【作者】 李德生；

【导师】 崔恒敏； 张和民；

【作者基本信息】 四川农业大学 ， 基础兽医学， 2012， 博士

【摘要】 流感病毒(Influenza virus)属于正黏病毒科流感病毒属成员,可分甲、乙、丙三个型,其基因组由分节段单股负链的RNA组成。流感病毒广泛流行于世界各国,对养殖业造成了巨大经济损失的同时,对人类的健康也构成了潜在的威胁,因此,对流感病毒的研究在公共卫生上也有相当重要的意义。对此,本研究通过鸡胚接种、MDCK细胞培养、血凝及血凝抑制实验和RT-PCR等方法,首次从大熊猫鼻腔分泌物中分离并鉴定了一株既能在鸡胚上稳定传代又能在MDCK细胞上稳定产生细胞病变,即以细胞颗粒增多、肿胀、漂落为特征的大熊猫HINI亚型流感病毒,命名为A/Panda/Sichuan/01/2011(H1N1)。大熊猫流感病毒A/Panda/Sichuan/01/2011(H1N1)全基因克隆和序列分析本文根据GenBank报道的流感病毒H1N1亚型基因序列,对分离的流感病毒A/Panda/Sichuan/01/2011(H1N1)八个基因片段设计了包括全部基因组的8对引物,通过RT-PCR的方法扩增病毒全基因序列,扩增的PCR产物连接pMD18-T载体,转化DH5a感受态细胞,经质粒PCR鉴定出阳性克隆菌后送上海生工公司测序。测序结果通过GenBank的BLAST比对分析,同时使用MEGA5等生物信息学软件对测序结果进行序列同源性分析,并在此基础上建立了分子进化的系统发育树。测序结果发现,流感病毒A/Panda/Sichuan/01/2011(H1N1)的八个节段全基因组共13595bp,其中3’与5’末端有一段较短的保守序列；通过全基因组序列分析,发现本株流感病毒与2009H1N1流感病毒同源性较高,核苷酸序列同源性高达99%,结果表明,A/Panda/Si chuan/01/2011(H1N1)来源于2009H1N1人流感病毒。大熊猫流感病毒A/Panda/Sichuan/01/2011(H1N1)反向遗传技术的建立本文在大熊猫流感病毒A/Panda/Sichuan/01/2011(H1N1)全基因序列测序的基础上,将三个分段扩增的聚合酶基因连接成完整的基因,并根据序列信息设计八对包含全部完整基因的引物,同时引入酶切位点BspQI,通过PCR扩增出八个完整的基因,将PCR产物回收并磷酸化后连接pMD18-T,转化DH5a感受态细胞,挑选阳性菌落抽质粒酶切,回收酶切产物；同时使用BspQI对表达与转录载体PBD进行酶切。将二者电泳回收后使用T4DNA酶进行连接反应,将连接产物转化DH5a感受态细胞,挑选阳性克隆菌抽质粒酶切鉴定后,送上海生工公司测序并进行序列分析。酶切结果显示,在pBD载体中正确插入八个流感病毒基因片段；测序结果表明本次八质粒共转染系统的构建是成功的。将构建的八质粒共转染系统质粒重组菌扩大培养,进行质粒抽提并进行纯化和浓度的检测；同时在细胞瓶里培养293T细胞和MDCK细胞；将所得到的八个重组质粒通过共转染的方法转染到293T细胞,培养48h后收冻293T细胞培养物,将转染产物接种MDCK细胞扩大培养,待细胞产生明显的细胞病变后收冻细胞培养物。将MDCK细胞培养物的上清接种9日龄SPF鸡胚,72h后取尿囊液进行HA和HI实验,将呈阳性的样品通过电镜负染的方法观察病毒粒子。HA和Hl实验结果表明,转染产物在鸡胚尿囊液里得到增殖,产生了重组的病毒离子,通过电镜观察到流感病毒粒子存在。一系列的实验证明,本次反向遗传操作获得成功,为今后流感病毒的研究乃至这类病毒的研究提供了一个优越的操作平台。M2和HA基因融合表达重组质粒免疫效力研究将M2的C端与HA基因融合构建真核表达重组质粒pM2HA,该质粒可在细胞内转录表达；pM2HA免疫小鼠后,可以检测到M2特异性抗体,HI抗体效价与pCI-HA组差异不显著,而中和抗体稍高于后者但差异不显著;小鼠经致死剂量病毒攻击后,pM2HA组实验小鼠体重轻微下降,攻毒保护率达100%,在遭受中等剂量攻毒后能减轻肺脏病理损害程度；同时对于不同亚型的流感病毒也有一定免疫保护力。M2e与HA的联合应用为新型流感疫苗的研究提供了一条新的思路。更多还原

