【作者】 朱战波；

【导师】 金宁一；

【作者基本信息】 吉林大学 ， 预防兽医学， 2012， 博士

【摘要】 新城疫（ND）是由新城疫病毒（NDV）引起的一种禽类动物的烈性传染病之一，给养禽业带来了巨大的经济损失。疫苗接种是特异性预防和控制ND的可靠和有效的手段。鸡传染性法氏囊病（Infectious Bursal Disease，IBD）又称甘波罗病，是由呼肠弧病毒引起的一种急性、高度传染性疾病。由于该病发病突然、病程短、死亡率高，且可引起鸡体免疫抑制，是危害当前养禽业的重要传染病。近年来，各地出现超强毒株（vvIBDV）及变异株，并能突破母源抗体的保护，给IBD的防制带来了巨大的困难。这两种病毒病常规灭活苗免疫原性低，弱毒疫苗存在易散毒、毒力返强等缺陷，因此，研究新型疫苗已经成为当前的热点。口蹄疫（Foot-and-Mouth disease，FMD）是由口蹄疫病毒（Foot-and-Mouthdisease virus，FMDV）引起偶蹄动物感染的烈性传染病之一。目前FMD呈世界范围流行趋势，其血清型有O、A、C、SAT1、SAT2、SAT3和Asia1型共7种。O型、Asia1型等FMD在我国周边国家或东南亚地区时有发生，对我国养殖业构成了严重的威胁。使用重组鸡痘病毒诱导鸡和哺乳动物细胞免疫应答抵抗外来抗原已经取得明显进步，鸡痘病毒的主要优势在于它允许插入较大量的外源基因。需要我们明确的是对于任何一种病毒表达哪些基因用作多价抗原。使用天然的鸡痘病毒启动子可能优化外源抗原的表达。也要对重组鸡痘病毒表达抗原（外源抗原或鸡痘病毒抗原）之间的竞争进行更多的认识，从而帮助构建有效预防多种病原体的多价重组体鸡痘病毒。联合免疫的深入研究有助于帮助我们认识进而选择不同疫苗形式（包括DNA、蛋白和传统的疫苗）的最佳组合。本实验以合成的痘病毒早晚期启动子PE/L及P7.5作为串联启动子的具有自主产权的鸡痘病毒转移载体为基础，选择NDV的F、HN基因，IBDV的VP0（VP243）、VP2基因，分别置于双向启动子下游，构建了双向表达的鸡痘病毒转移载体pMTB18-F-F、pMTB18-F-HN、pMTB18-F-VP0、pMTB18-VP0-VP2、pUTAL-F-VP2。应用脂质体转染法，将该重组鸡痘病毒中间转移载体与282E4株鸡痘病毒共转染鸡胚成纤维细胞(CEF)，经BrdU连续三次加压筛选，挑斑纯化后传代，对鸡痘病毒重组体在体外分别以PCR、RT-PCR和IFA对外源基因的重组、转录和蛋白表达及其生物学活性进行了鉴定。PCR检测结果表明转移载体构建均正确，同时在转录水平上进一步证实了重组病毒可以稳定携带外源基因并正确转录。将重组鸡痘病毒rFPV-F-VP0、rFPV-F-F、rFPV-F-HN、rFPV-F-VP2以抗NDV血清作为一抗，rFPV-F-VP0、rFPV-VP0-VP2、rFPV-F-VP2以抗IBDV血清作为一抗分别进行间接免疫荧光和免疫酶检测，分别检测到了特异性荧光和褐色的痘斑。研究证明获得了5株重组鸡痘病毒rFPV，能够有效的表达外源蛋白，表达产物具有相应的生物学活性。以SPF鸡作为动物模型，对之前构建的5株重组鸡痘疫苗进行了免疫研究，通过体液与细胞免疫指标检测，对疫苗的免疫原性进行了评估。结果显示，所构建的重组鸡痘病毒rFPV均能刺激SPF鸡产生抗NDV或IBDV的特异性ELISA抗体，疫苗接种后第2周抗体水平开始逐渐提高，在第5-6周达到高峰，其抗体水平均高于对照组。对脾T淋巴细胞的增殖能力和血清中Th1类细胞因子（IFN-γ和IL-2）、Th2类细胞因子（IL-4和IL-10）进行了检测。研究表明，重组鸡痘疫苗使SPF鸡产生了特异性体液和细胞免疫应答。攻毒试验结果显示，用重组鸡痘病毒rFPV-F-HN能在一定程度上抵抗新城疫强毒的攻击；重组鸡痘病毒rFPV-F-VP2、rFPV-VP0-VP2免疫的SPF鸡能够明显降低分离株IBDV-JL01/2007对法氏囊组织的损伤程度。本实验室先后成功构建并筛选出FMDV vUTAL3CP1（O型）、vUTAL-P1-2A-3C-IL18（Asia1型）、 vUTA2-P1-2A-3C （Asia1型）、vUTA2-P1-2A-IL18（O型）重组鸡痘病毒疫苗， FMDVGS115/pPIC9K-VP1-2A-CTE（O型）、GS115/pPIC9K-VP1-2A-CTE（Asia1型）酵母重组表达蛋白，pVIRIL18P1（O型）和pVAXI-P1-2A-3C（Asia1型）核酸疫苗。上述疫苗经小鼠单独免疫试验，表明均具有良好的免疫原性，可以激发针对FMDV的特异性体液和细胞免疫应答。据此，本研究利用上述疫苗，采用prime-boost策略免疫FMDV敏感动物豚鼠，并对其诱导的特异性体液和细胞免疫水平进行了检测，以此确定最佳疫苗和免疫策略。结果显示，O型疫苗中，核酸苗pVIRIL18P1首免，重组鸡痘病毒疫苗加强免疫的体液和细胞效果最好，Asia1型以重组鸡痘苗vUTA2-P1-2A-3C-IL18细胞免疫效果突出。研究表明，在针对目标抗原的初次免疫反应中DNA免疫是最有效的，重组痘病毒可加强免疫。同时能依靠能痘病毒自身佐剂的活性增强免疫反应。以猪为动物模型，应用prime-boost策略，评价了O型苗pVIRIL18P1/vUTAL3CP1、 vUTAL3CP1/vUTAL3CP1， Asia1型苗pVAXI-P12A3C/vUTA2-P12A3CIL18、vUTA2-P12A3CIL18/vUTA2-P12A3CIL18几种联合免疫方案的特异性体液和细胞免疫应答水平。核酸苗（500μg/头）采用壳聚糖包裹，鸡痘苗（107PFU）加入佐剂QS-21后与冻干保护剂混合。对35日龄仔猪间隔28d进行两次免疫，应用O型和Asia1型口蹄疫间接ELISA、LPB-ELISA、VP1结构蛋白抗体ELISA（O型）、ELISPOT、T淋巴细胞增殖试验、细胞因子检测等试验方法，证实了候选DNA和鸡痘重组疫苗均能诱导特异性的免疫应答。核酸苗首免，重组鸡痘病毒疫苗加强免疫的体液和细胞效果最优。总之，本研究以新的载体构建和联合免疫研究思路，探讨了重组鸡痘病毒在鸡新城疫病毒、传染性法氏囊病病毒、口蹄疫病毒新型疫苗的设计和免疫策略，对基于双向启动子的重组核酸疫苗构建与实验免疫进行了深入研究，为有效禽类和哺乳动物重组疫苗的研制与临床前实验研究奠定了基础。更多还原

