【作者】 张辉；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2007， 博士

【摘要】 伪狂犬病毒（Pseudorabies virus，PRV）可引起多种家畜和野生动物的伪狂犬病，尤其是猪伪狂犬病，给世界养猪业造成了巨大的经济损失。伪狂犬病毒基因组全长约150kb，可编码70-100种蛋白，与α-疱疹病毒亚科的其它成员一样，PRV具有潜伏感染和神经嗜性等α-疱疹病毒的典型特征。尽管早在上个世纪80年代中期就有学者开始了PRV重要功能基因的克隆与功能研究，但由于PRV基因组的G+C含量极高（平均G+C含量约为74％），迄今仍有相当一部分基因的功能特性不清楚，如UL4、UL7、UL14、UL24、US2等。毫无疑问，对这些未知基因的研究与分析将对整体上阐明PRV基因组的结构与功能具有重要意义。长期以来，基因缺失突变株的构建是研究疱疹病毒功能基因的主要策略，但在哺乳动物细胞中通过同源重组的方法构建基因缺失突变株是一项极为繁琐的工作。1997年，德国学者Messerle等首次以细菌人工染色体（Bacterial Artificial Chromosomes，BAC）为基础构建了鼠巨细胞病毒的感染性克隆。该技术允许病毒基因组以BAC质粒的形式在大肠杆菌中保存、增殖和遗传修饰，并且操作简便、快速，极大地促进了疱疹病毒的功能基因研究，并为疱疹病毒高效载体系统的开发提供了新的途径。鉴于上述研究背景，本研究探讨了PRV间质蛋白UL14的功能，并构建了PRVTK^-/gG^-的感染性克隆，主要研究内容如下：1．PRV Ea株BamHI第3片段的克隆与序列分析BamHI酶切PRV Ea株基因组DNA，回收长约17kb的BamHI第3片段并将其克隆到pBR322载体中，获得重组质粒pBRB₃。进一步对pBRB₃进行亚克隆，获得含7.5kb的SphI-KpnI片段的重组质粒pUCB₃，并进行序列分析，证实该片段包含UL13、UL14、UL16和UL17基因的完整编码区以及UL15的部分编码区。UL14基因全长480bp，编码159个氨基酸，与其它几种α-疱疹病毒的UL14进行比较，发现其N端的氨基酸序列高度保守，与HSV-1、HSV-2、BHV-1的同源性分别为62％、62％、72％，但C端同源性很小。2．PRV UL14基因的克隆及其在大肠杆菌和IBRS-2细胞中的表达PCR扩增UL14基因并将其克隆到pMD18-T载体中，获得重组质粒pT-UL14。进一步以pGEX-KG为载体，构建了UL14完整编码区与GST融合的表达质粒pGEX-UL14，经IPTG诱导在大肠杆菌BL21（DE3）plysS中获得高效表达，表达蛋白的分子量约为44kDa，主要以包涵体形式存在。将纯化的融合蛋白免疫新西兰兔，获得了针对UL14的多克隆抗体。将UL14基因插入真核表达载体pcDNA3.1（+）中，构建的表达质粒pcDNA-UL14转染IBRS-2细胞，G418筛选获得了克隆细胞系，Western blotting检测证实该细胞系能稳定表达UL14。3．PRV UL14的亚细胞定位及结构域分析将UL14基因插入pEGFP-C1中，构建了EGFP-UL14融合的表达质粒pC1-UL14。将pC1-UL14转染Hela细胞，并与对照质粒pEGFP-C1进行比较，发现EGFP-UL14融合蛋白在转染后24 h荧光主要分布在胞浆，但随时间延长，荧光逐渐向细胞核中转移，在转染后48 h完全定位于核中，并呈点状分布，而对照质粒pEGFP-C1表达的EGFP荧光始终呈弥散状分布。为了进一步分析UL14亚细胞定位的结构域，构建了UL14 C-端不同区域缺失的5个突变体（1-146aa、1-124aa、1-88aa、1-77aa、1-65aa）以及N-端不同区域缺失的6个突变体（14-159aa、37-159aa、63-159aa、85-159aa、106-159aa、121-159aa）。将上述突变体分别与EGFP融合，构建了UL14基因不同区域与EGFP融合的重组表达质粒，在脂质体介导下分别转染Hela细胞，通过荧光检测并与对照质粒pC1-UL14进行比较，发现缺失UL14 C-端94个氨基酸和N-端37个氨基酸均不影响UL14在细胞中定位，但缺失N-端62个氨基酸则导致荧光完全呈弥散性分布，推测UL14的37-65位氨基酸区域对其亚细胞定位具有重要作用。4．PRV感染细胞后UL14的表达时相分析在PRV-Ea感染IBRS-2细胞后不同时间收集细胞，RT-PCR检测UL14基因和晚期基因gD的cDNA，发现与gD基因一样，UL14在病毒感染后9 h才开始转录。Western blotting检测显示UL14在PRV感染后12 h才开始表达，表明PRV UL14为晚期表达蛋白。5．UL14缺失的PRV突变株的构建对pUCB3质粒进行亚克隆，获得含有UL14、UL16全基因和部分UL13、UL15基因的重组质粒pUC3.0。PCR扩增CMV启动子驱动的EGFP表达盒并将其插入pUC3.0中位于UL14基因编码区的StuI位点上，获得EGFP表达盒插入UL14基因失活的重组转移质粒pUC3.0-14／EGFP。将该转移质粒与PRV-Ea株基因组共转染IBRS-2细胞，通过EGFP荧光标志和噬斑纯化，获得了UL14缺失的重组病毒PRV-UL14D。PCR、Western blotting、荧光显微镜观察和流式细胞术检测证实重组病毒构建正确，并能稳定表达EGFP。6．PRV UL14的功能分析通过比较UL14缺失突变株PRV-UL14D与亲本株（PRV Ea）的多种体外生物学特性，包括侵入细胞的能力、形成噬斑的大小、一步增殖曲线及感染细胞后病毒粒子的形态，发现UL14的缺失不影响PRV侵入细胞和病毒的释放，但延缓了PRV在IBRS-2细胞上的增殖并导致形成的噬斑明显减小。同时，UL14缺失导致感染细胞中无囊膜化的核衣壳增多，说明UL14缺失影响了核衣壳的有效囊膜化，从而对病毒粒子的成熟造成影响。上述体外生物学缺陷在稳定表达UL14的细胞系中均能完全恢复，表明这些缺陷是由UL14的缺失所致。进一步的体内感染试验还发现：小鼠感染PRV-UL14D突变株的平均死亡时间明显长于亲本株（PRV Ea），说明UL14的缺失也影响了PRV在体内的增殖。此外，通过细胞凋亡检测和电子显微镜分析，发现PRV-UL14D和PRV-Ea均能诱导IBRS-2细胞发生凋亡，呈现染色质形态改变等早期凋亡的典型特征，而且稳定表达UL14的细胞系也不能抑制山梨醇诱导的细胞凋亡，说明UL14不具有抗凋亡活性，这与报道的HSV-1、HSV-2和BHV-1 UL14具有抗凋亡活性存在差异。7．PRV TK^-/gG^-突变株感染性克隆的构建将含大肠杆菌F因子的BAC载体骨架序列插入到质粒pgG-Uni中，得到重组转移质粒pSB301。将该质粒与PRV TK^-/gG^-／LacZ⁺基因组共转染PK-15细胞，获得在gG基因中插入BAC载体序列的重组病毒PRV TK^-／gG^-／BAC。提取重组病毒复制早期的坏状病毒基因组DNA并转化DH10B细胞，筛选含有PRV全长基因组的BAC细菌克隆，大量培养后，提取超纯的PRV BAC质粒DNA再转染PK-15细胞，可观察到典型的伪狂犬病毒所致的细胞病变，证实获得的BAC质粒DNA具有感染性。一步增殖曲线测定发现BAC质粒DNA转染所获得的病毒与亲本株TK^-／gG^-／LacZ⁺具有相似的增值特性，表明BAC载体序列的插入不影响病毒在细胞上的增殖。8．PRV TK^-/gG^-突变株BAC质粒DNA对小鼠的免疫原性将PRV BAC质粒DNA、转移质粒pSB301、重组病毒PRV TK^-／gG^-／BAC及亲本株PRV TK^-／gG^-／LacZ⁺分别免疫小鼠，检测PRV BAC质粒DNA的免疫原性。结果表明，PRV BAC质粒DNA诱导的PRV中和抗体水平与重组病毒TK^-／gG^-／BAC、亲本株TK^-／gG^-／LacZ⁺相当，且都能完全抵抗致死剂量PRV强毒的攻击，表明构建的感染性克隆具有良好的免疫原性，为进一步开展PRV免疫学基础研究和高效载体系统的开发奠定了基础。更多还原

