【作者】 王泽；

【导师】 丁壮；

【作者基本信息】 吉林大学 ， 预防兽医学， 2013， 博士

【摘要】 自1997年以来，在我国多数地区的鹅群中相继爆发和流行鹅副黏病毒病，经鉴定病原为血清Ⅰ型副黏病毒，即新城疫病毒，该病定名为鹅副黏病毒病，又可称为鹅新城疫。各种年龄的鹅对该病均易感，鹅群发病率平均为27.67%，死亡率个别可高达100%，平均死亡率为18.19%，且年龄越小其发病率和死亡率越高，给我国的养鹅业造成严重的经济损失。目前，鹅副黏病毒病防治主要以疫苗免疫为主，尽管目前有多种强毒疫苗和灭活疫苗用于免疫预防，但是仍然不能控制鹅副黏病毒病的发生。目前研究结果均表明，利用新城疫病毒强毒苗或灭活苗甚至是传统的LaSota弱毒疫苗来免疫预防鹅副黏病毒病均不能起到完全的免疫保护作用。因此，对于鹅副黏病毒病我们须寻求一种新的预防思路和技术手段。随着分子生物学及其技术方法的不断发展，基因阻断技术也得到了极大的发展，在哺乳动物或其他动物细胞中通过siRNA诱导RNAi效应来实现特定基因的有效抑制，该方法已经成为研究基因功能的重要工具。慢病毒载体介导的基因转导是近年来发展起来的技术，其突出的特点是具有较高的转染效率、能感染静止期细胞和较高的目的基因表达效率。本研究利用具有基因组整合特性和高侵染能力的慢病毒载体介导RNA干扰技术，研究其对鹅源新城疫病毒NA-1株在体外和体内复制增殖能力的影响，进而为禽类抗病毒研究奠定基础。首先针对鹅源新城疫病毒NA-1株NP、M基因保守区序列，分别设计3对shRNA，同时设计1对阴性对照shRNA序列，合成后退火形成双链，连接到双酶切线性化的带有pol Ⅲ U6启动子的慢病毒shRNA表达载体（pLKD-GFP），分别命名为：pLKD-shNP268、pLKD-shNP437、pLKD-shNP710、pLKD-shM168、pLKD-shM646、pLKD-shM1062及pLKD-control阴性对照，上述重组慢病毒干扰质粒经PCR及测序鉴定确认。将构建的不同重组慢病毒干扰质粒瞬时转染Vero细胞，48h后接种1000个TCID50/0.1mL的鹅源新城疫病毒NA-1株，分别于病毒感染的12h、24h、36h、48h，应用Real-time RT-PCR方法检测病毒基因mRNA表达量的变化，结果显示，重组慢病毒干扰质粒pLKD-shNP710及pLKD-shM646对病毒基因mRNA表达的抑制率分别为85.8%和84.6%；进一步测定TCID50/0.1mL，观察病毒滴度的变化，结果显示，转染重组慢病毒干扰质粒pLKD-shNP710，pLKD-shM646组其病毒的TCID50/0.1mL分别为10-4.16/0.1mL和10-5.15/0.1mL；应用间接免疫细胞化学方法进一步检测各干扰组及对照组中病毒在细胞中的复制增殖的情况，结果也显示出，转染重组慢病毒干扰质粒pLKD-shNP710，pLKD-shM646组较好的抑制病毒在细胞中复制，重复三次试验均获得一致结果。将重组慢病毒干扰质粒pLKD-shNP710，pLKD-shM646及阴性对照pLKD-control及pLKD-GFP分别与慢病毒包装系统的2个辅助质粒（pR8.2，pVSV-G）共转染293T细胞进行重组慢病毒假病毒的包装，分别于24h、48h收集培养上清，4000r/min离心10min，以去除细胞碎片，然后经0.45μm一次性滤器过滤，4℃50,000g离心2h，以获得浓缩的重组慢病毒，浓缩后的重组慢病毒应用Lastra法测定滴度，滴度为5×107TU/mL。将重组慢病毒分别命名为：LV-NP710、LV-M646、LV-control及LV-GFP。将获得的浓缩的重组慢病毒侵染Vero细胞（培养液中加入终浓度为8μg/mL polybrene，以提高包装的病毒对细胞的侵染能力），经嘌呤霉素抗性筛选，获得具有抗性的细胞，然后将细胞进行传代，传至第20代，并且取第5、10、15、20代的细胞提取基因组，应用PCR和DNA测序方法检测shRNA序列是否整合到细胞基因组DNA中，结果显示，每代细胞基因组DNA中均存在shRNA序列，且在细胞内绿色荧光蛋白呈现较高水平表达，证明成功建立了稳定表达shRNA的细胞系，将其分别命名为：NP710-Vero、M646-Vero、control-Vero及GFP-Vero。将稳定表达shRNA的Vero细胞系感染鹅源新城疫病毒NA-1株（1000个TCID50/0.1mL），应用Real time RT-PCR方法，对病毒感染12h、24h、36h、48h后，各细胞系干扰NDV基因mRNA表达情况进行检测，结果显示，对NP、M基因的抑制率分别为NP710-Vero组（86.8±0.35）%（P＜0.05），M646-Vero组（85.9±0.26）%（P＜0.05）；应用TCID50方法对病毒感染48h后，病毒滴度变化进行检测，结果显示，NP710-Vero细胞组TCID50为10-4.21/0.1mL，M646-Vero细胞组TCID50为10-5.17/0.1mL；应用间接免疫细胞化学方法检测病毒在各组细胞系中复制情况，结果表明，可稳定表达shRNA的细胞系（NP710-Vero组、M646-Vero组）均能明显抑制病毒在细胞中复制增殖。上述结果说明，稳定表达shRNA序列的Vero细胞系建立成功，并且对NDV的复制产生持续的、稳定的抑制作用。本研究进一步将重组慢病毒液（滴度为106TU/mL）注射入新鲜的鹅受精卵胚下腔制备可表达shRNA序列的嵌合体鹅胚，用经灭菌的附带蛋壳膜的合适大小的蛋壳封口，医用胶布固定，然后按标准鹅胚孵化程序进行孵化。提取胚胎基因组DNA，通过PCR鉴定，在检测的鹅胚胎基因组中均有shRNA序列和GFP基因的插入，说明获得的鹅胚为嵌合体胚胎。更多还原

【Abstract】 Since1997, Goose paramyxovirus disease had broken out and spread in themajority of our region, and was identified that the pathogen was paramyxovirusserotypeⅠ, the disease was named Goose paramyxovirus disease, also maybe calledNewcastle disease of goose. All ages goose are susceptible to the disease, the gaggleincidence rate of27.67%on average, the mortality rate can be as high as100%individual, an average rate of18.19%. This disease cause serious economic losses tothe goose industry in China. At present, the prevention of Goose paramyxovirus diseasewas mainly injected vaccine, although a variety of the attenuated vaccine andinactivated vaccine for immunization prevention, but still can not control theoccurrence of Goose paramyxovirus disease. The results showed that Newcastledisease virus