the mice vaccinated with nano-ESAT-6-FL plasmid DNA vaccine can enhance the cell immune responses.

关 键 词：壳聚糖纳米 ESAT-6抗原 FL 重组DNA疫苗 细胞因子 

分 类 号：R392.11[医药卫生—免疫学]