论文部分内容阅读
目的以300L发酵罐中试规模生产的冻干Mtb Ag85A质粒DNA疫苗为基础免疫小鼠,对该疫苗的免疫效能进行研究。方法将4～6周龄的BALB/c小鼠20只通过数字表法随机分为2组,一组免疫MtbAg85A质粒DNA疫苗(结核DNA疫苗免疫组),另一组免疫PBS作为阴性对照(PBS对照组);于免疫前采血,肌内注射免疫3次。在第3次免疫后3周摘眼球处死小鼠,收集血清,进行血清特异性抗体检测和细胞因子(IFN-γ、IL-4)检测;同时对每只小鼠取脾细胞进行特异性γ干扰素(IFN-γ)分泌检测和脾细胞CD4+、CD8+百分率检测。结果 (1)免疫前后免疫组与对照组抗体水平吸光度A值均小于0.2。(2)结核DNA疫苗免疫组血清细胞因子IL-4水平[(146.2±34.3)pg/ml]低于PBS对照组[(177.7±28.1)pg/ml],差异有统计学意义(t=2.244,P=0.038),而IFN-γ水平[(129.6±159.0)pg/ml]虽然高于PBS对照组[(76.5±21.5)pg/ml],但差异无统计学意义(t=-1.047,P=0.309)。(3)酶联免疫斑点检测结果表明结核DNA疫苗免疫组脾细胞特异性IFN-γ分泌水平(分泌IFN-γ细胞个数)(103.60±112.14)高于PBS对照组(5.78±5.83),差异有统计学意义(t=36.538,P=0.018)。(4)结核DNA疫苗免疫组CD4+细胞百分率均值(21.57%)高于PBS对照组(12.17%),差异有统计学意义(t=3.043,P=0.038);CD8+细胞百分率均值(13.70%)与PBS对照组(10.57%)相比,差异无统计学意义(t=0.847,P=0.445)。结论Mtb Ag85A质粒DNA疫苗主要刺激机体Th1型细胞免疫,而对诱导机体产生Th2型细胞免疫应答没有显著的促进作用。
OBJECTIVE To study the immunological efficacy of the vaccine by immunizing mice with the freeze-dried Mtb Ag85A plasmid DNA vaccine in a pilot-scale 300L fermenter. Methods Twenty BALB / c mice of 4 ~ 6 weeks old were randomly divided into two groups according to the digital table. One group was immunized with MtbAg85A plasmid DNA vaccine (TB DNA vaccine) and the other group was immunized with PBS as negative control (PBS Control group); before immunization blood, intramuscular immunization 3 times. The mice were sacrificed 3 weeks after the third immunization and the serum was collected for detection of serum specific antibodies and detection of cytokines (IFN-γ, IL-4). At the same time, spleen cells were taken from each mouse for specific γ Interferon (IFN-γ) secretion and splenocyte CD4 +, CD8 + percentage detection. Results (1) Before and after immunization, the antibody level of immunized group and control group were all less than 0.2. (2) The serum level of IL-4 in the group immunized with DNA vaccine was lower than that in the PBS control group [(146.2 ± 34.3) pg / ml] (177.7 ± 28.1 pg / ml, t = 2.244 (P = 0.038), while the level of IFN-γ [(129.6 ± 159.0) pg / ml] was higher than that of PBS control group (76.5 ± 21.5 pg / ml) P = 0.309). (3) The results of ELISA showed that splenocyte specific IFN-γ secretion (the number of IFN-γ secreting cells) (103.60 ± 112.14) was higher than that of PBS control group (5.78 ± 5.83) There was statistical significance (t = 36.538, P = 0.018). (4) The average percentage of CD4 + cells in immunized group was higher than that in PBS control group (21.17%) (P = 0.038), and the average percentage of CD8 + cells was PBS control group (10.57%), the difference was not statistically significant (t = 0.847, P = 0.445). Conclusion Mtb Ag85A plasmid DNA vaccine mainly stimulates Th1 type cellular immunity in vivo, but has no significant effect on inducing Th2 type cellular immune response in vivo.