肺炎链球菌荚膜多糖分子大小及分子量分析方法的比较

发布时间：2018-09-19 16:27

【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。
[Abstract]:Objective to analyze the molecular size and molecular weight of 10 serotype Streptococcus pneumoniae capsule polysaccharide (pneumococcal capsular polysaccharides,Pn Ps) samples by three methods and compare them. Methods Sepharose CL-4B gel exclusion chromatography (SEC) technique was used to detect the distribution coefficient (KD) and the sample composition characteristics of 3 batches of type 1, type 2, type 4, type 1, type 9, N, type 1, type 12F, type 14, type 1, type 18C, type 2, Pn Ps, and type 23 F, respectively, in the chromatographic column. The molecular weight standard curve of Pullulan standard product was established by high performance gel exclusion chromatography-differential refraction detector (high performance size-exclusion chromatography with refractive index,HPSEC-RI). The weight average molecular weight (weight-average molecular weight,Mw) of each Pn Ps sample was calculated and the Mw, number of each Pn Ps sample was detected by high performance gel exclusion chromatography-multi-angle laser light scattering spectrometer / differential refractive detector (high performance size-exclu-sion chromatography with multi-angle laser light scattering and refractive index,HPSEC-MALLS/RI). Average molecular weight (number-average molecular weight,Mn), polydispersing level (Mw/Mn) and root-mean-square rotation radius (root mean square ratio of gyration,Rg) were compared between HPSEC-RI and HPSEC-MALLS/RI methods for the detection of Pullulan molecular weight standard. Results the KD values of the same batch Pn Ps samples detected by different chemical methods by SEC technique were very close, and the main peak morphology was similar, and the peak shapes obtained by different chemical methods of individual batches of Pn Ps were different. The regression equation obtained by HPSEC-RI detection of Pullulan molecular weight standard product is yang-0.278x 10.13r2r2O0.998.The molecular weight of each Pn Ps sample is 1.812 脳 105N1.246 脳 106.HPSEC-MALLS/RI. The Mw distribution of each batch of Pn Ps samples is 9.898 脳 105N1.471 脳 105rg = 84.925.4 nm,. The Mw of different batches of samples with the same serotype is larger than that of Rg samples of the same serotype. There is a good consistency between the small relation and the size relation of Mw. Conclusion the molecular size and molecular weight of polysaccharide samples can be detected by various methods, and the properties of Pn Ps molecules can be understood more objectively and comprehensively.
【作者单位】：兰州生物制品研究所有限责任公司第三研究室甘肃省疫苗工程技术研究中心;
【分类号】：R378

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