目的  研究不同链长的6A型肺炎球菌荚膜多糖（type 6A pneumococcal capsular polysaccharide，Pn6A）稳定性差异及链长对结合物稳定性和免疫原性的影响。方法 采用高压均质机获得不同链长的Pn6A，并在1-氰基-4-二甲胺基吡啶四氟硼酸盐作用下与破伤风类毒素-己二酰肼衍生物形成共轭结合物。通过比较原始多糖、多糖降解物及结合物的核磁共振氢谱验证结构的正确性，通过硫酸蒽酮法和速率比浊法定量比较抗原性；通过高效液相-分子尺寸排阻层析-多角度激光散射仪比较不同链长多糖的稳定性；分析结合物物理化学（理化）性质，并通过重均相对分子质量（weight-average relative molecular weight，Mw）变化及游离多糖变化比较结合物稳定性；用原始多糖和结合物分别皮下免疫NIH小鼠3次，ELISA检测抗多糖IgG水平。结果 Pn6A经机械剪切3、8、20次后，Mw由14.01×10⁵ g/mol分别下降为1.55×10⁵、0.92×10⁵和0.58×10⁵ g/mol，其对应结合物理化数据相近；核磁共振结果表明，多糖降解物和结合物各特征质子的化学位移相比原始多糖未发生改变；硫酸蒽酮法和速率比浊法定量计算得到的不同多糖和结合物单位抗原值相近；在不同pH及温度条件下，长链多糖及其结合物的Mw和游离多糖变化均大于短链多糖。不同链长多糖结合物免疫小鼠后诱导的IgG抗体滴度均高于原始多糖（F=2.90，P=0.048），而不同结合物之间的免疫原性差异无统计学意义（F=1.39，P=0.267）。结论  机械剪切不会破坏Pn6A化学结构和抗原性；Pn6A链长越短，所获得的多糖及结合物越稳定；Pn6A经机械剪切后制备的结合物在小鼠体内可诱导良好的免疫应答。

Objective To explore the stability of type 6A pneumococcal capsular polysaccharide (Pn6A) with different chain lengths and effect of chain length on the stability and immunogenicity of conjugates. Methods Pn6As with different chain lengths were obtained by high pressure homogenizer, then activated by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate to form conjugates with tetanus toxoid-adipic acid dihydrazide derivatives. The structure was verified by proton nuclear magnetic resonance spectroscopy (¹H-NMR) comparison of the original polysaccharides, polysaccharides degradation products and conjugates, and the antigenicity was quantitatively compared by anthrone sulfate method and the rate nephelometry method. The stability of polysaccharides with different chain lengths was compared by high performance liquid-size exclusion chromatography-multiangle laser scatter. The physical chemistry properties of conjugates were analyzed, and the stability of conjugates was compared by changes in the weight-average relative molecular weight (Mw) and free polysaccharides. NIH mice were immunized subcutaneously with original polysaccharides and conjugates for 3 doses, respectively, and the IgG levels were determined by ELISA. Results The Mw of polysaccharides sheared 3, 8, 20 times decreased from 14.01×10⁵ g/mol to 1.55×10⁵, 0.92×10⁵ and 0.58×10⁵ g/mol, respectively, and the corresponding conjugates physical chemistry data were similar. The ¹H-NMR results showed that the chemical shift of each characteristic proton of polysaccharides degradation products and conjugates did not change from original polysaccharides. The unit antigen values of the polysaccharides and conjugates were similar. The changes of Mw and free polysaccharides of the long chain polysaccharides and conjugates were greater than those of short chain polysaccharides and conjugates under different pH and temperature conditions. The IgG levels of mice immunized with conjugates with different chain lengths were statistically significantly higher than that with original polysaccharide (F=2.90，P=0.048), but there was no statistically significant difference in immunogenicity among the different conjugates (F=1.39，P=0.267). Conclusions Mechanical shearing does not destroy the chemical structure and antigenicity of Pn6A. The shorter the Pn6A chain lengths are, the more stable the polysaccharide and conjugates are. The conjugates from Pn6A prepared by mechanical shear show good immune response in mice.