日本血吸虫复合表位DNA疫苗免疫保护作用的研究

The protective effect of the multi-epitope DNA vaccine against Schistosoma japonicum infection

目的研究日本血吸虫复合表位DNA疫苗诱导BALB/c小鼠抗血吸虫感染的免疫保护作用。方法将40只雌性BALB/c小鼠随机分为4组:pcDNA3.1组(对照组),每鼠经两侧股四头肌注射100μg pcDNA3.1质粒DNA,每侧50μg;TPI组,每鼠肌注100μg pcDNA3.1-TPI质粒DNA;TP组,每鼠肌注100μg pcDNA3.1-T-linker-P质粒DNA;PT组,每鼠肌注100μg pcDNA3.1-P-linker-T质粒DNA。每隔2周加强免疫1次,剂量和方法相同,共免疫3次。末次免疫后4周每鼠经腹部皮肤攻击感染(45±1)条日本血吸虫尾蚴,45d后剖杀,计数成虫及肝脏虫卵数。首次免疫前2 d及感染前2 d尾静脉采血,间接ELISA检测特异IgG及IgG1I、gG2a水平。末次免疫后3周,每组取2只小鼠制备脾细胞,双抗体夹心法检测脾细胞经ConA和rSjCTPI刺激后培养上清中的IL-2I、L-4和IFN-γ水平。结果TP组和PT组小鼠减虫率分别为34.76%和36.14%,显著高于TPI组(P<0.05)和对照组(P<0.01);减卵率分别为51.20%和50.79%,与TPI及对照组比较差异有显著性(P<0.05)。TP组和PT组小鼠血清特异性IgG水平均升高(P<0.05),IgG2a/IgG1的比值分别为4.23和4.34。脾细胞经ConA和rSjCTPI刺激后,IL-2水平TP组和PT组较对照组均升高。结论复合表位DNA疫苗能诱导小鼠产生抗血吸虫感染的免疫保护力,效果优于rSjCTPI疫苗。

Objective To study the protective efficacy of multi epitope DNA vaccine against Schistosoma japonicum infection in BALB/c mice. Methods Forty female BAI.B/c mice were randomly divided into 4 groups. In pcDNA3.1 group （control group）, each mouse was immunized with 100 μg of pcDNA3. 1 plasmid DNA by intramuscular injection; In TPI group, each mouse was immunized with 100 μg of pcDNA3.1 TPI plasmid DNA; In TP group, each mouse was immunized with 100 μg of pcDNA3.1-T-linker P plasmid DNA, In PT group, each mouse was immunized with 100 μg of pcDNA3.1-P-linker-T. All animals were boosted at week 2 and week 4. Four weeks after the 3rd immunization, all mice were challenged with 45±1 cercariae of S. japonicum by abdominal skin penetration. 45 days post-challenge, mice were perfused and the numbers of recovered worms and hepatic eggs were counted. Blood was collected from the tall vein of all mice 2 days before the 1st immunization and challenge respectively. Serum was prepared for detection of IgG, IgG1 and IgG2a by indirect ELISA. Three weeks after the 3rd inoculation, the spleen cells of 2 mice from each group were cultured and stimulated with ConA and rSjCTPI. The supernatant was collected to detect IL-2, IL-4, IFN-γ by DAB sandwich. Results The worm reduction rates in TP group and PT group were 34.76% and 36.14%, the hepatic egg reduction rates were 51.20% and 50.79% respectively compared with control group. The level of protection in TP group was equal that in PT group（P〉0.05）. ELISA results indicated that mice immunized with peDNA3.1-T-linker-P and pcDNA3.1-P-linker-T produced specific IgG to rSjCTPI, while mice immunized with pcDNA3.1 did not. Mice in TP group and PT group also produced IgG1 and IgG2a antibody isotypes, with the ratios of [gG2a/IgG1 4.23 and 4.34 respectively. In comparison with control, the levels of IL-2 and IFN-7 of mice immunized with pcDNA3.1-T-linker-P and peDNA3.1-P-linker-T were augmented. Conclusion This study indicates that multiepitope DNA vaccine could induce protective immunity in BALB/c mice significantly. The effect is stronger than that by rSjCTPI DNA vaccine, and is obviously correlated with Thl type responses.