virulent vaccine or inactivated vaccine even traditional LaSotaattenuated vaccine to prevent Goose Newcastle disease virus disease also can not playcompletely immune protective effect. Therefore, we need to find a new preventionideas and technical means for Goose Newcastle disease virus.With the development of molecular biology and its technical methods, the geneblocking technology has also been a great deal of development. The siRNA inducedRNAi effect in mammalian or other animal cells, to achieve effective inhibition ofspecific genes, this method has become an important tool for studies of gene function.Lentiviral vector-mediated gene transduction is a new technology, its advantages werethe high efficiency of transfection and can effectively infect quiescent cells andexpress the target gene. In this study, the use of genomic integration features and highinfection capacity of lentiviral vector and mediated RNA interference technology,research the effect of proliferative capacity of NA-1strains of Goose Newcastledisease virus in vitro and in vivo, and thus established the foundation for anti-virus research of poultry.First, three pairs shRNA sequences were designed for the conserved regionsequences of NP, M gene of Goose Newcastle disease virus NA-1strain respectivelywhile a pair negative control shRNA sequence was designed, then synthesized andannealing to form double-stranded, and connected to the lentivirus vector(pLKD-GFP)with Pol Ⅲ U6promoter which was linearized by double digestion. The recombinantvectors were named pLKD-shNP268, pLKD-shNP437, pLKD-shNP710,pLKD-shM168, pLKD-shM646, pLKD-shM1062and pLKD-control (negative)respectively. These recombinant lentiviral vector plasmid were confirmed by PCR andsequencing. The different recombinant lentivirus vector plasmids were transientlytransfected Vero cells, after48h, the cells were super-infected the NA-1strain ofGoose Newcastle disease virus. To determine whether NP and M gene expression ofthe NDV strain NA-1could be inhibited by lentivirus vector-mediated siRNA, therelative expression levels of the NP and M genes were evaluated by real-time RT-PCR.The results of the real-time RT-PCR indicated a significant inhibitory effect in therelative expression level of NP and M gene transcripts by85.8%and84.6%,respectively, when plasmids pLKD-shNP710and pLKD-M646were used, ascompared to the mock and pLKD-control transfected cells at48h p.i. PlasmidspLKD-shNP268and pLKD-shNP437down-regulated mRNA expression levels of theNP gene by55.6%and64.2%, while pLKD-shM168and pLKD-shM1062down-regulated mRNA expression levels of the M gene by56.5%and69.0%,respectively. To demonstrate the inhibitory effect of the different recombinantinterference plasmids further, the TCID50assay was used to titrate the NA-1virus inthe cell supernatants at48h p.i. The results show that the TCID50was10-4.16/0.1mLand10-5.15/0.1mL when cells were transfected with pLKD-shNP710and pLKD-M646,respectively. These values indicate significant viral titer reduction when compared tothe mock group, which had a TCID50/0.1mL of10-9.37/0.1mL. The TCID50values forthe other plasmids were as follows:10-7.83/0.1mL for pLKD-shNP268,10-7.12/0.1mLfor pLKD-shNP437,10-6.97/0.1mL for pLKD-shM168, and10-7.1/0.1mL forpLKD-shM1062. We