CAJ | 학술논문

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Western blot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。
목적건립분리、순화류감병독RNA취합매PB1、PB2화PA 3P단백적유효방법，위진일보연구3P단백적결구화공능전정기출。방법용감유、CsCl1급CsTFA불동개질적밀도제도초속리심법진행분리，용SDS－PAGE전영급Western blot법검측3P단백，용뇨소변성PAGE검측vRNA。결과용감유밀도제도리심，가종병독입자중분리유NP、3P화RNA조성적RNP。장순화적RNP용CsCl화감유쌍조분불련속밀도제도리심，가형성NP화3P－RNA량부분，사NP여3P－RNA분리。진일보장3P－RNA재CsTFA－감유쌍조분불련속밀도제도개질중진행초속리심，가유효지장3P여RNA분리，획득교순적3P단백。3P위우리심관적상반부분，이RNA칙위우리심관적근저단부분。결론용감유、CsCl－감유화CsTFA－감유분보초리심법가유효지종류감병독입자중순화출고순도적류감병독RNA취합매。
Objective　To establish the method for isolation and purification of the RNA polymerase PB1, PB2 and PA from the influenza virus in order to investigate the structure and the function of the polymerase. Methods　The RNA polymerase of influenza virus was isolated from virus particles by stepwise centrifugation in different salts. The 3P proteins were analyzed by SDS-PAGE and Western-blotting using monospecific antibodies against each P protein. The vRNA was detected using urea-denatured PAGE.Results　The RNA-dependent RNA polymerase of influenza virus was isolated from virus particles by stepwise centrifugation in different salts. The RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride(CsCl) gradient centrifugation.The P-RNA (P proteins-viral RNA) complexes were further dissociated into 3P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The three P proteins located in the upper part of the CsTFA gradient tube, while the RNA in the lower part of the CsTFA gradient tube. Conclusion　The RNA-dependent RNA polymerase of influenza virus could be purificated using glycerol, CsCl and CsTFA gradient centrifugation.