日本血吸虫SjC23 DNA疫苗基因枪免疫小鼠诱导免疫保护作用的研究

PROTECTIVE IMMUNE RESPONSE INDUCED WITH pcDNA3.1-SjC23 IMMUNIZED BY GENE GUN IN BALB/c MICE

目的 研究日本血吸虫中国大陆株 2 3k Da膜蛋白 DNA疫苗基因枪免疫诱导 BAL B/ c小鼠产生的抗血吸虫感染作用。方法 6 0只雌性 BAL B/ c小鼠随机分为 6组 :pc DNA3.1组 (对照组 ) ,每只小鼠用基因枪经腹部皮肤免疫 pc DNA3.1质粒 DNA2次 ,共 2 μg;Sj C2 3基因枪 (gg)组 ,每鼠用基因枪免疫 pc DNA3.1- Sj C2 3质粒 DNA2 μg;Sj C2 3肌注 (im)组 ,每鼠肌注 10 0 μg pc DNA3.1- Sj C2 3质粒 DNA;Sj C2 3/ Cp G(gg)组 ,每鼠用基因枪免疫 pc DNA3.1- Sj C2 3/ Cp G质粒 DNA2 μg;Sj C2 3/Cp G(im)组 ,每鼠肌注 10 0 μg pc DNA3.1- Sj C2 3/ Cp G质粒 DNA;联合免疫组 ,每只小鼠先肌注 10 0μg pc DNA3.1- Sj C2 3/ Cp G质粒 DNA,第 2天用基因枪免疫 2 μg。各组小鼠隔 2周加强免疫 1次 ,共 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 ,4 5 d后剖杀 ,计数成虫及肝脏虫卵数。首次免疫前 2天及感染前 2天经尾静脉采血检测 Ig G抗体水平及抗体亚类 Ig G1 、Ig G2 a。末次免疫后 3周检测脾细胞经 Con A和 r Sj C2 3- HD刺激后培养上清中鼠 IL- 2、IL- 4和 IFN- γ的水平。结果 Sj C2 3(gg)组、Sj C2 3/ Cp G(gg)组及联合免疫组小鼠所检成虫数均低于对照组 (P均 <0 .0 1) 。

Objective To study the protective effect of SjC23 DNA vaccine against Schistosoma japonicuminfection immunized by gene gun. Methods Sixty BALB/c mice were divided into 6 groups randomly. The mice in control group were immunized with pcDNA3.1 plasmid DNA by gene gun; in SjC23 group (gg) immunized with pcDNA3.1-SjC23 plasmid DNA by gene gun; in SjC23group (im) immunized with 100 μg of pcDNA3.1-SjC23 plasmid DNA intramuscularly; in SjC23/CpG group (gg) immunized with pcDNA3.1-SjC23/CpG plasmid DNA by gene gun; in SjC23/CpG group (im) immunized with 100 μg of pcDNA3.1-SjC23/CpG plasmid DNA intramuscularly; in co-immunized group immunized with pcDNA3.1-SjC23/CpG plasmid DNA by intramuscular injection and by gene gun subsequently. For immunization by gene gun, two shots per mouse were performed into abdominal skin with 1 μg plasmid DNA per shot. For intramuscular injection, 100 μg of plasmid DNA was injected into two sides of quadriceps muscle, 50 μg for each side. All the animals were boosted at week 2 and week 4. Four weeks after the 3rd immunization all mice were challenged with (45±1) cercariae by abdominal skin penetration, and 45 days post-challenge, all mice were perfused and the numbers of recovered worms and hepatic eggs were counted. Blood was collected from the tail vein of all mice 2 days before the 1st immunization and challenge respectively. Serum was prepared for detection of IgG, IgG 1 and IgG 2a. Three weeks after the 3rd inoculation, the spleen cells of 2 mice from each group were cultured and stimulated with ConA and rSjC23-HD peptide. The supernatant was collected for detection of IL-2, IL-4, IFN-γ.[WT5”HZ] Results The worm reduction rates in SjC23 group (gg), SjC23/CpG group (gg) and co-immunized group were 19.57%, 25.38% and 32.31% respectively compared with control group, the hepatic egg reduction rates being 16.92%, 19.56% and 27.73% in three experimental groups correspondingly. The worm reduction rates in SjC23 group (im) and SjC23/CpG group (im) were 28.07% and 35.14%, the hepatic egg reduction rates were 21.56% and 26.52% respectively. The level of protection in co-immunized group was higher than those in SjC23 group (gg) and SjC23/CpG group (gg). ELISA results indicated that mice in three experimental groups generated specific IgG for rSjC23-HD, while mice in the control group did not. The antibody isotype in serum immunized with pcDNA3.1-SjC23 plasmid DNA by gene gun was IgG 1 predominantly and IgG 2a not detected, whereas the ratios of IgG 2a/IgG 1 in SjC23/CpG group (gg) and co-immunized group were 2.01 and 5.18 respectively. Mice in SjC23 group (im) and SjC23/CpG (im) group also produced IgG 1 and IgG 2a antibody isotypes, with the ratios of IgG 2a/IgG 1 10.06 and 12.15 respectively. Splenocytes stimulated with rSjC23-HD, IL-2 in co-immunized group was detectable. The level of IL-4 in SjC23 group (gg) was higher than those in SjC23/CpG group (gg) and in co-immunized group. Reversely, the level of IFN-γ in SjC23/CpG group(gg) was higher than that in SjC23 group (gg).Conclusion pcDNA3.1-SjC23 immunized by gene gun could induce a low protection in BALB/c mice and the immune response induced in vivo appears to be Th2 type.