人呼吸道合胞病毒不同纯化方法的比较研究  

Comparative Study on Different Purification Methods of Human Respiratory Virus

作　　者：董莉娟[1] 李华[1] 蒋蕊鞠[1] 杨蓉[1] 龙润乡[1] 白慧珠[1] 崔萍芳[1] 杨婷[1] 谢忠平[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,昆明650118

出　　处：《医学研究杂志》2011年第4期73-76,共4页Journal of Medical Research

摘　　要：目的比较人呼吸道合胞病毒（HRSV）的2种纯化方法。方法 HRSV经超滤浓缩后,分别采用蔗糖密度梯度离心法和Sepharose 4FF凝胶过滤层析法纯化。对纯化产物进行感染性效价、SDS-PAGE还原电泳分析及Western blot检测,并取目的产物免疫ICR小鼠,检测免疫血清的中和抗体效价。结果经还原电泳,蔗糖密度梯度离心和Sepharose 4FF凝胶过滤层析的纯化产物都在60-70kDa间呈现一主要条带,前者证实为HRSV特异性F蛋白;但在Sepharose 4FF凝胶过滤层析3个样品（第2峰）中检测出目的病毒,感染性效价分别为4.50、4.25和6.25 lgCCID50/ml。两种方法所得纯化产物免疫ICR小鼠均能诱导产生中和抗体,几何平均效价（GMT）为1∶13.93-1∶4.00。含铝佐剂组的抗体阳性率和中和抗体GMT分别是不含铝佐剂组的1.5-2.0倍和1.0-1.4倍。β-丙内酯灭活组的抗体阳性率和中和抗体GMT分别是甲醛灭活组的1.0-1.3倍和1.0-1.1倍。结论蔗糖密度梯度离心法和Sepharose 4FF凝胶过滤层析法均能纯化出HRSV,后者效果更好、效率更高。加铝佐剂有助于提高抗体阳转率;β-丙内酯和甲醛2种灭活剂对免疫效果的影响差异不大。Objective To compare two purification methods of human respiratory syncytial virus（HRSV）.Methods HRSV was concentrated by ultrafiltration,and then purified by sucrose density gradient centrifugation and Sepharose 4FF gel filtration chromatography respectively.The purified product were determined for infectious titer and identified by SDS-PAGE under reducing conditions and Western blot.We immunized ICR mice with the target product and determined the serum neutralizing antibody titer.Immune effects of the product purified by two methods were compared.Results The purified product yielded by sucrose density gradient centrifugation and Sepharose 4FF gel filtration chromatography both showed a main band with molecular weight between 60 and 70kDa on reduction electrophoresis,and the former had been proved to be the specific F protein.But in the three samples of the second peak collected from Sepharose 4FF gel filtration chromatography,the purpose virus was detected,and their infections titer was 4.5,4.25 and 6.25lgCCID50/ml respectively.The purified product yielded by two methods could induce neutralizing antibody in immunized ICR mice,and geometric mean titers（GMTs） were 1∶13.93-1∶4.00.The antibody positive rate and GMTs of the vaccine containing aluminium adjuvant were 1.5-2.0 times and 1-1.4 times of the vaccine not containing aluminum adjuvant respectively.The antibody positive rate and GMTs of the vaccine inactivated by β-propionolactone were 1.0-1.3 times and 1.0-1.1 times of the vaccine formaldehyde respectively.Conclusion HRSV could be purified by sucrose density gradient centrifugation and Sepharose 4FF gel filtration chromatography,but the latter has a better effect and higher efficiency.Adding aluminium adjuvant to vaccine was helpful for improving the antibody positiving rate.β-propionolactone and formaldehyde as two inactivators have little difference in the influence on the immune effect.

关 键 词：人呼吸道合胞病毒 纯化 免疫原性 灭活剂 

分 类 号：R392.7[医药卫生—免疫学]