重组Sj14-3-3,SjGST及mIL-12/聚乳酸聚乙醇酸共聚物(PLGA)缓释微球对小鼠免疫保护性的研究

Protective immunity induced by recombinant signaling proteins Sj14-3-3 and SjGST within IL-12/polylactic-co-glycolic acid delayed release microspheres

目的研究聚乳酸聚乙醇酸共聚物(PLGA)微球给药系统在血吸虫病分子疫苗中的应用,并探讨rSj14-3-3,rSjGST及mIL-12作为疫苗的协同作用,及mIL-12刺激机体产生CTL和辅助性T细胞(Th)在抗血吸虫病中的作用。方法从pET28a/Sj14-3-3和GST重组质粒中诱导表达rSj14-3-3及rSjGST,过柱纯化。构建真核表达质粒pcDNA3.1(+)mIL-12,并共同包入PLGA缓释微球。对制备的微球进行体外释放,观察其释放速度。分组免疫BALB/c小鼠,进行尾蚴攻击感染实验。在攻击感染6w后,剖杀小鼠,计算各组的减虫率。结果各组的减虫率:单独rSj14-3-3组为27.6%,rSj14-3-3+PcDNA3.1(+)mIL-12组34.9%,rSj14-3-3+PcDNA3.1(+)mIL-12PLGA微球组37.5%,rSj14-3-3与rSjGST混合后+PcDNA3.1(+)mIL-12PLGA微球组38.8%;各组减卵率分别为(按以上组序)35.3%,49.1%,50.5%,和43.1%。结论PLGA微球给药系统可诱导并调节体液与细胞免疫从而增强了疫苗的抗感染,抗生殖作用。但rSj14-3-3和rSjGST抗原之间未表现出协同作用。mIL-12刺激机体产生CTL和辅助性T细胞(Th),在BALB/c鼠抗血吸虫攻击感染发挥作用,增强了疫苗保护作用。

In order to evaluate the role of polylactic-co-glycolic acid （PLGA）delayed release microspheres system in the studies of the molecular vaccines for schistosomiasis and the synergistic effect of recombinant Sj14-3-3, SjGST and mIL-12, as well as the effect of CTLs and the Th cells activated by mIL-12 against Schistosoma japonicum, the expressions of recombinant Sj14-3-3 and SjGST were induced from recombinant plasmid pET28a/Sj14-3-3 and SjGST and purified through His Band Purification Kit. To construct the eukaryotic expression plasmid pcDNA3. 1（＋）mIL-12, BALB/c mice immunized with recombinant Sj14-3-3 and SjGST and plasmid mIL-12/PLGA delayed release microsphere were challenged with cercaria. Six weeks after challenge, mice were sacrificed, and the worm and egg reduction rates were calculated afterwards. It was found that the worm reduction rates of single rSj14-3-3 group, rSj14-3-3 ＋ pcDNA3. 1 （＋） mIL-12 group, rSj14-3-3 ＋ pcDNA3. 1 （＋） mIL-12/PLGA delayed release microsphere group, rSj 14-3-3 ＋ rSjGST ＋ pcDNA3. 1（ ＋ ） mIL-12 ; PLGA/PLGA delayed release microsphere group were 27. 6 %, 34.9 %. 37.5% and 38.8% respectively, while the egg reduction rates in liver tissues of these 4 groups were 35.3%, 49. 1%, 50.5% and43.1% respectively. It is concluded that the PLGA delayed release microsphere system can induce and regulate the development of humoral and cell-mediated immunity so as to promote the anti-infectious immunity of vaccines against schistosomiasis, however, there was no synergistic effect between rSj14-3-3 and rSjGST.