通过CDAP活化多糖制备b型流感嗜血杆菌荚膜多糖-D蛋白疫苗初探
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摘要
目的用CDAP活化b型流感嗜血杆菌(Hib)荚膜多糖,制备b型流感嗜血杆菌荚膜多糖及D蛋白疫苗。方法采用CDAP法在不同p H值条件下活化Hib荚膜多糖,再通过乙二酰肼(ADH)与D蛋白共价结合,制备Hib荚膜多糖与其D蛋白结合物原液,并对三批结合物原液的各项指标进行检测。获得的结合物原液免疫BALB/c小鼠,应用ELISA法检测血清中的Ig G抗体水平,评价其免疫原性。结果三批结合物原液以上检测指标均符合药典要求,衍生物中ADH含量对结合物的免疫原性有重要影响。结论用CDAP活化工艺制备的Hib荚膜多糖-D蛋白结合物适宜制备结合疫苗,为以D蛋白为载体的Hib结合疫苗的制备及五价联苗的研制奠定实验基础。
Objective To prepare haemophilus influenzae type b( Hib) capsular polysaccharide- D protein conjugate vaccine by activation of polysaccharide with 1- cyano- 4- dinmethylaminopyridinium tetrafluoroborate( CDAP). Methods CDAP was used to activate the purified Hib capsular polysaccharide antigens under different level of p H. Though oxalyl hydrazine( ADH),Hib capsular polysaccharide binds covalently to D protein. The glycoprotein conjugations of three runs were tested successively on different indexes. The BALB / c mice were immunized with these conjugations. The level of Ig G in serum was analyzed by ELISA. Results The above indexes of three glycoprotein conjugations conformed to the requirements of the pharmacopoeia. ADH content in activated polysaccharide had important influence on immunogenicity of glycoprotein conjugations. Conclusion Hib capsular polysaccharide was activated,and t was bonded with carrier D protein. The polysaccharide derivatives and combination concentrate were measured and their quality according with the requirement of Chinese Pharmacopoeia 2010 edition. D protein works as the carrier protein of the conjugate vaccine preparation,and makes a good basis for five league vaccine in the future.
Objective To prepare haemophilus influenzae type b( Hib) capsular polysaccharide- D protein conjugate vaccine by activation of polysaccharide with 1- cyano- 4- dinmethylaminopyridinium tetrafluoroborate( CDAP). Methods CDAP was used to activate the purified Hib capsular polysaccharide antigens under different level of p H. Though oxalyl hydrazine( ADH),Hib capsular polysaccharide binds covalently to D protein. The glycoprotein conjugations of three runs were tested successively on different indexes. The BALB / c mice were immunized with these conjugations. The level of Ig G in serum was analyzed by ELISA. Results The above indexes of three glycoprotein conjugations conformed to the requirements of the pharmacopoeia. ADH content in activated polysaccharide had important influence on immunogenicity of glycoprotein conjugations. Conclusion Hib capsular polysaccharide was activated,and t was bonded with carrier D protein. The polysaccharide derivatives and combination concentrate were measured and their quality according with the requirement of Chinese Pharmacopoeia 2010 edition. D protein works as the carrier protein of the conjugate vaccine preparation,and makes a good basis for five league vaccine in the future.
引文
1 Pitman M.A Type d strain of hemophilus influenzae previously designated provisionally as type d(2)and ty Pe g[J].Bacteriol,1947,53(4):499
2 Watt JP,Wolfson LJ,O'Brien KL,et al.Burden of disease caused by Haemophilus influenzae type b in children younger than 5 years:global estimates[J].Lancet,2009,374(9693):903-911
3 Chong CY,Lim WE,Heng JT,et al.The changing trend in the Patten of infective etiologies in childhood acute lower respiratory tract infection[J].Acta Paediatr Jpn,1997,39(3):317-321
4 赵铠,张以浩,李河民.医学生物制品学[M].2版.北京:人民卫生出版社,2007:540-560
5 Douglas E,Shafera,Richard F,et al.Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrauoroborate(CDAP)for use inprotein-polysaccharide conjugate vaccines and immunologicalreagents.II.Selective crosslinking of proteins to CDAP-activated Polysaccharides[J].Vaccine,2000,18:1273-1281
6 中华预防医学会.吸附无细胞百白破、灭活脊髓灰质炎和b型流感嗜血杆菌(结合)联合疫苗(DTa P-IPV/Hib五联疫苗)应用技术指南[J].华南预防医学,2011,37(2):67-71
7 Lees A,Nelson BL,Mond JJ.Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents[J].Vaccine,2000,14(3):190-198
8 Forsgern A,Riesbeck K,Janson H.Protein D of haemophilus influenza:a protective nontypeable H.influenzae antigen and a carrier for pneumococcal conjugate vaccines[J].Clin Infect Dis,2008,46(5):726-731
9 Andrew L,Brett LN,James JM.Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents[J].Vaccine,1996,14(3):190-198
2 Watt JP,Wolfson LJ,O'Brien KL,et al.Burden of disease caused by Haemophilus influenzae type b in children younger than 5 years:global estimates[J].Lancet,2009,374(9693):903-911
3 Chong CY,Lim WE,Heng JT,et al.The changing trend in the Patten of infective etiologies in childhood acute lower respiratory tract infection[J].Acta Paediatr Jpn,1997,39(3):317-321
4 赵铠,张以浩,李河民.医学生物制品学[M].2版.北京:人民卫生出版社,2007:540-560
5 Douglas E,Shafera,Richard F,et al.Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrauoroborate(CDAP)for use inprotein-polysaccharide conjugate vaccines and immunologicalreagents.II.Selective crosslinking of proteins to CDAP-activated Polysaccharides[J].Vaccine,2000,18:1273-1281
6 中华预防医学会.吸附无细胞百白破、灭活脊髓灰质炎和b型流感嗜血杆菌(结合)联合疫苗(DTa P-IPV/Hib五联疫苗)应用技术指南[J].华南预防医学,2011,37(2):67-71
7 Lees A,Nelson BL,Mond JJ.Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents[J].Vaccine,2000,14(3):190-198
8 Forsgern A,Riesbeck K,Janson H.Protein D of haemophilus influenza:a protective nontypeable H.influenzae antigen and a carrier for pneumococcal conjugate vaccines[J].Clin Infect Dis,2008,46(5):726-731
9 Andrew L,Brett LN,James JM.Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents[J].Vaccine,1996,14(3):190-198