本文選題：Hib莢膜多糖　切入點：核磁共振波譜　出處：《昆明醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：b型流感嗜血桿菌(Haemophilus influenzae type b,簡稱Hib)是引起小兒細菌性腦膜炎和急性呼吸道傳染病的的主要致病菌,也能引起其他嚴重的侵襲性疾病,包括肺炎、敗血癥、會厭炎、蜂窩組織炎、心包炎、脊髓炎等疾病。據(jù)WHO統(tǒng)計的194個國家和地區(qū)中,97%已經(jīng)或計劃將Hib疫苗納入國家免疫規(guī)劃。早期Hib疫苗是以提純莢膜多糖制備而成的,但對小于18月齡者無保護效果。為此,20世紀80年代研發(fā)了多糖-蛋白結合疫苗,從而實現(xiàn)多糖抗原類型的轉變。在Hib結合疫苗研發(fā)和生產(chǎn)過程中,其莢膜多糖的質(zhì)量控制至關重要,其中包括血清型鑒定,核糖含量,蛋白含量,磷含量的測定等。 核磁共振波譜除了用于化合物的結構測定外,也是定量分析的一種重要手段,且測定不需要事先分離混合物中的各個組分,故待測物的樣品制備和處理步驟簡單,而且不會破壞樣品中的組分,實驗時間相對較短,可以同時測定混合物中多個目標分析物。 本文運用NMR技術中的1H-NMR譜,對經(jīng)發(fā)酵,提取,純化3批Hib莢膜多糖進行了型別鑒定、多糖質(zhì)子H排布一致性鑒定；對Hib多糖生產(chǎn)工藝中試劑的殘留做了定性檢測,以確定多糖純度,并對多糖衍生后,游離的未反應試劑的去除進行檢測,以確定最佳透析時間,有利于多糖蛋白載體的有效結合。除此之外,還利用NMR技術中1H-NMR譜和31P-NMR譜建立了測定核糖和磷含量的方法,并驗證了其精密度、重復性及精確度,與《中國藥典》中的化學檢定方法做了比較,利用最優(yōu)條件參數(shù)測定Hib多糖中的核糖和磷含量。研究結果表明核磁共振波譜精密度高,重現(xiàn)性好,準確度高,可作為細菌多糖質(zhì)控指標檢測的一個簡單、準確、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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R445.2;R392

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