【作者】 蒋大良；

【导师】 余兴龙；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2013， 博士

【摘要】 本文以CSFV蛋白在宿主细胞中表达为研究基础,分别研究：各蛋白在感染细胞中表达情况；在细胞中各蛋白真核表达情况；在细胞中真核表达的各蛋白对宿主细胞的影响；以及在细胞中真核表达的各蛋白对病毒感染的影响。为研究猪瘟病毒蛋白在感染细胞中表达情况,将原核表达的各个CSFV蛋白纯化后分别免疫昆明小鼠制备多克隆抗体血清。结果获得除P7蛋白外11个重组CSFV蛋白并制备获得高滴度抗体的免疫血清。Western-Blotting能检测到病毒各蛋白片段与预期大小的一致,其中E0、E1、E2和NS5A还以蛋白聚合体形式存在。间接免疫荧光分析(Indirect Immuno-fluorescence Assay IFA)检测显示Npro主要定位于细胞核；C、E0和NS2在细胞核亦有分布,但主要是位于细胞质中；其他蛋白则位于细胞质中；E0蛋白还在细胞膜表面有分布。结果分析表明,分布于细胞核上的CSFV蛋白可能对细胞有调控作用,而分布于细胞质内的蛋白则主要参与病毒复制、组装和增殖。将CSFV C株各蛋白基因以pEGFP-N1为载体构建成真核表达质粒,转染ST细胞来研究各蛋白在细胞中真核表达情况。PCR检测结果显示目的蛋白基因成功转染进入细胞,荧光显微镜观察表明除pEGFP-E1外各转染细胞中均表达了目的融合蛋白。以绿色荧光蛋白(EGFP)为标签观察重组蛋白表达情况发现各细胞间荧光分布存在一定的差异性：所有转染细胞细胞质中均能观察到荧光,但在pEGFP-N1、pEGFP-Npro、pEGFP-C和pEGFP-NS2等4个转染细胞的细胞核也有荧光分布；pEGFP-EO和pEGFP-NS3转染细胞荧光信号在细胞核周围富集,同时pEGFP-EO细胞在细胞膜表面有聚合状态荧光信号。将转染细胞进行克隆后成功获取了11个CSFV蛋白稳定表达细胞系(pEGFP-E1和pEGFP-NS3除外),Western-Blotting检测显示：Npro、C、P7、NS2和NS5B与EGFP融合表达后会发生完全切割,△E0、E0、E2和NS5A蛋白与EGFP融合表达后出现部分切割,NS4A和NS4B蛋白与EGFP未发生切割。结果分析表明,Npro和NS2通过白裂解酶的作用进行切割；NS4A和NS4B蛋白蛋白切割需要病毒蛋白酶的作用；C和NS5B则通过细胞蛋白酶作用与EGFP裂解,C和NS5B蛋白3、端含有一个细胞蛋白酶位点；△E0、E0、 E2和NS5A蛋白通过细胞蛋白酶作用会发生部分切割。细胞增殖活力检测结果发现：与正常ST细胞比较pEGFP-NS4B细胞增殖能力明显降低;pEGFP-NS2、pEGFP-NS5A和pEGFP-P7细胞增殖能力有所下降；而pEGFP-N1、pEGFP-Npro、pEGFP-EO、pEGFP-E2和pEGFP-NS4A细胞增殖正常；而pEGFP-NS5B细胞前期正常而后期下降。结果分析表明,部分病毒蛋白对宿主细胞增殖具有调控作用,主要表现为抑制作用。细胞周期测定发现pEGFP-NS4B和pEGFP-NS5B细胞出现S期停滞现象,表明NS4B和NS5B蛋白表达后会导致细胞周期S期延长。分别用CSFV C-株和INLY-2011野毒株感染上述蛋白细胞系,采用实时荧光定量RT-PCR方法测定不同代次细胞系中病毒含量,从而研究CSFV蛋白对病毒感染的调控作用。首先以β-Actin基因为内参成功建立起基于SYBR-Green I实时荧光定量RT-PCR方法来检测病毒含量。根据在病毒增殖过程中的作用可以将10个蛋白细胞系分为促进作用、抑制抑制和双向调控作用三类：pEGFP-Npro、pEGFP-E0和pEGFP-NS5B细胞系有促进作用,尤其是pEGFP-E0细胞；pEGFP-C、pEGFP-E2、pEGFP-NS2、pEGFP-NS4B和pEGFP-NS5A细胞有抑制作用；pEGFP-P7和pEGFP-NS4A细胞系表现双向调控作用。同时根据不同毒株在细胞系中增殖的情况差异,又可以分成特异性、非特异性及相对特异性作用三类：其中C和NS4B蛋白为特异性作用；E0、E2、P7、NS2和NS4A蛋白为非特异性作用；Npro、NS5A和NS5B蛋白为相对特异性作用。表明在病毒感染过程中CSFV蛋白可以通过病毒蛋白-病毒蛋白以及病毒蛋白-宿主细胞之间多种相互作用途径来调控病毒增殖,使病毒与宿主细胞之间的达到平衡状态而利于病毒持续性感染存在。通过荧光显微镜可以观察到E0-EGFP融合蛋白在细胞膜上分布,并分泌释放到细胞外,其可以通过离心方式从细胞培养基中沉淀下来。Western-Blotting检测显示,信号肽决定E0蛋白是否能够加工成熟为高度糖基化的蛋白,糖基化后的E0蛋白具有相互聚合作用和分泌功能,从而形成E0蛋白聚合物释放到细胞外。进一步研究发现,CSFV病毒粒子之间也可以相互聚形成病毒粒子聚合体,并可以通过离心方式从感染细胞培养基中沉淀下来。我们分析并推断,在CSFV感染过程中病毒粒子之间是通过E0蛋白的聚合作用而形成病毒聚合体,再依赖E0蛋白的分泌作用通过高尔基体以囊泡的形式从感染细胞中释放到细胞外,这对研究CSFV释放机制和途径具有重要意义。更多还原

【Abstract】 In this study, we focused on the expression of CSFV proteins in host cells, which either were infected with CSFV or transfected with eukaryotic expression systems for expressing CSFV proteins. In addition, influences of CSFV proteins, which were expressed with eukaryotic expression systems, on host cells as well as virus infection were investigated.In order to observate CSFV proteins in infected cells, the recombinant proteins by prokaryotic expression