犬细小病毒VP1蛋白入核转运的分子机制研究

The worldwide prevalent virus, canine parvovirus, is a significant lethal virus of dog. Entering nucleus and reproducing inside is necessary for the virulence of canine parvovirus. Research has shown that, NLSp1, the nuclear localization signal of VP1, can be recognized by the nuclear transport receptors, and help VP1 transport across the nuclear pore complexes into the nucleus, by way of temperature and energy dependence. But there is no research to elucidate the nuclear transport receptors and the nuclear transport ways. Our early studies found that, in canine parvovirus infected cells, the expression level of importin alpha 1 rise significantly, and proteins including RanBP1 and Ran, which participate in importin nuclear transport way, are also expressed differently. On this basis, we intend to construct recombinant plasmids, by immunofluorescence, confocal laser, co-immunoprecipitation and western blot, using the competition experiment, the inhibition experiment, the nuclear transport model in vitro and the cytosol reconstruction experiment, to clarify the molecular mechanism of nuclear transport of VP1 from 3 aspects: the nuclear localization signal NLSp1, nuclear transport receptors and nuclear transport ways, to provide a new strategy for constructing genetic engineering vaccines by modifying NLSp1, and to found the theoretical basis for developing drugs against canine parvovirus disease by blocking the nuclear transporting and assembling of viral capsids.

犬细小病毒是世界范围内流行的重要的犬致死性病毒，进入细胞核完成增殖是其致病的前提。研究表明，CPV衣壳蛋白VP1上的核定位信号NLSp1，可被转运受体识别，介导VP1通过温度、能量依赖的途径，经核孔复合体入核，但尚无研究阐明VP1入核的转运受体及途径。课题组前期研究发现，犬细小病毒感染后，宿主细胞表达的入核转运受体中，impα1的表达水平显著上调，且RanBP1、Ran等参与importin入核途径的蛋白也存在差异表达。在此基础上，本研究拟构建重组质粒，利用免疫荧光、激光共聚焦、免疫共沉淀和蛋白质印记等技术，通过竞争、抑制、体外入核转运和细胞溶质重构等实验手段，从核定位信号、入核转运受体及入核转运途径3方面，研究VP1入核转运机制，为从改造NLSp1角度构建新型基因工程疫苗提供策略，为从阻断病毒衣壳入核装配角度开发治疗药物奠定理论基础。

犬细小病毒（Canine Parvovirus, CPV）是世界范围内流行的重要的犬致死性病毒，进入细胞核完成增殖是其致病的前提。以往研究表明，NLSp1可能是CPV衣壳蛋白VP1上的有效核定位信号（Nuclear Location Signal, NLS），NLSp1与BSA形成的重组蛋白可以通过能量、温度、转运受体依赖的途径，经核孔复合体进入细胞核，目前VP1蛋白入核利用的核定位信号，入核转运受体及入核转运途径尚不明确。. 本研究构建了定位于核内的重组质粒模型pEGFP-N1-GST-VP1以及定位于核外的阴性对照重组质粒pEGFP-N1-GST，通过预测VP1上的核定位信号，在VP1上突变了可能发挥作用的核定位信号NLSp1和NLM，通过激光共聚焦及免疫共沉淀实验发现，NLSp1和NLM均不是唯一的VP1上的有效核定位信号；通过构建impα1、impα3、impα4、impα5、impα6、impα7及impα8的重组质粒，通过激光共聚焦和免疫共沉淀实验，揭示参与VP1入核转运的转运受体为impα7；通过免疫共沉淀和体外入核转运实验初步得出VP1利用的入核转运途径是impα依赖，impβ非依赖的过程。. 该研究为从改造CPV的角度构建新型基因工程疫苗提供了参考，为从阻断病毒衣壳入核装配角度开发治疗药物奠定理论基础。本研究还存在很多不足，对VP1入核转运机制的阐释还不够完整，本研究将分段表达VP1，进一步探讨VP1上有效的核定位信号，并利用siRNA干扰impα7的表达，以及敲除impα7的F81细胞株，研究VP1的入核转运受体，进一步阐明VP1的入核转运途径。