大规模纯化脊髓灰质炎病毒的蔗糖垫层离心法的建立

Development of sucrose cushion centrifugation method for large-scale purification of poliovirus

目的建立可大规模纯化脊髓灰质炎病毒(poliovirus,PV)的蔗糖垫层离心法。方法病毒大量增殖后,收集病毒液,经PEG浓缩、三氟三氯乙烷抽提后,采用蔗糖垫层或Cs Cl密度梯度离心法纯化PV(Ⅰ、Ⅱ、Ⅲ型),电镜下观察病毒形态,进行核酸及感染性滴度的检测。将纯化的Ⅰ、Ⅱ、Ⅲ型PV免疫豚鼠,心脏采血,分离血清,微量中和试验法检测血清中和抗体滴度。结果蔗糖垫层离心法纯化的各型PV,病毒颗粒形态典型,背景清晰,除完整的光滑型病毒颗粒外,还可见大量空心病毒颗粒样品,纯化效果较好;Cs Cl密度梯度离心法纯化的PV样品,病毒形态也较典型,但完整的病毒颗粒较多。两种纯化方法获得的病毒基因组核酸特征显著,背景清晰,无明显差异。蔗糖垫层离心法纯化的各型PV的感染性滴度及免疫后豚鼠血清中和抗体滴度均高于Cs Cl密度梯度离心法。结论蔗糖垫层离心法纯化的PV纯度高,免疫原性好,可用于大规模纯化PV或其他病毒。

Objective To develop a sucrose cushion centrifugation method for large-scale purification of poliovirus（PV）.Methods PV liquid was harvested after propagation in a large quantity, concentrated with PEG, extracted with trifluorotrichloroethane, then purified by sucrose cushion centrifugation and cesium chloride density gradient centrifugation.Guinea pigs were immunized with PV of types Ⅰ, Ⅱ and Ⅲ respectively, of which serum samples were collected and determined for neutralizing antibody titer by micro-neutralization test. Results The particles of PV purified by sucrose cushion centrifugation showed typical morphology, of which the background was clear. In addition to the intact and smooth virus particles, a large quantity of empty virus particles were observed, indicating a satifactory purification effect. However,though the PV purified by cesium chloride density gradient centrifugation also showed typical morphology, intact virus particles were in a large quantity. The genomic nucleic acids were of obvious characters, of which the background was clear. No obvious difference was observed between the genomic nucleic acids of PV purified by two methods. The infec-tious titers and neutralizing antibody titers induced by PV of various types purified by sucrose cusion centrifugation were higher than those by cesium chloride density gradient centrifugation. Conclusion The PV purified by sucrose cushion centrifugation showed high purity and immunogenicity, indicating that the method was suitable for large scale purification of PV or other viruses.