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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 purified by sucrose density gradient centrifugation and Sepharose 4FF gel filtration chromatography respectively. The infectious product titer, SDS-PAGE reduction electrophoresis analysis and Western blot detection, and the desired product was immunized with ICR mice to detect immune serum neutralizing antibody titer. Results The purified products from 60 kDa to 70 kDa by SDS-PAGE, sucrose density gradient centrifugation and Sepharose 4FF gel filtration chromatography showed a major band of HRSV-specific F protein. However, in the presence of Sepharose 4FF gel filtration chromatography The virus of interest was detected in 3 samples (2nd peak) with infectivity titers of 4.50, 4.25 and 6.25 lg CCID50 / ml, respectively. The purified products from both methods were able to induce neutralizing antibodies in ICR mice. The geometric mean titer (GMT) was 1:13.93 ~ 1: 4.00. The antibody positive rate and neutralizing antibody GMT of the aluminum-containing adjuvant group were 1.5-2.0 times and 1.0-1.4 times higher than that of the aluminum-free adjuvant group, respectively. The positive rate of antibody and neutralizing antibody GMT in β-propiolactone inactivated group were 1.0 to 1.3 times and 1.0 to 1.1 times that of formaldehyde inactivated group respectively. Conclusion HRSV could be purified from both sucrose density gradient centrifugation and Sepharose 4FF gel filtration chromatography, the latter being more effective and more efficient. Addition of aluminum adjuvant will help to improve the antibody positive rate; β-propiolactone and formaldehyde two kinds of inactivators on the immune effect of little difference.