【作者】 张振杰；

【导师】 崔治中；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2014， 博士

【摘要】 马立克氏病毒(Marek′s disease virus, MDV)是α疱疹病毒的成员，基因组为180kb左右的双股DNA，可引起鸡的淋巴细胞瘤。GX0101株MDV是国内外从鸡体内分离到的第一株整合有网状内皮组织增生症病毒(Reticuloendotheliosis virus, REV)长末端重复序列(Long terminal repeat, LTR)的重组野毒株。本实验室前期研究中已经构建了GX0101的细菌人工染色体(Bacterial artificial chromosome, BAC)克隆，并构建了敲除肿瘤基因meq的突变株GX0101meq病毒。该基因缺失性MDV不但丧失对SPF鸡的致病性，而且不再引起肿瘤以及胸腺和法氏囊萎缩。本文利用敲除meq基因的GX0101株MDV作为载体表达AIV-H9N2和NDV主要保护抗原基因，构建了2株敲除meq致肿瘤基因且同时表达2种不同外源基因的重组MDV。通过体内和体外连续传代分析这些重组MDV在细胞水平和动物体内的复制能力、探讨插入片段在重组MDV基因组中的稳定性、通过免疫SPF鸡这些重组MDV在体内诱导鸡的免疫应答的能力，从而为研发用于预防MDV和H9N2更好的疫苗提供候选疫苗株及相关的技术支持。研究主要从以下几个方面展开:1.荧光定量PCR检测1型MDV在不同感染阶段各组织中病毒含量的变化和分布。感染马立克病毒的鸡持续携带病毒一段时间后，可以增加器官肿瘤的发生率和提高横向传播的能力。鸡的马立克病毒含量与恶性肿瘤的发生率和作为感染性病毒的颗粒的唯一来源的羽毛囊上皮细胞中病毒传播水平呈有正相关的关系。本研究建立了一种SYBR Green双重实时荧光定量PCR的方法，通过选择病毒的meq基因和管家基因-鸡卵铁转蛋白ovo来检测和量化发病鸡的马立克病毒的含量和不同组织病毒的分布。研究中分别用马立克基因组的BAC克隆，BAC-GX0101和携带管家基因-鸡卵铁转蛋白特异性基因片段的质粒(pMD18T-ovo)来定量病毒和宿主基因组。该方法具有1-8logs动态变化范围，其变化的平均值的批间和批内系数小于2%，并且MDV最小的检测含量为10拷贝。利用该方法绝对定量检测感染1000PFU的SDWJ1302菌株发病鸡的分别于7、14、21、28、40、60和90天取不同样品组DNA，空白组的鸡也作同样处理，并与常规的PCR检测方法的灵敏度进行比较。结果表明，感染鸡的免疫器官在感染7天后就可以检测到马立克基因的拷贝数，鸡的血液和不同器官在感染28天后是检测到马立克基因拷贝的高峰期，但40天后逐渐下降。在淋巴细胞，肝脏和脾脏的病毒载量水平均高于其它器官，并且在不同阶段羽毛囊上皮细胞的含量最高。研究数据进一步证明涉及病毒感染的多器官和免疫器官的主要分布，从而导致免疫抑制和免疫细胞的转化。这是首次对早期潜伏感染和发病后阶段的不同组织和器官的马立克基因组的拷贝数进行了绝对定量，应用此方法能有利于促进我们对马立克氏病的发病机机理、传播、诊断、遗传特性和疫苗控制的进一步认识。2.共表达H9N2-NA和NDV-F的meq缺失性重组马立克病毒的构建及其生物学特性研究本实验室以前的研究报道表明，在马立克氏病病毒(MDV)基因组的pp38基因和1.8kb mRNA之间的305bp是一个双向启动子，分别启动MDV的1.8kb mRNA转录子和pp38基因的表达（两个方向作用的启动子分别P1.8kb和Ppp38表示），该双向启动子可被MDV自身表达的pp38/pp24二聚体作为反式转录因子激活。为了构建能同时表达禽流感病毒H9N2株的NA基因和鸡新城疫病毒(NDV)的F基因的meq基因缺失型重组I型MDV，首先以真核表达性质粒pcDNA3.1为骨架，构建了重组表达性载体质粒pPpp38-NA/1.8kb-F。在该质粒中，NDV-F基因位于双向启动子上游，由P1.8kb启动；AIV-NA基因位于该双向启动子下游，由Ppp38启动。为了提供作为反式转录因子pp38/pp24二聚体，将重组质粒pPpp38-NA/1.8kb-F与包含pp38/pp24表达质粒pBud-pp38-pp24共转染CEF。用针对NDV-F和AIV-NA的小鼠血清，对转染细胞做间接免疫荧光反应（IFA），证明了在重组质粒pPpp38-NA/1.8kb-F中该双向启动子确实可同时调控这两个外源基因的表达。为了将NA和F基因共表达盒插入MDV US2的非必要区，分别将MDV插入位点两侧序列并作为同源重组的两同源臂整合到NA和F基因共表达盒的两侧，构建成转基因重组载体质粒pPpp38-NA/1.8kb-F。PCR扩增同源重组大片段，将敲除了meq基因的GX0101(GX0101Δmeq)作为亲本病毒,在EL250大肠杆菌中通过同源重组将NA和F基因共表达盒插入GX0101Δmeq的US2非必要区，构建并筛选到重组MDV (MZC13NA/F)。IFA、ELISA和免疫印迹分析表明，重组病毒MZC13NA/F可在双向启动子的控制及MDV自身pp38/pp24二聚体激活下同时表达NDV-F和AIV-NA基因。MZC13NA/F的体外生长特性也说明，MZC13F/NA与其亲本病毒GX0101的CEF无显著差异。同时也表明,在1.8-kb mRNA转录方向的启动子活性高于pp38基因方向。用MDV的这个自身双向启动子构建并成功同时表达二个外源基因的重组MDV，不仅表明它可以作为一个有潜在利用价值的启动子，而且可在构建的重组MDV中减少外源DNA的插入。3.共表达H9N2-HA和-NA的meq缺失性重组马立克病毒的构建及其生物学特性研究为了构建能够表达来源于低致病性禽流感H9N2HA和NA，将野毒株GX0101作为亲本毒，通过同源重组的方法将强启动子CMV和SV40分别启动HA和NA共表达的串联大片段插入到GX0101基因组的meq位点取代meq基因，得到的病毒命名为MZC12HA/NA。体外进行rMDV和亲本毒GX0101生物学活性比较发现MZC12HA/NA与GX0101在CEF细胞的生长动态无明显差异。Southern bolt表明共表达盒成功插入到meq基因的双拷贝位点，meq基因完全被敲除。RT-qPCR显示HA和NA的mRNA转录水平显著高于MDV自身pp38mRNA的转录。ELISA检测显示HA和NA蛋白的表达也远远高于pp38蛋白的表达。IFA和western bolt分析表明HA和NA能够同时表达，且检测不到meq基因的表达。结果表明，作为AIV-H9N2与MDV重组得到的MZC12HA/NA致肿瘤meq基因被删除，且能够高水平表达H9N2的抗原基因HA和NA。4.同时表达AIV-H9N2的HA和NA基因的MZC12HA/NA重组MDV的免疫原性MZC12HA/NA免疫SPF和海兰褐鸡，在鸡体内均能良好地复制，但复制能力弱；重组病毒免疫SPF鸡后能诱导滴度为2.0左右的H9N2抗体；重组病毒和H9N2对海兰褐的免疫应答有明显协同作用；免疫后SPF鸡H9N2的排毒实验，对H9N2的保护率为30%-35%，所以该重组病毒将来可能应用于抵抗MDV和H9N2感染的重组疫苗的筛选。更多还原

