【作者】 张涛；

【导师】 朱涛；

【作者基本信息】 天津科技大学 ， 轻工技术与工程（发酵）（专业学位）， 2015， 硕士

【摘要】 肺炎链球菌所引发的疾病在全球的发病率和死亡率非常高,人数最多的是老人和儿童。已有实验证明其荚膜多糖是TI抗原,再次免疫不能使降低的抗体达到初次免疫的水平,不能诱导免疫记忆细胞的产生。若将其共价连接到蛋白却使之成为TD抗原,能够对婴幼儿和老人产生抗体,免疫效果会得到提高。因此研究肺炎链球菌多糖蛋白结合疫苗具有重要意义。论文以3型肺炎链球菌荚膜多糖蛋白结合工艺的研究为对象,通过对3型荚膜多糖水解预处理,分子筛,氧化醛基,超滤,共价结合CRM197蛋白,SDS-PAGE电泳,液相纯化工艺流程研制结合疫苗,并通过动物免疫实验观察免疫原性。首先研究了多糖水解预处理方法,从不同水解温度和不同水解时间两个方面,通过分子筛的测定,发现随着温度和时间的增加,分子量变得越来越小。在85℃下用0.2 mol/L乙酸溶液水解1.0 h,可以得到分子量较大的结合产物,游离蛋白很少；其次多糖活化过程中发现活化反应16h以后,多糖溶液中高碘酸钠基本已经反应完全。随着加入高碘酸钠的浓度升高,活化度降低,超滤后的多糖回收率也逐渐降低。加入0.4 g/L高碘酸钠,活化度为10左右,活化多糖的回收率在78%左右；然后结合过程中,通过从反应体系容器,结合反应时间,投料比,多糖和蛋白回收率四个方面的优化研究结合疫苗,发现在4 mL体系中选用50 mL离心管,多糖和蛋白的投料比为20：10,加入7倍醛基含量的氰基硼氢化钠,在37℃下反应48 h时,利用SDS-PAGE电泳,液相纯化和超滤可以获得收率较高,没有游离蛋白的大分子量结合产物；最后纯化后的结合产物用高压灭菌的生理盐水和5 mmol/L pH 5.8琥珀酸缓冲液配制成疫苗,通过BALB/C小鼠免疫实验研究发现,85℃下水解1.0 h多糖,活化度10左右时,多糖蛋白投料比为20：10时获得的结合疫苗免疫原性相对来说最好。更多还原

【Abstract】 The morbidity and mortality rates of disease caused by Streptococcus pneumoniae are extremely high all over the world, especially in the elder and children. The decreased antibodies cannot reach the level of primary immunity response in case that the capsular polysaccharide is TI-Ag, that it cannot induce immune cell. The effect of produced TD-Ag have enhanced when Streptococcus pneumoniae were united with protein, then the antibodys for the elder and children produced. So the research of streptococcus pneumoniae conjugate vaccine had a great significance.The research discussed mainly the process of type 3 streptococcus pneumoniae capsular polysaccharide unite with protein. The conjugate vaccine were prepared through the hydrolysis pretreament of capsular polysaccharide, molecular sieving, aldehyde oxidized, ultra-filtration, covalently bonded with CRM197 protein, SDS-PAGE and AKTA FPLC system. Then its immunogenicity tested by animal immune experiment.The research studied the pretreament of capsular polysaccharide in the effect of hydrolysis temperature and hydrolysis time. It proved that, with increasing temperature and time, molecular weight was becoming more and more small. The larger combination molecular weight can be achieved by hydrolyzing in acetic acid at 85 ℃, with few free protein; The sodium periodate in polysaccharides pretty reacted primitively, after the polysaccharide activation reaction for 16 h. Along with the concentrations of sodium periodate increasing, activated degree and recovery rate of the polysaccharides were both decreasing. The recovery rate of the polysaccharides reached around 78%, as added 0.4 g/L sodium periodate and activated degree is about 10; Through the four optimized aspects, combined reaction time, rate of charge, the recovery rate of the polysaccharides and proteins and reaction container, were discussed in the resesch of conjugate vaccine. It proved the 4 ml system in 50 mL centrifuge tube, rate of charge is 20:10,7 times the aldehyde content in sodium borohydride, reacted at 37 ℃for 48 h, The yield of conjugate vaccine is much higher, it has no free protein in reaction media; The vaccine was prepared with puried conjugate production, high-pressure sterilizated physiological saline and 5 mmol/L succinic acid buffer at pH 5.8. Through the BALB/C mice immune test found the polysaccharides which was hydrolyzed for 1h in the condition of 85 ℃, activated degree is about 10, rate of charge is 20:10, The immunogenicity of conjugate vaccine was relatively efffective.更多还原