【作者】 唐青海；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2010， 博士

【摘要】 猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起的一种高度接触性传染病,被世界动物卫生组织列为必须上报的动物传染病之一。近年来,国内外对猪瘟的研究多数集中在诊断方法和新型疫苗的研发方面,而对猪瘟病毒致病机理研究较少。在CSFV编码的非结构蛋白中,NS2的蛋白功能尚不清楚。本文以揭示CSFV NS2蛋白的功能为目标,开展了NS2蛋白的降解特性、亚细胞定位特点、NS2蛋白对宿主细胞周期与增殖的影响及NS2蛋白对宿主细胞炎症因子的表达调控等方面的研究,取得了如下研究结果:(1)采用RT-PCR方法成功扩增到了CSFV NS2基因。生物信息学分析表明,CSFV NS2基因全长为1371bp,共编码457个氨基酸,编码蛋白质的分子质量约为53 ku;CSFV NS2蛋白的N-端极有可能具有5个跨膜区,N-端具有极强的疏水性,而C-端则表现出良好的亲水性;CSFV NS2蛋白N-端第1-170位氨基酸形成的结构多以β-折叠为主,而C-末端则以α-螺旋为主;磷酸化分析表明,NS2蛋白第223位和第224位的丝氨酸的磷酸化可能性很高;第37位和第47位的苏氨酸、第204位、第332位和第392位的酪氨酸也有很强的磷酸化特性。(2) CSFV NS2基因与绿色荧光蛋白报告基因进行融合表达,Western blot分析发现CSFV NS2蛋白的分子质量约为53 ku,该蛋白通过蛋白酶体途径快速降解,这一降解过程可以被特异性的蛋白酶体抑制剂MG132所抑制。共定位研究表明,CSFV NS2蛋白定位于细胞的内质网上,而且至少含有2个内部信号肽序列、4个跨膜区。(3)细胞增殖能力试验表明,CSFV NS2蛋白对宿主细胞的增殖活性具有一定的抑制作用。流式细胞术分析发现,CSFV NS2蛋白的表达诱导宿主细胞周期阻滞于S期,但并不是通过诱导细胞的凋亡来抑制细胞的增殖。mRNA水平和蛋白水平分析表明,CSFV NS2蛋白能够促进Cyclin A蛋白被蛋白酶体快速降解,但是并不影响该基因的转录。(4) CSFV NS2蛋白能够引起宿主细胞内质网应激反应,激活核转录因子NF-кB,CSFVNS2蛋白及Cyclin A蛋白被蛋白酶体所快速降解可能与内质网应激反应有关。(5)采用间接免疫荧光、RT-PCR等方法检测CSFV在血管内皮细胞中的敏感性及增殖状况。结果表明,CSFV E2蛋白主要分布在细胞质中,CSFV在血管内皮细胞中的增殖良好,而且CSFV的感染能够显著上调血管内皮细胞表明黏附分子整合素β3表达的表达,深入的研究发现,CSFV NS2蛋白在其中起着重要的作用。(6) CSFV NS2蛋白的表达通过激活NF-кB来诱导血管内皮细胞中IL-8的表达上调,MG132却能够通过抑制NF-кB的活性来抑制IL-8的表达,而CSFV NS2蛋白的表达能够对抗MG132对NF-кB的活性和IL-8表达的影响;(7)蛋白酶体抑制剂MG132能够诱导正常细胞的凋亡,但是,表达CSFV NS2蛋白的细胞表现出对MG132诱导凋亡作用的抗性,深入的研究表明,CSFV NS2蛋白能够上调宿主细胞中抗凋亡蛋白Bcl-2的表达。综上所述,本研究对CSFV NS2蛋白的特性及其与宿主细胞之间的相互作用进行了一系列的研究。首次发现了其降解特性与亚细胞定位特征,通过体外试验揭示了该蛋白对细胞周期与细胞增殖的调控机制,明确了CSFV NS2蛋白的表达对血管内皮细胞表面黏附分子整合素β3、趋化因子IL-8、抗凋亡蛋白Bcl-2的表达的调节作用与机制。本项研究所取得试验结果为CSFV NS2蛋白的功能增添了新的内容,为更加深入了解CSFV致病的分子机制提供了一些新的理论资料。更多还原

【Abstract】 Classical swine fever(CSF) is a highly contagious and often fatal disease of pigs that caused by classical swine fever virus (CSFV ) which is classified as a notifiable (previously List A) disease by the World Organization for Animal Health (OIE). Recently, almost all the researchers were focused on the development of the detection methods and novel vaccines, only few reports pay attention to the molecular pathology of CSF. To elucidate the function of CSFV nonstructure protein 2 (NS2), for which is poorly understand, in the present study, the degradation characterization, subcellular localization of CSFV NS2 protein, as well as the effect of CSFV NS2 protein on the cell proliferation and cell cycle, the regulation of CSFV NS2 protein on the inflammatory factors expressed in the swine vascular endothelial cells were extensively investigated.From the experiments, the following results were obtained:(1)The CSFV NS2 gene was successfully amplified by reverse transcription polymerase chain reaction (RT-PCR), the sequences analysis showed that, CSFV NS2 gene was 1371 bp encoding 457 amino acids (aa), of which, the molecular weight is 53 ku. Bioinformatics analysis showed that there were probably five trans-membrane regions in the N-terminal of CSFV NS2 protein which showed high possibility of hydrophobicity, however, the C-terminal showed great hydrophilicity. From the first to the 170th aa, the secondary structure is mainly ofβ-lap, to the C-terminal, it is mainly ofα-coil. The Ser223 and Ser224 showed great possibility of phosphorylation, as well as the Thr37, Thr47, Thy204, Thy332 and Thy392.