【Abstract】 Marek’s disease virus (MDV) is a member of the Alphaherpesvirinae subfamily, andits genome contains a linear double-stranded DNA of about180kb. MDV causes Marek’sdisease in chickens with lymphomas. GX0101is the first recombinant MDV field straincontaining the reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert isolatedfrom chicken both at home and abroad. In previous research in our laboratory, GX0101bacterial artificial chromosome and its mutant strain GX0101LTR with a deletion of theREV-LTR were constructed and demonstrated that REV-LTR insert increased thehorizontal transmission ability of GX0101. Mutant strain GX0101meq with a deletion ofmeq oncogene was demonstrated to have lost its pathogenicity in SPF chicken, and nolonger induced tumors and atrophy of thymus and bursa of fabricius. With the widespreaduse of molecular biology, it is promising for MD recombinant vaccine. MDV vaccine strainis considered as one of the most potential virus vectors, which could prevent poultry diseasethrough constructing multivalent live vaccine of exogenous gene antigen. The articlediscusses whether the expression of pp38/pp24heterodimers are the required activators forthe expression of the foreign genes. The confirmed function of the bi-directional promoterprovides better feasibilities to insert multiple foreign genes in MDV genome based vectorsk,and try to analyze rMDV replication ability in cells and animal bodies through serialpassage in vivo and in vitro. And discuss the stability of rearrange pieces in MDV genometo provide research direction for rearrange virus special biology activity. And study theimmune response ability of rMDV through inoculating SPF chickens, discuss synergisticeffect between inactivated vaccine rMDV and H9N9through co-inoculating chickens. Andprovide screening vaccine strain for the prevention of MDV and H9N9. And providetheoretical and technical support for constructing rearrange MDV virus vaccine in future.1. Absolute quantification of MDV dynamic loads and distributions in different tissuesusing duplex real-time PCR assayChickens infected with Marek’s disease virus (MDV) carry the virus consistently for a long time, which increases the incidence and rate of virus-induced multi-organ tumors in vivoand increases its potential for horizontal transmission. There is a positive correlation betweenMDV loads and the incidence of malignant tumors and the level of viral transmission in thefeather follicle epithelia of chickens serving as the sole sources of infectious virus particles.The present study was aimed at developing a SYBR Green duplex real-time quantitative PCR(q-PCR) assay to detect and quantify MDV loads and distributions in different tissues,targeting the Meq gene of the virus and the house-keeping ovotransferrin (ovo) gene ofchickens. A bacterial artificial chromosome (BAC) clone of the MDV genome, BAC-GX0101,and a plasmid (pMD18T-ovo) bearing a fragment of the chicken ovo gene were used toquantify virus and host