【作者】 李凯；

【导师】 王笑梅；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2014， 博士

【摘要】 网状内皮组织增生病（Reticuloendotheliosis，RE）是由网状内皮组织增生病病毒（Reticuloendotheliosis virus，REV）群的反转录病毒引起的鸡、鸭、火鸡和其他禽类的一群病理综合征。动物感染REV后主要引起慢性肿瘤、矮小综合症等，还可以引起免疫器官的萎缩，导致机体免疫功能显著下降，从而造成免疫失败并使感染动物继发其它传染病。REV与马立克氏病病毒混合感染时，经常因其基因重组而增强马立克氏病病毒的致病性和变异率。近年来，REV已在全球范围内流行，国内鸡群中REV的感染也非常严重，分布日益广泛。REV通过免疫抑制、混合感染及对疫苗的污染等对养禽业造成了严重的危害，市场和现地迫切期望RE能像其他动物传染病一样有适宜、安全、有效的疫苗来进行防控。然而，目前国内外还没有相应的商品化疫苗用于RE的预防。本研究首先对实验室分离的一株REV病毒（HLJR0901株）在不同日龄SPF鸡上的致病特性进行了系统的研究，建立了REV在SPF鸡上的动物感染模型。通过对攻毒后感染鸡临床症状、体重、器官指数、病理变化、病毒载量的检测表明，1日龄SPF雏鸡感染REV后出现生长迟缓，法氏囊、胸腺萎缩，肝脏、脾脏和腺胃肿大的症状；大日龄鸡（5周龄）感染REV后上述症状均不明显；然而，不同日龄的SPF鸡感染REV后在法氏囊、胸腺、肝脏、脾脏和腺胃中均可检测到病毒存在，并出现病毒血症。另外，1日龄SPF雏鸡感染REV后出现了严重的免疫抑制，使AIV灭活疫苗和NDV弱毒疫苗的免疫效果明显降低。REV动物感染模型的建立为REV疫苗的效力评价奠定了基础，并为深入研究REV致病机理和免疫抑制机制提供了材料。将REV HLJR0901毒株的保护性抗原gp90蛋白在毕赤酵母表达系统中进行了分泌表达，并对构建的重组酵母菌株在宿主菌、基因拷贝数、启动子和表达条件等方面进行了优化。结果表明，重组gp90蛋白在毕赤酵母中成功获得分泌表达，在酵母宿主菌SMD1168中的表达量显著高于GS115和KM71；gp90重组蛋白的表达量与酵母菌株中gp90基因表达盒的插入拷贝数呈正相关；联合使用诱导型启动子AOX1和组成型启动子GAP使gp90蛋白在酵母中的表达量进一步提高了41.1%；共表达毕赤酵母分子伴侣PDI和Ubi4降低了gp90重组蛋白在发酵过程中的蛋白酶降解；经过对表达条件的优化，表明重组酵母菌株在28°C和pH6.0下表达量最高，在发酵培养基中加入1%的干酪素进一步提高了gp90蛋白的表达水平。经过对表达菌株和发酵条件的优化，重组酵母菌株SMD1168/GA90在摇瓶中的表达量平均可达到200mg/L，在发酵罐中进行发酵培养后的表达量平均可达1.0g/L。将发酵蛋白进行Western blot分析，结果表明，发酵表达的gp90重组蛋白可以与gp90单克隆抗体发生特异性反应；将该重组gp90蛋白免疫鸡后可以诱导产生REV特异性抗体，说明酵母表达的gp90蛋白具有良好的抗原性。以毕赤酵母分泌表达的重组gp90蛋白作为免疫原制备了RE重组亚单位疫苗。为了对RE亚单位疫苗的免疫效力和安全性进行评价，在SPF雏鸡上进行了最小免疫剂量和免疫程序的研究，确定了该疫苗免疫后的抗体消长规律、免疫持续期以及抗体滴度与攻毒保护的相关性；并进一步在SPF蛋种鸡和商品蛋种鸡上进行了免疫试验，研究了RE亚单位疫苗对种鸡的免疫效力以及母源抗体对子代雏鸡的免疫保护作用。试验结果表明，该重组亚单位疫苗对雏鸡的最小免疫剂量为20微克/只；免疫程序为3周龄首免，间隔三周后二免；免疫鸡血清具有良好的中和活性；当ELISA抗体滴度达到2800时可以对REV感染造成的病毒血症提供完全保护；疫苗免疫持续期为6个月。种鸡的免疫试验表明，RE亚单位疫苗可以诱导种鸡产生高水平的抗体反应，子代鸡通过被动免疫获得的母源抗体可以抵抗REV早期感染造成的病毒血症和生长迟缓。以上结果表明，RE亚单位疫苗对雏鸡和种鸡均具有良好的免疫效力。另外，对研制的RE亚单位疫苗进行单剂量免疫、单剂量重复免疫和大剂量免疫后，试验鸡均未出现任何不良临床反应，对种鸡的生产性能也没有明显的影响，说明该重组亚单位疫苗对雏鸡和种鸡是安全可靠的。本研究还同时构建了表达REV保护性抗原gp90的DNA疫苗，并就其免疫效力与亚单位疫苗进行了比较研究。结果表明，本研究构建的DNA疫苗免疫鸡后可以诱导试验鸡产生体液免疫和细胞免疫应答，目的基因gp90的密码子优化和土拨鼠肝炎病毒转录后调控元件（WPRE）的使用可以有效提高目的基因的表达量和DNA疫苗的免疫原性。免疫100微克双重优化后的DNA疫苗pCAGWoptigp90后获得了87%的保护率，而40微克重组gp90蛋白免疫组可以达到完全保护；DNA疫苗诱导产生的抗体反应同样弱于亚单位疫苗。试验进一步表明，DNA疫苗首免-亚单位疫苗加强的联合免疫方式比单独使用DNA疫苗或亚单位疫苗免疫两次诱导产生了更高水平的体液免疫和细胞免疫应答，提示了该联合免疫方式在REV预防中的潜在应用价值。更多还原

