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目的研究日本血吸虫中国大陆株磷酸丙糖异构酶基因(TPI基因)密码子优化后的DNA疫苗增强免疫保护作用的效果。方法60只BALB/c雌性小鼠随机均分为A(pcDNA3.1空质粒对照组)、B(pcDNA3.1-TPI组)、C(pcDNA3.1-TPI-mHSP70组)、D(pcDNA3.1-TPI.opt组)、E(pcDNA3.1-TPI.opt-mHSP70组)等5组。每鼠肌肉注射相应的纯化质粒DNA100μg,每隔3周免疫1次,共3次。末次免疫后4周,每鼠经腹部皮肤攻击感染日本血吸虫尾蚴(40±1)条,42d后剖杀,计数成虫及肝脏虫卵数。首次免疫前2d及感染前2d经尾静脉采血,检测IgG及IgG1、IgG2a的水平。攻击感染前2d取脾脏,制备单个脾细胞,用流式细胞仪检测白细胞介素2(IL-2)、IL-4、IL-5、γ干扰素(IFN-γ)及肿瘤坏死因子(TNF)的水平。结果B、C、D、E组小鼠血清均检测到特异性IgG及IgG2a与IgG1抗体,IgG2a/IgG1的比值分别为1.73、2.06、2.44、3.09。D、E组的IL-2、IFN-γ、TNF含量较B、C组均有不同程度地升高。D、E组减虫率分别为36.03%、39.03%,减卵率分别为41.71%、46.85%,均显著高于B、C组(P<0.01)。结论TPI基因密码子优化后的DNA疫苗相对于未优化TPIDNA疫苗能诱导小鼠产生较高的免疫保护作用,且诱导宿主产生较强的,及以Th1为主的免疫应答。
Objective To study the effect of DNA vaccine of codon optimized DNA vaccine of Schistosoma japonicum triose triose phosphate isomerase gene (TPI gene) in Chinese mainland. Methods Sixty BALB / c female mice were randomly divided into A (pcDNA3.1 empty plasmid control group), B (pcDNA3.1-TPI group), C (pcDNA3.1 -TPI-mHSP70 group) and D (pcDNA3. 1-TPI.opt group) and E (pcDNA3.1-TPI.opt-mHSP70 group). Each mouse intramuscular injection of the corresponding purified plasmid DNA 100μg, immunized once every 3 weeks, a total of 3 times. Four weeks after the last immunization, each mouse was challenged with cercariae of Schistosoma japonicum (40 ± 1) by abdominal skin challenge. After 42 days, the mice were sacrificed and the number of adult and liver eggs was counted. Two days before the first immunization and 2 days before infection, the tail vein blood was collected to detect the levels of IgG, IgG1 and IgG2a. The spleens were harvested 2 days prior to challenge to prepare single splenic cells. The levels of interleukin 2 (IL-2), IL-4, IL-5, IFN- γ and tumor necrosis factor (TNF) were measured by flow cytometry )s level. Results The specific IgG, IgG2a and IgG1 antibodies were detected in the serums of mice in groups B, C, D and E, and the ratios of IgG2a / IgG1 were 1.73, 2.06, 2.44, 3.09, respectively. The levels of IL-2, IFN-γ and TNF in group D and group E were increased to some extent compared with those in group B and C, respectively. The worm reduction rates in groups D and E were 36.03% and 39.03%, respectively. The egg reduction rates were 41.71% and 46.85%, respectively, which were significantly higher than those in groups B and C (P <0.01). Conclusion The DNA vaccine optimized by codon optimized TPI gene can induce higher immunoprotection in mice than the unoptimized TPIDNA vaccine and induce the host to produce stronger and predominant Th1-based immune response.