【Abstract】 Pseudorabies virus （PRV） is the causative agent of Pseudorabies （Aujeszky’s disease）of many domestic and wild animals. Especially, porcine Pseudorabies has become one ofthe most economically important diseases in worldwide swine industry. PRV genome is adouble-stranded DNA of approximately 150 kb and encodes 70-100 proteins. Like otheralpha herpesvirus, PRV has some typical characteristics of alpha herpesvirus such aslatent infection and neurotropism. Although cloning and sequencing of some importantfunctional genes of PRV were carried out in the middle of 1980s, full-length sequence ofPRV genome has not been reported because of high content of G+C of PRV genome （themean content of G+C is 74%）. Functions of some genes still remain unclear, such as UL4,UL7, UL14, UL24 and US2. It is significant to study and analyze unknown genes, whichcontributes to clarify entirely the structure and function of PRV genome.For a long time, the main method studying on the functional genes of herpesviruswas to construct genetic-deleting mutants. However, it was tedious to producerecombinant mutants using homologous recombinant in infected cells. In 1997, Messerleet al., German researchers, firstly constructed infectious clone of MCMV based onbacterial artificial chromosomes （BACs）. This technique actualized that the genomes ofinfectious virus could be stored, propagated or modificated by BAC-plasmid pattern in E.coli, and it was convenient and rapid to manipulate the technique. This technique lead toadvancement in studying on gene functions of herpesvirus, and provided new ways todevelope high-performance herpesviral-vetor system.Based on the above mentioned researchs background, the present study investigatedthe functions of PRV tegument protein UL14, and constructed PRV TK^-/gG^- straininfectious clone. The main projects are as following:1. Cloning and sequence analysis of PRV Ea strain BamHI-3 fragmentThe BamHI-3 fragment was isolated from PRV genome DNA digested with BamHI,and cloned into pBR322 to generate the recombinant plasmid pBRB₃. A 7.5kb unknownfragment was isolated from the plsmid pBRB₃ digested with BamHI and KpnI to generatethe plasmid pUCB₃. Sequences analysis showed that the UL13-UL17 fragment containedcomplete coding-region of UL13, UL14, UL16 and UL17 and partial coding-region ofUL15. UL14 gene with 480bp encodes 159 amino acid residues. Compared with aminoacid residues of UL14 gene of other Alphaherpesviruses, it was found that N-terminalamino acid residues sequence of UL14 of PRV is highly conservative and there was 62%,62%, 72% homology with HSV-1, HSV-2, BHV-1, respectively, but the homology ofC-terminal is low. 2. Cloning and expression of PRV UL14 gene in E.coli and IBRS-2 cellsPRV UL14 gene was amplified by PCR and cloned into pMD18-T vector to obtainthe recombinant plasmid