【作者】 陈君彦；

【导师】 申之义； 李海燕； 关平原；

【作者基本信息】 内蒙古农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 本论文对2001年中国华南及东北地区的15株H1N1亚型猪流感病毒（SIV）分离株进行了全基因序列测定，并对15株病毒全基因组（8个基因）的核苷酸序列和推导的氨基酸序列的同源性进行了分析和比较，同时应用遗传进化树理论对各基因的来源及其分子遗传衍化规律进行研究。本实验所用15株H1N1亚型SIV分离株均在SPF鸡胚中扩增培养，从病毒感染的鸡胚尿囊液中直接提取核酸。应用Oligo（6.0版本）引物设计分析软件设计H1N1亚型SIV 所有8个基因片段的特异性PCR引物，通过反转录－聚合酶链式反应（RT-PCR）方法扩增15个毒株各基因片段的cDNA，PCR产物经分离纯化后克隆到pMD 18-T载体中，重组质粒经PCR鉴定纯化后在DNA序列分析仪上进行序列测定，然后应用DNAstar软件进行序列的拼接、序列信息编辑和分析。同时从GenBank中下载了H1N1亚型猪流感古典型代表株和类禽型代表株、人(类猪) 流感代表株以及禽流感代表株，分析不同毒株各基因同源性，绘制进化树，以阐明中国内地H1N1亚型SIV分离株的基因来源和遗传衍化规律。研究结果发现，15株H1N1亚型SIV分离株中，大部分毒株（11株）的8个基因皆属于古典型H1N1猪谱系，而且与人流感病毒的进化关系密切。SW/GD/714/01和SW/GD/711/01的NA基因属于类禽型H1N1谱系，共同起源于禽H1N1流感病毒，而其他基因（HA和内部基因）均属于古典型H1N1猪谱系，说明这2株病毒是古典型H1N1 SIV和类禽型H1N1 SIV的重组病毒。经分析还发现，SW/GD/718/01的PB1基因也同样来源于禽流感病毒。另一个有意义的发现是，黑龙江分离株SW/HLJ/395/01的内部基因均与类禽型H1N1亚型代表株(SW/GER/2/81)高度同源，都属于类禽型H1N1谱系，而其HA和NA基因属于古典型H1N1谱系，是一株由古典型H1N1亚型SIV的内部基因和类禽型H1N1亚型SIV的外部基因进行重组的病毒。总之，本研究系统地阐明了目前中国内地H1N1亚型SIV分离株的基因来源和分子遗传衍化情况，为今后H1N1亚型SIV的进一步研究和猪流感的防制奠定了基础，为中国乃至全球（人）流感疫情发生的可能性和发展趋势提供一定的理论依据。更多还原

【Abstract】 Fifteen H1N1 subtype Swine Influenza viruses (SIVs) isolated from the mainland of China were sequenced and molecular evolutionarily analyzed in this study. These viruses in 2001 were isolated from swine in south and northeast of China.The nucleic acids of the all swine influenza viruses conducted in the research were extracted from the viruses propagated in specific-pathogen-free chicken embryos. All of the eight segments were amplified by RT-PCR (Reverse transcription-polymerase chain reaction). The cDNAs were cloned into pMD18-T vector and identified. The sequencing products were purified and run gel on CEQTM 8000 Genetic Analysis system DNA sequencing machine. Finally, using DNAStar software was used for data analysis, sequence translation, phylogenetic tree drawing and molecular evolution analysis.The phylogenetic trees revealed that all of the genes of eleven of the 15 H1N1 SIV isolates conducted in this study belonged to classical swine lineage. Furthermore, they were closely related to human strains. The result of this investigation displayed that two H1N1 SIV isolates SW/GD/714/01 and SW/GD/711/01 were possibly reassortants of classical swine H1N1 subtype and avian virus. The neuraminidase (NA) genes of two isolates were of avian origin, while the hemagglutinin (HA) and internal genes of them were of swine origin. However, PB1 genes of one isolate (SW/GD/718/01) were originated from avian influenza A virus lineage.The research showed that SW/HLJ/395/01 isolated from HeiLongjiang was reassortant of classical swine H1N1 and avianlike H1N1 viruses. The hemagglutinin (HA) and neuraminidase (NA) genes belonged to classical H1N1 subtype swine lineage, while the internal genes were located in avianlike swine lineage. In conclusion, this study elucidated the molecular evolution of H1N1 SIVs isolated from the mainland of China, clarified the category of the recent epidemical strains of H1N1 SIVs circulating in the mainland of China, and made a foundation for the further investigation. Of particular significance, these would provide the theory support for the possibility and development trend of influenza occurring in China even the whole globe.更多还原