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纳米氢氧化铝吸附抗淋病LTB-PorB重组蛋白疫苗的免疫活性研究

发布时间:2018-08-05 13:47
【摘要】:目的: 制备纳米氢氧化铝佐剂,吸附抗淋病PorB、LTB-PorB重组蛋白疫苗,通过鼻饲途径免疫雌性BALB/c小鼠,分析其免疫活性,探讨纳米氢氧化铝佐剂能否辅佐蛋白疫苗诱导产生更有效的特异性体液免疫和细胞免疫,尤其是高效的黏膜免疫,为寻找经黏膜途径感染病原体的疫苗递送载体进行有益探索和提供实验依据。 方法: 1.采用微乳液法制备纳米氢氧化铝颗粒,检测其粒径大小并进行透射电镜测定,同时进行细胞毒性试验和动物毒性试验以评价其作为疫苗载体的安全性; 2.将课题组已构建的原核表达重组质粒pET-30a/LTB、pET-30a/porB和pET-30a/LTB-PorB在大肠杆菌BL21中诱导表达重组蛋白,鉴定后大量制备并纯化; 3.吸附试验比较纳米氢氧化铝和常规铝佐剂吸附重组蛋白后的稳定性; 4.纳米氢氧化铝颗粒吸附重组疫苗蛋白,鼻饲途径免疫雌性BALB/c小鼠,检测重组蛋白诱导的体液免疫和细胞免疫水平。 结果: 1.采用微乳液法成功制备了纳米氢氧化铝颗粒,经检测其粒径分布范围为100-200nm,峰值为148.12nm。经细胞及动物毒性试验分析,制备的纳米氢氧化铝颗粒符合本次试验的要求; 2.纳米氢氧化铝(Nano)分别吸附的PorB(Nano-P)和LTB-PorB(Nano-L-P)重组蛋白通过鼻饲途径免疫雌性BALB/c小鼠,其血清特异性IgG和生殖道黏膜特异性sIgA水平随免疫时间的增加呈上升趋势;第3次免疫后(第6w) IgG A450值分别为0.724±0.076,0.854±0.146,明显高于PBS组、Nano组、LTB组、PorB组、LTB-PorB组五组,有统计学意义(P0.01);第6周生殖道灌洗液sIgA450值分别为0.487±0.052,0.673±0.122,明显高于其他五组,有统计学意义(P0.01);Nano-P组和Nano-L-P组脾淋巴细胞诱生的IL-4水平均明显高于其他五组,有统计学意义(P0.01); IFN-γ水平与其他五组比较无统计学意义(P0.05);脾淋巴细胞刺激指数与其他五组比较有统计学意义(P0.05);Nano-P和Nano-L-P之间比较无统计学意义(P0.05);IgG2a比IgG1比值均小于1,提示纳米氢氧化铝吸附重组蛋白以诱导Th2型体液免疫应答为主。 结论: 1.纳米氢氧化铝作为疫苗递送载体,通过鼻饲途径免疫小鼠,能促进PorB和LTB-PorB重组蛋白产生较高水平的特异性体液免疫,尤其是生殖道的黏膜免疫。
[Abstract]:Objective: to prepare nano-aluminum hydroxide adjuvant, to adsorb the recombinant protein vaccine against gonorrhea, and to immunize female BALB/c mice by nasal feeding. To investigate whether nano-aluminum hydroxide adjuvant can induce more effective specific humoral and cellular immunity, especially mucosal immunity. In order to search for the vaccine delivery vector which infects pathogens through mucosal pathway and provide experimental basis. Methods: 1. Nanometer aluminum hydroxide particles were prepared by microemulsion method. The particle size was measured and the transmission electron microscopy (TEM) was carried out. Meanwhile, the cytotoxicity test and animal toxicity test were carried out to evaluate its safety as vaccine carrier. 2. The recombinant plasmids pET-30a / L LTBN pET-30a / porB and pET-30a/LTB-PorB were induced to express recombinant proteins in E. coli BL21, and the recombinant proteins were prepared and purified in large quantities after identification. The stability of recombinant protein adsorbed by nano-aluminum hydroxide and conventional aluminum adjuvant was compared. 4. The recombinant vaccine protein was adsorbed by nano-aluminum hydroxide particles and the female BALB/c mice were immunized by nasal feeding. The humoral immunity and cellular immunity induced by recombinant protein were detected. Results: 1. Nanometer aluminum hydroxide particles were successfully prepared by microemulsion method. The particle size distribution ranges from 100 to 200 nm and the peak value is 148.12 nm. According to the analysis of cytotoxicity test and animal toxicity test, the prepared nano aluminum hydroxide particles meet the requirements of this test. 2. The recombinant proteins of PorB (Nano-P) and LTB-PorB (Nano-L-P) adsorbed by nano-aluminum hydroxide (Nano) were immunized by nasal feeding to female BALB/c mice. The levels of serum specific IgG and reproductive tract mucosal specific sIgA increased with the increase of immune time. The value of IgG A450 was 0.724 卤0.0766 卤0.146 after the third immunization, which was significantly higher than that in the PBS group and PBS group (P 0.01), and the sIgA450 value of the reproductive tract lavage fluid was 0.487 卤0.0520.673 卤0.122 in the 6th week, which was significantly higher than that in the other five groups. The levels of IL-4 induced by splenic lymphocytes in Nano-P group and Nano-L-P group were significantly higher than those in other five groups (P0.01). There was statistical significance (P0.01), IFN- 纬 level was not significant compared with other five groups (P0.05), spleen lymphocyte stimulating index was statistically significant compared with other five groups (P0.05) there was no significant difference between P05 and Nano-L-P (P0.05) the ratio of IgG2a to IgG1 was less than 1, indicating that the ratio of IgG2a to IgG1 was lower than that of other five groups (P0.05). The recombinant protein adsorbed by nanometer aluminum hydroxide mainly induced Th2 humoral immune response. Conclusion: 1. Nano aluminum hydroxide as a vaccine delivery carrier immunized mice by nasal feeding could promote the production of specific humoral immunity of PorB and LTB-PorB recombinant proteins especially the mucosal immunity of reproductive tract.
【学位授予单位】:南华大学
【学位级别】:硕士
【学位授予年份】:2011
【分类号】:R392

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