目的　建立分离、纯化流感病毒RNA聚合酶PB1、PB2和PA 3P蛋白的有效方法 ,为进一步研究 3P蛋白的结构和功能奠定基础。方法　用甘油、CsCl1及CsTFA不同介质的密度梯度超速离心法进行分离 ,用SDS -PAGE电泳及Westernblot法检测 3P蛋白 ,用尿素变性PAGE检测vRNA。结果　用甘油密度梯度离心 ,可从病毒粒子中分离由NP、3P和RNA组成的RNP。将纯化的RNP用CsCl和甘油双组分不连续密度梯度离心 ,可形成NP和 3P -RNA两部分 ,使NP与 3P -RNA分离。进一步将 3P -RNA在CsTFA -甘油双组分不连续密度梯度介质中进行超速离心 ,可有效地将 3P与RNA分离 ,获得较纯的 3P蛋白。 3P位于离心管的上半部分 ,而RNA则位于离心管的近底端部分。结论　用甘油、CsCl-甘油和CsTFA -甘油分步超离心法可有效地从流感病毒粒子中纯化出高纯度的流感病毒RNA聚合酶 Objective To establish an effective method for the isolation and purification of the influenza virus RNA polymerase PB1, PB2 and PA 3P proteins and lay a foundation for further study on the structure and function of 3P protein. Methods The density gradient ultracentrifugation method was used to separate the different media of glycerol, CsCl1 and CsTFA. The 3P protein was detected by SDS-PAGE and Western blot. The vRNA was detected by urea denaturing PAGE. Results Centrifugation with a glycerol density gradient enabled the isolation of RNPs composed of NP, 3P and RNA from virions. The purified RNP was centrifuged with a two-component discontinuous density gradient of CsCl and glycerol to form NP and 3P-RNA fractions, separating the NP from the 3P-RNA. 3P-RNA was further subjected to ultracentrifugation in a CsTFA-glycerol two-component discontinuous density gradient medium to efficiently separate 3P and RNA to obtain a pure 3P protein. 3P is located in the upper half of the tube while RNA is located in the near-bottom part of the tube. Conclusion The stepwise ultracentrifugation with glycerol, CsCl-glycerol and CsTFA-glycerol can effectively purify the influenza virus RNA polymerase of high purity from influenza virus particles