Hib PRP-TT结合疫苗的制备及其免疫原性研究

发布时间：2018-11-03 20:30

【摘要】： 流感嗜血杆菌是引起婴幼儿呼吸系统原发性感染和病毒性感染时引起继发性感染的重要病原性细菌。根据荚膜多糖的组成成分和抗原性,分为a、b、c、d、e、f和一组不可分型的流感嗜血杆菌;其中b型致病力最强,可引起败血症,由此导致脑膜炎、肺炎、会厌炎、关节炎、蜂窝组织炎、心包炎、中耳炎等。主要感染5岁以下的儿童。尤其是2岁以下的婴幼儿,其中2/3的感染发生在18个月以下的婴幼儿。多年来一直采用抗生素治疗,随着耐药菌株的增加及多重耐药性的出现,用疫苗预防该菌导致的疾病是十分必要的。本试验首先对Hib 49247菌种特性及传代稳定性进行试验观察;然后对其发酵及提糖条件进行优化,所得到纯化的Hib荚膜多糖抗原通过己二酸二肼(ADH)与破伤风类毒素(TT)蛋白共价结合,制备b型流感嗜血杆菌结合物。试验结果表明,Hib 49247菌种具有其菌种特性,发酵葡萄糖,不发酵乳糖;且传代稳定;发酵Hib时培养基中生长因子的加量为Hemin 2mg/100mL,NAD1.5mg/100mL,初始pH值应为7.2,种子液应在OD_(550nm)下光吸收值0.4～0.6之间,发酵过程中溶氧应控制在20%,培养6～8小时为宜,通过100L发酵罐的中试对所得参数进行验证发酵参数具有良好的重复性和稳定性;Hib多糖纯化中,采用冷酚抽提4次,30%乙醇沉核酸,65%乙醇沉糖的条件最佳;对Hib多糖衍化工艺选择最有水平为pH11.0±0.2,活化时间为30min,溴化氰加0.5mg/mL;结合反应多糖与蛋白质量比为1∶1所得到的结合物各项指标均合格且较稳定;并通过以12.5μg多糖和含2.5μg多糖的结合物经腹腔免疫Balb/c小鼠,用ELISA法检测小鼠血清PRP抗体水平;并对Hib PRP-TT结合物对其过敏原性及TT毒性进行观察。免疫原性试验结果表明结合物免疫小鼠能诱导出高水平的血清PRP抗体,而单独用多糖免疫小鼠未能诱导高水平的抗体;过敏原性实验表明制备的PRP-TT结合物不会引起过敏性症状;TT毒性逆转实验结果表明TT均未出现逆转。本试验为进一步临床评价结合物的体内保护性效果提供了实验基础。
[Abstract]:Haemophilus influenzae (Haemophilus influenzae) is an important pathogenic bacterium that causes primary respiratory infection and secondary infection in infants. According to the composition and antigenicity of capsular polysaccharides, it was divided into two groups: Haemophilus influenzae (Haemophilus influenzae) and Haemophilus influenzae (Haemophilus influenzae). Type b is the most virulent, leading to septicemia, which can lead to meningitis, pneumonia, epiglottis, arthritis, cellulitis, pericarditis, otitis media and so on. It mainly infects children under 5 years of age. Especially in infants under 2 years of age, 2 / 3 of infections occur in infants under 18 months of age. Antibiotic therapy has been used for many years. With the increase of resistant strains and the emergence of multidrug resistance, it is necessary to prevent the disease caused by this bacterium with vaccine. In this experiment, the characteristics of Hib 49247 strain and the stability of its passage were observed. The purified Hib capsule polysaccharide antigen was prepared by covalent binding of dihydrazine adipate (ADH) and tetanus toxoid (TT) protein to Haemophilus influenzae type b. The results showed that the strain of Hib 49247 had its own characteristics, fermentation of glucose, no fermentation of lactose, and stable passage. When Hib was fermented, the amount of growth factor was Hemin 2 mg / 100 mL NAD 1.5 mg / 100 mL, the initial pH value should be 7.2, the light absorption value of seed solution should be between 0.4 and 0.6 under OD_ (550nm), and the dissolved oxygen should be controlled at 20% during fermentation. The optimum culture time was 6g / 8h, and the fermentation parameters were proved to have good reproducibility and stability by the medium scale experiment of 100L fermenter. In the purification of Hib polysaccharides, 4 times of cold phenol extraction, 30% ethanol precipitation of nucleic acid, 65% ethanol precipitation of sugar were the best conditions, and the optimum conditions for the extraction of Hib polysaccharides were as follows: pH11.0 卤0.2, activation time 30 min, cyanide bromide plus 0.5 mg / mL; When the ratio of polysaccharide to protein was 1:1, all the indexes of the conjugate were qualified and stable. Balb/c mice were immunized intraperitoneally with 12.5 渭 g polysaccharides and 2.5 渭 g polysaccharides. The serum PRP antibody levels were detected by ELISA method, and the allergen and TT toxicity of Hib PRP-TT conjugates were observed. The results of immunogenicity test showed that the conjugate immunized mice could induce high level of serum PRP antibody, but the mice immunized with polysaccharides alone could not induce high level of antibody. The anaphylaxis test showed that the PRP-TT conjugate prepared did not cause anaphylactic symptoms, while the TT toxicity reversal test showed no reversal of TT. This experiment provides the experimental basis for further clinical evaluation of the protective effect of the conjugate in vivo.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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