b型流感嗜血杆菌结合物纯化过程不同部分的生化及免疫学特性

Biochemical and Immunological Characters of Different Fractions During Purification of Haemophilus influenzae Type b(Hib) Conjugate

目的分析Hib结合物纯化过程不同部分的生化及免疫学特性。方法制备的Hib结合物经SepharoseCL4B层析柱纯化,从中选5个不同位点取样,检测其生化特性及免疫原性。结果1、2和3部分多糖蛋白质比例在0.4左右,高分子结合物含量均在80%以上,经高压液相分析,这3部分的相对分子质量均在1400000以上,免疫小鼠后均能产生较好的免疫应答。第4部分高分子结合物含量在70%左右,相对分子质量约在670000,免疫小鼠后也能产生较好的免疫应答。第5部分高分子结合物含量为23.7%,相对分子质量在50000左右,基本是以游离状态存在的多糖和载体蛋白,免疫小鼠后仅能产生很低的免疫应答。结论1、2和3部分是Hib结合物纯化中需收集的部分,第4部分虽也以结合状态存在,但相对分子质量相对较低,第5部分主要是游离的多糖和蛋白质。

Objective To analyze the biochemical and immunological characters of different fractions during the purification of Haemophilus influenzae type b（Hib） conjugate. Methods Purify Hib conjugate by Sepharose CL-4B column chromatography and collect five different fractions for the determination of biochemical and immunological characters. Results The ratios of polysaccharide to protein in fractions 1,2 and 3 were about 0. 4 ,and the high molecular conjugate contents were more than 80%. HPLC proved that all the relative molecular weights of the three fractions were more than 1 400 000. Good immune responses were induced in the mice immunized with the three fractions. The fraction 4,with a high molecular conjugate content of about 70% and a relative molecular weight of about 670 000,also induced good immune response in mice. However,the fraetlon 5 ,with a high molecular conjugate content of 23.7% and a relative molecular weight of 50 000, was mainly polysaccharlde and vector protein in free form and only induced low immune response in mice. Conclusion The fractions 1,2 and 3 were needed to be collected during purification of Hib conjugate. The fraction 4 was conjugate with a low relative molecular weight. However,the fraction 5 was mainly free polysaccharide and protein.