【作者】 张明明；

【导师】 秦爱建；

【作者基本信息】 扬州大学 ， 预防兽医学， 2008， 博士

【摘要】 腺病毒最早发现于1953年,由于它们倾向于感染上皮细胞而被命名为腺病毒。腺病毒属腺病毒科(Adenoviridae),共分为四个属即哺乳动物腺病毒属(Mastadenovirus)、禽腺病毒属(Aviadenovirus)、腺胸腺病毒属(Atadenovirus)和唾液腺病毒属(Siadenovirus)。禽腺病毒是禽类常见的病毒之一,大部分在禽体内复制却不致病,少数引起轻微的病症。对禽腺病毒的流行和变异国外有很多研究工作,国内的报道较少。腺病毒作为载体,宿主范围广,致病性低,在机体内复制不发生整合,安全性高;能在增殖和非增殖细胞中感染和表达基因;能有效进行增殖,病毒滴度高;腺病毒载体应用方便,载体疫苗可经口服途径接种。因此以腺病毒作为病毒载体具有独特的优势。许多学者在禽类腺病毒载体方面做了很多研究工作,国内研究较晚,但是所研制的基因工程疫苗离临床应用还有一定距离。目前,对禽腺病毒载体研究主要使用CMV启动子,获得了较好的表达效果,也有使用病毒自身的晚期启动子/先导序列(MLP/LS)的报道,但是尚未见到对两个启动子的重组病毒的免疫效果进行比较的报道。本研究从外表健康禽群中随机采取泄殖腔拭子,根据禽腺病毒CELOV的全序列设计引物,PCR扩增了病毒基因组右末端1.5Kbp的ITR片段。结果表明:禽腺病毒在禽群中的流行比较广泛,鸡群的阳性率为23.8%(30/126),鸭群的阳性率为51.7%(15/29),鹅群的阳性率为17.3%(10/58)。从鸡源、鸭源、鹅源ITR扩增阳性的样品中各选取3株,将ITR片段的PCR扩增产物克隆测序,并与CELOV、FAV-9、EDSV以及FAV-JS株的相应序列进行了比较。结果表明:本次分离的9株禽腺病毒与血清1型的代表株CELOV和我们实验室早期分离株FAV-JS株在ITR片段上的同源性最高可达99.9%,最低也为94.7%;而与D亚群的FAV-9和禽腺病毒Ⅲ群的EDSV相比较,同源性较低。在此基础上,根据已经发表的CELOV的全基因组序列设计了引物,对分离株的保守序列ORF8进行了扩增和序列测定工作,结果表明所有分离株、CELOV、FAV-9、EDSV以及FAV-JS株的ORF8片段均具有高度同源性,符合腺病毒的基因组结构特征。根据以上序列分析结果,本研究证明了这些分离到的禽腺病毒属于Ⅰ群禽腺病毒。同时,禽腺病毒在鸡群、鸭群、鹅群中非常普遍,有必要对这些病毒的生物学特性进行研究。为了更好的了解分离株的生物学特性以便探讨其作为活病毒疫苗载体的可能性,通过病毒凝集实验、电镜形态观察、细胞病变、动物攻毒实验等方法对上述疑为Ⅰ群禽腺病毒3个分离株进行了研究。电镜观察病毒粒子,结果发现病毒为球型、无囊膜,直径为70-80nm,具有腺病毒典型的二十面体结构。红细胞凝集试验证明,分离到的病毒均不能凝集鸡的红细胞,与Ⅰ群禽腺病毒的特征一致。细胞培养表明:病毒分离株不能引起鸡胚成纤维细胞(CEF)的细胞病变,而在鸡胚肾细胞(CEK)上可以产生典型的禽腺病毒的细胞病变,表现为细胞变圆,折光性增强等病变。鸡胚接种试验未见明显肉眼可见的病变,鸡胚生长发育正常,这与Ⅰ群禽腺病毒分离株CELOV的特性不完全一致。通过大剂量皮下注射和口服两种途径接种1日龄的SPF鸡,进行人工致病性试验。结果发现,试验鸡在感染病毒后,未出现任何明显临床症状。在10日龄时,皮下注射和口服攻毒的小鸡的体重都明显轻于对照组,而在20、30日龄时,各组和健康对照组之间体重没有明显差异,表明该分离株对SPF鸡的体重影响不大,呈一过性。在10日龄时,攻毒组小鸡主要免疫器官重量略低于对照组,实验后期各组免疫免疫器官的重量基本一致。组织病理学切片观察结果表明,部分脏器有轻微病变,大部分脏器基本没有病变,后期组织器官均正常。这表明该病毒对免疫器官没有明显的生长抑制和可见的病理损伤。以上体内外实验均表明病毒分离株致病性低,生物安全性高,可能是较好的禽腺病毒载体。前期研究中筛选了FAV-JS的复制非必需区,将CMV调控外源基因表达的表达盒插入的禽腺病毒FAV-JS株左右臂构建了通用转移载体pFACMV3.1。在此研究基础上,从本实验室分离的所有禽腺病毒中选择FAV-JS进行重组禽腺病毒的构建。本研究根据CELOV全基因序列设计引物,从FAVI-JS基因组中PCR扩增出腺病毒的主要晚期启动子(MLP),将扩增产物克隆测序,并与CELOV的MLP进行了序列比较,两者同源性为99%。在此基础上,将FAVI-JS株MLP片段克隆至pcDNA3.1/zeo(+),然后再将pFACMV3.1中含有部分L片段和完整R片段的L#-R片段克隆至同一pcDNA3.1/zeo(+)中,随后将含有MLP片段、部分L片段和完整R片段的大片段克隆至pFACMV3.1,将pFACMV3.1中的CMV启动子替换为MLP启动子,而其它部分不变,从而构建了MLP启动子控制的通用转移载体pFAMLP 3.1。在此基础上插入eGFP报告基因,构建了MLP启动子控制表达eGFP的正向转移载体pFAMLP-eGFP,以用于重组病毒的构建。将构建的pFAMLP-eGFP转移载体转染原代鸡胚肾细胞(CEK)细胞,证实所克隆的MLP启动子具有启动子活性。在此研究基础上,将pFAMLP-eGFP转染已被wtFAVI-JS分离株感染的原代鸡胚肾细胞(CEK),进行细胞内同源重组。转染后的细胞及细胞上清混合物经过冻融和离心处理,取上清进行稀释,感染96孔细胞培养板上的CEK单层,结果连续传了三代后,仍然能观察到绿色荧光蛋白的表达,表明成功构建了表达eGFP的重组禽腺病毒,为探讨晚期启动子在重组病毒中是否可以高效的表达外源蛋白,重组禽腺病毒基因工程疫苗的启动子对疫苗免疫效果的影响提供了条件。更多还原

【Abstract】 Adenovirus was found in 1949, which is tendency to infect epithelial cell. Adenoviridae is divided into two genuses, the Mastadenoviridae and the Aviadenoviridae in the past. However, Atadenoviridae and Siadenoviridae were added to this family in the ICTV 8th report, 2005. Duck adenovirus A is classified as members of Atadenoviridae while HEV as Siadenoviridae, an intermediate between the Mastadenoviridae and the Aviadenoviridae because both encode sialidase in the right structure of the genome. It would be worth notice that EDSV is called Duck adenovirus A and