日本血吸虫rSjLAP和rSjFBPA重组蛋白抗原疫苗的保护性免疫效果评价

发布时间：2018-03-20 12:15

本文选题：日本血吸虫　切入点：重组抗原　出处：《安徽医科大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的:利用rSjLAP和rSjFBPA加佐剂分组免疫BALB/c小鼠,探讨基于2-DE和血清蛋白质组筛选的上述二种重组抗原的免疫保护效果;通过福氏佐剂和纳米C60佐剂在增强免疫效果方面的比较,观察纳米C60佐剂在血吸虫分子疫苗研发中的价值。方法:从重组质粒中诱导表达rSjLAP和rSjFBPA,分别用亲和层析法(His-Bind Purification Kit)纯化,并用Bradford法分别检测二者的含量。将6周龄BALB/c小鼠随机分为7组,每组6只。除对照组外,每组均给予rSjLAP或rSjFBPA 100μg/次/鼠,腹部皮下多点注射,共免疫3次,每次间隔2周。按以下分组免疫:①对照组:皮下注射等体积的生理盐水;②rSjLAP+福氏佐剂组;③rSjFBPA+福氏佐剂组;④rSjLAP+ rSjFBPA+福氏佐剂组;⑤rSjLAP+纳米C60佐剂组;⑥rSjFBPA+纳米C60佐剂组;⑦rSjLAP+ rSjFBPA+纳米C60佐剂组。免疫5周后,收集小鼠血清,用ELISA法测定小鼠血清IFN-γ和IL-4的含量(pg/ml)。用尾蚴感染BALB/c小鼠,30±2条/鼠,尾蚴感染6周后,处死小鼠,收集成虫,计算减虫率;收集肝组织虫卵,计算减卵率。结果:不同实验组BALB/c小鼠,测得2、3及4组重组蛋白加福氏佐剂组诱导的减虫率分别为:35.5%(P0.05); 33.0%(P0.05)、38.1%(P0.05);5、6及7组重组蛋白加纳米C60佐剂诱导的减虫率分别为:43.1%(P0.05)、39.1%(P0.05)、45.2%(P0.05); 2、3及4组诱导的减卵率分别为:40.2%(P0.05),42.6%(P0.05),48.9%(P0.05),5、6、及7组诱导的减卵率分别为41.6%(P0.05),45.4%(P0.05),46.9%(P0.05)。ELISA法测定对照组、rSjLAP+福氏佐剂组、rSjFBPA+福氏佐剂组各组IFN-γ水平(pg/ml)分别为:20.4,20.1,19.6;IL-4水平(pg/ml)分别为:192.0,382.0,518.0。结果表明:rSjLAP和rSjFBPA加佐剂均能够明显提高蛋白对BALB/c小鼠抗血吸虫感染的保护性免疫作用。结论:rSjLAP和rSjFBPA,加福氏佐剂或纳米C60佐剂免疫BALB/c小鼠,获得了一定的免疫保护作用;福氏佐剂和纳米C60佐剂均增强了重组蛋白的免疫效果,实验结果表明福氏佐剂的免疫保护效果和纳米C60佐剂差别不大,但低剂量的纳米C60佐剂对细胞不造成损伤,危害较小。以上结果表明,rSjLAP和rSjFBPA有可能作为日本血吸虫病的候选疫苗分子,为其开发应用研究打下了基础。各实验组小鼠IFN-γ水平无显著变化,IL-4水平变化明显,表明日本血吸虫感染BALB/c小鼠伴随蛋白抗原免疫,主要表现为Th2细胞应答反应。
[Abstract]:Objective: to immunize BALB/c mice with rSjLAP and rSjFBPA plus adjuvant to investigate the immunoprotective effects of these two recombinant antigens screened based on 2-DE and serum proteome, and to compare the effects of Freund's adjuvant and nano-C60 adjuvant in enhancing the immune response. To observe the value of nano-C60 adjuvant in the development of Schistosoma japonicum molecular vaccine. Methods: rSjLAP and rSjFBPA were induced from recombinant plasmid and purified by affinity chromatography. The 6-week-old BALB/c mice were randomly divided into 7 groups with 6 mice in each group. Except for the control group, each group was given rSjLAP or rSjFBPA 100 渭 g / time for 3 times. Each time interval 2 weeks. According to the following groups: control group: subcutaneous injection of normal saline 2rSjLAP Freund's adjuvant group 3rSjFBPA Freund's adjuvant group 4rSjLAP rSjFBPA Freund's adjuvant group 5rSjLAP nano-C60 adjuvant group 6rSjFBPA nano-C60 adjuvant group 7rSjLAP rSjFBPA nano-C60 adjuvant group. After 5 weeks of immunization, The contents of IFN- 纬 and IL-4 in serum of mice were measured by ELISA method. Cercariae infected BALB/c mice with 30 卤2 mice. After 6 weeks of cercariae infection, the mice were killed, the adult worms were collected, the rate of worm reduction was calculated, and the liver tissue eggs were collected. Results: different experimental groups of BALB/c mice, The results showed that the worm reduction rates induced by recombination proteins of 2t3 and 4 groups with Fowler's adjuvant group were: 1: 35.5P 0.05; 33.0 + P0.05 + 38.1and 7groups of recombinant proteins plus nano-C60 adjuvant were induced by recombination proteins and 7 groups of recombinant proteins and nano-C60 adjuvants, respectively. The worm reduction rates of groups 2 and 4 were 43.1P0.05P0.05P0.05 and 42.20.2N 42.6P 0.05N 48.9P 0.05p 0.05A, respectively, and the rate of egg reduction induced by 2C3 and 4 groups were 40.20.2ng / 42.6P 0.05 and 48.9p 0.05g / 5 respectively. The induced egg reduction rates of groups 2 and 4 were 40.20.2n and 42.6P 0.05g / 48.9p 0.05a respectively, and the rate of reduction of eggs induced by groups 2 and 4 were 42.20.2P 0.05 and 42.6P 0.05P 0.05a, respectively. The egg reduction rates of 41.6% P0.05 and 45.45% P0.05 and 46.9% P0.05U. Elisa were used to determine the level of IFN- 纬 in the control group, rSjFBPA adjuvant group and the control group, respectively. The level of PG / 纬 in the control group was 20.420.119.6IL-4 and the level of IL-4 was 192.0382.0518.0.The results showed that both the rSjFBPA adjuvant group and the control group could significantly increase the sensitivity of protein to schistosomiasis in BALB/c mice. The results showed that both the rSjLAP and rSjFBPA adjuvant could significantly increase the sensitivity of protein to Schistosoma japonicum in BALB/c mice. Conclusion BALB/c mice were immunized with 1% rSjLAP and rSjFBPA, plus Freund's adjuvant or nano-C60 adjuvant. Both Freund's adjuvant and nano-C60 adjuvant enhanced the immune effect of recombinant protein, and the results showed that the protective effect of Freund's adjuvant was not different from that of nano-C60 adjuvant. However, the low dose of nano-C60 adjuvant did not damage the cells and did little harm. These results suggest that rSjLAP and rSjFBPA may be the candidate vaccine molecules for schistosomiasis japonicum. The results showed that the level of IFN- 纬 did not change significantly in mice of all experimental groups, indicating that the immune responses of Th2 cells to the associated protein antigen in BALB/c mice infected with Schistosoma japonicum were mainly manifested by the response of Th2 cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【相似文献】