【Abstract】 Influenza virus is a member of the genus Orthomyxovirus, family Orthomyxoviridae, which divided three types from the difference of protein M and NP, and influenza virus contains eight piece of negative sense single-stranded RNA. Currently, influenza virus circulate and be epidemic in different animal populations throughout of the world countries, and do great damage to the farms, and also threat the human health at all the time. So it is very important to do deeply research in the influenza virus. In order to do that, the author collected some samples of the Pandas and firstly, we isolated and identificated a strain Panda influenza virus which can be cultured stably both in MDCK cells and9-11day eggs by HA, HI and RT-PCR assays from a Sichuan pig samples, and the virus was named A/Panda/Sichuan/0]/20]1(H1N1).Influenza A virus is one of negative sense single-stranded RNA, and its genome is composed of eight solo genes. PB1. PB2and PA are coded by fragments1,2and three genes, and their function is about the virus genome synthesis when the virus infects the cells. HA and NA protein are the most important surface protein, and concerned with the infecting cells and the antigencity of the virus. NP protein is the cod of the genome, and composed of vRNPs with the PB2, PB1and PA protein. M1and M2protein are the peplos of the genome, and it is one of the evidence of the type of the virus. NS protein is the non-construction protein of the virus, and also, it is relation to the early synthesis, but now, human are not very clear about it’function. The each gene5’end and3’end of the virus is very conservative, so it give us a chance to design the primers at the end of the3’end and the5’end, according to the reference strain reported in NCBI, we design11pairs of primers for the virus’genome, and amplified the genome with RT-PCR assays, linked the8fragments to vector pMD18-T, and transfer to the DH5a cells. After the identification of the PCR of plasmids, we sent the recombinant plasmids to shanghai shenggong to determine the sequences of the virus genome. We have constructed the homology and the phylogenetic trees to analyses of H1N1Panda influenza a virus isolates from the world. As the result, A/Panda/Sichuan/01/2011(H1N1) is composed of13595bp and5’end and3’end is very conservative. Based on the phylogenetic analyses of H1N1Panda influenza virus, we found the virus genome is both closed to2009H1N1influenza virus strain.The future of reverse genetics to generate vaccine candidates is lactiferous. The influenza reverses genetics on plasmid DNA include two system12plasmids and8plasmids. The8plasmids system is a bi-directional two-expression construct that contains cDNA virus gene flanked by an RNA polymerase1(pol1) promoter for vRNA synthesis and an RNA polymerase Π (pol Π)promoter for mRNA synthesis. The key point is to screen influenza virus strain that is high yield on MDCK, and clone its whole genome. In this study, we amplified the virus genome cDNA and cloned the eight fragments to pMD18-T vectors, after digestion of BspQl, the fragments was sub-cloned into the vector pBD, then the eight plasmids based on the pBD vectors was constructed, then rescue the A/Panda/Sichuan/01/2011(H1N1).strain which have a high-growth property and the antigen characteristic of the circulating strain. Used HA and HI assays, the recombinant virus was detected, and the viruses also be detected in the electron microscope,The plasmid pM2HA construct expressing a fusion protein of M2joined to the N terminus of the HA could transcripe and translate. The special antibody of M2e of mouse immunized pM2HA show remarkable higher titer than pCI-M2immunized group, and almost no difference to pCl-HA immunized group in HI antibody but a slightly higher neutralization antibody but not significant. After challenged by lethal dose virus, pM2HA immunized group show a satisfied effectiveness according to body weight change and survival rate(100%) and own a good heterologous protecting capacity; After challenged by moderate dose virus, pM2HA group could alleviate lesion of lung compare to control group. It is that fusion HA and M2e provied a new method and ideal to research-develop a new type SI vaccine.更多还原