Turkey adenovirus A is designated as new species after this report. Fowl adenoviruses are divided into three groups by differences in serotype, most of which only replicate in birds and don’t cause clinical symptoms or only have low pathogenicity. But most of them will influence the health of birds if there are other inducing factors or they superinfect with other pathogens while some fowl adenoviruses themselves are pathogenic. There are some reports about the prevalence or variation of fowl adenovirus (FAV) in foreign countries, but little in China.With the development of molecular biology, many viral vectors are being used for gene therapy, vaccination or transfer. Differently from retrovirus, adenovirus does not depend on host cell division for its replication, and its chromosome rarely integrates into the cell genome, remaining episomal in most cases. Adenoviral vectors have a broad spectrum of cell infectivity that includes virtually all post-mitotic and mitotic cells, and also can be produced in high titers. Therefore, it is convenient to use adenoviral vectors for gene therapy or transfer by many routs. It has been found that birds were reinfected with the same strain after 8 week, eliciting a secondary response neutralizing and precipitating antibodies; virus excretion also occurred despite of humoral antibody. So, if the virus can be constructed into a vector to express foreign protein, it would induce strong immunity against foreign protein in chickens for a long time. Up to now, there is some preliminary research on FAV vector in foreign countries, while there was little research progress in China. The CMV promoter is used more popularly than the major late promoter of FAV in the construct of recombinant virus, but which one is more efficient to express the foreign gene is unknown.In this study, fowl adenoviruses (FAV) distributions in flocks of chickens, ducks and geese were investigated by PCR. The results show that FAV was extensively popular in chickens, geese and ducks, with the positive rate of 51.4%, 51.7%, and 17.3%, respectively. Three positive samples were selected separately from chickens, ducks and geese, and then the open reading frame 8 (ORF8) and the right terminal of viral genome (ITR), were amplified, sequenced and analyzed. All nine isolates were 94.7%-99.9% homologous to FAVI when compared with that of CELOV, FAVI-JS, but shared low nucleotide sequence homology to FAV-9 and EDSV. They all converged into the same lineage with CELOV, FAVI-JS, while the viruses from group III shared the other one. The sequence of the ORF8 from all the above fowl adenoviruses shared very high homology to each other, which is in accordance with the genomic structural feature of adenovirus. From these results, we can presume that the fowl adenoviruses isolates belong to FAV group I.In order to investigate the possibility of FAV as live vaccine vector, the biological characterization of FAVs isolates were further studied with technology of electron microscope, virus morphology, cytopathogenic