蔗糖密度梯度离心法纯化流感病毒工艺的优化

Optimization of procedure for purification of influenza virus by sucrose density gradient centrifugation

目的优化蔗糖密度梯度离心法纯化流感病毒的工艺参数。方法在不同进样量（600、700和800 ml）时,检测经蔗糖密度梯度离心纯化后各紫外检测峰的糖浓度、血凝滴度及卵清蛋白含量,并对血凝滴度大于1∶480的检测峰样品进行蛋白质含量和血凝素含量检测,确定最佳收峰位置、糖度范围及病毒浓缩液进样量;在不同比例的30%和50%的蔗糖溶液、不同离心时间（2、3和4 h）条件下对流感病毒浓缩液进行蔗糖密度梯度离心纯化,检测纯化后的病毒纯化液蛋白质、血凝素及卵清蛋白含量,计算蛋白质含量与血凝素含量比值及血凝素回收率,确定最佳离心时间。结果确定最佳收峰位置为第三峰,糖度范围为30%~50%之间;当病毒浓缩液进样量为600~700 ml时,30%蔗糖溶液进液量为450~550 ml,55%蔗糖溶液进液量为400~500 ml,转速为90 639×g离心3 h后,可获得最佳纯化效果。结论确定了蔗糖密度梯度离心法纯化流感病毒的最佳工艺参数,为进一步提高流感病毒的纯化效果提供了参考。

Objective To optimize the technological parameters of purification procedure for influenza virus by sucrose density gradient centrifugation. Methods The influenza virus at various sample loadings were purified by sucrose density gradient centrifugation, of which the ultraviolet absorption peaks were determined for sucrose concentration, hema-gglutination titer and ovalbumin content. The peaks at hemagglutination titers of more than 1 ∶ 480 were determined for protein and hemagglutinin contents, based on which the position for harvest of absorption peak, sucrose concentration range and sample loading of concentrated virus were optimized. The concentrated virus was purified by sucrose density gradient centrifugation for 2, 3 and 4 h respectively, and determined for protein, hemagglutinin and ovalbumin contents,based on which the ratio of protein content to hemagglutinin content as well as recovery rate of hemagglutinin were calculated.and the time for centrifugation was optimized. Results The optimal position for harvest of absorption peak was peak 3, while the sucrose concentration range was 30% ~ 50%. The optimal sample loadings of concentrated virus, 30%sucrose solution and 55% sucrose solution were 600 ~ 700, 450 ~ 550 and 400 ~ 500 ml, while the optimal rotating rate and time for centrifugation were 90 639 × g and 3 h, respectively. Conclusion The optimal technological parameters of purification procedure for influenza virus were determined, which provided a reference for increasing the purification efficacy of influenza virus.