CAJ | 학술논문

目的探讨以壳聚糖包裹的PotD DNA(Chitosan-potD)纳米微粒经鼻黏膜免疫小鼠对肺炎链球菌鼻咽部定植的保护作用.方法将制备的Chitosan-potD微粒、裸pVAX1-potD重组质粒(裸potD)及pVAX1分别鼻腔免疫小鼠,收集黏膜和血清标本用以检测特异性抗体水平;分离并培养脾淋巴细胞,检测IL-17A、IL-4及IFN-γ的分泌水平;于免疫小鼠鼻腔进行肺炎链球菌攻击,通过菌落计数评估各免疫组对小鼠肺炎链球菌鼻咽部定植的保护作用.结果 Chilosan-potD纳米微粒在小鼠体内诱导产生的抗PotD特异性血清IgG、IgG1、IgG2a、黏膜IgA抗体水平及IL-17A、IL-4、IFN-γ水平与裸potD组及pVAX1组相比均明显升高,且差异具有统计学意义(P<0.05);菌落计数结果显示,Chitosan-potD微粒组在鼻咽部定植的肺炎链球菌数量显著减少(P<0.05).结论壳聚糖包裹PotD DNA微粒对肺炎链球菌鼻咽部定植具有免疫保护作用.
목적 탐토이각취당포과적PotD DNA(Chitosan-potD)납미미립경비점막면역소서대폐염련구균비인부정식적보호작용.방법 장제비적Chitosan-potD미립、라pVAX1-potD중조질립(라potD)급pVAX1분별비강면역소서,수집점막화혈청표본용이검측특이성항체수평;분리병배양비림파세포,검측IL-17A、IL-4급IFN-γ적분비수평;우면역소서비강진행폐염련구균공격,통과균락계수평고각면역조대소서폐염련구균비인부정식적보호작용.결과 Chilosan-potD납미미립재소서체내유도산생적항PotD특이성혈청IgG、IgG1、IgG2a、점막IgA항체수평급IL-17A、IL-4、IFN-γ수평여라potD조급pVAX1조상비균명현승고,차차이구유통계학의의(P<0.05);균락계수결과 현시,Chitosan-potD미립조재비인부정식적폐염련구균수량현저감소(P<0.05).결론 각취당포과PotD DNA미립대폐염련구균비인부정식구유면역보호작용.
Objective To prepare the chitosan-potD nanoparticles and to evaluate its protective efficacy against pneumococcal nasopharyngeal colonization. Methods potD gene was amplificated from pneumococcal genome and was inserted into pVAX1 expression vectors to construct pVAX1-potD recombinant plasmid which was then transfected into 293T cell using LipofectAMINE 2000 to analyze transient potD gene expression in vitro by RT-PCR and Western blot. Chitosan-potD nanoparticles were freshly prepared by coacervation methods at each time and the characterizations of the nanoparticles were then evaluated. BALB/c mice were immunized with chitosan-potD, naked potD DNA or pVAX1 for 4 times at two-week intervals. Anti-PotD IgG, IgG1 and IgG2a levels in serum and IgA levels in nasal washes, bronchoalveolar lavage fluids (BALF) and middle ear lavages(MEL) were detected by indirect enzyme-linked immunosorbent assay (ELISA). IL-17A, IL-4 and IFN-γ levels in splenocytes were determined by double sandwich ELISA. Mice were intrannsally challenged with Streptococcus pneumoniae ATCC6303, and Pneumococci were recovered from the nasopharyngeal niche at the fifth day after challenge. Results potD gene was successfully amplificated by PCR and the sequence was confimed to be consistent with that in the Genbank. The pVAX1-potD recombinant plasmid was successfully constructed and was expressed in eukaryocytes in vitro. The mean size and zeta potential of chitosan-potD nanoparticles was 430 nm and + 20.5 mv, respectively. Chitosan-potD nanoparticles were not digested by DNase Ⅰ , while naked potD DNA was completely digested. The levels of antibodies inculding IgG, IgG1, IgG2a, IgA and cytokines including IL-17A, IL-4 and IFN-γ were significantly higher in mice immunized with chitosan-potD nanoparticles than mice with naked potD or pVAX1 ( P <0.05) only. More importantly, much less Pneumococci were recovered from mice immunized with chitosan-potD nanoparticles than the other groups(P <0.05). Conclusion Chitosan-potD nanoparticles significantly enhanced the immunogenicity and protection efficacy of DNA vaccines by intranasal immunization and could be used as a potential mucosal vaccine to prevent pneumococcal infection.