effect (CPE) and experiments in ovo and in vivo. The results of these experiments showed that the virus particles are spherical, non-enveloped, and about 70-80nm in diameter and couldn’t agglutinate the erythrocytes of chickens. The virus couldn’t cause CPE in chicken embryo fibroblast cells (CEFs), but cause typical CPE of FAV in chicken embryonic kidney cells (CEKs), which became round with stronger light refraction. No gross lesions and growth retardation was observed in chicken embryo inoculated with the isolates, which are not completely in conformity with the characterization of CELOV (Chicken embryo lethal orphan virus).To evaluate the safety of isolated viruses, SPF chickens were inoculated with the FAVI-G24-0605 at 1-day-old by subcutaneous injection or oral way. The weight of body and some immune organs were measured when a couple of chicken was euthanized at 10d, 20d, and 30d post infection (p.i). The results showed that the inoculated chickens were all healthy with no gross lesions during the animal experiment and there were no serious effects on the body weight of SPF chickens, although the body weight of chickens was lower than the control at 10d p.i and became normal at 20d and 30d p.i. The same changes also occurred in the weight of major immune organs of chickens inoculated. Most of tissue samples had no histopathological changes except some minor lesions at the early stage. These results indicate that the FAVI-G24-0605 strain may be a good candidate for recombinant FAV.The non-essential region for replication of FAVI-JS was determined and the plasmid pFACMV3.1, a universal transfer vector for gene transferred to FAVI-JS, has been constructed by our studying team. On the basis of this research, FAVI-JS was selected from all the fowl adenovirus isolated in our lab for recombination. The major late promoter (MLP) of FAVI-JS, which belongs to group I avian adenovirus, were amplified by PCR and the sequence of MLP has high homology (99%) with that of CELOV. Then the MLP, together with a fragment from pFACMV3.1, containing the R fragment and the large part of L fragment, were cloned into pcDNA3.1/zeo (+). The above large fragment, digested with another two restrict enzymes, was subcloned to pFACMV3.1 to produce pFAMLP3.1, an universal transfer vector controlled by the MLP. By the same way, the ORF of eGFP gene was cloned into pFAMLP3.1 to construct pFAMLP-eGFP as a transfer vector with eGFP controlled by the MLP.CEK was transfected with pFAMLP-eGFP plasmid. Expression of eGFP was found at 48h post transfection. Then CEK infected with wt-FAVI-JS was transfected with pFAMLP-eGFP to produce recombinant virus. The transfected cells and supernatant was freezed, thawed and centrifuged after 3-4d. And the diluted supernatant was re-infected CEK. The expression of eGFP was observed for at least 3 passages. Thus the recombinant fowl adenovirus rFAMLP-eGFP was successfully constructed and provided the basis for study the influence of the promoter on the immune efficiency of FAV.更多还原