肺炎链球菌荚膜多糖分子大小及分子量分析方法的比较
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摘要
目的 3种方法分析10个血清型肺炎链球菌荚膜多糖(pneumococcal capsular polysaccharides,Pn Ps)样品的分子大小及分子量,并对这3种方法进行比较。方法采用Sepharose CL-4B凝胶排阻层析(SEC)技术检测1型、2型、4型、8型、9N型、12F型、14型、18C型、19A型和23F型Pn Ps各2~3批次样品在色谱柱中的分配系数(KD)和样品组分特征;采用高效凝胶排阻色谱-示差折光检测器(high performance size-exclusion chromatography with refractive index,HPSEC-RI)法建立Pullulan标准品分子量标准曲线,计算各Pn Ps样品的重均分子量(weight-average molecular weight,Mw);采用高效凝胶排阻色谱-多角度激光光散射仪/示差折光检测器联用技术(high performance size-exclu-sion chromatography with multi-angle laser light scattering and refractive index,HPSEC-MALLS/RI)检测各Pn Ps样品的Mw、数均分子量(number-average molecular weight,Mn)、多分散水平(Mw/Mn)及均方根旋转半径(root mean square ratio of gyration,Rg);对HPSEC-RI法和HPSEC-MALLS/RI法检测Pullulan分子量标准品的结果进行比较。结果 SEC技术不同化学方法检测的同批次Pn Ps样品的KD值十分接近,且主峰形态也比较近似,个别批次Pn Ps不同化学方法获得的峰形有差别。经HPSEC-RI检测Pullulan分子量标准品获得的回归方程为y=-0.278 x+10.13,r2=0.998,各Pn Ps样品的分子量为1.812×105~1.246×106。HPSEC-MALLS/RI联用技术检测的各批Pn Ps样品的Mw分布于9.898×105~1.471×105,Rg为84.9~25.4 nm,同一血清型不同批次样品Rg间的大小关系与其Mw间的大小关系具有较好一致性。结论结合多种方法检测多糖样品的分子大小和分子量,可更客观、全面地了解Pn Ps分子的各种性质。
Objective To compare three methods for determination of molecular size and molecular weight of pneumococcal capsular polysaccharides(Pn Ps). Methods Pn Ps of serotypes 1, 2, 4, 8, 9N, 12 F, 14, 18 C, 19 A and 23 F,2 ~ 3 batches for each, were determined for distribution coefficient(KD) value and composition specificity by sizeexclusion chromatography(SCE) with Sepharose CL-4B media. Standard molecular weight distribution curve was plotted by high performance size-exclusion chromatography-differential refractive index detector(HPSEC-RI) with Pullulan standard to calculate the weight-average molecular weight(Mw) of various Pn Ps samples. The Mw, number-average molecular weight(Mn), Mw/ Mnand root mean square ratio of gyration(Rg)of various Pn Ps samples were determined by HPSEC-mutiple angle laser light scattering apparatus(MALLS) / RI. The determination results of Pullulan standard by HPSEC-RI and HPSEC-MALLS / RI were compared. Results The KDvalues and shapes of major peaks of Pn Ps samples of the same batch determined by various methods were similar. However, the peak shapes of samples of individual batches determined by various methods were different. The regression equation of Pullalan standard determined by HPSEC-RI were y =-0. 278 x + 10. 13,r2 = 0. 998, while the molecular weights of various Pn Ps samples were 1. 812 × 105 ~ 1. 246 ×106. The Mwand Rgof Pn Ps samples of various batches determined by HPSEC-MALLS / RI were 9. 898 × 105~ 1. 471 ×105and 84. 9 ~ 25. 4 nm respectively. The Rgof samples of the same serotypes of various batches were consistent with their Mw. Conclusion Determination of molecular size and weight of Pn Ps samples by various methods was helpful to understanding the properties of Pn Ps molecules more objectively and more comprehensively.
