【作者】 藏玉婷；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 鹅副粘病毒(Goose Paramyxovirus，GPMV)与新城疫病毒(New castle disease virus，NDV)基因核苷酸同源性来看，GPMV与NDV属于同种病毒，GPMV是NDV的一个变异，而不是一个新种病毒，是NDV毒株在水禽上引起发病，且病毒毒力加强。但是，GPMV与传统的NDV又有不同，差异主要在血凝素-神经氨酸酶蛋白(HN)及融合蛋白(Fusion protein，F)上，常规的NDV疫苗不能提供有效的保护，因此从分子生物学水平上对该病进行研究具有重要的意义。HN和F蛋白是GPMV诱导体液免疫的重要靶抗原，在抗病毒免疫中起着重要作用，无疑HN和F基因已成为研制GPMV基因工程苗及免疫监测试剂的首选基因。本试验根据GenBank所发表的HN及F基因的cDNA序列，利用Primer5.0软件，参照昆虫细胞杆状病毒表达系统的质粒图谱，设计引物，以本实验室所保存的pMD18-T-HN及pMD18-T-F阳性质粒为模板扩增出HN及F基因，分别命名为pMD18-T-GPMV-HN及pMD18-T-GPMV-F。将其克隆至昆虫杆状病毒转移载体pBlueBacHis2A的XhoⅠ和EcoRⅠ之间的多克隆位点上，分别命名为pBlue-GPMV-HN及pBlue-GPMV-F。纯化该重组质粒并与杆状病毒共转染Sf9昆虫细胞，4d后获得重组病毒，名为P1。将经过3轮蚀斑筛选纯化，PCR鉴定获得重组病毒，分别命名为rBv-HN及rBv-F。将重组病毒反复扩毒3次，获得高浓度的重组病毒名为P3，接种Sf9单层细胞上，4d后收获病变细胞，经SDS-PAGE电泳鉴定，表达F蛋白分子量约为60ku，表达HN蛋白分子量约为66ku，与理论值相符。表达产物HN蛋白的Western blot和Dot-ELISA结果表明其可与鸡抗GPMV血清发生特异性抗原反应，证实所表达的HN蛋白有较好的反应原性。本研究将株的HN及F基因利用昆虫杆状病毒表达系统中表达，并获得了HN主要抗原位点基因的表达，对GPMV的基础性研究、预防控制和病毒诊断试剂研制奠定重要的物质基础。更多还原

【Abstract】 Goose Paramyxovirus(GPMV)is homologous with New castle disease virus(NDV) at a greatsignificance, GPMV was revealed to be a variant of NDV but not a new virus. One strain of NDVinfect waterfowl and the virulence was enhanced. However, GPMV was not absolutely differentwith NDV, hemagglutinin-neuraminidase(HN) and fusion protein(F)protein were mutant and resultin the invalidation of traditional vaccine. Therefore, the study on the virus at molecular degree wasof great significance. HN and F proteins were the primary target antigens of humoral immuneresponse against virus. HN and F proteins have become the preferred genes for production ofgenetically engineering vaccine and pathogen diagnosis.In this research, one set of primers was designed based on the sequence of HN and F gene ofGPMV of GenBank and the sequence of eukaryotic transfer vector pBlueBachis2A using primer 5.0. HN and F gene was amplified from pMD18-T-HN and pMD18-T-F respectively and designatedas pMD18-T-GPMV-HN and pMD18-T-GPMV-F. They were ligated into the transfer vectorpBlueBachis2A with XhoⅠand EcoRⅠrestriction sites, and the recombinant plasmids weredesignated as pBlue-GPMV-HN and pBlue-GPMV-F. Co-transfection of Sf9 insect cell wasperformed using purified recombinant plasmids and Bacmid-N-blue DNA. Recombiant virusnamed P1 was recovered 4d after transfection. After 3 serial passges of plaque assay screenfollowed by PCR confirmation, the recombinant virus were designated as rBv-HN and rBv-F. After3 passages of propagation of the recombinant virus, high-titer virus named P3 was used to inoculateSf9 cells followed by harvest after 4d. The F and HN proteins were confirmed to be 60ku and 66kuin length which were consistent with the speulated size as judged by SDS-PAGE seperation. HNprotein could be specifically recognized by chicken-anti-GPMV antibodies as investigated byWestern blot and Dot-ELISA assays. All the results proved the better reactionogenicity of HNprotein.In this research, HN and F proteins of GPMV/JS/1/97/Go strain were expressed withbaculovirus expression system. The expression of the antigenic sites of HN protein wasconfirmed. The results of this study provided data for fundamental research on GPMV, preventivecontrol and virus diagnosis.更多还原