核磁共振波谱技术在b型流感嗜血杆菌多糖结合疫苗质控中的应用

发布时间：2018-03-19 20:06

本文选题：Hib荚膜多糖　切入点：核磁共振波谱　出处：《昆明医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：b型流感嗜血杆菌(Haemophilus influenzae type b,简称Hib)是引起小儿细菌性脑膜炎和急性呼吸道传染病的的主要致病菌,也能引起其他严重的侵袭性疾病,包括肺炎、败血症、会厌炎、蜂窝组织炎、心包炎、脊髓炎等疾病。据WHO统计的194个国家和地区中,97%已经或计划将Hib疫苗纳入国家免疫规划。早期Hib疫苗是以提纯荚膜多糖制备而成的,但对小于18月龄者无保护效果。为此,20世纪80年代研发了多糖-蛋白结合疫苗,从而实现多糖抗原类型的转变。在Hib结合疫苗研发和生产过程中,其荚膜多糖的质量控制至关重要,其中包括血清型鉴定,核糖含量,蛋白含量,磷含量的测定等。核磁共振波谱除了用于化合物的结构测定外,也是定量分析的一种重要手段,且测定不需要事先分离混合物中的各个组分,故待测物的样品制备和处理步骤简单,而且不会破坏样品中的组分,实验时间相对较短,可以同时测定混合物中多个目标分析物。本文运用NMR技术中的1H-NMR谱,对经发酵,提取,纯化3批Hib荚膜多糖进行了型别鉴定、多糖质子H排布一致性鉴定；对Hib多糖生产工艺中试剂的残留做了定性检测,以确定多糖纯度,并对多糖衍生后,游离的未反应试剂的去除进行检测,以确定最佳透析时间,有利于多糖蛋白载体的有效结合。除此之外,还利用NMR技术中1H-NMR谱和31P-NMR谱建立了测定核糖和磷含量的方法,并验证了其精密度、重复性及精确度,与《中国药典》中的化学检定方法做了比较,利用最优条件参数测定Hib多糖中的核糖和磷含量。研究结果表明核磁共振波谱精密度高,重现性好,准确度高,可作为细菌多糖质控指标检测的一个简单、准确、快捷的方法。
[Abstract]:Haemophilus influenzae type b is the leading cause of bacterial meningitis and acute respiratory infections in children. It also causes other serious aggressive diseases, including pneumonia, septicemia, epiglottitis, cellulitis, and cellulitis. Pericarditis, myelitis and other diseases. According to WHO statistics, 97% of 194 countries and territories have or plan to include Hib vaccines in national immunization programs. Early Hib vaccines were prepared from purified capsular polysaccharides. In 80s of the 20th century, a polysaccharide protein-binding vaccine was developed to change the type of polysaccharide antigen. In the process of development and production of Hib binding vaccine, The quality control of its capsule polysaccharide is very important, including the identification of serum type, the content of ribose, the content of protein, the determination of phosphorus content, etc. Nuclear magnetic resonance spectroscopy (NMR) is not only an important means of quantitative analysis, but also an important means of quantitative analysis. In addition, it does not need to separate the components of the mixture in advance, so the preparation and treatment of the samples to be tested is simple. Moreover, it can not destroy the components of the sample, and the experimental time is relatively short, so it can be used to determine multiple target analytes in the mixture at the same time. In this paper, three batches of Hib capsule polysaccharides by fermentation, extraction and purification were identified by using 1H-NMR spectra of NMR technique, and the consistency of proton H distribution of Hib polysaccharides was identified, and the residue of reagents in the production process of Hib polysaccharides was determined qualitatively. In order to determine the purity of polysaccharides, and to detect the removal of free unreacted reagents after polysaccharide derivation, to determine the best dialysis time, which is conducive to the effective binding of polysaccharide protein carriers. A method for the determination of ribose and phosphorus was established by using 1H-NMR and 31P-NMR spectra of NMR. The precision, reproducibility and accuracy of the method were verified and compared with the chemical method in the Chinese Pharmacopoeia. The content of ribose and phosphorus in Hib polysaccharides was determined by the optimum condition parameters. The results showed that the NMR spectrum had high precision, good reproducibility and high accuracy. It could be used as a simple, accurate and quick method for the determination of bacterial polysaccharides quality control index.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R445.2;R392

【共引文献】