genomes, respectively. The assay had a dynamic range of8logs, amean inter-and intra-assay coefficient of variation (CV) of <2%and minimum detection limitof10MDV genome copies. The sensitivity of the assay was compared with that of bothconventional PCR and viremia assay. The q-PCR was validated using different tissue DNApreparations derived from chickens infected with1000plaque forming units (PFUs) of theSDWJ1302strain and sampled on days7,10,15,21,28,40,60and90post-infection (p.i.),along with uninfected chickens. The resulted indicated that the MDV genome was almostquantifiable in mmune organs of infected chickens by day7p.i., and the number of MDVgenome copies in the blood and different organs peaked by day28p.i., but then graduallydecreased by day40p.i.. The levels of viral loads in the lymphocytes, liver and spleen wereall higher than that in other organs, and that in the feather follicles was the highest at differentstages of MDV infection. The data provided further evidence of viral infection involvingmulti-organ distribution and mainly involving immune organ proliferation, resultingin immunosuppression and lymphocyte transformation. This is the first report of absolutequantification of MDV genomes in different tissues and organs during the early latentinfected and later incidence stages, and application of this assay could significantly furtherour understanding of the pathogenesis, spread, diagnosis, genetic resistance and vaccinalcontrol of Marek’s disease.2. Biological characteristics of rMDV lacking the meq oncogene and co-expressingAIV-H9N2-NA and NDV-F genes under control of MDV’s own bi-directionalpromoter Our previous studies reported a bi-directional promoter of only305bp between pp38geneand1.8kb mRNA family on Marek’s disease virus (MDV) genome, its transcriptionalfunction could be activated by pp38/pp24hetero-dimers as trans-acting transcriptional factors.To qualitatively analyze and evaluate its bi-directional transcriptional function in bothtransient and transgenic systems, several different plasmids were constructed and recombinantMDV type1strain GX0101was developed to co-express a NA gene from Avian InfluenzaVirus H9N2strain and a F gene from the Newcastle disease virus (NDV).The two foreign genes, NDV-F gene and AIV-NA gene, were inserted in the plasmiddriven by the bidirectional promoter. NDV-F gene was located in the upstream of thepromoter, while the AIV-NA gene was located in the downstream of the promoter. To testwhether the expression of pp38/pp24heterodimers are the required activators for theexpression of the foreign genes, the recombinant plasmid pPpp38-NA/1.8kb-F containingexpression cassette for the two foreign genes was co-transfected with pp38/pp24expressionplasmid, pBud-pp38-pp24in chicken embryo fibroblast (CEF) cells. Alternatively, plasmidpPpp38-NA/1.8kb-F was transfected with GX0101-infected CEF which pp38/pp24wasexpressed via virus infection. Moreover, a recombinant MDV was constructed containing theexpression cassette of pPpp38-NA/1.8kb-F. To do so, a Meq-deleted