【作者】 黄兵；

【导师】 陈溥言；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2005， 博士

【摘要】 腺病毒因其粘膜免疫优势而被应用于病毒载体的研究中。鸭腺病毒1型病毒是产蛋下降综合征病毒(EDSV)或类似该病毒的一类腺病毒，它的宿主范围很广，致病力相差很大。选用无致病力的鸭腺病毒1型病毒作为载体在基因药物上具有很大的潜力。高致病性禽流感病毒(AIV)感染禽群发病急，应用全病毒灭活疫苗刺激产生的抗体极为缓慢，且不利于疫情抗体监测。AIV表面结构蛋白血凝素(HA)和神经氨酸酶决定了疫苗的免疫效力，很显然，禽流感基因工程标记性疫苗的研究是一个很好的方向。本文通过生物学特性和病毒纤突蛋白分析，筛选到一株低致病力的鸭腺病毒1型病毒，通过突变分析确定了其复制非必需区。AIV的HA是病毒重要的保护性抗原，将其作为靶蛋白构建重组禽腺病毒载体疫苗，并利用Cre-loxP系统的位点特异性重组特性将重组病毒基因组中的GFP报告基因自动敲除，最终获得了只含H5血凝素基因表达盒的禽腺病毒重组体疫苗。同时建立了相应的鉴别重组病毒疫苗与野外感染的AIV或引起产蛋下降的EDSV的核酸检测方法。本文研究主要从以下几个方面展开：1．禽腺病毒QU分离株的生物学特性及纤突蛋白分析将鹌鹑源腺病毒QU分离株的纤突蛋白基因进行扩增克隆测序，结果显示与鸡源HS株和AV127株腺病毒的同源性极高。该病毒接种雏鸡未出现临床病症及生长障碍，不致死鸡胚，对鸭胚的致死率明显比引起产蛋下降的HS株低。病毒在鸡胚肝细胞、鸭胚成纤维细胞及鸡胚成纤维细胞上生长良好，且在鸡胚肝细胞上的增殖滴度最高。结果表明该毒株系一株类似产蛋下降综合征病毒的低毒力腺病毒毒株，可以用作禽用基因疫苗或基因治疗的候选病毒载体。2．禽腺病毒QU株基因组突变分析参照鸭腺病毒1型病毒基因组右侧E4区附近序列设计引物，扩增出禽腺病毒QU株DNA的2.7kb片段。将pEGFP-C1质粒上含绿色荧光蛋白基因表达盒的MluⅠ/AseⅠ酶切片段插入QU DNA扩增片段的BglⅡ/StuⅠ酶切位点，构建了含GFP基因的转移质粒载体pADGFP。用脂质体将其与QU株共转染CEF细胞，用96孔板稀释法筛选得到纯化的表达绿色荧光蛋白的重组QU病毒株rQUGFP。该重组病毒的复制效率与亲本毒一致，连续传代后病毒滴度稳定。结果表明，QU株基因组右侧的E4附近区域为病毒的1个复制非必需区。3．禽流感病毒H5血凝素基因检测方法的建立参照AIV的HA基因序列设计1对引物，对H5亚型AIV核酸进行了扩增，产物大小为176bp。经测试，该引物不与新城疫病毒等鸡的其它传染性病原及鸡肌肉组织的核酸发生交叉反应。敏感性分析发现，从50pg的AIV总RNA中亦能扩增到目的条带。此次建立的方法可以方便地用于以后的AIV血凝素重组禽腺病毒构建的筛选纯化，同时，此对引物亦可扩增到H9亚型AIV的血凝素基因的579bp大小的片段，适用于在一次反应中同时将H9和H5亚型AIV进行快速鉴别。4．表达禽流感病毒H5血凝素的禽腺病毒重组体的构建将AIV H5血凝素基因插入鸭腺病毒1型QU株基因组，构建了H5血凝素重组禽腺病毒疫苗。利用pEGFP-C1质粒的CMV启动子和polyA尾信号序列，将禽流感病毒H5血凝素全基因的RT-PCR扩增片段插入其中构建了H5血凝素基因表达盒pHAE。通过PCR延伸获得了一段含双loxP的序列，并将GFP表达盒基因插入其中得到了loxP-GFP-loxP顺序的报告基因pLOXH。将pHAE和pLOXH的表达盒片段串连插入pAD16中，构建了含H5血凝素和GFP表达盒的转移质粒载体pHAGFP。在脂质体介导下将其与病毒DNA共转染CEF细胞，获得了表达H5和GFP的腺病毒重组体rQUHAGFP。通过二次转染，利用Cre-loxP位点特异性重组自动敲除了重组病毒中的GFP基因，最终获得了只含H5血凝素基因表达盒的重组腺病毒rQUHA。试验结果表明，rOUHA的一步生长曲线与亲本毒OU一样，经过连续传代后重组病毒仍然稳定复制并表达HA血凝素。5．禽流感病毒H5血凝素重组禽腺病毒的免疫及监测对AIVH5血凝素重组禽腺病毒疫苗的免疫效力进行了测试。将SPF鸡经口鼻途径分别接种rQUHA重组病毒和QU株病毒，免疫6d后重组病毒免疫鸡群的抗AIV和鸭腺病毒1型病毒抗体开始上升，12d后分别达到5.31og2和7.51og2，此时QU株免疫鸡群的抗腺病毒HI抗体滴度达7.51og2。结果表明，重组病毒可以刺激机体产生HA特异抗体，而自身的HI抗体产生水平未受影响。根据AIV核蛋白基因序列和禽腺病毒的目的基因插入位置的缺失序列设计引物，对鸭腺病毒1型和AIV的特异扩增片段分别为151bp和219bp，建立了AIV的一步法RT-PCR和鸭腺病毒1型的PCR检测方法，利于禽群中使用的重组禽腺病毒疫苗与野外感染的AIV或EDSV快速鉴别。更多还原

【Abstract】 Adenovirus was widely applied into virus vector construction because of its advantageous mucous immunity. Duck adenovirus type 1, including egg drop syndrome virus (EDSV) or EDSV-alike fowl adenovirus, have large range of host, and the virulence of the isolates or strains varied. Then apathogenic duck adenovirus type 1 virus is a promising delivery vector in gene pharmacy for poultry. Genetic marker vaccine against avian influenza virus (AIV) is a trend, because poultry inoculating with-inactivated AIV whole virus vaccine can produce slow antibody and have no help for infection information by surveillance of antibody. The immune efficacy of vaccine are decided mainly by surface glycoprotein of hemagglutinin (HA) and neuraminase of AIV, apparently, genetic marker vaccine is a potential one for control AIV. Now, in this paper, one low virulent duck adenovirus type 1 virus strain was screened through