【作者】 李继东；

【导师】 才学鹏；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2014， 博士

【摘要】 口蹄疫（foot and mouth disease，FMD）、小反刍兽疫（peste des petits ruminants，PPR）和羊痘（capripox，CP）是危害山羊、绵羊等小型反刍动物的3种重大疫病，均为世界动物卫生组织（OIE）发布的A类烈性传染病。3种疫病的病原分别是口蹄疫病毒（foot and mouth disease virus，FMDV）、小反刍兽疫病毒（peste des petits ruminants virus，PPRV）和羊痘病毒（capripoxvirus，CPV）。目前这3类疫病的防控主要依靠灭活苗或弱毒苗，在实际应用中存在着免疫持续期短、热稳定性差、病毒毒力有返强风险等诸多缺陷。因此，需要研制更为安全高效的新型疫苗。病毒载体是现代生物科学研究的热点之一，诸多病毒载体已成功构建，并成为研制新型疫苗的候选工具。羊痘病毒活载体就是其中之一。在上述3种感染羊的病毒中，PPRV的H蛋白和F蛋白是刺激机体产生中和抗体的主要抗原；而FMDV的P1-2A在3C的作用下可以形成病毒的核衣壳，是中和抗体产生的主要诱导物，其中P1蛋白中的VP1片段是抗原决定簇最集中的区域，因此，PPRV的H和F基因、FMDV的VP1基因和P12A3C片段是研制亚单位疫苗和重组疫苗的主要候选基因。本研究利用DNA重组技术，构建用于羊痘病毒重组的基因工程转移载体，通过转移载体和病毒之间的同源重组，将PPRV和FMDV的抗原基因分别插入到羊痘病毒基因组中，形成重组羊痘病毒，作为转运外源蛋白的活载体，旨在研制一种能有效预防PPR、羊FMD、羊痘的高效疫苗。获得结果如下：1.构建了重组转移质粒ptkpp，并通过延长重组同源臂、插入报告基因GFP和压力筛选基因gpt、引入多克隆位点，优化了其功能，最后形成了可重复使用的转移质粒pftkpgigp和pftkpgigpi；验证了重组转移质粒启动子p7.5和p11的功能，为此系列质粒的正常使用奠定了基础。2.将PPRV和O型FMDV的抗原基因分别插入转移质粒pftkpgigp和pftkpgigpi的多克隆位点中，构建了6个重组质粒：pftkpgigp-H、pftkpgigp-F、pftkpgigp-VP1、pftkpgigp-P12A3C、pftkpgigp-H-i-P12A3C、pftkpgigp-F-i-P12A3C，以用于和羊痘病毒的同源重组。3.用脂质体转染法将6个重组转移质粒分别导入预先感染羊痘病毒的羔羊睾丸细胞中，经同源重组获得了6株重组羊痘病毒第一代混悬液。然后用选择培养液反复培养筛选纯化，通过GFP的表达、细胞病变效应以及PCR法检测，初步鉴定形成了4株重组GPV，分别命名为rGPV/FMDV-VP1（携带FMDV VP1基因）、rGPV/PPRV-F（携带PPRV F基因）、rGPV/PPRV-H（携带PPRV H基因）、rGPV/PPRVF-FMDVP12A3C（携带PPRV F基因和FMDV的P12A3C片段）。4.4株rGPV分别感染LT细胞，经RT-PCR检测mRNA的转录状况，后用间接免疫荧光技术检测外源基因的表达。结果显示，所有插入的外源基因得到了正常转录，其表达产物与特异性抗体之间产生了良好的免疫反应。5.4株rGPV按105TCID50剂量经皮内接种山羊，分别诱导产生了较高水平的抗GPV、FMDV和PPRV抗体；产生的抗GPV抗体效价和GPV疫苗差异不显著，抗FMDV抗体效价和疫苗组差异显著，抗PPRV效价在加强免疫后和疫苗组差异不显著。更多还原

