A群和C群脑膜炎球菌荚膜多糖提制工艺研究

Purification of Group A and Group C Meningococcal Polysaccharides

研究一种不使用苯酚的荚膜多糖提制工艺,用于疫苗制备。采用液体罐培养A群和C群脑膜炎球菌,杀菌后离心取上清液,用十六烷基三甲基溴化铵沉淀多糖,用氯化钙溶液解离后,加乙醇至25%去除核酸等杂质,经乙醇沉淀获得粗多糖。将粗多糖溶解后,加乙醇至一定浓度沉淀蛋白,离心获得上清液,经超滤浓缩和乙醇沉淀,获得纯化多糖。纯化的多糖生化检测结果符合WHO和《中国药典》(2010年版)规定,豚鼠和小鼠异常毒性检查合格,豚鼠过敏试验未见过敏反应,1H NMR检测显示多糖结构具有一致性。该研究建立的A群和C群脑膜炎球菌荚膜多糖提制工艺为应用于疫苗制备奠定了基础。

To study a method avoiding and to use phenol to purify capsular polysaccharides（PS） for vaccines,Neisseria Meningitidis（Nm） group A and group C capsular polysaccharides（PS） are important antigens against meningococcal infection.A new purification method employing multiple ethanol precipitation to avoid toxic phenol was described in this paper.Nm group A or group C were cultivated and the supernatants were collected by continuous flow centrifugation after killing of bacteria.The PSs were precipitated by Cetavlon and recovered by centrifugation.After re-suspended in CaCl2 solution,the nucleic acid was removed by centrifugation after precipitation at a final 25% ethanol solution.The crude PSs were collected after ethanol precipitation.The protein in the redissolved rude PSs was removed at a certain concentration of ethanol solution.The debris was removed by centrifugation and the purified PSs were recovered after ultra filtration and ethanol precipitation.Both group A and group C PSs showed the characteristics required by WHO and Chinese pharmacopoeia（2010）.The tests for abnormal toxicity in guinea pigs and mice were qualified.No allergic reactions were observed in the guinea pig test,and the molecular identities were determined by 1H NMR.The established purification method for group A and group C meningococcal PSs could be used in vaccine preparation.