pathogenicity analysis and fiber protein gene sequencing, and then its non-essential region for replication was decided by genome mutation assay. The recombinant adenovirus vector was constructed with insertion of the protective antigen Hemagglutinin gene of AIV subtype H5 and the enhance green fluorescence protein gene. The recombinant vaccine only expressing H5 HA of AIV was obtained by site-specific recombination of Cre-loxP system to self-excise the GFP reporter gene in recombinant virus genome. In addition, the nucleotide detection methods were developed for distinction of the recombinant vaccine from field infection of AIV or EDSV causing egg drop.These studies include:1.The biological feature and fiber protein analysis of fowl adenovirus QUstrainThe fiber protein gene of quail-original adenovirus QU strain was amplified by polyerase chain reaction and sequenced, the result showed that the target fragment is highly homologous with those of chicken original HS strain and AV127 strain. The young specific-pathogen-free chicken, oro-nasally inoculated with the QU strain, presented no clinical sign and growth withdrawing, no lethal to chicken embryo and lower motility rate to duck embryo than HS strain that causes egg drop of laying chickens. The QU strain adapted to chicken embryos liver cell, duck embryos fibroblast cell and chicken embryos fibroblast cell, and the virus titer was highest in chicken embryos liver cell. Above data suggested that the QU strain was apathogenic adenovirus in chicken and some characteristic of the virus assembles egg drop syndrome virus. So, the fowl adenovirus virus may be a candidate vector for poultry gene engineering vaccine or gene therapy.2. Mutation analysis of genome of fowl adenovirus QU strainThe primers were designed for amplification of 2.7kb of fragment on the basis of E4-closly region sequence of duck adenovirus type 1 genome, pADGFP was constructed, in which enhance green fluorescence protein gene cassette of pEGFP-C1 plasmid digested with mluⅠand AseⅠcutter, and inserted into the amplified fragment through the BglⅡand StuⅠendonuclease. The rQUGFP recombinant virus was gained by co-transfection of QU virus and pADGFP plasmid. The one step growth curve of the rQUGFP virus was identical with that of parent QU strain and the titers of serially-passenging recombinant virus were stable. These data suggested that the E4 nearby region of QU strain genome is nonessential for virus replication, and the inserted foreign gene into virus genome could be expressed efficiently.3. Development of Detection Method for Hemagglutinin Gene of Avian Influenza Virus Subtype H5One pair of primers were designed on the basis of hemagglutinin (HA) gene of avian influenza virus(AIV), allowing detection of AIV subtype H5, and the specific amplicon was 176bp in length. The primers were tested to be specific for AIV with no cross-reaction to RNA from the chicken muscle and other chicken pathogens such as Newcastle disease virus etc. The