MDV GX0101(GX0101Δmeq) was used as parental virus, the AIV-NA gene and NDV-F gene co-expression cassettewas inserted at the nonessential region of MDV US2to form a new recombinant MDV(MZC13NA/F) through homologous recombination. Indirect fluorescence antibody (IFA) test,ELISA and Western blot analyses indicated that F and NA genes were expressedsimultaneously under control of the bi-directional promoter, but in opposite directions. Theexpression of both foreign genes was activated by pp38/pp24dimers either via virus infection,or transient expression. The CEFs transfected with pPpp38-NA/1.8kb-F alone had noexpression. The data also indicated that the activity of the promoter in the1.8-kb mRNAtranscript direction was higher than that in the direction for the pp38gene. In vitro growthproperties of MZC13NA/F were also inspected and concluded that the MZC13F/NA had thesame growth kinetics in CEF cultures as its parental wild type virus GX0101.The pp38/pp24dimers expressed by pBud-pp38-pp24in co-transfected cells or byGX0101in infected cells were critical to activate the bi-directional promoter for expression of two foreign genes in two directions. The bi-directional promoter could be used as a potentialpromoter with some advantages in construction of recombinant MDVs to express more thanone foreign gene.3. Biological characteristics of rMDV lacking the meq oncogene and co-expressingAIV-H9N2-HA and NA genes under control of exogenous promotersTo develop a recombinant Marek’s disease virus (rMDV1) co-expressing thehemagglutinin gene (HA) and Neuramidinase gene (NA) from a low pathogenic AvianInfluenza virus (LPAIV) H9N2strain and lacking the Meq oncogene that shares homologywith the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101was used asparental virus, the HA and NA genes co-expression cassette under control of the CMV andSV40early promoters was inserted at two Meq sites of GX0101to form a new complete lackof oncogenicity of a Meq knock-out mutant MDV (MZC12HA/NA) through homologousrecombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC-MDVDNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purifiedMZC12HA/NA was obtained after four rounds of plaque purification and proliferation. Invitro growth properties of recombinant virus were also inspected and concluded that theMZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virusGX0101. Southern bolt indicated that co-expression cassette was successfully inserted at twocopies sites of Meq gene, so two Meq genes were knocked-out completely. RT-qPCR showedtranscription and expression levels of the HA ana NA genes were both significantly higherthan that of GX0101own pp38gene. Indirect fluorescence antibody (IFA) test, and Westernblot analyses indicated that HA and NA genes were co-expressed simultaneously undercontrol of the different promoters but Meq genes were not. These results herald an new andeffective recombinant Meq-deleted MDV-based AIV-H9N2vaccine may be useful inprotecting chickens from very virulent MDV and H9N2challenges.更多还原