system were used to immunize Kunming mice for preparation of polyclonal antibody against CSFV proteins. Eleven recombinant CSFV proteins were successfully recovered except P7, and a series of sera, which contained high titer antibodies against CSFV proteins, were obtained. The target viral proteins were detected in infected cells by Western-Blotting, and proteins including E0, E1, E2, and NS5A were also checked as polymeric proteins. Indirect immunofluorescence assay (IFA) revealed that Npro is located at nucleus, and C, EO and NS2both in cytoplasm and at nucleus, while the remaining proteins in cytoplasm. Unusually, Erns was also distributed on cellular membrane. These findings implied that proteins distributed at nucleus were related to a function of modulating cellar life cycle or function, while those in cytoplasm were implicated in viral replication, particle assembly, and proliferation.Eukaryotic expression vectors containing protein genes of CSFV C-strain were conducted with pEGFP-Nl vector to investigate the expressions of CSFV protein in ST cells. The results of PCR detection showed that the target protein genes were successfully transfected into ST cells, and the expression of recombinant CSFV proteins was observed under fluorescent microscope except pEGFP-E1cells. Taken EGFP as flag, there exited differences of the location and intensity among CSFV protein cells:EGFP was detected in cytoplasm of all positive cells, while also detected at nucleus of pEGFP-N1, pEGFP-Npro, pEGFP-C and pEGFP-NS2cells. Enrichment of recombinant protein surround the nucleus was observed in pEGFP-EO and pEGFP-NS3cells. Additionally, EO-EGFP alone can secrete out and distribute on cellular membrane. Eleven cell lines, including Npro, C,△EO, E0, E2, P7, NS2, NS4A, NS4B, NS5A, and NS5B, were obtained, while El and NS3failed. As the thesults of Western-Blotting revealed, recombinant proteins in pEGFP-Npro, pEGFP-C, pEGFP-NS2and pEGFP-NS5B cell lines were cleaved into viral proteins and EGFP completely, and those in pEGFP-AEO, pEGFP-EO, and pEGFP-E2cleaved partly, while in pEGFP-NS4A and pEGFP-NS4B detected to be un-cleaved. These results demonstrated as followed:NS4A and NS4B were cleaved by viral protease during CSFV infection; and the self protease is responsible for cleaving Npro-EGFP and NS2-EGFP; and C-EGFP and NS5B-EGFP was digested by a cellular protease, which occurred at the3-terminal of C or NS5B; while△EO-EGFP, EO-EGFP, E2-EGFP and NS5A-EGFP were partly cleaved by cellular protease, however the mechanism of regulation remains un-known. Cell proliferation detection by CCK-8kit demonstrated that, compared with ST cells, the proliferation of pEGFP-NS4B cells was obviously