【Abstract】 Reticuloendotheliosis virus (REV) is a member of gammaretrovirus with a variety of strains, whichcauses an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts includingchickens, ducks, turkeys and some other bird species. REV causes immunosuppression in infectedchickens, resulting in an increased susceptibility to concurrent or secondary bacterial and viralinfections and poor immune responses to other vaccines. REV fragments could integrate into thegenome of Marek’s disease virus (MDV) and increase its virulence when co-infecting with MDV. REVis widely distributed around the world and also highly prevalent in China, causing severe damages toour poultry industry. The oncogenicity and the immunosuppressive ability of REVs, their co-infectionwith other infectious viruses and their presence as contaminants in poultry biologics warrantdevelopment of a suitable vaccine. However, no commercially effective vaccine is available forpreventing this disease until now.The pathogenicity of REV HLJR0901strain in different ages of specific-pathogen-free (SPF)chickens was investigated, and the infection model of REV HLJR0901strain was established. Theinfected chickens were monitored weekly after infection for signs of illness and pathological changes.Decline in body weight, atrophy in bursa and thymus, tumefaction in liver, spleen and proventriculuswere observed after REV infection in1-day-old SPF chickens. The chickens infected with REV at5-week-old showed no or mild clinical signs. However, both the early infected chickens and the lateinfected chickens showed viremia and high proviral loads in multiple organs including bursa, thymus,liver, spleen and proventriculus. Additionally, the antibody responses to AIV and NDV vaccines weresignificantly inhibited by REV early infection compared with the control chickens. The detailedinfection model of REV in SPF chickens could be used to evaluate the efficacy of potential vaccines andantiviral agents.For the development of the recombinant subunit vaccine against REV, the gp90protein wasexpressed in yeast Pichia pastoris. The construction of recombinant Pichia strains and the expressionconditions were optimized in order to enhance the gp90production. The results showed that the REVgp90protein was secretory expressed in P. Pastoris successfully and SMD1168was the optimal hoststrain for gp90expression than the GS115and KM71strains. An increase in gp90copy number canimprove the yield of gp90protein in P. pastoris. The combined usage of both constitutive promoterGAP and inducible promoter AOX1further enhanced the gp90expression by41.1%. The results alsoindicated that co-expression of gp90protein with the yeast chaperones PDI and Ubi4reduced theproteolytically degradation of the gp90protein. In addition, the fermentation culture was optimized, and28°C and pH6.0were found to be the optimal conditions for gp90expression. The gp90productionwas further improved by adding1%casanimo acids in the fermentation medium. Following large-scalefermentation, the average gp90production level from strain SMD1168/GA90reached up to200mg/L inflask cultures, and1.0g/L after fermentation. The recombinant gp90protein could react with thegp90-specific MAb and induced high levels of REV antibody in immunized chickens, which showed that the yeast-expressed gp90protein had good antigenicity.To evaluate the efficacy and safety of the recombinant gp90as a subunit vaccine against REV,3-week-old SPF chickens as well as the SPF and commercial layer breeders were immunized with thegp90protein. The results showed that the minimum immunizing dose of the recombinant gp90proteinwas20μg; the optimal immunizing procedure was two shots at3week intervals, and the protectiveantibody duration was six months. The minimum antibody titer that could confer protection from REVinfection was2800as detected by ELISA. The immunization assay further showed that the recombinantsubunit vaccine induced high levels of antibody responses in SPF and commercial breeders, and thematernal antibody of the new-born chicks protected them from REV-induced viremia and runtingsyndrome. The results demonstrated that the chickens immunized with the recombinant gp90protein didnot show any abnormal clinical signs, and the immunization of the subunit vaccine made no influenceon the egg production rate of the breeder chickens. Overall, the yeast-expressed recombinant gp90protein was capable of inducing high levels of protective immune responses in both SPF chickens andlayer breeders with good safety.The DNA vaccines expressing the gp90protein of REV was constructed and also evaluated in SPFchickens in this study. The results showed that the DNA vaccines containing gp90gene induced bothhumoral and cellular immune responses and were efficacious in conferring protection against REVchallenge in chickens. Further, codon optimization and woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE) were very powerful approaches in improving the immunogenicity of theDNA vaccines. Immunization of100μg of the optimized plasmid pCAGWoptigp90provided87%protection against REV challenge, lower than that conferred by40μg recombinant gp90protein whichconferred full protection against REV-induced viremia. Additionally, a DNA prime-protein booststrategy produced higher levels of humeral and cellular immune respinses in chickens than did either ofthe component parts of this vaccine alone. These findings highlight the potential value of combinationusage of both DNA and subunit vaccines for the prevention of REV infection.更多还原