next used an indirect immunocytochemistry assay to detect theexpression of NA-1in Vero cells. The results indicated that the expression of NA-1 was significantly lower in cells transfected with pLKD-shNP710and pLKD-M646than in the Mock Vero and pLKD-control groups. Indeed, the replication of NA-1wassignificantly inhibited in vitro.Based on the promising results above, we generated recombinant lentivirus thatcontained a U6Pol III promoter and the NP and M gene siRNA cassette targeting the710-731nt (NP gene) and646-667nt (M gene) regions. The recombinant-defectivepseudotype lentiviruses carried an EGFP reporter gene for quantification by FACSanalysis. These lentiviruses containing the NP and M gene siRNAs were titrated inVero cells, which were found to contain5×107TU/mL. We transduced Vero cells withrecombinant lentivirus stocks LV-NP710, LV-M646, LV-GFP, LV-control andscreening them by puromycin, then we can obtain the resistant cells. These resistantcells were passaged to the20th generations. And extracted the genomic of5th,10th,15th,20thgeneration cell and detected the shRNA sequences by PCR and DNAsequencing methods, the results show that each generation of the cell genomecontaining the shRNA and GFP gene sequences, and the green fluorescent proteinexpression to a higher level. These results proved that it is successful to establish astable cell lines, the cell lines were named that NP710-Vero, M646-Vero, GFP-Veroand control-Vero.The different Vero cell lines which can stably expressing shRNA were infectedwith NA-1strain of Goose Newcastle disease virus (1000TCID50/0.1mL). The relativeexpression levels of the NP and M genes were evaluated by real-time RT-PCR. Theresults of the real-time RT-PCR indicated a significant inhibitory effect in the relativeexpression level of NP and M gene transcripts by86.8%and85.9%, respectively inNP710-Vero and M646-Vero cell lines. TCID50assay was used to titrate the NA-1virus in the cell supernatants at48h p.i. The results showed that the TCID50/0.1mLvalues were10-4.21/0.1mL and10-5.17/0.1mL when cells were transduced withLV-NP710and LV-M646, respectively. The mock Vero TCID50/0.1mL was10-9.43/0.1mL, and the LV-control TCID50/0.1mL was10-9.18/0.1mL. The results were similar tothe results of Vero cell transfected the recombinant lentivirus plasmids. We also nextused an indirect immunocytochemistry assay to detect the expression of NA-1indifferent stable Vero cell lines. The results indicated that the expression of NA-1was significantly lower in cells transduced with LV-NP710and LV-M646than in the MockVero and LV-control groups. Indeed, the replication of NA-1was significantlyinhibited in vitro.To further research the inhibitive effect of recombinant lentivirus expressingshRNA for Goose Newcastle disease virus in vivo, we take the fresh goose fertilizedegg, recombinant lentivirus solution (titer to106TU/mL) injected into the fresh goosefertilized egg embryo inferior vena, and capped with sterilized the eggshell with shellmembrane, and fixed with medical tape, and then hatched the embryo in accordancewith the standard incubator program. Extracted the genomic DNA of embryomic, andidentified the integration of the shRNA sequence, the results showed that the embryois a chimera embryo.更多还原