sensitivity of this assay was about 50pg of total RNA of AIV subtype H5. Our data showed that the establishment of this method was simple and feasible for screening and purification of the following AIV HA-expressing recombinant fowl adenovirus vaccine. In addition, the primer was specific for 579bp of fragment of AIV subtype H9,and available for simultaneous subtyping these two subtype AIV by one-step reverse transcription-polyerase chain reaction.4. Construction of recombinant fowl aflenovirus expressing hemagglutinin of avian influenza virus subtype H5The amplicon of 2.7kb in length was cloned as pAD16 according to E4-close region of duck adenovirus type 1 QU strain genome. The pHAE plasmid was constructed, the hemagglutinin gene of avian influenza virus type H5 was amplified by RT-PCR and inserted in pEGFP-C1 for its CMV promoter and polyA singal tail. The loxP-GFP-loxP fragment from ploxH, which constructed through GFP cassette gent inserted the double loxP sequence using PCR extended, and the H5 hemagglutinin cassette gene from pHAE plasmid were inserted in pAD16 plasmid, the delivery vector pHAGFP was constructed. The redcombinant virus rQUHAGFP was gained through co-trnasfection of pHAGFP with genome DNA of QU strain. After second co-transfeetion of Cre recombinase plasmid with rQUHAGFP, the GFP reporter gene in the recombinant virus genome was removed self-excisely and the recombinant rQUHA virus only involving H5 hemagglutinin gene cassette was obtained finally. The resulted showed that the recombinant virus was stable for replication by one step growth curve test and. also serially passenging recombinant vaccine can consistently expressed the H5 hemagglutinin.5. Immunization and surveillance of avian influenza virus H5 hemagglutinin-expressing recombinant fowl adenovirusThe immune efficacy of a recombinant fowl adenovirus vaccine expressing H5 bemagglutinin(HA) of avian influenza virus was confirmed. The specific pathogen flee chickens were inoculated oro-nasally with recombinant rQUHA virus and fowl adenovirus QU strain. The antibodies against H5 HA of avian influenza virus and duck adenovirus virus increased six days after immunization and the antibodies arrived at the level of 5.31og2 and 7.5log2 respectively after 12 days. At the time the antibodies to QU strain were 7.53log2 HI titer. The results demonstrated that the recombinant vaccine might activate chicken to produce antibodies against H5 HA as well as QU virus antigen. According to the nucleoprotein gene of avian influenza virus and the deletion region of duck adenovirus type 1 virus genome through insertion of target gene, two oligonucleotide primers were designed, the 219bp of fragment specific for AIV and 151 bp for duck adenovirus type 1. The development of detection method of both one step RT-PCR for AIV and PCR for duck adenovirus type 1 were useful for differentiation of the recombinant vaccine from field egg drop syndrome virus or avian influenza virus infection in poultry.更多还原