(2)The CSFV NS2 protein was expressed as fused to the green fluorescent protein (GFP), Western blot analysis showed that the molecular weight of CSFV NS2 was approximately 53 ku, this protein was a short-live protein for its dynamically degradation through the proteasomal degradation pathway which was inhibited by proteasomal inhibitor MG132. Co-localization suggested that CSFV NS2 protein localized in the endoplasmic reticulum (ER), at least including 2 internal signal sequences and 4 trans-membrane regions.(3)Cell proliferation assay results showed that CSFV NS2 was capable of inhibiting of the host cells proliferation through inducing the cell cycle arrested in the S-phase rather than inducing apoptosis. The mRNA and protein level analysis of cyclinA revealed that CSFV NS2 expression promoted the proteasomal degradation of Cyclin A protein rather than suppressing the transcription of Cyclin A.(4)CSFV NS2 induced the ER stress and activated the nuclear factor kappaB (NF-кB) which may strongly contribute to the accelerated proteasomal degradation of Cylin A and CSFV NS2 protein.(5)The susceptibility and propagation of CSFV in swine vascular endothelial cells was investigated by indirect immunofluorescence assay and RT-PCR, the results showed that swine vascular endothelial cells was susceptible to CSFV which propagated efficiently. Further study showed that the infection of CSFV dramatically up-regulated the cell surface adhesion molecule integrinβ3 expressed in vascular endothelial cells, and the CSFV NS2 played an important role in this process.(6)The CSFV NS2 significantly induced the interleukin-8 (IL-8) expressed in vascular endothelial cells through the activation of NF-кB. The proteasomal inhibitor MG132 was able to suppress the NF-кB activity and subsequently inhibited the transcription of IL-8, however CSFV NS2-expressing cells showed antagonizes to the inhibition induced by MG132.(7)The proteasomal inhibitor MG132 induced the apoptosis of the normal vascular endothelial cells, however, CSFV NS2-expressing cells showed antagonizes to the apoptosis effect induced by MG132. Further study showed that CSFV NS2 protein up-regulated the expression of the anti-apoptotic protein Bcl-2(B-cell leukemia-lymphoma-2) in the host cells, which possibly contributed to the CSFV NS2-expressing cells showing anti-apoptosis effect.In conclusion, in the present study, the characterization of CSFV NS2 protein and the interaction between CSFV NS2 protein and vascular endothelial cells were mainly investigated. This is the first time to reveal the short-live characterization and the ER-localization of CSFV NS2 protein in the host cells. CSFV NS2 protein played an important role in the regulation of cell proliferation and cell cycle, as well as in the up-regulation of integrinβ3, chemotatic factor IL-8 and the anti-apoptotic protein Bcl-2 expressed in vascular endothelial cells. All the results from this study provided novel information to the function of CSFV NS2 protein, and, may be helpful for us to understand the molecular pathogenesis of CSFV.更多还原