Luciferase报告基因禽流感病毒小基因组的构建及其在病毒聚合酶功能研究中的应用
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摘要
H5N1亚型禽流感的暴发给世界养禽业造成毁灭性打击,而且“97香港禽流感事件”及东南亚陆续发生的暴发禽流感并再次导致数人感染致死充分证明,H5亚型高致病性禽流感对人类公共卫生构成巨大潜在威胁。H5亚型高致病性禽流感病毒如何跨越禽-哺乳动物宿主种间屏障,仍是一个重大课题。
通过反向遗传技术证实DKGX22、DKGX35毒株对哺乳动物致病性的差异是由于PB2基因引起,但PB2基因究竟在病毒对哺乳动物致病性的那个环节影响流感病毒的感染性仍不清楚,本实验针对这一点进行研究。
实验利用反向遗传操作系统,在真核细胞中构建DKGX /35 /01,DKGX/22/01两毒株聚合酶蛋白复合体(RNPs)。萤光素酶报告基因载体同双向转录pBD质粒pBD-PB1、pBD-PB2、pBD-PA、pBD-NP共转染,通过检测荧光活性值报告病毒聚合酶复合体的活性。荧光检测证实在293T细胞中可以构建出有活性的流感病毒聚合酶复合体。
通过单个基因替换,构建DKGX/35/01聚合酶复合体背景的重组复合体和DKGX/22/01聚合酶背景的重组复合体,通过荧光检测证实,PB2基因是导致流感病毒聚合酶复合体活性不同的关键基因;两两替换转染中病毒聚合酶复合体的基因片段,双基因替换结果显示凡是含有DKGX/22/01的PB2基因的组合,聚合酶活性显著降低;通过点突变基因替换证实,PB2蛋白的699位点和701位点都可以改变流感病毒聚合酶复合体的活性,但以701位点影响最大。
实验结果证实,PB2基因对流感病毒聚合酶复合体活性影响与对应的流感病毒对哺乳动物致病性影响相符合。由遗传规律可知,DNA-蛋白质-蛋白质功能-生物活性:在PB2基因影响流感病毒致病性方面,首先影响了流感病毒聚合酶复合体的活性,进一步导致流感病毒对哺乳动物的致病性显著不同。
H5N1 highly pathogenic avian influenza is responsible for one of the most devastating diseases in domestic poultries. H5N1 avian influenza virus caused several infected humans in South Asia suggested this subtype viruses pose severe threat for pubic health. However, the mechanisms for H5N1 avian influenza virus to cross the avian-mammalian host barrier are still not clear..
Using an 8-plismid reverse genetics system, Zejun Li discovered that the PB2 gene of the DKGX/22 virus dramatically attenuated the DKGX/35 virus (MLD50, 6.3log10EID50), but the point is in which step does the attenation happens .
We constructed RNPs in 293T cell by transfection with pBD-PB1, pBD-PB2, pBD-PA, pBD-NP. The constructed-vector pPolI - Luci-T were co-transfected 293 T cells. The fluorescent light was tested through fluorescence detector test kit and the result showed that functional protease was assembled.
Single gene was replaced in RNPs. We constructed recombinant virus polymerase complex with 3 genes from DKGX/35/01 RNPs and 1 gene from DKGX/22/01 RNPs, and 3 genes from DKGX/22/01 RNPs and 1 gene from DKGX/35/01 RNPs. The fluorescence was measures by fluorescence detector, the result showed PB2 gene plays a key rule in determing the activity of RNPs . The test of 2 genes re-assort RNPs activity also confirmed the conclusion. Polymerase activity was significantly reduced through the replacement PB2 protein containing mutation in 699 and 701 Aa, but to a greater impact on 701 points.
We can conclude that: the activity of protease was on the line with the the ability of influenza virus pathogencity in mammals. From the Genetic law, DNA-protein - protein function - biological activity: the PB2 gene of influenza virus affects the polymerase complex functions, thereby the activity of RNPs affects the pathogenicity of the influenza virus to mammals markedly.
通过反向遗传技术证实DKGX22、DKGX35毒株对哺乳动物致病性的差异是由于PB2基因引起,但PB2基因究竟在病毒对哺乳动物致病性的那个环节影响流感病毒的感染性仍不清楚,本实验针对这一点进行研究。
实验利用反向遗传操作系统,在真核细胞中构建DKGX /35 /01,DKGX/22/01两毒株聚合酶蛋白复合体(RNPs)。萤光素酶报告基因载体同双向转录pBD质粒pBD-PB1、pBD-PB2、pBD-PA、pBD-NP共转染,通过检测荧光活性值报告病毒聚合酶复合体的活性。荧光检测证实在293T细胞中可以构建出有活性的流感病毒聚合酶复合体。
通过单个基因替换,构建DKGX/35/01聚合酶复合体背景的重组复合体和DKGX/22/01聚合酶背景的重组复合体,通过荧光检测证实,PB2基因是导致流感病毒聚合酶复合体活性不同的关键基因;两两替换转染中病毒聚合酶复合体的基因片段,双基因替换结果显示凡是含有DKGX/22/01的PB2基因的组合,聚合酶活性显著降低;通过点突变基因替换证实,PB2蛋白的699位点和701位点都可以改变流感病毒聚合酶复合体的活性,但以701位点影响最大。
实验结果证实,PB2基因对流感病毒聚合酶复合体活性影响与对应的流感病毒对哺乳动物致病性影响相符合。由遗传规律可知,DNA-蛋白质-蛋白质功能-生物活性:在PB2基因影响流感病毒致病性方面,首先影响了流感病毒聚合酶复合体的活性,进一步导致流感病毒对哺乳动物的致病性显著不同。
H5N1 highly pathogenic avian influenza is responsible for one of the most devastating diseases in domestic poultries. H5N1 avian influenza virus caused several infected humans in South Asia suggested this subtype viruses pose severe threat for pubic health. However, the mechanisms for H5N1 avian influenza virus to cross the avian-mammalian host barrier are still not clear..
Using an 8-plismid reverse genetics system, Zejun Li discovered that the PB2 gene of the DKGX/22 virus dramatically attenuated the DKGX/35 virus (MLD50, 6.3log10EID50), but the point is in which step does the attenation happens .
We constructed RNPs in 293T cell by transfection with pBD-PB1, pBD-PB2, pBD-PA, pBD-NP. The constructed-vector pPolI - Luci-T were co-transfected 293 T cells. The fluorescent light was tested through fluorescence detector test kit and the result showed that functional protease was assembled.
Single gene was replaced in RNPs. We constructed recombinant virus polymerase complex with 3 genes from DKGX/35/01 RNPs and 1 gene from DKGX/22/01 RNPs, and 3 genes from DKGX/22/01 RNPs and 1 gene from DKGX/35/01 RNPs. The fluorescence was measures by fluorescence detector, the result showed PB2 gene plays a key rule in determing the activity of RNPs . The test of 2 genes re-assort RNPs activity also confirmed the conclusion. Polymerase activity was significantly reduced through the replacement PB2 protein containing mutation in 699 and 701 Aa, but to a greater impact on 701 points.
We can conclude that: the activity of protease was on the line with the the ability of influenza virus pathogencity in mammals. From the Genetic law, DNA-protein - protein function - biological activity: the PB2 gene of influenza virus affects the polymerase complex functions, thereby the activity of RNPs affects the pathogenicity of the influenza virus to mammals markedly.
引文
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