Objective To compare three methods for determination of molecular size and molecular weight of pneumococcal capsular polysaccharides(Pn Ps). Methods Pn Ps of serotypes 1, 2, 4, 8, 9N, 12 F, 14, 18 C, 19 A and 23 F,2 ~ 3 batches for each, were determined for distribution coefficient(KD) value and composition specificity by sizeexclusion chromatography(SCE) with Sepharose CL-4B media. Standard molecular weight distribution curve was plotted by high performance size-exclusion chromatography-differential refractive index detector(HPSEC-RI) with Pullulan standard to calculate the weight-average molecular weight(Mw) of various Pn Ps samples. The Mw, number-average molecular weight(Mn), Mw/ Mnand root mean square ratio of gyration(Rg)of various Pn Ps samples were determined by HPSEC-mutiple angle laser light scattering apparatus(MALLS) / RI. The determination results of Pullulan standard by HPSEC-RI and HPSEC-MALLS / RI were compared. Results The KDvalues and shapes of major peaks of Pn Ps samples of the same batch determined by various methods were similar. However, the peak shapes of samples of individual batches determined by various methods were different. The regression equation of Pullalan standard determined by HPSEC-RI were y =-0. 278 x + 10. 13,r2 = 0. 998, while the molecular weights of various Pn Ps samples were 1. 812 × 105 ~ 1. 246 ×106. The Mwand Rgof Pn Ps samples of various batches determined by HPSEC-MALLS / RI were 9. 898 × 105~ 1. 471 ×105and 84. 9 ~ 25. 4 nm respectively. The Rgof samples of the same serotypes of various batches were consistent with their Mw. Conclusion Determination of molecular size and weight of Pn Ps samples by various methods was helpful to understanding the properties of Pn Ps molecules more objectively and more comprehensively.
引文
[1]Morens DM,Taubenberger JK,Fauci AS.Predominant role of bacterial pneumonia as a cause of death in pandemic influenza:implications for pandemic influenza preparedness[J].J Infect Dis,2008,198(7):962-970.
[2]Han F,Cao X,Hu P,et al.Development of 23-valent pneumococcal capsular polysaccharide vaccine[J].Int J Biologicals,2010,33(3):134-138.(in Chinese)韩菲,曹欣,胡鹏,等.23价肺炎链球菌荚膜多糖疫苗的研制[J].国际生物制品学杂志,2010,33(3):134-138.
[3]Bednar B,Hennessey JP Jr.Molecular size analysis of capsular polysaccharide preparations from Streptococcus pneumonia[J].Carbohydr Res,1993,243(1):115-130.
[4]World Health Organization.Recommendations to assure the quality,safety of pneumococcal conjugate vaccine[S].Geneva:WHO,2010.
[5]Chai YJ,Zuo ZJ,Ma B,et al.Optimization of condition for the fermentation process of Streptococcus pneumoniae serotype 5[J].Prog in Mod Biomed,2012,12(9):1660-1664.(in Chinese)柴雁菁,左智洁,马波,等.肺炎链球菌5型发酵工艺的优化[J].现代生物医学进展,2012,12(9):1660-1664.
[6]Chinese Pharmacopoeia Commission(Ch PC).Pharmacopoeia of People's Republic of China(VolⅢ)[S].Beijing:China Med Sci Press,2010:Append.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2010:附录.
[7]European Directorate for the Quality of Medicines and Health Care.European Pharmacopoeia 7.0[S].Strasbourg:Council of Europe,2010.
[8]Nagy DJ.Universal calibration in aqueous size exclusion chromatography with on-line differential viscometry using commercial TSK-PW columns[J].J Liq Chromatogr,1990,13(4):677-691.
[9]Lesec J,Volet G.Data treatment in aqueous GPC with on-line viscometer and light scattering detectors[J].J Liq Chromatogr,1990,13(5):831-849.
[10]施良和.凝胶色谱法[M].北京:科学出版社,1980:4-7.
[11]于世林.高效液相色谱方法及应用[M].2版.北京:化学工业出版社,2010:157-159.
[12]Mac Nair JE,Desai T,Teyral J,et al.Alignment of absolute and relative molecular size specifications for a polyvalent pneumococcal polysaccharide vaccine(PNEUMOVAX 23)[J].Biologicals,2005,33(1):49-58.
[13]Wang X,Ren KM,Chen XH,et al.Application of NMR spectrum in identification of pneumococcal capsular polysaccharides[J].Prog in Microbiol Immunol,2013,41(3):18-24.(in Chinese)王玺,任克明,陈晓航,等.核磁共振波谱法在肺炎球菌荚膜多糖检定中的应用[J].微生物学免疫学进展,2013,41(3):18-23.
[14]Wu K,Dai ZY,Ma B,et al.A improved anthrone colorimetry for determination of polysaccharide concentration in Streptococcus pneumoniae capsular polysaccharide conjugate[J].Chin J Biologicals,2007,20(7):536-538,549.(in Chinese)吴凯,代泽友,马波,等.改进的蒽酮法检测肺炎链球菌荚膜多糖结合物中多糖浓度[J].中国生物制品学杂志,2007,20(7):536-538,549.
