【作者】 王海燕；

【导师】 赵振华； 曹殿军；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2001， 硕士

【摘要】 将NDV F₄₈E₉株接种鸡胚进行病毒增殖，并对发病鸡胚作病理学观察，可见病胚发育迟滞、胚体弱小、体表潮红、多处有出血点，胃、肠、肝、脑、肾、心、骨骼肌等重要器官出现不同程度的充血、淤血、水肿、出血和实质细胞的变质性变化，所收集的病胚的尿囊液纯化后经电镜观察可见NDV粒子。 根据NDV P蛋白和M蛋白已知基因核苷酸序列设计并合成了两对引物PU1/PL1和PU2/PL2。提取F₄₈E₉株RNA，以RT-PCR法扩增出大小约1.1kb和1.6kb的目的基因片段，两者之间约有100bp左右的基因重复，将这两个片段分别克隆到pMD18-T载体上，对阳性重组子经酶切分析、PCR鉴定及序列测定证明是正确的。根据测序结果，再利用限制性内切酶EcoRⅠ、HindⅢ、MseⅠ对两个重组质粒分别进行了酶切处理并回收目的片段进行连接，所得的阳性重组质粒经酶切鉴定后再进行序列测定，结果证实我们得到了NDV F₄₈E₉株P基因完整序列的克隆。通过序列分析发现F₄₈E₉株P蛋白基因核苷酸序列与La Sota株的P蛋白基因核苷酸同一性为87％，氨基酸同源性为91％，表明F₄₈E₉株与La Sota株有一定亲缘关系。同时对其编码氨基酸的抗原性值分析，表明在20-28、113-122、190-200、220-230位氨基酸等四个区域存在抗原表位。 根据NDV F₄₈E₉株P基因核苷酸序列和杆状病毒转移载体pF_ASTB_AC1多角体蛋白基因阅读框架，将重组质粒pMD18-T-P经HindⅢ作部分酶切，再经EcoRⅠ酶切，然后与用EcoRⅠ/HindⅢ双酶切的杆状病毒转移载体pF_ASTB_AC1相连接，使P蛋白基因克隆到pF_ASTB_AC1的多角体启动子下游。经酶切分析、PCR鉴定及序列测定，证实获得了含P蛋白基因的转移载体pF_ASTB_AC1-P。然后将该转移载体转化到DH_10BAC感受态细胞中，经蓝白斑筛选、挑菌、质粒提取，通过杆状病毒多角体蛋白基因通用引物及P基因特异引物进行PCR鉴定，证实得到了重组杆状病毒DNA。将此重组组杆状病毒DNA转染sf9昆虫细胞，于感染后72小时收获重组病毒粒子。再将重组病毒感染对数生长期的sf9昆虫细胞，于感染后24、48、72、96小时收获感染细胞，经SDS-PAGE电泳分析，证明NDV F₄₈E₉株P蛋白基因在重组杆状病毒系统中获得了表达，其分子量大小约为56kD。更多还原

【Abstract】 Chick embryos were inoculated with NDV F43E9 strain and the pathomorphological changes of the sick embryos were examined. The results showed all the sick embryos were hypoevolutism, weak, flush and there were many hemorrhagic spot in the surfaces of the sick body, Hyperemia, congestion, edema, hemorrhage and parenchymatous degeneration were found in stomach, intestine,liver, brain, kidney, heart and skeletal muscles, the NDV particle was observed though electron microscope in the purificated allantoic liquid of sick embryos. Two pairs of oligonucleotid primers PU1/PLI and PU2/PL2 were designed according to the published P protein and M protein gene sequences of NDV.Two fragment of 1.1kb and 1.6kb were amplified by RT-PCR from RNA of F48E9 strain,and cloned into pMD18-T vector respectively. The recombinant plasmids were digested by restriction enzyme EcoRI, HindIII and MseI,and subcloned into pMD18-T vector again, as a result, we obtained complete P protein gene clone of NDV F48E9 strain. It was found that the P has 87% and 91% homologies with La sota in nudeotide and amine acide level ,respectively.It was concluded that strain F48E9 is genetically closer to strain La sota There were four epitopes across the squence of the P,they are 20-28,113-112,190-200 and 220-230 amine acid residues. The recombinant plasmid pMD1S-T-P was digested with EcoRI/HindIII and subcloned into PFASTBAC1 digested with EcoRI/HindIII. The 時esulting recombinant PFASTBACI-P contains P gene at downstream of the polybedrine gene initiatioin codon. The recombinant PFASTBAC1-P was transposed into the DH1OBAc competent cell and the recombinant bacmid DNA was isolated and subjected to identification by PeR. The recombinant baculoviruses were obtained by transfection of sf9 cells with the the recombinant bacmid DNA and CELLFECTIN. The infected insect cells were collected at different time post-infection, analysed by SDS-PAGE. The results showed that the expressed product had a molecular mass of approximately 56kD.更多还原