inhibited, and pEGFP-NS2, pEGFP-NS5A and pEGFP-P7cells slightly lagged behind, and pEGFP-N1, pEGFP-Npro, pEGFP-E0, pEGFP-E2and pEGFP-NS4A cells equally, however pEGFP-NS5B cells were equally in early stage then backward at late. These results illustrated the cellular growth can be regulated by some CSFV proteins with an inhibition. Cell cycle arrest at S-phase was observed in pEGFP-NS4B and pEGFP-NS5B cells based on cell cycle analysis by flow cytometry, and it can be concluded that both CSFV NS4B and NS5B have a capability to modulate cell cycle by prolonging cellular S-phase.The CSFV protein cell lines were separately infected with CSFV C-strain or HNLY-2011isolate, and the viral yield was detected by quantitative real-time RT-PCR, and then the regulation of viral proliferation by CSFV proteins was studied. A SYBR-Green I based quantitative real-time RT-PCR, which taken β-Actin as an internally control, was successfully developed for detecting CSFV yield. According to the effect on viral proliferation, these cell lines could be classified as three types:pEGFP-Npro, pEGFP-EO and pEGFP-NS5B cell lines have a promoting action (pEGFP-EO cells especially), and pEGFP-C, pEGFP-E2, pEGFP-NS2, pEGFP-NS4B and pEGFP-NS5A exhibit a negative effect, while pEGFP-P7and pEGFP-NS4A present positive and negative feedback control. Additionally, based on the difference of proliferation between CSFV C-strain and HNLY-2011strain, CSFV proteins were further clustered into three groups:C and NS4B presented as specific action, E0, E2, P7, NS2and NS4A as non-specific action, meanwhile Npro, NS5A and NS5B as relatively specific action. So it can get a conclusion that the viral proliferation can be modulated by both viral-viral and viral-celllular protein interaction networks, which may be favourable for viral intracellular survival.The reconbinant protein EO-EGFP was observed on the cellular manbrane of pEGFP-EO cell line, and also secreted into cells’culture, which can be precipated by centrifugation. The result of Western-blotting revealed that the signal peptide is crucial for processing EO mature and glycosylation, and the glycosylated EO has a property of polymerization to form protein aggregates and secretion out of cells. The further experiment revealed that CSFV virions can adsorb each other and aggregate into virosomes, which can be precipated from culture by centrifugation same as EO. Taken together, these results demonstrated that CSFV EO has a function of clustering virions to assemble virus clumps, and then virus clumps can be secreted out of infected cells through Golgi apparatus by means of EO secretion. This may laid a foundation for further studies of CSFV release.更多还原