【Abstract】 Newcastle disease virus (NDV) infection results in Newcastle disease (ND), ahighly epidemic and most costly disease, which greatly can threaten poultry. Vaccineinoculation is a safe and effective way to prevent NDV. Infectious bursal disease(IBD) caused by IBD virus is highly cute and contagious disease of chook. Recently,emerge of very-virulent IBDV (vvIBDV) and variant isolate bring out enormousdifficulties because of breaking through protection of parent antibody. Due to lowimmuogeniciy of inactivated vaccine and disperse and returning virus of live vaccine,current focus is development of promising vaccine.Foot and mouth disease (FMD), as a violently artiodactylous infection, iscaused by Foot and mouth disease virus (FMDV). At present, FMD is prevalent inthe world and the FMD virus exists in the form of seven different serotypes: O, A, C,Asia1, and South African Territors1(SAT1), SAT2and SAT3. Historically, FMDSerotype O and Asia1had been spread in part of China. Now it has serious threatthat still occasionally happen in the many countries around China and Southeast Asiahappen.Poxvirus, which permit large exogenous gene be inserted, displays obviousadvantage and distinct progress show that poxvirus can induce immune response ofchook and mammalian against invaded antigen. The selection of what genes can beused for antigen expressed in virus vectors need to be made a decision. A possibleoptimizing heterogenic expression route is to use natural poxvirus promoter. Inaddition, further knowledge for competition among expressed antigens inserted inpoxvirus will help construct multivalent rFPV to prevent various pathogens. We canselect various formatted vaccine containing DNA, protein and inactivated vaccinefollowing to going to investigate co-immunization.The aim to establish to fowl pox virus shuttle transfer plasmid pMTB18-F-F, pMTB18-F-HN, pMTB18-F-VP0, pMTB18-VP0-VP2and pUTAL-F-VP2, the fourgene, F and HN gene of NDV, VP0and VP2of IBDV, was be inserted downstreamof bi-directed promoter of reconstructive poxvirus transfer vector carrying linedpromoter PE/L and P7.5. Above recombinant cassettes were cotransfected with poxvirus282E4, respectively. After serial passage of positive recombinant virusconstruct, expression and biological activity of fusion protein was identified by PCR,RT-PCR and IFA. Exogenous transcription of these construct virus was constantlydetermined by PCR. Indirect immunofluorescence and immune enzymes detection ofrFPV-F-VP0, rFPV-F-F, rFPV-F-HN and rFPV-F-VP2were carried out by usingserum of anti-NDV as the first antibody. Correspondingly, the serum of anti-IBDVwas used for the same detection of rFPV-F-VP0, rFPV-VP0-VP2and rFPV-F-VP2.Five rFPV can effectively express and product has promising biological activity.In present study, this five recombinant poxvirus vaccine was investigated byhumoral immunity and cellular immunity index detection and assessed on vaccineimmunogenicity. These results show that our constructed poxvirus rFPV inducedSPF chicken producing special ELISA antibody against NDV or IBDV. Antibodylevel increased following to