日本血吸虫23kDa膜蛋白DNA疫苗保护性免疫及IL-12免疫佐剂作用的研究

PROTECTIVE IMMUNITY INDUCED WITH SjC23 DNA VACCINE AND THE ENHANCING EFFECT OF IL-12 AS AN ADJUVANT

目的 研究日本血吸虫中国大陆株 2 3k Da膜蛋白 (Sj C2 3) DNA疫苗诱导 BAL B/ c小鼠免疫保护作用以及 IL- 12的免疫增强效果。方法 5 6只 BAL B/ c雌性小鼠随机分为 A、B、C、D4组 ,每组 14只。 A组 (对照组 ) ,于每鼠两腿股四头肌注射 10 0 μg pc DNA3.1质粒 DNA;B组 (Sj C2 3组 ) ,注射 10 0μg pc DNA 3.1- Sj C 2 3质粒 DNA;C组 (Sj C 2 3+IL - 12 DNA组 ) ,注射 10 0μg pc D-NA3.1- Sj C 2 3、10 0μg pc DNA 3.1- p35、10 0μg pc DNA3.1- p40质粒 DNA的混合液 ;D组 (Sj C2 3+r IL- 12蛋白组 ) ,免疫剂量同 Sj C2 3组。共免疫 3次 ,每次免疫间隔 2周 ,剂量和方法同第 1次。于末次免疫后 1个月 ,每鼠攻击感染 (4 5± 2 )条日本血吸虫中国大陆株尾蚴。但 D组的小鼠在攻击感染当天 ,及感染后第 2、4、6天经腹腔注射 0 .2 μg/鼠 r IL- 12蛋白。攻击后 45 d剖杀小鼠 ,计数成虫及肝内虫卵数。于末次免疫后 2周对照组及 Sj C2 3组用 51 Cr释放法测定 Sj C 2 3介导的特异性细胞毒作用 ;用脾细胞培养法检测经重组日本血吸虫 2 3k Da膜蛋白亲水区大片段融合蛋白 (GST- HD)刺激后 ,在免疫后及攻击后小鼠脾细胞 IL - 2、IL - 4、IL - 10和 IFN -γ的水平。分别于攻击前后 2周经小鼠尾静脉采血 ,用 EL

Objective To investigate the protective efficacy of 23 kDa membrane protein DNA vaccine of Schistosoma japonicum Chinese strain and the enhancing effect of IL-12 as anadjuvant in infected BALB/c mice.Methods 56 female BALB/c 5~6 weeks old mice were divided into four groups(A?B?C?D),14 mice each group. Of group A(control group) , each mouse was injected with 100 μg of pcDNA3.1 plasmid DNA; of group B(SjC23), each mouse was injected with 100 μg of pcDNA3.1-SjC23 plasmid DNA; of group C(SjC23+IL-12 DNA), each mouse was injected with the mixture of 100 μg of pcDNA3.1-SjC23,100 μg of pcDNA 3.1-p35 and 100 μg of pcDNA3.1-p40 plasmid DNA ; of group D(SjC23+rIL-12 protein),each mouse was injected with 100 μg of pcDNA3.1-SjC23 plasmid DNA. Each mouse was immunized for three times at week 0?2?4 with the same dose and method ( but of group D each mouse was injected with recombinant mouse IL-12 by intraperitoneal at the day of challenge and day 2?4?6 after challenge ). Four weeks after the third immunization each mouse was challenged with 45 of cercariae of Schistosoma japonicum Chinese strain. At day 45 after challenge, all mice were sacrificed and perfused, the numbers of recovered worms and hepatic eggs were counted. The cytotoxic activity mediated by SjC23 was detected with 51 Cr release assay. By the culture of spleen cells, the production of IL-2,IL-4,IL-10 and IFN-γ with the stimulation of the fusion protein of GST and recombinant hydrophilic domain of SjC23(GST-HD) was observed two weeks before and after challenge. ELISA was performed for detection of anti-SjC23 antibody. Results The cytotoxic activity in the SjC23 group was 56 4%, while in the control group was 25 8%. Compared with control group, the obvious rising of IL-2 and IFN-γ in SjC23 group were detected, but IL-4 and IL-10 could not be found with any changes before and after challenge. Detection of anti-SjC23 antibody with ELISA showed that all the serum samples from the control group were negative; 4 of 10 sera from B group and 5 of 10 sera from C group and 5 of 10 sera from D group were positive after the third immunization. The worm reduction rates in group B?group C and group D were 28 1%?40 1% and 41 7%; the hepatic egg reduction rates were 37 9 %?53 3% and 51 7% respectively in comparison with the control group. Conclusion pcDNA3.1-SjC23 DNA vaccine could induce partial protection against Schistosoma japonicum in infected BALB/c mice and IL-12 as an adjuvant could enhance the protective immune effect of SjC23 DNA vaccine.