Hib PRP-TT結(jié)合疫苗的制備及其免疫原性研究
發(fā)布時(shí)間：2018-11-03 20:30
【摘要】： 流感嗜血桿菌是引起嬰幼兒呼吸系統(tǒng)原發(fā)性感染和病毒性感染時(shí)引起繼發(fā)性感染的重要病原性細(xì)菌。根據(jù)莢膜多糖的組成成分和抗原性,分為a、b、c、d、e、f和一組不可分型的流感嗜血桿菌;其中b型致病力最強(qiáng),可引起敗血癥,由此導(dǎo)致腦膜炎、肺炎、會(huì)厭炎、關(guān)節(jié)炎、蜂窩組織炎、心包炎、中耳炎等。主要感染5歲以下的兒童。尤其是2歲以下的嬰幼兒,其中2/3的感染發(fā)生在18個(gè)月以下的嬰幼兒。多年來(lái)一直采用抗生素治療,隨著耐藥菌株的增加及多重耐藥性的出現(xiàn),用疫苗預(yù)防該菌導(dǎo)致的疾病是十分必要的。 本試驗(yàn)首先對(duì)Hib 49247菌種特性及傳代穩(wěn)定性進(jìn)行試驗(yàn)觀察;然后對(duì)其發(fā)酵及提糖條件進(jìn)行優(yōu)化,所得到純化的Hib莢膜多糖抗原通過(guò)己二酸二肼(ADH)與破傷風(fēng)類(lèi)毒素(TT)蛋白共價(jià)結(jié)合,制備b型流感嗜血桿菌結(jié)合物。 試驗(yàn)結(jié)果表明,Hib 49247菌種具有其菌種特性,發(fā)酵葡萄糖,不發(fā)酵乳糖;且傳代穩(wěn)定;發(fā)酵Hib時(shí)培養(yǎng)基中生長(zhǎng)因子的加量為Hemin 2mg/100mL,NAD1.5mg/100mL,初始pH值應(yīng)為7.2,種子液應(yīng)在OD_(550nm)下光吸收值0.4～0.6之間,發(fā)酵過(guò)程中溶氧應(yīng)控制在20%,培養(yǎng)6～8小時(shí)為宜,通過(guò)100L發(fā)酵罐的中試對(duì)所得參數(shù)進(jìn)行驗(yàn)證發(fā)酵參數(shù)具有良好的重復(fù)性和穩(wěn)定性;Hib多糖純化中,采用冷酚抽提4次,30%乙醇沉核酸,65%乙醇沉糖的條件最佳;對(duì)Hib多糖衍化工藝選擇最有水平為pH11.0±0.2,活化時(shí)間為30min,溴化氰加0.5mg/mL;結(jié)合反應(yīng)多糖與蛋白質(zhì)量比為1∶1所得到的結(jié)合物各項(xiàng)指標(biāo)均合格且較穩(wěn)定;并通過(guò)以12.5μg多糖和含2.5μg多糖的結(jié)合物經(jīng)腹腔免疫Balb/c小鼠,用ELISA法檢測(cè)小鼠血清PRP抗體水平;并對(duì)Hib PRP-TT結(jié)合物對(duì)其過(guò)敏原性及TT毒性進(jìn)行觀察。免疫原性試驗(yàn)結(jié)果表明結(jié)合物免疫小鼠能誘導(dǎo)出高水平的血清PRP抗體,而單獨(dú)用多糖免疫小鼠未能誘導(dǎo)高水平的抗體;過(guò)敏原性實(shí)驗(yàn)表明制備的PRP-TT結(jié)合物不會(huì)引起過(guò)敏性癥狀;TT毒性逆轉(zhuǎn)實(shí)驗(yàn)結(jié)果表明TT均未出現(xiàn)逆轉(zhuǎn)。本試驗(yàn)為進(jìn)一步臨床評(píng)價(jià)結(jié)合物的體內(nèi)保護(hù)性效果提供了實(shí)驗(yàn)基礎(chǔ)。
[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 鹵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.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392
本文編號(hào)：2308946
[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 鹵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.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392
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