[15]Shi JC,Luo SQ,Li MG,et al.Structure and molar mass analysis of pneumococcal capsular polysaccharides by 1H NMR and HPSEC-MALLS[J].Chin J Microbiol Immunol,2013,33(9):700-705.(in Chinese)石继春,罗树权,李茂光,等.核磁共振氢谱和高压液相分子排阻层析-十八角激光散射仪对肺炎球菌荚膜多糖的结构及分子质量分析[J].中华微生物学和免疫学杂志,2013,33(9):700-705.
[2]Han F,Cao X,Hu P,et al.Development of 23-valent pneumococcal capsular polysaccharide vaccine[J].Int J Biologicals,2010,33(3):134-138.(in Chinese)韩菲,曹欣,胡鹏,等.23价肺炎链球菌荚膜多糖疫苗的研制[J].国际生物制品学杂志,2010,33(3):134-138.
[3]Bednar B,Hennessey JP Jr.Molecular size analysis of capsular polysaccharide preparations from Streptococcus pneumonia[J].Carbohydr Res,1993,243(1):115-130.
[4]World Health Organization.Recommendations to assure the quality,safety of pneumococcal conjugate vaccine[S].Geneva:WHO,2010.
[5]Chai YJ,Zuo ZJ,Ma B,et al.Optimization of condition for the fermentation process of Streptococcus pneumoniae serotype 5[J].Prog in Mod Biomed,2012,12(9):1660-1664.(in Chinese)柴雁菁,左智洁,马波,等.肺炎链球菌5型发酵工艺的优化[J].现代生物医学进展,2012,12(9):1660-1664.
[6]Chinese Pharmacopoeia Commission(Ch PC).Pharmacopoeia of People's Republic of China(VolⅢ)[S].Beijing:China Med Sci Press,2010:Append.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2010:附录.
[7]European Directorate for the Quality of Medicines and Health Care.European Pharmacopoeia 7.0[S].Strasbourg:Council of Europe,2010.
[8]Nagy DJ.Universal calibration in aqueous size exclusion chromatography with on-line differential viscometry using commercial TSK-PW columns[J].J Liq Chromatogr,1990,13(4):677-691.
[9]Lesec J,Volet G.Data treatment in aqueous GPC with on-line viscometer and light scattering detectors[J].J Liq Chromatogr,1990,13(5):831-849.
[10]施良和.凝胶色谱法[M].北京:科学出版社,1980:4-7.
[11]于世林.高效液相色谱方法及应用[M].2版.北京:化学工业出版社,2010:157-159.
[12]Mac Nair JE,Desai T,Teyral J,et al.Alignment of absolute and relative molecular size specifications for a polyvalent pneumococcal polysaccharide vaccine(PNEUMOVAX 23)[J].Biologicals,2005,33(1):49-58.
[13]Wang X,Ren KM,Chen XH,et al.Application of NMR spectrum in identification of pneumococcal capsular polysaccharides[J].Prog in Microbiol Immunol,2013,41(3):18-24.(in Chinese)王玺,任克明,陈晓航,等.核磁共振波谱法在肺炎球菌荚膜多糖检定中的应用[J].微生物学免疫学进展,2013,41(3):18-23.
[14]Wu K,Dai ZY,Ma B,et al.A improved anthrone colorimetry for determination of polysaccharide concentration in Streptococcus pneumoniae capsular polysaccharide conjugate[J].Chin J Biologicals,2007,20(7):536-538,549.(in Chinese)吴凯,代泽友,马波,等.改进的蒽酮法检测肺炎链球菌荚膜多糖结合物中多糖浓度[J].中国生物制品学杂志,2007,20(7):536-538,549.
[15]Shi JC,Luo SQ,Li MG,et al.Structure and molar mass analysis of pneumococcal capsular polysaccharides by 1H NMR and HPSEC-MALLS[J].Chin J Microbiol Immunol,2013,33(9):700-705.(in Chinese)石继春,罗树权,李茂光,等.核磁共振氢谱和高压液相分子排阻层析-十八角激光散射仪对肺炎球菌荚膜多糖的结构及分子质量分析[J].中华微生物学和免疫学杂志,2013,33(9):700-705.