【Abstract】 Foot and mouth disease (FMD), peste des petits ruminants (PPR) and capripox (CP) are threehighly contagious and economically important diseases affecting goats, sheep and other smallruminants, caused by foot and mouth disease virus (FMDV), peste des petits ruminants virus (PPRV)and capripoxvirus (CPV) respectively. The three diseases are published as class A of infectiousdiseases by the world organization for animal health (OIE). At present, the prevention and control ofthese three diseases mainly rely on the inactivated vaccine and attenuated vaccine. The vaccines havethe deficiency of short immune duration, poor thermal stability and reversion of virulence in thepractical application. Therefore, it becomes the objective need to develop more effective and safer newgeneration vaccine.Virus vector is one of the hotspots in the field of modern biology research. A number of viralvectors have been successfully constructed, and become a new vaccine candidate. Capripoxvirusvector is one of these virus vectors. Among three viruses infected goat and sheep, fusion protein (F)and hemagglutinin (H) of PPRV are major antigen inducing organism to produce neutralizing antibody;P1-2A of FMDV can form viral nucleocapsid in the presence of3C, which is the main inducement toproduce neutralizing antibody, and the VP1fragment of P1protein occupies the most concentratedarea of antigenic determinants. Therefore, VP1gene or P12A3C fragment of FMDV and H gene or Fgene of PPRV is the main candidate antigen gene of recombinant vaccines and subunit vaccines. In thisstudy, according to DNA recombination technique, the transferring vectors containing antigen genes ofPPRV or/and FMDV were constructed to develop recombinant capripoxvirus. Finally, severalrecombinant capripoxviruses were generated by inserting the antigen genes of FMDV or/and PPRV tocapripoxvirus genome in the process of homologous recombination between the transferring vectorand the virus. As a live vector transporting exogenous protein, the target of recombinant capripoxvirusis to develop an efficient vaccine that can effectively prevent the PPR, FMD and CP. The results wereas following:1. A transferring plasmid named ptkpp was constructed to use in the recombination ofcapripoxvirus. Furthermore, its function was improved by lengthening homologous flanks, insertingreport gene (GFP) and pressure selection gene (gpt) and introducing multiple cloning site (MCS). Thetransferring plasmids named pftkpgigp and pftkpgigpi were constructed eventually and these plasmidscan be used repeatedly in the capripoxvirus recombination. Moreover, the function of promoter p11and p7.5in these plasmids were demonstrated to ensure their proper work in practice.2. The antigen genes of PPRV or/and FMDV were inserted into the transferring plasmid pftkpgigpor pftkpgigpi and the six recombinant plasmids were constructed and were named pftkpgigp-H,pftkpgigp-F, pftkpgigp-VP1, pftkpgigp-P12A3C, pftkpgigp-H-i-P12A3C and pftkpgigp-F-i-P12A3Crespectively according to the foreign gene. These plasmids can be used in the following experiments.3. The above six plasmids were transfected into the lamb testis (LT) cells infected capripoxvirus previously using Lipofectamine respectively. Consequently, six recombinant virus suspension of firstgeneration were obtained after homologous recombination. However, four recombinantcapripoxviruses were generated by gpt drug selection and GFP selection repeatedly and were identifiedby cytopathic effect (CPE) and PCR method. These recombinant viruses were namedrGPV/FMDV-VP1, rGPV/PPRV-F, rGPV/PPRV-H and rGPV/PPRVF-FMDVP12A3C respectivelyaccording to its exogenous gene contained.4. The transcription and expression of the exogenous genes in the rGPVs was demonstrated byRT-PCR method and immunofluorescence assay at the level of mRNA and protein. The results showedthat all foreign genes were transcribed successfully and their products could be reacted with thespecific antibody, demonstrating the immunogenic activity of the expressing products.5. The goats were inoculated to rGPV with a dose of105TCID50intradermally and high levels ofanti GPV, FMDV and PPRV antibody were induced respectively. There was no significant difference atthe titers of anti-GPV antibody between rGPV and GPV vaccine; however, there was significantdifference at the titers of anti-FMDV antibody between rGPV and FMDV vaccine. Furthermore, therewas no significant difference at the titers of anti-PPRV antibody between rGPV and PPRV vaccine afterbooster immunization.更多还原