pT-UL14. The UL14 gene was removed from pT-UL14 andcloned into prokaryotic expression vector pGEX-KG resulting in the recombinant plasmidpGEX-UL14, which expressed GST-UL14 fused protein. After induction by IPTG, therecombinant fused protein with about 44kDa was highly expressed in E.coliBL21（DE3）plysS and expressing mainly in the form of inclusion body. The GST-UL14fused protein was used to immunize rabbits to generate polyclonal antibodies againstUL14. UL14 gene was inserted into vector pcDNA3.1（+） resulting in the eukaryoticexpression plasmid pcDNA-UL14. After transfection, UL14-expressing IBRS-2 cell linewas obtained by G418 screening. Western blotting analysis demonstrated that this IBRS-2cell line could stably express UL14.3. Construction of PRV UL14 mutants and analysis of function domainsUL14 gene was inserted into vector pEGFP-C1 resulting in the eukaryoticexpressing plasmid pC1-UL14 expressing EGFP-UL14 fused protein, pC1-UL14 wastransfected into Hela cells, and the fluorescence mainly distributed in cytoplasm at 24 hposttransfection, but as time went on, the fluorescence imaging occured to transfer andaggregated in nucleus at 48h posttransfection. In order to analyze UL14 function domains,a series of C- （1-146aa, 1-124aa, 1-88aa, 1-77aa, 1-65aa） or N-（14-159aa, 37-159aa,63-159aa, 85-159aa, 106-159aa, 121-159aa） terminal truncated mutants were obtained,and EGFP-fused expressing plasmids were constructed. When transfected into Hela cells,different fluorescence imaging in cells transfected with pC1-UL14 at various timesposttransfection were observed. The deletion of the C-terminal 94aa or N-terminal 37aasequences did not affect the intracellular localization of UL14, whereas, 62aa-deletion atthe N-terminal sequences resulted in the diffusedly cytoplasmic distribution. The resultssuggested that the 37aa to 65aa sequence of UL14 played an important role onintracellular localization.4. Expression analysis of UL14 after infecting cells with PRVCells were collected at various time after PRV-Ea infection, RT-PCR was performedto amplify cDNA of UL14 and late gene gD of PRV. Like gD gene, the transcriptionproduct of UL14 was observed at 9 h postinfection. Meanwhile, Western blottingdemonstrated that UL14 protein was expressed at 12 h postinfection. The resultsconfirmed that PRV UL14 protein was expressed with late kinetics.5. Construction of PRV UL14 mutant strainEGFP expression cassette drived by CMV promoter was amplified by PCR, andinserted into StuI site of UL14 gene of plasmid pUC3.0 containing entire UL14, UL16 gene and partial UL13, UL15 gene to generate transfer plasmid pUC3.0-14/EGFP. Thenthis plasmid was co-transfected into IBRS-2 cells with PRV-Ea genome. After cytopathiceffect occurred in cells, recombinant virus PRV-UL14D expressing EGFP was screenedfrom the cytopathic cells by EGFP fluorescence marker and plaque purification. Therecombinant virus was verified by PCR, Western blotting and Flow Cytometry （FCM）,and can stably express EGFP protein.6. Function analysis of PRV UL14The penetration kinetics, plaque size, one step growth curves, and morphogonesis ofPRV-UL14D mutant were investigated