the second week and reach the peak at the five-six weekafter vaccine inoculation. The data of antibody level is higher compared with thecontrol group. In the test of multiplication capacity of T lymphocyte of spleen andTh1-like cytokine (IFN-γ and IL-2) and Th2-like cytokine (IL-4and IL-10), therFPV can induce SPF chicken to produce special immune response. The challengeexperiment showed that rFPV-F-HN could partially prevent SPF chicken againstNDV and SPF chicken could reduce damage degree of IBDV-JL01/2007.In our lab, we successively construct FMDV vUTAL3CP1(O serotypev),UTAL-P1-2A-3C-IL18(Asia-I serotype), vUTA2-P1-2A-3C (Asia-Ⅰserotype) andvUTA2-P1-2A-IL18(O serotype) foxvirus vaccines and FMDVGS115/pPIC9K-VP1-2A-CTE (O serotype). GS115/pPIC9K-VP1-2A-CTE (Asia-Iserotype) recombinant protein expressed in yeast and nucleic acid vaccinepVIRIL18P1(O serotype) and pVAXI-P1-2A-3C (Asia-I serotype). Above vaccinecould induce special humoral and cellular immunity of FMDV in mice and hadefficient immunogenicity. Accordingly, we immunize guinea pig and detected level of humoral and cellular response level by using prime-boost strategy in this study.These results showed that the best effect is to employ the protocol of nucleic acidvaccine pVIRIL18P1as the first immunization and recombinant poxvirus as boostimmunization. The obvious cellular effect can be found in the test of immunizationof recombinant vUTA2-P1-2A-3C-IL18. in our experiment, DNA vaccine extendedits most effective response in the first immunization and recombinant poxvirus canbe used to boost immunization. At the same time, we found that poxvirus couldincrease immune response by self-adjvant activity.We evaluated humoral and cellular response by combined immunization withseveral vaccine O serotype vaccine pVIRIL18P1/vUTAL3CP1and vUTAL3CP1/vUTAL3CP1, Asia-I serotype vaccine pVAXI-P12A3C/vUTA2-P12A3CIL18andvUTA2-P12A3CIL18/vUTA2-P12A3CIL18in animal model of pig. Nucleic acidvaccine(500μg/portion) was coated with chitosan and poxvirus vaccine(107PFU)added adjuvant QS-21made a mixture with freeze-drying protective agent. Both thedifferent vaccine were used for two inoculation of28days interval in35days piglet.Subsequently, we confirm their good immune response by serial detection containingindirect ELISA, LPB-ELISA, ELISPOT, T lymphopoiesis and cytokine detection.The further investigation confirmed the immune protocol is best by DNA vaccine asfirst immunization and recombinant poxvirus boost immunization. In conclusion, weprobed into novel vaccine design and immune strategy aiming to NDV, IBDV andFMDV based on reconstructed vector and co-immunization in present study. Inaddition, recombinant DNA vaccine carrying bi-directed promoter and immune testwere as deep research objects. These studies provide good foundation ofdevelopment of recombinant vaccine and of pre-clinical experiment for poultry andmammalian.更多还原