and compared with the parent virus, PRV Ea ininfected cells. The results showed that the penetration and release of PRV wereindependent of UL41, but the UL41 deleted mutant exhibited a delayed growth andsignificantly small plaque size. Meanwhile, electron microscopic examination indicatedthat the nonenveloped particles obviourly increased in the PRV-UL14D-infected cells.The results illuminated that UL 14-deletion affected the virus’s enveloping, and furtherlyinfluence the mature of PRV particles. But these defects above in vitro can be completelyrecovered in UL14-expressing IBRS-2 cells, which indicated that the UL 14-deletion maybe responsible for these changes. In further researches, we found that the average deathtime of BALB/c mice inoculated with PRV-UL14D was obviously delayed, comparedwith the same dose of PRV-Ea. The results suggested that the UL14-deletion also affectedthe replication of PRV in vivo. In additional, anti-apoptotic activity of UL14 wasinvestigated by cell apoptosis detection and electron microscopic examination. The resultsindicated that UL14 expressing cell lines could not be resistant to apoptosis induced bysorbitol treatment. Electron microscopy showed that there were no obviously differencesof apoptotic phenomenon between cells infected with PRV-Ea and PRV-UL14D, whichsuggested that UL14 protein did not share anti-apoptotic function, contrast to HSV-1,HSV-2 and BHV-1 UL14 as reported previously.7. Construction of PRV TK^-/gG^- infectious cloneBAC vector sequence containing F-factor was cloned into plasmid pgG-Uni togenerate transfer plasmid pSB301. And then, this plasmid was co-transfected into IBRS-2cells with PRV TK^-/gG^-/LacZ⁺ genome. When eytopathie effect occurred in cells,recombinant virus PRV TK^-/gG^-/BAC was screened from the cytopathic cells by plaquepurification. Replieative intermediate （covalently closed circular） viral DNA extractedfrom cells infected with the recombinant virus was electroporated into E.coli DH10B cells.The BAC plasmid DNA isolated from E.coli harboring PRV full-length BAC DNA wastransfected into PK-15 cells, and cytopathic effect was visible, suggested that PRV BACDNA possess infection. Furthermore, the results of one step growth curves of PRV TK^-/gG^- infectious clone showed that the insertion of BAC vector sequence had noinfluence on the propagation of recombinant viruses in cells.8. Immunogenicity of PRV TK^-/IgG^- BAC plasmid DNA in miceImmunogenicity of PRV BAC plasmid DNA was detected in mice, and comparedwith transfer plasmid pSB301, recombinant PRV TK^-/gG^-/BAC and parent strain PRVTK^-/gG^-/LacZ⁺. The results showed that PRV neutralization antibodies induced by PRVBAC plasmid DNA corresponded with that induced by TK^-/gG^-/BAC, TK^-/gG^-/LacZ⁺.Moreover, mice immunized with BAC plasmid DNA could resist the challenge of lethalPRV and displayed the same protective efficacy with that vaccinated with TK^-/gG^-/BACor TK^-/gG^-/LacZ⁺. The results indicated that PRV TK^-/gG^- infectious clone had favourableimmunogenicity, and lay foundation for further research on immunology and thedevelopment of perfect viral-vector system of PRV.更多还原